DIFFERENTIATION AND DEVELOPMENT OF THE BOVINE BLASTOCYST

By

VERÓNICA M. NEGRÓN-PÉREZ

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2017

© 2017 Verónica M. Negrón-Pérez

To my grandparents, Efraín & Nérida, Puca & Carmina, my parents, Mariano & Nerybelle and my siblings, Efraín, Mariano, Martín & Cecilia, for being my inspiration,

my teachers and my biggest cheerleaders

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ACKNOWLEDGMENTS

I am extremely grateful for all the people that were involved in the years I have

spent as a graduate student. I thank my mentor and advisor, Dr. Peter J. Hansen, for

giving me the opportunity to be part of his team and his academic family tree, and, for

all the positive feedback. Dr. Hansen’s outstanding mentoring allowed me to think more

critically, to grow as a scientist and to be a leader. I truly admire Dr. Hansen’s devotion

to teach, to ensure that we reach our goals and to maintain a big and productive lab

while keeping a positive environment. I also expanded my networks and met many of

the stars in reproduction because of Dr. Hansen and his connections. He is very

demanding but also ensures that he has time to meet his students on a weekly basis

and he saves time to socialize and enjoy the little things in life. I truly hope that I can be

as enthusiastic, dedicated and productive as he has been. I would also like to thank my

advisory committee members, Dr. John Driver, Dr. Stephanie Wohlgemuth, and Dr.

Paul Cooke, for their time, contributions and support throughout these four years. Also,

thanks to my forever mentors, Dr. Rocío Rivera, Dr. Esbal Jiménez and Dr. Melvin

Pagán, for teaching me from afar, being a phone call away and for believing in me. I still

keep in mind the statements “hang in there” and “go for the gold” from Rocío, who got

me through my master’s and touched bases with me every so often during my PhD.

I would like to express my gratitude to the University of Florida, Department of

Animal Sciences and the Animal Molecular and Cellular Biology Graduate Program for

allowing me to complete my doctoral degree. I thank Joan Fischer (in memorium),

Renee Parks, Pam Krueger and Joyce Hayen, the graduate advisors, secretaries and

friends, for their help, patience and advice. I also want to thank Sarah McLemore and

Dr. Tyisha Hathorn from the Office of Graduate Minority Programs for helping, advising

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and double checking on me every semester. Also, Charles E. Jackson and Dr.

Lawrence Morehouse from the McKnight Doctoral Fellowship Program of the Florida

Education Fund. Charles and Dr. Morehouse have an incredible, positive and

contagious vibe that reminded us, McKnight Fellows that no matter what was going on,

someone was there for us and everything was going to be alright. And, Dr. Sally K.

Williams, who is also a McKnight Alumni and a faculty at the Department of Animal

Sciences, because she would also make sure that I was doing alright in the department

and the program.

Thanks also to William Rembert and Eddie Cummings for ovary collection, and

thank owners and employees of Central Beef Packing Co. (Center Hill, FL), Adena Meat

Products L.P. (Fort McCoy, FL), and Florida Beef Inc. (Zolfo Springs, FL) for providing

ovaries. I am thankful for the assistance from personnel of the Interdisciplinary Center

for Biotechnology Research, University of Florida: Mei Zhang, Antoinette Noel and

Linda Green; Doug Smith and the McKnight Brain Institute Cell Tissue and Analysis

Core of the University of Florida; Pablo Ross of the University of California-Davis and

Joseph Kramer of the University of Florida; and Marc Rothenberg and Melissa Mingler

from the Division of Allergy and Immunology, Department of Pediatrics, Cincinnati

Children’s Hospital Medical Center. Without these collaborators, my research would

have not been possible.

Thank you to my previous and current lab mates for their help and friendship

during these years; Kyle Dobbs, Anna Denicol, Sofia Ortega, Paula Tríbulo, Jasmine

Khannampuzha-Francis, Antonio Ruiz, Adriana Zolini, Liz Jannaman, Gulnur

Jumatayeva, Eliab Estrada and William Ortiz. In addition, I want to thank the visiting

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professor, Khoboso Lehloenya, for allowing me to share my knowledge with her and,

visiting student, Luana Teixeira Rodrigues, for helping me with my research.

I specially thank Anna and Sofia for teaching me how things were done at the

beginning. And, I deeply thank Paula (my Argentinian twin) for always being so

optimistic, for being my study partner and for being there when things were difficult. I

also want to thank Jim Moss because he is probably the only lab tech that cares this

much for the students. Besides always being willing to help, Jim and Gail Moss opened

their house for us, allowed us to have pool parties to de-stress from the lab and took us

in when we needed a roof for a few days.

I am also appreciative for my extended family here in Gainesville, from the

department: Sossi Iacovides, Natalia Martínez, Rachel Piersanti, Fernanda Ferreira,

Renata Ramos, Marcos Zenobi and Eduardo Ribeiro. My roommates, study-mates and

partners in crime: Melissa Cruz, Lorena Maldonado, Claribel Nuñez and Oscar Paulin.

My close friends: Dorianmarie Vargas, Johnny Muñíz, Jose Vega, Carla Rodríguez,

Lorraine Martínez, Angie Rivera, Camilo Velez, Christian Rojas, Gabriel Miranda,

Glenda Díaz, Katy Otero and Rene Zamot, for all the dances, the dinners, the laughs,

the get togethers, the moral support and all the love which made me feel at home.

My friends and family from home also deserve my deepest thank you for being

there with me through so many moments. My childhood friends, Camila Espina, Carmen

Espina, Adriana Ramírez, Victoria Cano, Daniel Toraño, and Gaby Ortiz. My friends

from my time as a master’s student at Mizzou: Zhiyuan Chen and Angie Rost. For

reminding me of my strengths and cheering for me. My grandparents: Efraín Pérez,

Nérida Rosas, Puca Negrón and Carmen Delgado, my parents: Nerybelle Pérez and

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Mariano Negrón; and my siblings: Efraín, Mariano, Martín, and Cecilia Negrón Pérez.

For supporting me with life decisions, helping me understand and walking me through

hard times, for being a phone call away on the desperate nights, for welcoming home

with a warm plate of my favorite food, for inspiring me to always do better, for asking me

questions about what I do and for never giving up on me. I would have not made it this

far without your guidance, your love and support.

To all, thank you for the encouragement and for being part of my success.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES .......................................................................................................... 11

LIST OF FIGURES ........................................................................................................ 12

LIST OF ABBREVIATIONS ........................................................................................... 14

ABSTRACT ................................................................................................................... 16

CHAPTER

1 LITERATURE REVIEW .......................................................................................... 18

Introduction ............................................................................................................. 18

Overview of Preimplantation Development in the Cow ........................................... 21 Mechanisms for Key Events in Preimplantation Development ................................ 25

Embryonic Genome Activation ......................................................................... 25

Compaction and Polarization of the Morula ...................................................... 28

Formation of the Blastocyst - Introduction ........................................................ 31

Key Transcription Factors Involved in Formation of the ICM and TE ............... 32 Mouse Models to Explain How Cells in the Morula are Chosen for ICM or

TE ................................................................................................................. 35 Role of the Hippo Signaling Pathway in Formation of ICM and TE in the

Mouse Embryo .............................................................................................. 38

Role of FGF and its Receptor in Formation of the Hypoblast ........................... 40 Key Transcription Factors Involved in Differentiation of the ICM into Epiblast

and Hypoblast ............................................................................................... 41 Hatching From the Zona Pellucida ................................................................... 43

Goals and Significance of the Current Investigation ............................................... 45

2 ANALYSIS OF SINGLE-CELL GENE EXPRESSION OF EPIBLAST, HYPOBLAST AND TROPHECTODERM CELLS OF THE BLASTOCYST ............. 51

Introduction ............................................................................................................. 51 Materials and Methods............................................................................................ 54

In Vitro Production of Embryos ......................................................................... 54 Preparation of cDNA from Single Blastomeres ................................................. 56 Gene Expression Analysis ................................................................................ 58 Statistical Analysis ............................................................................................ 59

Results .................................................................................................................... 60

Identification of Cell Populations Using Cluster Analysis .................................. 60 Identification of Cell Subpopulations as Epiblast, Hypoblast, and TE ............... 60 Other Genes Overexpressed in Epiblast and Hypoblast .................................. 62

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Other Genes Overexpressed in All Subpopulations of TE ................................ 63 Other Genes Overexpressed in Some TE Subpopulations .............................. 63 Genes Whose Expression Did Not Differ Between Subpopulations ................. 64

Discussion .............................................................................................................. 64

3 ROLE OF YES-ASSOCIATED PROTEIN 1, ANGIOMOTIN AND MAP KINASE IN BLASTOCYST DEVELOPMENT IN THE PREIMPLANTATION EMBRYO ........ 89

Introduction ............................................................................................................. 89 Materials and Methods............................................................................................ 92

In Vitro Production of Bovine Embryos ............................................................. 92

Immunofluorescent Analysis of Embryos ......................................................... 93

RNA Isolation ................................................................................................... 95 Quantitative Real-time PCR (qPCR) ................................................................ 95 Gene Expression Analysis Using High Throughput RT-PCR ........................... 96 Experiment 1: Developmental Changes in Immunoreactive YAP1 and CDX2 . 97

Experiment 2: Inhibition of Interactions between YAP1 and TEAD4 ................ 98 Experiment 3: Knockdown of YAP1 .................................................................. 99 Experiment 4: Knockdown of AMOT .............................................................. 100

Experiment 5: Inhibition of MAP2K1/2 ............................................................ 100 Statistical Analysis .......................................................................................... 101

Results .................................................................................................................. 102

Experiment 1: Developmental Changes in YAP1 and CDX2 .......................... 102

Experiment 2: Inhibition of YAP1-TEAD Interactions by Treatment with Verteporfin ................................................................................................... 102

Experiment 3: YAP1 knockdown .................................................................... 103 Experiment 4: AMOT Knockdown .................................................................. 104

Experiment 5: Inhibition of the MAP2K1/2 ...................................................... 105

Discussion ............................................................................................................ 106

4 ROLE OF CC CYTOKINES IN SPATIAL ARRANGEMENT OF THE INNER CELL MASS OF THE BLASTOCYST ................................................................... 123

Introduction ........................................................................................................... 123

Materials and Methods.......................................................................................... 125 In Vitro Production of Embryos ....................................................................... 125 Developmental Changes in mRNA for CCL24, CCR3 and CCR5 .................. 126

Production of Antisera to CCL24 .................................................................... 128 Immunolocalization of CCL24 and CDX2 ....................................................... 129

Consequences of Inhibition of CCR3 for Localization of GATA6+ Cells in Hypoblast .................................................................................................... 130

Consequences of Knockdown of CCL24 for Localization of GATA6+ Cells in Hypoblast .................................................................................................... 132

Statistical Analysis .......................................................................................... 134

Results .................................................................................................................. 135 Developmental Changes in Expression of CCL24 in the Bovine Embryo ....... 135

Immunolocalization of CCL24 ......................................................................... 135

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Consequences of Inhibition of CCR3 for Localization of GATA6+ Cells in Hypoblast .................................................................................................... 136

Consequences of Knockdown of CCL24 for Localization of GATA6+ Cells in Hypoblast .................................................................................................... 136

Expression of CCR3 and CCR5 ..................................................................... 137

Discussion ............................................................................................................ 137

5 THE BOVINE EMBRYO HATCHES FROM THE ZONA PELLUCIDA THROUGH EITHER THE EMBRYONIC OR ABEMBRYONIC POLE .................. 151

Introduction ........................................................................................................... 151

Materials and Methods.......................................................................................... 153

In Vitro Production of Embryos ....................................................................... 153 Immunolocalization of Cells Labeled with Epiblast, Hypoblast and TE

Markers ....................................................................................................... 153 Identification of Cell Types and Embryonic Poles ........................................... 155

Statistical Analysis .......................................................................................... 156 Results .................................................................................................................. 156

Discussion ............................................................................................................ 157

6 GENERAL DISCUSSION ..................................................................................... 165

LIST OF REFERENCES ............................................................................................. 176

BIOGRAPHICAL SKETCH .......................................................................................... 203

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LIST OF TABLES

Table page 2-1 Information about genes selected for gene expression analysis ........................ 76

3-1 Effects of verteporfin on characteristics of blastocyst development in the bovine embryo .................................................................................................. 111

3-2 Consequences of YAP1 knockdown in the bovine embryo .............................. 112

3-3 Effects of AMOT knockdown on the bovine embryo ......................................... 113

3-4 Effects of treatment with MAP2K1/2 inhibitor on development of bovine embryos to the blastocyst stage ....................................................................... 114

5-1 Percent and frequency of embryos hatching from the embryonic or abembryonic pole at Days 7 and 8 ................................................................... 160

5-2 Proportion of hatched cells that were inner cell mass (ICM) and trophectoderm (TE) as affected by hatching pole ............................................. 161

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LIST OF FIGURES

Figure page 1-1 Polarization of the outer cells of the morula. ....................................................... 47

1-2 Overview of the three models proposed for the first lineage differentiation. ....... 48

1-3 Hippo signaling pathway in the mammalian embryo. .......................................... 49

1-4 Schematic representation of the second differentiation event in blastocysts to epiblast and hypoblast as controlled by interactions between FGF4 and FGFR2 activation of the MAPK pathway. ........................................................... 50

2-1 Identification of embryonic cell populations. ....................................................... 79

2-2 Suggested epiblast markers used to identify blastocyst cell populations.. .......... 80

2-3 Suggested hypoblast markers used to identify blastocyst cell populations. ........ 81

2-4 Suggested trophectoderm markers used to identify blastocyst cell populations. ........................................................................................................ 82

2-5 Additional epiblast and hypoblast differentially expressed genes. ...................... 83

2-6 Additional trophectoderm genes equally expressed in all four TE. ..................... 84

2-7 Trophectoderm-1 and trophectoderm-2 differentially expressed genes. ............. 85

2-8 Trophectoderm-3 and trophectoderm-4 differentially expressed genes. ............. 86

2-9 Transcripts whose expression was not significantly different among cell populations. ........................................................................................................ 87

2-10 Model for cell specific expression amongst cell populations. .............................. 88

3-1 Immunolocalization of CDX2 and YAP1 in the bovine oocyte and early embryo. ............................................................................................................ 115

3-2 Representative images of blastocysts in absence (vehicle) or presence of verteporfin from Days 5-9.5 of development. .................................................... 116

3-3 Amounts of YAP1 mRNA, CDX2, YAP1, GATA6 and NANOG as affected by treatment with YAP1 targeting GapmeR. .......................................................... 117

3-4 Knockdown of YAP1 alters gene expression of 23 transcripts in blastocysts at Day 8.5 of development as determined by quantitative real-time PCR data. 118

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3-5 Effect of AMOT knockdown on expression of genes associated with blastocyst differentiation of epiblast and hypoblast. .......................................... 119

3-6 Expression of genes in blastocyst at Day 8.5 of development that were not affected by YAP1 knockdown as determined by quantitative real-time PCR data. ................................................................................................................. 120

4-1 Schematic representation of methodology used for determination of number of cells located in inner and outer regions of the ICM. ...................................... 143

4-2 Developmental changes in CCL24 expression. ................................................ 144

4-3 Representative examples of patterns of immunoreactive CCL24 in the Day 7 and Day 8 blastocyst as determined by epiflourescent microscopy.. ................ 145

4-4 Differential immunolocalization of CCL24 in the ICM and TE of Day 7 and Day 8 blastocysts.. ........................................................................................... 146

4-5 Confocal z-stack projections of representative Day 8 blastocysts after inhibition of CCR3.. ........................................................................................... 147

4-6 Inhibition of CCR3 affects the location of GATA6+ cells at Day 8 of development.. ................................................................................................... 148

4-7 Confocal z-stack projections of representative Day 8 blastocysts as affected by morpholino treatment. .................................................................................. 149

4-8 Injection of a morpholino against CCL24 affects the location of GATA6+ cells. 150

5-1 Representative images of embryos hatching through the embryonic or abembryonic pole.. ........................................................................................... 162

5-2 Examples of immunolocalization of inner cell mass (ICM) and trophectoderm (TE) in blastocyst experiencing hatching. ......................................................... 163

5-3 Analysis of a blastocyst hatching through the embryonic pole using confocal microscopy.. ..................................................................................................... 164

6-1 Schematic representation of a new model for development of the bovine preimplantation embryo to the blastocyst stage of development.. .................... 174

6-2 Gene expression specific for epiblast, hypoblast and specific trophectoderm (TE) cell populations in the cow. ....................................................................... 175

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LIST OF ABBREVIATIONS

Official gene and protein symbols are used without definition following rules of

the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/gene/).

Exceptions are made for selected molecules where the name is spelled out (e.g. E-

cadherin; β-catenin) or where the gene symbol is defined at first use (i.e. E-cadherin;

Cdh1). Other abbreviations used in the dissertation are listed below.

5hmeC 5-hydroxymethylcytosine

5meC 5-methylcytosine

AJ Adherens junctions

AP Animal pole

AQP Aquaporin

ART Assisted Reproductive Technologies

BSA Bovine serum albumin

cDNA Complementary deoxyribonucleic acid

CSF2 Colony stimulating factor

DAPI 4’,6-Diamidino-2-phenylindole

DEPC Diethylpyrocarbonate

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DPBS Dulbecco’s phosphate-buffered saline

EGA Embryonic genome activation

Epi Epiblast

FITC Fluorescein isothiocyanate

GJ Gap junctions

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Hypo Hypoblast

ICM Inner cell mass

IgG Immunoglobulin G

IVF In vitro fertilized

IVP In vitro produced

MAPK Mitogen-activated protein kinase

Mural TE Mural trophectoderm

PVP Polyvinylpyrrolidone

qPCR Quantitative real time polymerase chain reaction

RNA Ribonucleic acid

SOF-BE2 Synthetic oviductal fluid-bovine embryo 2

TALP Tyrode’s albumin lactate pyruvate

TE Trophectoderm

TJ Tight junctions

VP Vegetal pole

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

DIFFERENTIATION AND DEVELOPMENT OF THE BOVINE BLASTOCYST

By

Verónica M. Negrón-Pérez

August 2017

Chair: Peter J. Hansen Major: Animal Molecular and Cellular Biology

Differentiation of the blastocyst into epiblast, hypoblast and trophectoderm (TE)

are key to establishment of the cell lineages that form fetal, extraembryonic endoderm

and placental tissues. The aims were to use the cow as a model to develop markers for

specific cell lineages in the blastocyst, understand the role of key molecules in

blastocyst differentiation, and gain understanding of the spatial orientation of the

processes for formation of the hypoblast and hatching from the zona pellucida. Using

RT-PCR of single blastomeres, gene markers were identified for epiblast (AJAP1,

DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2), hypoblast

(ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17) and TE

(ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN). Moreover,

TE contained four subpopulations varying in expression of multiple genes including the

TE markers IFNT and EOMES, suggesting that cells were at different degrees of

differentiation. In other experiments, it was found that blastocyst differentiation was

regulated by two proteins involved in Hippo signaling, YAP1 and AMOT, as well as by

the MAPK signaling pathway. Knockdown of either YAP1 or AMOT disrupted blastocyst

function as determined by number of TE cells, hatching from the zona pellucida and

17

gene expression. Moreover, knockdown of YAP1 or AMOT affected either number of

epiblast or hypoblast cells or expression of genes characteristic of these cells. Addition

of a MAPK inhibitor increased the number of epiblast cells without affecting numbers of

hypoblast cells. In other experiments, the chemokine CCL24 was identified as being

important for spatial organization of the hypoblast since addition of either receptor

antagonists or knockdown of CCL24 mRNA decreased the percent of GATA6+ cells

(i.e., hypoblast) that were located on the outer portion of the inner cell mass. Finally, it

was shown that the blastocyst has no preference for zona hatching through the

embryonic or the abembryonic pole. Overall, new markers for studying blastocyst

differentiation were identified, heterogeneity of the TE was established and

experimental evidence was obtained to show that members of the Hippo signaling

pathway, MAPK and selected chemokines play a role in regulating differentiation of the

bovine blastocyst.

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CHAPTER 1 LITERATURE REVIEW

Introduction

Understanding the processes controlling development of the preimplantation

embryo is important for optimization of reproductive function in cattle and for exploiting

new reproductive technologies. In cattle, there are four essential periods that are

associated with the majority of the embryonic loss the first trimester of gestation

(Wiltbank et al. 2016): 20-50% of the embryos are lost by Day 7 after insemination

(Sartori et al. 2010; Wiltbank et al. 2016) and nearly 25% of the embryos that are able to

reach the blastocyst stage die sometime during pregnancy and prior to calving (Hansen

2011). Moreover, about 30% of the embryonic losses occur between Days 8 and 27,

~12% are lost between Days 28-60 and nearly 2% of the embryos are lost between

Days 60-90 (Wiltbank et al. 2016). Thus, understanding the processes involved in

development to the blastocyst stage at Day 7 after insemination could lead to

improvements in fertility.

Early embryonic development is also important for setting up the developmental

program that affects phenotype of a new individual after birth. Alterations in maternal

environment during the preimplantation period can have effects on behavior,

cardiovascular function, growth rate, fatness and glucose homeostasis in a variety of

species (Kwong et al. 2000; Watkins et al. 2008; Williams et al. 2011). The

preimplantation period is also important for programming postnatal phenotype in cattle.

In one experiment, embryos produced in vitro using female sex-sorted semen were

treated with colony stimulating factor 2 (CSF2) or vehicle from Day 5 to Day 7 and then

transferred into recipient cows (Kannampuzha-Francis et al. 2015). Birth weight of the

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resultant calves did not differ between treatments and neither did weight in the first 3 mo

of life. Thereafter, though, the calves that were exposed to CSF2 during pre-

implantation development grew faster than the control calves.

Another example of developmental programming in the preimplantation period is

a study where the performance of cows that were produced using a series of assisted

reproductive technologies were evaluated (Siqueira et al. 2017). Embryos were

produced either by artificial insemination, superovulation, in vitro fertilization using

conventional (non-sexed) semen followed by embryo transfer, or in vitro fertilization

using female-reverse sex-sorted semen (sex-sorted upon thawing) followed by embryo

transfer. Treatments did not differ in weight at birth, had similar age at first calving and

had similar amount Days open to first lactation. However, the milk yield at first lactation

was lower for cows that were produced by in vitro fertilization using reverse sex-sorted

semen than for the other three groups. The same was true for the fat yield and protein

yield. In the same study, it was found that parity of the recipient had an effect on milk

production; when embryos were transferred into heifers, these cows produced less milk

than the cows that developed from embryos transferred into the uterus of a multiparous

female (Siqueira et al. 2017). Taken together, the environment of the preimplantation

embryo and the uterine environment throughout pregnancy can have effects on the

offspring that are expressed after birth.

It is also important to understand preimplantation development because of the

growing importance of assisted reproductive technologies (ART) in cattle. There has

been an increase in the use of ART in cattle over the last decade, with a 16.7%

increase in the number of embryos transferred from 2011 to 2013 (Perry 2014). Most

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importantly, the use of ART has been increasing to produce offspring with improved

genetic merit for economically-important traits (Thomasen et al. 2016; Kaniyamattam et

al. 2017). Embryo transfer can also be used to increase pregnancy rates in repeat-

breeder cows and cows exposed to heat stress (Hansen 2014). Unfortunately,

technologies used for ART are not fully optimized and embryos can experience a

number of abnormalities that cause embryonic death in utero. Compared to in vivo

derived embryos, embryos produced in vitro have abnormal gene expression (Corcoran

et al. 2006), altered lipid content (Crosier et al. 2001), abnormal methylation (Niemann

et al. 2010) and affected ultrastructure (Rizos et al. 2002). Most (60-70%) of the in vitro

produced (IVP) embryos die during the first week of development coincident with

blastocyst formation (Lonergan et al. 2006). It is possible that some of the abnormalities

lead to poor lineage specification that later result in embryonic death. Approximately 9%

of the embryos that are recovered by flushing the uterine horn, at Day 14, do not have

an epiblast (Berg et al. 2010) and 15-20% fail to form the embryonic disc (Fischer-

Brown et al. 2004; Block et al. 2007).

This literature review will be used to summarize current knowledge of

development of the mammalian embryo through the blastocyst stage of development.

Emphasis will be placed on mechanisms controlling the first and second lineage

differentiation events. Because it is the most studied model, the mouse will be used as a

prototype to develop concepts underpinning our understanding of preimplantation

development. Comparisons with embryonic development in the bovine will be pointed

out to emphasize similarities and differences between species and to provide current

knowledge of processes involved in preimplantation development of the cow. It should

21

be pointed out that, despite the fact that morphological characteristics of early

embryonic development are conserved in evolution, the mechanisms involved in

development have undergone significant evolutionary change. Although some genes

show similar functions in the cow embryos, there is divergence between species in

some of the fundamentally-important functions described. In a study in which embryonic

gene expression was compared between mouse, bovine and human embryos, it was

found that 40% of the genes expressed in all three species had species-specific

expression patterns (Xie et al. 2010). It was suggested that mutations in the promoter

genes were responsible for species divergence as well as consequences of relocation

of transposable elements. Thus, even though the mouse can serve as a framework for

understanding development in other species, it is still important to map out mechanisms

involved in preimplantation development in the particular species of interest.

Overview of Preimplantation Development in the Cow

The morphological characteristics of preimplantation development in the cow is

similar to that of other species. Fertilization of the matured oocyte in the upper third of

the oviduct (Hunter and Wilmut 1984) is followed by a series of cell divisions in which

daughter cells become progressively smaller while remaining totipotent until compaction

at the morula stage of development (Betteridge and Fléchon 1988; Van Soom et al.

1997). The embryo resides in the oviduct until Day 4-5 of development and then enters

the uterine lumen (Betteridge and Fléchon 1988). Compaction refers to the process

where the total volume of the embryo decreases and individual cell boundaries become

obscured. The process involves acquisition of tight junctions and development of

polarity in the outer cells of the embryo (Betteridge and Fléchon 1988; Koyama et al.

1994; Barcroft et al. 1998). Compaction takes places at about Day 5 (>32-cell stage) or

22

Day 6 when the embryo is about 32-64 cells (Betteridge and Fléchon 1988). The outer

cells differentiate into the trophectoderm (TE) that is the precursor of placental tissues

while the inner cells remain pluripotent and give rise to the inner cell mass (ICM).

Blastocyst formation, in which the embryo has acquired a fluid filled space called the

blastocoel and the TE and ICM can be readily identified, occurs by Day 7 of

development. Subsequently, the ICM undergoes a second differentiation event to form

the epiblast (precursor of fetal tissues) and hypoblast (precursor of extraembryonic

tissues) (Kuijk et al. 2012; Denicol et al. 2014). By Day 9, cells of the hypoblast migrate

to the periphery of the ICM to form a second epithelial-like layer of cells from which the

yolk sac and extraembryonic tissues will arise (Maddox-Hyttel et al. 2003).

At the blastocyst stage and approximately coincident with differentiation of ICM

into epiblast and hypoblast, the embryo escapes the zona pellucida. The bovine

blastocyst has been reported to hatch through the zona pellucida without a preference

through either the embryonic or abembryonic pole (Niimura et al. 2010). To promote

hatching, blastomeres secrete the urokinase-type plasminogen activator (PLAU) that

weakens the zona pellucida (Berg and Menino, Jr. 1992; Coates and Menino 1994). In

addition, the embryo undergoes cycles of expansion and contraction to exert

mechanical pressure to break the zona pellucida (Massip and Mulnard 1980; Massip et

al. 1982).

The bovine embryo does not implant immediately after hatching; instead, it floats

in the uterus for an additional 12 Days with apposition between trophoblast and

endometrial epithelium first occurring around Day 20 (King et al. 1981; Blomberg et al.

2008). Around the Days 8-10, the cell types in the embryo change position to prepare

23

the embryo for formation of the three germ layers. The epiblast and hypoblast cells

change from being scattered within the ICM to situation where the hypoblast forms an

inner lining below the epiblast and TE (Maddox-Hyttel et al. 2003). When both ends of

the hypoblast meet, the primitive yolk sac is formed. The TE cells continue to proliferate

during and after hatching and, overall, the embryo continues to grow in size and cell

number. From Days 9-12, the bovine embryo goes through a change in structure from a

sphere to an ovoid shape and from an ovoid to a tubular shape (Betteridge and Fléchon

1988; Maddox-Hyttel et al. 2003). Then, the trophectoderm grows massively and

elongates rapidly; the embryo can go from 0.5 mm at Day 8 to 160 mm by Day 16

(Betteridge and Fléchon 1988).

The portion of TE cells on top of the epiblast (i.e. polar TE or Rauber’s layer)

degenerates to expose the epiblast to the surface (Maddox-Hyttel et al. 2003). At the

onset of elongation (Day 12-14) the epiblast also rearranges from a circular to an ovoid

shape (Vejlsted et al. 2005). The epiblast together with the hypoblast are referred to as

the embryonic disk (Vejlsted et al. 2006a). On Days 14-16, endoderm and mesoderm

layers begin to form in the epiblast. To do this, an additional basal layer of cells is

formed, a subset of cells go through a process of ingression to form the mesoderm and

another subset of cells form the endoderm by displacing the hypoblast and are inserted

between the hypoblast (Maddox-Hyttel et al. 2003). On each side of the embryonic disk

the trophectoderm appears to fold on itself; these folds are referred to as amniotic folds

(Vejlsted et al. 2006a). This stage (~Day 14-16) of embryo development, as demarked

by the formation of the amniotic folds, mesoderm and endoderm, is referred to as the

primitive streak stage. Then, as these folds continue to grow and enclose the embryo

24

proper (i.e. embryonic disk plus amniotic folds), on the midline of the embryo proper, the

neural groove (Talbot et al. 2002) and the initial appearance of various organs can be

observed (Days 20-21) (Maddox-Hyttel et al. 2003; Vejlsted et al. 2006a). Coinciding

with the formation of the neural groove, the remaining cells of the epiblast proceed to

form the last of the three germ layers, the ectoderm (Vejlsted et al. 2006a). Before

neural groove formation endoderm, mesoderm and ectoderm cells are pluripotent as

indicated by POU5F1 expression but, after the neural groove formation, POU5F1

expression decreases for differentiation (Vejlsted et al. 2006b). Only a portion of cells,

the putative primordial germ cells, maintain pluripotency and expression of POU5F1

(Vejlsted et al. 2006a). After formation and extension of the neural groove from the

mesoderm, somites form and the embryo enters the somite stage. By this stage (Day

21), the only pluripotent cells are the primordial germ cells (Vejlsted et al. 2006b) which

migrate to the yolk sac and the hindgut (Vejlsted et al. 2006a). When primordial germ

cells migrate to the developing gonadal ridge, between Days 32-39 in the cow, sex

determination of the gonads is taking place (Erickson 1966).

The developing embryo is also in communication with the maternal endometrium

through secretion of proteins and cytokines. One of the main cytokines secreted from

the TE of the embryo is interferon-τ (IFNT) which blocks luteolysis and thus maintains

production of progesterone from the corpus luteum (Robinson et al. 2008). This cytokine

can be detected as early as Day 6 when the morula is compacting to later form a

blastocyst and levels of IFNT increase as the embryo grows in size (Kubisch et al.

1998). Around Day 20-22, in cattle, the elongating embryo attaches to the uterus

(Blomberg et al. 2008). Unlike other mammalian species with hemochorial placenta (i.e.

25

mouse and human), the bovine embryo does not fully invade the epithelium of the

endometrium to implant. Instead, the bovine embryos form an epitheliochorial placenta

(i.e. the TE does not penetrate the uterine epithelium) by having trophoblast embryonic

cells fuse with epithelial cells from the uterus. The bovine placenta diffuses through the

entire endometrium while other trophoblast cells (binucleate cells) migrate into the

uterine epithelium and fuse with maternal cells without breaking the basement

membrane (Wooding and Wathes 1980). This diffusion of cells is to increase surface

area and allow more efficient nutrient exchange between the cow and the fetus.

Mechanisms for Key Events in Preimplantation Development

The mechanisms involved in the processes leading to development of the

blastocyst have been worked out in most detail in the mouse. In the following sections,

key mechanisms required for successful development will be reviewed, using a

comparison of the mouse with the cow when data are available.

Embryonic Genome Activation

Following fertilization, the embryo spends a period of time when blastomeres are

transcriptionally silent and the embryo depends upon transcripts that were stored in the

oocyte before fertilization for new protein synthesis (Schier 2007; Fair 2010).

Maternally-derived transcripts are rapidly degraded in the embryo [see (Li et al. 2010)

for mouse and (Graf et al. 2014) for cow]. Embryonic genome activation (EGA) results

in newly synthesized transcripts from the embryonic genome being available for protein

synthesis. Embryonic genome activation occurs in two stages – a round minor of EGA,

where only a few genes are transcribed, and a second round of more global

transcription. The minor EGA occurs at the 1-cell stage in mice and 4-cell stage in cattle

while major EGA occurs at the 2-cell stage in mice (Schultz 1993; Wang and Dey 2006)

26

and 8-cell stage in cow (Vigneault et al. 2009; Graf et al. 2014). Many of the genes

initially transcribed are transcription factors required for major genome activation

(Vigneault et al. 2009).

The activation of embryonic transcription has been studied in depth in the mouse.

Synthesis of embryonic transcripts is activated by a series of mechanisms that include

degradation of maternal transcripts by loss of RNA masking proteins (Medvedev et al.

2008) and targeting of mRNAs by microRNAs (Bianchi and Sette 2011), epigenetic

changes in the chromatin structure that allows transcription in accessible regions, and

acquisition of transcription factors and other transcriptional machinery that favor EGA

(Bianchi and Sette 2011; Li et al. 2013a). Moreover, the transcripts that are initially

synthesized during EGA are part of the transcriptional machinery required for the major

genome activation (Stitzel and Seydoux 2007). One of the RNA masking proteins that is

lost is Msy2 [also known as Y box protein 2 (Ybx2) in cow]. This protein is synthesized

in the oocyte and binds specific populations of RNA to stabilize them; if Msy2 is

knocked down in the mouse oocyte, transcript abundance decreases (Medvedev et al.

2008). During EGA, Msy2 is phosphorylated and targeted for proteosomal degradation

and the maternal transcripts that were being stabilized by Msy2 decrease as well.

Simultaneous with the minor EGA, an increase in miRNAs is observed (Bianchi and

Sette 2011).

Epigenetic changes in chromatin structure also have a high influence in gene

expression [reviewed by (Messerschmidt et al. 2014)]. After fertilization and during

embryonic development, there are two rounds of DNA demethylation and re-methylation

that reset the DNA methylation marks in the developing embryo. The first round of

27

global DNA demethylation coincides with EGA at the 2-cell stage in the mouse embryo.

The DNA or paternal origin gets actively demethylated (Santos et al. 2002) by action of

Tet (tet methylcytosine dioxygenase) proteins (Gu et al. 2011) and independent of the

presence of Dnmt1 [maintenance DNA methyltransferase; DNA methyltransferase

(cytosine-5) 1]. The Tet proteins oxidize and convert 5-methylcytosine (5meC) to 5-

hydroxymethylcytosine (5hmeC) making it unrecognizable by Dnmt1. Meanwhile, the

maternal DNA remains methylated for a longer period due to passive demethylation in

absence of Dnmt1 (Monk et al. 1987, 1991). As a result of this round of DNA

demethylation, the promoter region of many genes now becomes accessible for

transcription during EGA [reviewed by (Messerschmidt et al. 2014)].

Chromatin accessibility or repression has also been linked to modifications in of

the core histones (Marlow 2010). Such modifications include acetylation, for example,

which would allow the zygote chromatin to be open for transcription when EGA is

occurring. Another example of a chromatin remodeler is the brahma-related gene-1

(Brg1), the catalytic subunit of the chromatin remodeling complex Swi/Snf

(Switch/Sucrose nonfermentable) (Marlow 2010). This complex allows for nucleosome

sliding along the DNA backbone and thereby change the regions of DNA that are

accessible for transcription. Experiments in mouse embryos have shown that knockout

of Brg1 is embryonic lethal (Bultman et al. 2006). When Brg1 is conditionally knocked

out in the oocyte, the resultant embryos cannot pass the 2-cell stage and EGA does not

occur (Bultman et al. 2006).

The cow seems to undergo similar changes for preparation of the transcriptional

machinery and changes in chromatin structure that are involved in EGA at the 4- cell

28

stage (minor EGA) and 8-cell stage (major EGA) (Vigneault et al. 2009). The TATA box

binding protein (TBP) transcript is part of the RNA polymerase II complex that serves as

an example in the bovine embryo as one of the transcripts that is initially synthesized

during the minor EGA and is required for the major genome activation (Vigneault et al.

2009). In bovine embryos, a number of miRNAs, including miR-130a and miR-21,

increase in abundance from the zygote to the 8-cell stage coinciding with EGA (Mondou

et al. 2012). Other overrepresented genes at the 8-cell stage in the cow are involved in

RNA processing, transcriptional regulators, protein biosynthesis, mitochondrial activity,

protein modification/transport and protein degradation (Graf et al. 2014). Using

immunofluorescence to quantify 5meC, Dobbs et al. (2013) observed a decrease in

methylation between the 2-cell and 6-8 cell stages before DNA methylation gradually

increased to the blastocyst stage. Similarly, the tri-methylation of lysine 27 in histone 3

(H3K27me3) that is associated with silencing of transcription is lost concurrent with

EGA and regained during blastocyst formation (Ross et al. 2008).

Compaction and Polarization of the Morula

At the onset of formation of the morula, all cells look alike, individual cell

boundaries are easily identifiable and all cells are totipotent (Chen et al. 2010).

Compaction, which begins at about the 8-cell stage in the mouse and 16 to 32-cell

stage in the cow, involves formation of tight junctions between outer cells of the morula

so that cells flatten and individual blastomeres are no longer distinguishable (Calarco

and Brown 1969; Ducibella et al. 1977). Following compaction, the outer cells become

polarized (Fleming and Pickering 1985) and outer and inner cells of the embryo are

exposed to different environments. Following the activation of cell polarization

complexes, the cell begins to form tight junctions on the apical domain which form an

29

impermeable seal between the outer cells. Also, the cells go through molecular changes

that lead to the loss of totipotency; the outer cells differentiate and the inner cells remain

pluripotent (Ducibella et al. 1975; Levy et al. 1986; Nikas et al. 1996; Suwińska et al.

2008). At the end of compaction, the blastomeres are connected through adherens

junctions (AJ, anchoring junctions), tight junctions (TJ, impermeable junctions), gap

junctions (GJ, communicating junctions) and desmosomes (Bell et al. 2008; Eckert and

Fleming 2008). As a result, the outer cells are exposed to one microenvironment and

the inner cells to another. The differences in polarity and the differences in

microenvironments are important for the first differentiation that results in the formation

of the first two cell types, the ICM and the TE (Fleming 1987; Watson and Barcroft

2001).

The cellular changes that are involved in compaction and polarization begin with

the relocalization of E-cadherin from the entire membrane to the basolateral domain

[reviewed by (Eckert and Fleming 2008)]. E-cadherin (also known as Cdh1) is a Ca2+-

ion dependent cell adhesive molecule and a component of adherens junctions found in

cell to cell contact sites (Larue et al. 1994; Riethmacher et al. 1995; Stephenson et al.

2010). As compaction is activated, mechanisms that trigger a change in cell polarity are

activated as well (Figure 1-1). The outer cells develop an apical pole facing the uterine

lumen and a basolateral domain between cells and facing the blastocoel [reviewed by

(Eckert and Fleming 2008)]. Overall, there is a change in the cytoskeleton of the cells

and the distribution of organelles in the cytoplasm: F-actin becomes localized to the

apical domain of the outer cells and the nucleus moves towards the basal domain. Also,

30

the cells acquire cuboidal shape similar to that of an epithelium and the apical surface

becomes rich in microvilli.

Formation of intercellular junctions occurs in a series of steps briefly described

(Bell et al. 2008; Eckert and Fleming 2008). By actions of atypical protein kinase C

(aPKC), E-cadherin relocates to the basolateral domain and α-catenin (also known as

CTNNA1) is modified to β-catenin (also known as CTNNB1) while forming the adherens

junctions. As the cells continue to flatten, the cell to cell contact area increases and the

Par complex (Par-3/ Par-6/aPKC and Cdc42) mediates the initiation of cellular

polarization and formation of tight junctions. Activation of Cdc42 is crucial for regulation

of actin cytoskeleton and cell polarization (Macara 2004; Wu et al. 2007). The proteins

of the Par complex become localized to different parts of the membrane to exert their

role as polarity regulators (Ahringer 2003; Macara 2004). Par-6 and aPKC are moved to

the apical pole and are colocalized with F-actin while Par-3 is initially also on the apical

domain but by the blastocyst stage is moved to the lateral domain where it is

colocalized with tight junctions. Immediately, zona occludens 1 (ZO-1α) and the

transmembrane occludins and claudins assemble to form the mature tight junctions in

between cells.

Concurrent with the formation of the apical and basolateral domain and the cell

junctions, Na+/K+-ATPase pumps are polarized to the basolateral membrane to facilitate

ion, amino acids and metabolite transport in the blastocyst (Wiley 1984; Donnay and

Leese 1999). Aquaporins (Aqp) are also polarized to the apical (Aqp9) or basolateral

(Aqp3 and Aqp8) domain (Offenberg et al. 2000; Barcroft et al. 2003) to promote water

influx into the blastocoel to maintain osmolarity (Donnay and Leese 1999; Watson and

31

Barcroft 2001). Meanwhile, coincident with tight junctions in the outer cells, gap

junctions (e.g. connexin-43) appear in inner cells (Becker and Davies 1995; Kidder and

Winterhager 2001). The role of these proteins has been confirmed with knockdown or

knockout experiments in the mouse embryo; in most cases, deletion of specific proteins

mentioned above prevented the embryo from becoming a blastocyst or surviving the

preimplantation period. For example, knockout of members of the Par complex result in

disrupted TE formation (Plusa et al. 2005; Hirate et al. 2013). Further, embryos lacking

E-cadherin are able to initiate the differentiation process to form ICM and TE but are not

able to form blastocysts (Larue et al. 1994; Riethmacher et al. 1995; Stephenson et al.

2010).

In the cow, compaction and polarization have not been studied so extensively.

Differences in distribution of microvilli have been observed for individual blastomeres of

16-cell stage embryos (Koyama et al. 1994), indicating that there are polarity

differences preceding compaction at the 32-cell stage. E-cadherin and β-catenin appear

to distributed unevenly throughout the cell membrane coinciding with compaction at the

32-cell stage (Betteridge and Fléchon 1988; Van Soom et al. 1997; Barcroft et al. 1998).

The role of E-cadherin in the bovine may not be crucial. Additionally, for embryos

derived in vivo, the marker for gap junctions, connexin-43 (also known as GJA1), is

detected from the zygote to the blastocyst but, in in vitro derived embryos, connexin-43

is undetectable at the morula and blastocyst stage (Wrenzycki et al. 1996).

Formation of the Blastocyst - Introduction

Mouse embryos reach the blastocyst stage around Day 3.5 (Arnold and

Robertson 2009) and the bovine embryo becomes a blastocyst at Day 7 (Betteridge and

Fléchon 1988). The blastocyst is composed of an ICM and a TE and, around 12-24 h

32

later, the ICM goes through the second lineage differentiation process to form the

epiblast and hypoblast (also known as primitive endoderm in the mouse) (Arnold and

Robertson 2009; Kuijk et al. 2012; Morris and Zernicka-Goetz 2012). There are a

number of transcription factors that are involved in signaling pathways that play a role in

both cell fate determination events. Moreover, there are three models that have been

linked to the first cell fate decision event and three additional hypotheses have been

proposed to understand how the hypoblast cells localize to the periphery of the ICM

after the second differentiation (Morris and Zernicka-Goetz 2012). The following

sections will develop ideas from the mouse regarding transcriptional factors and

hypothetical models for mechanisms guiding cell lineage commitment of the blastocyst.

Key Transcription Factors Involved in Formation of the ICM and TE

In the mouse several transcription factors have been identified as specific

regulators of one of the three cell lineages, epiblast, hypoblast and trophectoderm. The

main pluripotency promoting factors upregulated in the ICM and epiblast are Pou5f1,

Nanog and Sox2. The hypoblast differentiation promoting factors are Fgfr2, Gata6,

Sox17, Hnf4a and Pdgfra. Transcription factors involved in TE are Cdx2, Tead4, Gata3

and Eomes. Previous studies have described a role for most of these transcription

factors by evaluating the effects of gene knockdown or knockout on phenotypic

characteristics such as ICM and TE formation, abnormal localization in the membrane

of polarized proteins, and changes in localization of cytoplasmic or nuclear proteins in

specific cells (i.e. inner/outer) of the embryo.

During and after compaction, Cdx2 begins to appear in the nuclei of the outer

cells of the embryo and marks them for differentiation. Meanwhile, most of the cells of

the embryo express Pou5f1 to maintain pluripotency as the morula forms and cells lose

33

totipotency. After the first cell fate determination takes place and the ICM and TE are

formed, Pou5f1 is restricted to the ICM while Cdx2 is restricted to the TE. In studies

where Pou5f1 or Cdx2 have been eliminated, the embryos die around the time of

implantation (Le Bin et al. 2014; Jedrusik et al. 2015). As a result of the absence of

Pou5f1, the embryos are not able to form a proper epiblast (Le Bin et al. 2014). When

Cdx2 is knocked out, the mutant embryos form a blastocoel cavity but show abnormal

localization of tight junctions, increased apoptosis and the embryo is not able to

maintain the TE (Strumpf et al. 2005).

Working in conjunction with Cdx2 and Pou5f1 are Tead4 and Sox2. The

DNA/RNA binding and transcription factor Sox2 is another promoter of pluripotency.

Sox2 is present in the oocyte, decreases in amount during EGA and increases again at

the morula and blastocyst stage where it is restricted to the ICM (Avilion et al. 2003).

Sox2 can bind to the promoter regions of several genes including Pou5f1 (Lodato et al.

2013). Mutant embryos lacking Sox2 can reach the blastocyst stage and form an ICM

but are not able to develop further because the epiblast is not formed (Marikawa and

Alarcón 2012). Tead4 precedes Cdx2 expression and is required for Cdx2 transcription

and TE maintenance (Yagi et al. 2007). This transcription factor is expressed

throughout development and becomes limited to nuclei of the outer cells of the morula

and blastocyst stage embryo. Tead4 homozygous knockout embryos are unable to

implant, have aberrant or no TE differentiation (i.e. no CDX2 detected at Days 2.5-3.5)

and disappear by Day 6.5 (possibly due to reabsorption) (Yagi et al. 2007; Nishioka et

al. 2008).

34

Other factors participate in the TE differentiation process. Gata3 is expressed in

the early morula and promotes Cdx2 transcription (Home et al. 2009). In turn, Cdx2

reinforces Gata3 transcription to maintain the positive feedback of Cdx2 transcription

(Home et al. 2009). Eomes is involved in TE function after the TE forms; it controls TE

proliferation and is expressed highly in trophoblast stem cells (Probst and Arnold 2017).

When Eomes is knocked out, TE differentiation is absent or abnormal and embryos

cannot implant into the uterus (Russ et al. 2000).

Transcription factors involved in formation of the ICM and TE of the cow embryo

are conserved to a certain extent with the mouse but there are also important

differences. Most striking is the regulation of POU5F1 and CDX2. While CDX2 is a TE

marker in both species (Guo et al. 2010; Ozawa et al. 2012) as well as others (Kuijk et

al. 2008; Schuff et al. 2012; Yan et al. 2013), expression of the pluripotency gene

Pou5f1 is distinct from the mouse as compared to bovine and other species (Berg et al.

2011). The mouse embryo TE has acquired a regulatory element in the promoter region

that allows trophectoderm AP2 factor to represses Pou5f1 so that expression is limited

to ICM. In other species, the regulatory element is not found in the TE and high levels of

Pou5f1 can be detected in both the ICM and TE (Berg et al. 2011).

Other differences between the bovine and mouse embryo are genes that are

indispensable for development in the mouse but whose expression is not important for

development in the cow. SiRNA studies in which TEAD4 was decreased by 80% did not

affect the percent of embryos that became blastocysts, the number of CDX2+ cells or

the ration of ICM cells to TE cells (Sakurai et al. 2016). Similarly, decreasing zygotic

CDX2 does not inhibit blastocyst formation (Berg et al. 2011) but GATA3 expression is

35

decreased (Sakurai et al. 2016) and maintenance of TJ is negatively affected (Goissis

and Cibelli 2014) in embryos with decreased CDX2. Also, the embryos deficient for

CDX2 produce IFNT equal to that of the control counterparts but, are unable to elongate

(Berg et al. 2011). EOMES is poorly expressed or non-detectable in the bovine embryo

(Berg et al. 2011; Ozawa et al. 2012), suggesting that it is not required for bovine TE

differentiation and proliferation.

Mouse Models to Explain How Cells in the Morula are Chosen for ICM or TE

Three models have been proposed in the mouse to explain how cells are

destined to develop into ICM or TE as the embryo develops from a compact morula to a

differentiated blastocyst. These models have been termed the pre-patterning model

(Gardner 2001; Piotrowska et al. 2001; Piotrowska and Zernicka-Goetz 2002;

Piotrowska-Nitsche et al. 2005), the inside-out or positional model (Tarkowski and

Wróblewska 1967) and the polarity model (Johnson and Ziomek 1981; Jedrusik et al.

2008). Each of these models is illustrated diagrammatically in Figure 1-2.

The pre-patterning model posits that the oocyte cytoplasm is not uniform and that

the cell fate of each blastomere is determined beginning at the first cleavage after

fertilization by inheritance of molecules in the oocyte cytoplasm. The best data

supporting this model comes from the laboratory of Magdalena Zernicka-Goetz at

Cambridge. Work from this group indicates that the first cleavage division always occurs

meridional to the sperm entry point. After cell division, characteristics and fate of

subsequent blastomeres are distinct and depend on position relative to sperm entry and

plane of cleavage (Piotrowska and Zernicka-Goetz 2001; Piotrowska-Nitsche and

Zernicka-Goetz 2005; Piotrowska-Nitsche et al. 2005). Moreover, several epigenetic

markers were found to be different in each blastomere of the 4-cell embryo depending

36

on the cell origin relative to the position of sperm entry (Torres-Padilla et al. 2007). In

contrast, others who have analyzed single blastomeres of the early mouse and human

embryo found little variation in transcript abundance before compaction of the morula

(Guo et al. 2010; Lorthongpanich et al. 2012).

In contrast to the pre-patterning model, both the positional and polarity models

assume that all blastomeres are equivalent prior to differentiation. The proposition of

the positional model is that the position of each blastomere at the morula stage

influences the cell type it would become (Tarkowski and Wróblewska 1967). In

particular, different microenvironments caused by tight junctions between outer cells of

the morula cause outer cells of the morula to become TE and the inner cells to become

ICM. The hypothesis has been supported by studies showing that when marked inner

cells were placed on the outside of the embryo, recovered cells were differentiated; the

same was true vice-versa (i.e. when marked outer cells were placed on the inside of the

embryo, recovered cells were pluripotent) (Hillman et al. 1972; Suwińska et al. 2008).

The importance of junctional complexes in differentiation is indicated by experiments

showing that disruption of E-cadherin (required for tight junctions) did not prevent

embryos from initiating differentiation process since embryos displayed ICM- and TE-

like cells but the embryos failed to become blastocysts (Stephenson et al. 2010). Also,

another study involved a transcriptomic analysis of single cells after mechanical

dissociation of blastomeres at different stages from the 2-cell to the 32-cell stage. In the

late-morula (~32-cells), inner and outer cells remained in a non-differentiated state.

However, the cells that were located towards the outside showed several TE-like

transcripts suggesting that the cells were preparing for future differentiation. Thus,

37

results supported the hypothesis that the position of the blastomere influences the cell-

fate decision (Lorthongpanich et al. 2012).

The polarity model proposes that it is the polarization of the outer cells that

directs their fate to the TE lineage (Johnson and Ziomek 1981; Jedrusik et al. 2008).

Polarization of the outer cells in the mouse embryo occurs at the 8-cell stage and the

fate of their daughter cells depend on the orientation of the division axis. If the division

axis is parallel to the polarity axis, symmetric division occurs, both daughter cells share

similar polarity features, and both become part of the TE. If, on the contrary, the division

axis is perpendicular to the polarity axis, then an asymmetric division occurs, one

daughter cell would become polarized and become part of the TE while the other, non-

polarized, cell would become part of the ICM (Ducibella and Anderson 1975).

According to the polarity model, the apical and basal domain of outer cells play

important roles in differentiation. Four of the main proteins involved in cell polarity

arrangement are F2r [also known as Par1], Pard3, Pard6a/b and aPKC. Recent studies

show that these proteins are also crucial for the formation of the TE as differentiation is

disrupted in knockout embryos (Plusa et al. 2005). The Par1 is localized on the basal

domain while the Pard3-Pard6-aPKC complex is located on the apical domain after

polarization in TE cells. In Pard6 knockout embryos, CDX2 was lower and, in Par1

knockout embryos, AMOT was found in the basolateral domain instead of polarized to

the apical domain (Hirate et al. 2013), thus disrupting differentiation. These results

support the hypothesis that polarity plays an important role in the first cellular

differentiation.

38

Role of the Hippo Signaling Pathway in Formation of ICM and TE in the Mouse Embryo

The Hippo signaling pathway was originally described for its role in determination

of organ size through regulation of cell proliferation and apoptosis [reviewed by (Chan et

al. 2011)]. The pathway, which is illustrated in Figure 1-3, received its name because of

the protein kinase Hippo in Drosophila (homolog of MST1/2 in mammals). The Hippo

pathway was first studied in Drosophila in 1995 after a mutation of warts (Wts, homolog

of mammalian Lats) caused abnormal tissue growth (Justice et al. 1995; Xu et al. 1995).

Later Hippo signaling was characterized in mouse (Paramasivam et al. 2011; Hirate et

al. 2013) and is now known to be highly conserved among species (Varelas 2014). In

addition to playing roles in cell proliferation and apoptosis, experiments in mice indicate

the pathway is involved in differentiation of ICM and TE (Basu et al. 2003; Zhao et al.

2007; Lei et al. 2008; Heallen et al. 2011; Lorthongpanich and Issaragrisil 2015).

The most studied components of the Hippo pathway are the kinases, Mst1/2 and

Lats1/2, which are involved in inactivating the downstream target Yap1 (Nishioka et al.

2009; Oh et al. 2009; Song et al. 2010; Li et al. 2013b). However, there are other

components that have been recently characterized as mediators of the Hippo pathway.

These mediators include the Pard3-Pard6-aPKC complex (Wells et al. 2006; Robinson

et al. 2010; Sun and Irvine 2011; Hirate et al. 2013), E-cadherin (Stephenson et al.

2010), Amot (Ernkvist et al. 2006, 2009), Amotl2 (Ernkvist et al. 2006, 2009), Nf2

(merlin) (Gladden et al. 2010; Zhang et al. 2010; Chunling et al. 2011), Kibra (also

known as Wwc1; WW and C2 domain containing 1) (Xiao et al. 2011), Tead4 (Nishioka

et al. 2009; Home et al. 2012) and regulators of cell polarity such as Rho-Rock (Ernkvist

39

et al. 2009; Kono et al. 2014), Crb1 (Robinson et al. 2010; Varelas et al. 2010), and

scribbled planar cell polarity protein (Scrib) (Verghese et al. 2012; Mohseni et al. 2014).

Activation of the pathway is initiated by cell-to-cell contact which activates Kibra

and Nf2. These molecules in turn act together to phosphorylate Lats1/2. In parallel,

Mst1 and Mst2 are activated by unknown mechanisms and induce additional

phosphorylation of Lats1/2 (Li et al. 2013b). Lats1/2 phosphorylation actives proteins

that phosphorylate Yap1 at S127 to result in cytoplasmic retention by a protein first

called 14-3-3 and now referred to as stratifin (Sfn). An additional phosphorylation at

S381 causes Yap1 to be targeted for proteosomal degradation (Basu et al. 2003; Zhao

et al. 2010). In addition, Lats1/2 phosphorylates Amot at S176 and, phosphorylated

Amot strongly interacts with Lats1/2 for additional activation of Lats1/2 and inactivation

of Yap1 (Paramasivam et al. 2011; Hirate et al. 2013). Amot also plays a role in

inactivating Yap1 by binding to Yap1 when the Amot is associated with adherens

junctions through interactions with E-cadherin and Nf2 (Leung and Zernicka-Goetz

2013).

Phospho-Yap1 cannot enter the nucleus of the cell (Basu et al. 2003; Zhao et al.

2010) so that Yap1 genes promoting TE are not expressed and so, instead, genes that

promote pluripotency are transcribed (Lian et al. 2010). In the mouse embryo, inhibition

of Yap1 occurs in the inner cells of the developing embryo, hence all ICM cells remain

pluripotent (Lorthongpanich et al. 2012). When the Hippo signaling pathway is

inactivated in outer cells of the embryo, Yap1 is not phosphorylated and, instead it is

translocated into the nucleus where it associates with members of the TEAD family,

mainly Tead4, and promotes transcription of anti-apoptotic genes and cellular

40

differentiation genes (Nishioka et al. 2008, 2009). Inactivation of Hippo signaling in outer

cells is due, to loss of gap junctions (Ducibella and Anderson 1975; Lo and Gilula 1979;

Togashi et al. 2015) as well as polarization of outer blastomeres. Polarization results in

asymmetric distributions of several proteins and in transporting Amot to bind to F-acting

filaments so that Yap1 remains active (non-phosphorylated) and can go in the nucleus

(Hirate et al. 2013).

Role of FGF and its Receptor in Formation of the Hypoblast

In the mouse embryo, activation of the MAPK pathway by actions of FGF4 on

Fgfr2 in cells destined to be hypoblast is responsible for differentiation of ICM cells to

hypoblast. This pathway is illustrated in Figure 1-4. The first cells that occupy the ICM

highly express Fgf4 while the daughter cells (i.e. cells that enter the ICM in the next

cellular division) that are derived from this original group of cells express Fgfr2 (Morris

et al. 2010, 2013; Yamanaka et al. 2010; Kang et al. 2013). Activation of the Fgfr2 and

the MAPK pathway leads to transcription of the transcription factor Gata6 by the group

of Fgfr2+ cells. In these cells, Gata6 blocks expression of the pluripotency-promoting

transcription factor, Nanog and cells differentiate into hypoblast (i.e. primitive

endoderm). Those cells which express high amounts of Fgf4 become committed to the

pluripotent epiblast lineage due to low MAPK activity and low Gata6 expression in

conjunction with high Nanog expression (Morris et al. 2010).

Like for the mouse, inhibitor studies in bovine embryos indicate that the MAPK

pathway plays a role in regulating epiblast and hypoblast formation in a similar manner

to the mouse (Kuijk et al. 2012; Brinkhof et al. 2015). When bovine embryos are treated

with a MAPK inhibitor, cell differentiation is skewed to favor the epiblast and number of

cells positive for NANOG increases. Furthermore, treatment of embryos with FGF4 and

41

heparin (cofactor for FGF4 activation) or FGF2, the ICM differentiates to favor the

hypoblast (Yang et al. 2011; Kuijk et al. 2012). This observation and, the fact that cells

positive for GATA6 appear early in the blastocyst TE as well as the hypoblast (Kuijk et

al. 2012; Denicol et al. 2014), suggests that the mechanism regulating epiblast and

hypoblast formation involves the FGF4-FGFR2 and MAPK pathway in combination with

an unknown pathway or regulator.

Key Transcription Factors Involved in Differentiation of the ICM into Epiblast and Hypoblast

In the mouse, the formation of the epiblast and hypoblast from precursor cells in

the ICM is guided by activation of Fgf4 and Fgfr2, activation of the MAPK pathway and

nuclear Gata6 and Nanog (Lanner and Rossant 2010). Briefly, expression of nuclear

Gata6 or Nanog is regulated by Fgf4 interaction with Fgfr2 which are expressed in the

epiblast and hypoblast, respectively. Binding of Fgf4-Fgfr2 in precursor cells of

hypoblast activates the MAPK pathway which results in nuclear Gata6 expression and

downregulation of Nanog. In precursor cells of the epiblast, Fgfr2 is not present, the

MAPK pathway is not activated and, as a result, Nanog is transcribed to promote

pluripotency of the epiblast (Morris et al. 2010, 2013). Embryos lacking Fgf4 or Nanog

(Feldman et al. 1995; Mitsui et al. 2003) or Fgfr2 or Gata6 (Arman et al. 1998; Morrisey

et al. 1998; Koutsourakis et al. 1999) die during mid-gestation.

Other markers for hypoblast differentiation in the mouse are Sox17, Pdgfra,

Hnf4a and Gata4. These four interact with each other to favor the formation of the

hypoblast. Proteins of the HNF family are required for hypoblast formation in the mouse

(Duncan et al. 1994; Artus et al. 2011). Hnf4a is detected in the primitive endoderm and

yolk sac from Days 5.5 – 8.5 and embryos lacking Hnf4a die prior to birth due to

42

abnormal gastrulation (Chen et al. 1994). Pdgfra is one of the earliest markers for

hypoblast (Plusa et al. 2008; Artus et al. 2010) and mutant embryos without Pdgfra die

during mid-gestation as a result of underdeveloped lungs (Boström et al. 1996). Sox17

is a transcription factor involved in hematopoiesis (Kim et al. 2007) and vascularization

(Matsui et al. 2006; Sakamoto et al. 2007), and, thus, when absent, mouse embryos die

at Day 10.5. Gata4 is a later marker of the hypoblast (Frankenberg et al. 2011) and,

when knocked out, embryos are not able to develop post-implantation (Xenopoulos et

al. 2012).

In cow embryos, NANOG and GATA6 are epiblast and hypoblast makers,

respectively (Kuijk et al. 2012) but the mechanism of transcription activation for NANOG

and GATA6 differs slightly from that in the mouse. When single cells of ~64-cell

blastocysts are observed, Nanog expression is specific for epiblast and low in hypoblast

and, Gata6 is specific for hypoblast and absent in epiblast (Guo et al. 2010). This

agrees with the direct block of Nanog when Gata6 is present and vice-versa (Morris et

al. 2010). We have observed in our laboratory that NANOG expression in bovine

embryos fluctuates along with embryo differentiation, being high in the morula, low in

the early blastocyst and increasing again in the late blastocyst (Denicol et al. 2015).

Moreover, we have observed that GATA6 expression actually decreases with

differentiation. Immunoreactive GATA6 can be detected in the TE of blastocysts that are

negative for NANOG but, after epiblast and hypoblast differentiation, the intensity of

GATA6 detection is weak in TE and strong in hypoblast cells (Denicol et al. 2014). This

suggests that regulation might differ from the mouse, and it is still unclear how NANOG

and GATA6 are regulated in the bovine embryo. As mentioned in the section above, it is

43

likely that the FGF and the FGF receptor pathway are involved in regulation of epiblast

and hypoblast but, possibly an unknown pathway is also regulating transcription of

GATA6 and NANOG.

Hatching From the Zona Pellucida

The process that the embryo goes through to exit the zona pellucida is known as

hatching and was first described by observing rabbit embryos (Lewis and Gregory

1929). Embryo hatching is a crucial event for increasing embryo-uterus communication,

for establishment of the first physical contact of the embryonic trophectoderm with the

endometrium and for establishment of pregnancy (Gonzales and Bavister 1995).

Studies in human (Carney et al. 2012) and bovine (Taniyama et al. 2011) embryos have

shown assisted hatching in vitro can improve pregnancy rates after embryo transfer.

The duration of blastocyst hatching is approximately the same for smaller

(mouse, hamster and rabbits; 8-12 h) and larger species (pig, human and cow; 24-36 h)

but the timing of hatching is species dependent: mouse Day 4 – 4.5; (Cole 1967; Hurst

and MacFarlene 1981; Sawada et al. 1990), hamster Day 3 – 4 (Gonzales and Bavister

1995; Mishra and Seshagiri 2000), rabbit Day 4 – 5 (Lewis and Gregory 1929), pig Day

6 – 7 (Yoshida et al. 1990), human Day 5.5 – 6.5 (Cohen et al. 1990; Sathananthan et

al. 2003; Iwata et al. 2014) and cow Day 8 – 10 (Fléchon and Renard 1978; Berg and

Menino, Jr. 1992; Niimura et al. 2010).

Three mechanisms have been implicated in hatching: mechanical actions of the

embryo on the zona pellucida (Cole 1967; Massip and Mulnard 1980; Massip et al.

1982), release of proteolytic enzymes from the embryo (Sawada et al. 1990; Berg and

Menino, Jr. 1992; Mishra and Seshagiri 2000) to weaken the zona pellucida at a specific

point through which the embryo will escape, and protrusion of TE cells that penetrate

44

the zona pellucida (Gonzales et al. 1996; Seshagiri et al. 2009). Blastocysts of many

mammalian species including bovine (Massip and Mulnard 1980; Massip et al. 1982),

mouse (Cole 1967) and hamster (Kane and Bavister 1988) exhibit a series of blastocoel

expansions and contractions that apply mechanical pressure on the zona pellucida. The

importance of this mechanism is not fully understood because in hamster, for example,

the blastocyst shrinks prior to hatching instead of expanding (Seshagiri et al. 2009). The

proteolytic enzymes that weaken the zona pellucida differ among species. Mouse

embryos secrete a trypsin-like enzyme, serine 28 (Prss28, a.k.a. Isp1) (Perona and

Wassarman 1986; Sawada et al. 1990; O’Sullivan et al. 2001), hamster embryos

produce a number of cathepsins (Mishra and Seshagiri 2000) and bovine embryos

secrete PLAU (Berg and Menino, Jr. 1992; Coates and Menino 1994). Although these

enzymes may be produced in the ICM and the TE, in the mouse embryo, Isp1 was

localized to cells on the abembryonic pole (opposite to the ICM) (Perona and

Wassarman 1986; Sawada et al. 1990; O’Sullivan et al. 2001). Results from RNA-seq

experiments indicate PLAU is expressed more in the TE when compared to the ICM

(Ozawa et al. 2012; Nagatomo et al. 2013).

In cattle, hatching can occur from either of the poles of the embryo, i.e.,

embryonic pole, adjacent to the ICM, or abembryonic pole, opposite to the ICM

(Gonzales et al. 1996; Niimura et al. 2010). In mice (Perona and Wassarman 1986)

and human (Sathananthan et al. 2003), in contrast, occurs mainly through the mural

trophectoderm (mural TE). It has been hypothesized that hatching occurs through the

embryonic pole because this is where implantation occurs in these species (Qi et al.

2014). The embryo implants with the ICM facing the uterine lumen and the first cells to

45

invade the uterus are those that arose from the mural TE (Dickson 1963; Copp 1978). In

the bovine, the embryo is free in the uterus until Day 20 after fertilization (King et al.

1981) and thus, the hatching pole may not be as crucial.

Goals and Significance of the Current Investigation

To date, there is a gap in knowledge on the mechanisms used by the bovine

embryo to dictate cell fate in the early embryo and due to differences between species

(Berg et al. 2011; Denicol et al. 2014, 2015), it is uncertain if the markers involved are

the same as those in mouse development. Additionally, it remains unknown how the

bovine embryo escapes the zona pellucida and how this can be linked to the type of

placentation. This dissertation is focused on understanding key biological events in the

developing blastocyst.

The first experiment described, in Chapter 2, was performed to identify

transcripts that are overexpressed in epiblast, hypoblast and TE subpopulations of the

blastocyst. The goal was to identify the cell populations present in the bovine blastocyst,

identify new cell type specific genes and to understand how differential gene expression

of key genes could be involved in regulate cell differentiation. This is the first experiment

where epiblast and hypoblast gene markers were identified in the bovine. This

information can be used to study more in depth how the hypoblast and TE form and

develop, and to elucidate if the TE forms all at once or if it is a gradual process.

In Chapter 3, experiments were conducted to study if the Hippo signaling

pathway is involved in the first two differentiation events in the bovine blastocyst. It was

hypothesized that, like in the mouse, the Hippo pathway is inactivated in cells destined

to form TE and hypoblast For Chapter 4, it was tested whether chemokines participate

46

in differentiation and production of the hypoblast. Previously, it was observed that

CCL24, which is involved in cell migration (White et al. 2000; Provost et al. 2013),

CCL24, is upregulated in ICM during Days 7-9 of development (Ozawa et al. 2012;

Nagatomo et al. 2013; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al. 2016).

Because formation of the hypoblast involves movement of Gata6+ cells to the edge of

the ICM to form an epithelial layer (Maddox-Hyttel et al. 2003), it was hypothesized that

CCL24 is involved in controlling spatial distribution of the epiblast and hypoblast. Finally,

molecular markers were used for an experiment in Chapter 5 to described how the

bovine blastocyst escapes the zona pellucida and whether, as found previously

(Niimura et al. 2010), there is no preference for hatching to occur from a particular

embryonic pole. One outcome of the study was identification of the cells which first

escape the zona pellucida. These cells would be the first to come in physical contact

with the uterine endometrium and is possible that they could modify endometrial gene

expression as has been suggested (Lonergan and Forde 2014; Gómez and Muñoz

2015).

47

Figure 1-1. Polarization of the outer cells of the morula. After compaction, the outer cells change shape to become cuboidal and develop an apical and basolateral domain. The organelles are shifted to one of the sides of the cell; in this representation the nucleus is towards the basal domain. The apical domain is characterized with movement of F-actin and development of microvilli on the surface facing the perivitelline space. The Par complex (PAR6-αPKC-Par3) becomes active as a result of CDC42 and RHO-ROCK signaling to trigger cell polarization activation and formation of an apical domain. Adherens junctions (anchoring junctions composed of cadherins and catenins) and tight junctions (composed of claudins) form between cells and actin filaments are rearranged. Then, ion channels, amino acid (aa) transporters and aquaporins (AQP3, AQP8) are localized to the basolateral domain (except AQP9 which is localized on the apical domain) of the cells facing the blastocoel cavity to pump water and produce the energy required for blastocyst formation.

48

Figure 1-2. Overview of the three models proposed for the first lineage differentiation. (A) The pre-patterning model proposes that there is unequal distribution of molecules in the zygote so that, as early as the 2-cell stage, the daughter cells from one pole (animal pole, AV) are destined to become the inner cell mass (ICM) and the daughter cells from the other pole (vegetal pole; VP) are destined to become part of the trophectoderm (TE). (B) The inside-out model posits that all cells are equivalent in the early embryo but that compaction of the morula causes cells on the outside to become TE and the cells towards the inside to become the ICM. (C) The cell polarity model is similar except that it is proposed that outer cells of the morula polarize to develop apical and basal domains. Depending on the division plane and the symmetry of the cell division, either one daughter cell becomes ICM and the other becomes TE, or else, both daughter cells become TE.

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Figure 1-3. Hippo signaling pathway in the mammalian embryo. Top- shows a

trophectoderm cell with the Hippo pathway inactive. The presence of tight junctions (TJ, characterized by claudins) and lack of E-cadherin (E-cad) and cell-to-cell communication through gap junctions result in inactivation of kinases. Accordingly, YAP1 remains unphosphorylated and active so that YAP1 enters the nucleus and associates with TEAD4 to promote CDX2 transcription and differentiation of TE. This phenotype is strengthened by transport of AMOT by the PAR complex to the apical domain where it is restrained by F-actin. Then, unphosphorylated and inactive AMOT allow YAP1 to remain unphosphorylated. Bottom- shows an inner cell mass cell with the active Hippo pathway. The kinases NF2 and KIBRA activate and phosphorylate STK 1/2 and LATS 1/2 kinases; these latter two then phosphorylate YAP1. Phosphorylated YAP1 is restrained in the cytoplasm by 14-3-3 or targeted for degradation. Thus, YAP1 is unable to enter the nucleus and transcription of pluripotent genes, SOX2 and POU5F1 is maintained. AMOT is phosphorylated and activated by NF2 and LATS 1/2, and coupled with E-cadherin throughout the membrane. Active AMOT further phosphorylates and inhibits YAP1 to further maintenance of pluripotency. AJ= adherens junctions characterized by cadherins and catenins. GJ= gap junctions characterized by connexins. Arrows indicate activation direction, white boxes are active proteins, gray boxes are inactive proteins, purple circles with a P indicate phosphorylation and grouped proteins represent formed complexes or restrained proteins.

50

Figure 1-4. Schematic representation of the second differentiation event in blastocysts to epiblast and hypoblast as controlled by interactions between FGF4 and FGFR2 activation of the MAPK pathway. (A) The left panel shows how in the wave of division of the 8-16 cell embryo most cells express FGF4 but, for division of 16-32 cell embryos a subset of daughter cells of the first division begin to express FGFR2. Then, secretion of FGF4 from neighboring cells activates FGFR2 on the hypoblast precursor cells and this in turn activates the MAPK pathway (bottom panel A). This results in upregulation of NANOG in epiblast precursor cells and upregulation of GATA6 in hypoblast precursor cells. (B) In the late blastocyst, hypoblast cells move to line the epiblast near the blastocoele. At this time, epiblast is characterized by expression of FGF4 and NANOG and hypoblast is characterized by expression of FGFR2, GATA6, SOX17 and PDGFRA.

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CHAPTER 2 ANALYSIS OF SINGLE-CELL GENE EXPRESSION OF EPIBLAST, HYPOBLAST AND

TROPHECTODERM CELLS OF THE BLASTOCYST

Introduction

Preimplantation embryonic development involves a series of cleavage divisions

and cell differentiation processes that lead to the formation of the three primary cell

types in the embryo: epiblast (precursor of fetus), hypoblast (precursor of

extraembryonic endoderm including yolk sac) and trophectoderm (precursor of

placenta) (Arnold and Robertson 2009). Upon compaction of the morula, the outer cells

become polarized and mechanisms for tight junction formation are triggered (Levy et al.

1986; Nikas et al. 1996; Van Soom et al. 1997). As a consequence, the outer cells

begin to differentiate into TE cells while the cells of the ICM maintain pluripotency and

cell-to-cell communication through gap junctions (Ducibella and Anderson 1975; Lo and

Gilula 1979; Togashi et al. 2015). In the mouse, this first lineage differentiation event is

regulated in part by the Hippo signaling pathway which maintains pluripotency of the

cells when turned on (i.e., in the ICM) or favors differentiation of the cells when turned

off (i.e., in the TE cells) (Paramasivam et al. 2011; Hirate et al. 2013). The presence of

gap junctions in the ICM allows activation of a phosphorylation cascade that activates

Nf2, Mst1 and Lats1/2. The last molecule in this cascade phosphorylates and

inactivates Yap1to favor activation of pluripotency factors such as Pou5f1 (Lian et al.

2010) and Sox2 (Lorthongpanich et al. 2012). In the absence of gap junctions in the

outer cells of the embryo, the phosphorylation cascade is inactive, Yap1 remains

dephosphorylated and the protein undergoes nuclear translocation and interaction with

Tead4 to promote transcription of differentiation promoting factors such as Cdx2 and

Gata3 (Nishioka et al. 2008; Ralston et al. 2010). An additional player of the Hippo

52

pathway is Amot, which inactivated Yap1 by interacting with Yap1 and membrane

bound E-cadherin to prevent nuclear translocation of Yap1. In the outer cells of the

embryo, Amot is restricted to the apical domain of cells by binding to F-actin and is

thereby unable to inactivate Yap1 (Hirate et al. 2013; Leung and Zernicka-Goetz 2013).

Transcription factors involved in later TE differentiation include Elf5 (Ng et al. 2008;

Pearton et al. 2011) and Eomes (Russ et al. 2000).

As the blastocyst continues to develop, the ICM differentiates into the epiblast

and hypoblast. This second differentiation event in the mouse is accomplished in part

through activation of the Fgf4-Fgfr2 pathway which regulates transcription of Gata6 and

Nanog (Kang et al. 2013). It is thought that the first group of cells to enter the ICM

express Fgf4 but lack Fgfr2 so that expression of Nanog is maintained (Morris et al.

2010, 2013). The cells from the second round of cell division in the ICM begin to

express Fgfr2 while Fgf4 from neighboring cells activates Fgf4-Fgfr2 signaling to induce

expression of Gata6 and repression of Nanog. Thus, the first group of cells become

precursors of the epiblast and the second group are precursors of the hypoblast (Morris

et al. 2010, 2013).

The processes for formation and early differentiation of the blastocyst have been

well described in the mouse but not in other species. Indeed, mechanisms for

development during the preimplantation period are not completely conserved between

mammalian species. There are large-scale species differences in orthologous gene

expression patterns during the preimplantation period caused in large part to mutations

in transcription factor binding sites and insertion of transposons containing cis-

regulatory elements (Xie et al. 2010). Among the resultant differences in regulatory

53

processes between three of the most studied species – mouse, bovine and human –

include the use of the transcription factor CDX2 to downregulate the pluripotency factor

Oct4 (i.e., Pou5f1) in ICM in mice but not the other species (Berg et al. 2011) and the

role of CCL24 in movement of hypoblast cells in the bovine but not mouse (Chapter 4).

Understanding the mechanisms for differentiation and function of cell lineages in

the bovine embryo has been hampered by the limited repertoire of markers for each cell

type. The only marker identified for epiblast is NANOG (Kuijk et al. 2012; Denicol et al.

2014). One marker for ICM in mouse, Pou5f1 is expressed by both ICM and TE in

bovine (Berg et al. 2011; Ozawa et al. 2012). GATA6 can serve as a marker for

hypoblast in bovine (Kuijk et al. 2012; Denicol et al. 2014) but interpretation of

differences in gene expression for this transcription factor is complicated by the fact that

expression can also occur for TE (Kuijk et al. 2012; Denicol et al. 2014). The most

commonly used marker for TE in bovine is CDX2 (Dobbs et al. 2013; Schiffmacher and

Keefer 2013; Denicol et al. 2014) but the observation that outer cells of the blastocyst

only gradually become committed to the TE lineage (Berg et al. 2011) is suggestive that

there is heterogeneity within this cell population that could be elucidated if additional

markers were available.

Here we took advantage of recently-developed microfluidics-based technology to

isolate RNA from individual cells to identify markers of epiblast, hypoblast and TE in the

bovine blastocyst. The approach was to assess expression of 96 genes that included

those used as markers for epiblast, hypoblast and TE in other species (Chazaud et al.

2006; Guo et al. 2010; Yan et al. 2013; Hermitte and Chazaud 2014; Blakeley et al.

2015; Boroviak et al. 2015), genes differentially expressed between ICM and TE in the

54

bovine (Ozawa et al. 2012; Nagatomo et al. 2013; Brinkhof et al. 2015; Hosseini et al.

2015; Zhao et al. 2016), as well as genes involved in important processes for the

developing embryo including the Hippo signaling pathway, epigenetic regulation, tight

junction formation, cell polarity and chemokine signaling. The result was identification of

markers for each cell lineage of interest, evidence that the TE is a heterogeneous tissue

in the developing blastocyst, and identification of markers that can be used to

distinguish between subtypes of TE.

Materials and Methods

In Vitro Production of Embryos

Production of embryos involved use of culture media described elsewhere

(Ortega et al. 2017). Oocytes and sperm were from cattle representing B. taurus and an

admixture of B. taurus and B. indicus. Ovaries collected at an abattoir were the source

of oocytes. Oocytes were collected by bisecting follicles 3-8 mm in diameter using a

scalpel and, after all follicles on an ovary were bisected, vigorously washing the ovary in

oocyte collection medium (BoviPRO ™, MOFA Global, Verona, WI, USA) to dislodge

cumulus-oocyte complexes (COC). Fluid containing the COC was then filtered and COC

rinsed with fresh collection medium. The COC were then retrieved using a wiretrol

pipette (Drummond Scientific Company, Broomall, PA, USA) while visualizing under a

dissecting microscope. The COCs were washed another three times in fresh medium.

An average of 12-15 COCs (having at least one layer of cumulus cells and containing

homogeneous cytoplasm) were collected from each ovary. The COCs were pooled in

groups of 10 and matured for 22-24 h in oocyte maturation medium (Tissue Culture

Medium-199 with Earle’s salts supplemented with 2% (v/v) bovine steer serum, 100

U/mL penicillin-G, 0.1 mg/mL streptomycin, and 1 mM glutamine) in 50 µL microdrops

55

covered with mineral oil (Sigma-Aldrich, St. Louis, MO, USA) and in an atmosphere of

5% (v/v) CO2 in humidified air at 38.5°C.

Matured COC were pooled, washed three times in HEPES-buffered synthetic

oviductal fluid (HEPES-SOF) and placed in 1.7 mL synthetic oviductal fluid for

fertilization (SOF-FERT) in groups of up to 300 COCs. For fertilization, frozen-thawed

semen from three bulls was pooled, purified with an Isolate® gradient [Irvine Scientific,

Santa Ana, CA, USA; 50% (vol/vol) and 90% (vol/vol) isolate], washed in HEPES-SOF

and diluted with SOF-FERT. Then, 120 µL of semen plus 80 µL of 0.5 mM

penicillamine, 0.25 mM hypotaurine, and 25 µM epinephrine solution were added to the

COC for a final sperm concentration of 1x106 sperm/mL. Fertilization dishes were

incubated in an atmosphere of 5% (v/v) CO2 in humidified air at 38.5°C for 16-18 h.

Presumptive zygotes were collected and exposed to hyaluronidase (1000 U/mL

in approximately 0.5 ml HEPES-SOF) to remove cumulus cells and washed three times

in HEPES-SOF prior to culture. Embryos were pooled in groups of 25-30 and cultured at

38.5°C in 50 µL microdrops of BBH7 (Cooley Biotech, Gainesville, Florida, USA)

covered with mineral oil in a humidified environment consisting of 5% (v/v) O2, 5% (v/v)

CO2 and the balance nitrogen.

The proportion of embryos that cleaved was assessed at Day 3 after fertilization

and the proportion that became blastocysts assessed at Day 7. At Day 8.75 (207-209

hours post-insemination), all blastocysts (including non-expanded, expanded, hatching

and hatched blastocysts) were collected and subjected to blastomere dissociation.

Embryos were collected in a total of three replicates. A replicate was defined as a single

in vitro fertilization procedure consisting of insemination of 900-1,200 COC. A total of

56

eight bulls were used in the three replicates. The cleavage rate for the three replicates

averaged 80% and the percent of inseminated oocytes becoming blastocysts averaged

20% on Day 7 and 29.8% on Day 8.75.

Preparation of cDNA from Single Blastomeres

cDNA was prepared from individual blastomeres using the C1 Single-Cell Auto

Prep IFC (integrated fluidic circuit) system from Fluidigm (South San Francisco, CA,

USA) using manufacturer instructions.

For each replicate, single-cell suspensions were prepared from the blastocysts

collected at 207-209 h post-insemination. The number of blastocysts processed for

each replicate ranged from 227-336. Blastocysts were washed three times in

Dulbecco’s phosphate-buffered saline (DPBS) containing 0.1% (w/w)

polyvinylpyrrolidone (DPBS-PVP; Kodak, Rochester, NY, USA), incubated in 0.1% (w/v)

protease from Streptococcus griseus (Sigma-Aldrich, St. Louis, MO, USA) in DPBS until

the zonae dissolved, and then washed another three times in fresh DPBS-PVP.

Embryos were then incubated in 50 µL drop of TrypLE Select Enzyme 10X

(ThermoFisher Scientific, Waltham, MA, USA) for 15 min at 38.5°C to disaggregate

cells. Finally, blastomeres were transferred to a 1.7 mL tube, vortexed for 2 min,

resuspended in 500 µL DPBS-PVP and centrifuged for 5 min at 6000 x g. The

supernatant was removed and the cells were resuspended in 10 µL DPBS-PVP.

Following blastocyst disaggregation, cell viability and concentration were

measured using the Countess Automated Cell Counter (Life Technologies, Carlsbad,

CA, USA) and the concentration was adjusted to 300-400 cells/µL. Viability in each

replicate ranged from 33%-82%. A subset of the cells was subjected to a buoyancy test

as part of the quality control process for the C1 system to optimize probability of cell

57

capture probability. The cells were mixed with C1 Cell Suspension Reagent at a ratio of

7:1, 7:2 and 7:3 (v:v) and placed in a well of the C1 IFC plate. The buoyancy check was

performed under 10X magnification immediately and after 10 min waiting time.

Buoyancy was determined by looking at three planes of the well (top, middle and

bottom) under the microscope and noting where the majority of the cells were at time 0.

This was repeated after the 10 min incubation period. The goal was to have the majority

of cells suspended in the middle plane for better capture. The best ratio of suspension

reagent to cell suspension was found to be 7:2; at this ratio 78% of cells were in the

middle plane.

The C1 Single-Cell Auto Prep IFC for Preamp (10-17 µm) was primed following

the manufacturer’s instructions. Because the cells were 13 µm in diameter on average,

the plate and protocol used for priming was C1 DNA Seq IFC: Prime (1773x). The cell

suspension was loaded into the C1 plate for individual cell capture, lysis for RNA

extraction and cDNA pre-amplification. A viability cell staining was performed for one of

the replicates using the LIVE/DEAD Viability/ Cytotoxicity kit from Fluidigm to have an

idea of the proportion of live/dead cells that had been captured. In this replicate, 54 cells

were captured including 11 dead cells and 43 live cells. Only the cDNA from the 43

viable cells was used for further analysis. On average 79% (43/54) of the captured cells

were viable and used for gene expression analysis. Overall, the success of the system

for capturing individual cells was low. The number of cells collected for each replicate

were 6, 19 and 43 for a total of 68 cells analyzed. The C1 plates containing single-cell

cDNA were stored at -20°C until gene expression analysis.

58

Gene Expression Analysis

The Fluidigm® qPCR microfluidic device BiomarkTM HD system was used for

gene expression assays. Primers were designed by Fluidigm® Delta GeneTM assays

(Fluidigm Co., San Francisco, CA, USA) and validated by Miami Center for AIDS

Research (CFAR) at the University of Miami Miller School of Medicine. A description of

the genes analyzed and the rationale for including in the analysis are in Table 2-1.

Primers were designed for 2 housekeeping genes: ACTB and GAPDH: 9 epiblast

markers from one or more of 3 species; 9 hypoblast markers; 11 trophectoderm

markers, 16 genes involved in chemokine signaling, 9 genes involved in Hippo

signaling, 13 genes involved in epigenetic modification, 11 genes involved in tight

junction formation and cell polarity, and, another 16 genes of interest. Initially, three

genes, SDHA, H2AFZ and HPRT1, were considered to be housekeeping genes but

later were analyzed as part of the non-housekeeping gene set because they showed

unequal gene expression among cell populations.

The procedure for quantitative PCR (qPCR) using the BiomarkTM system

(Dominguez et al. 2013) was as follows. Primer-probe sets and samples were loaded on

an IFC plate and placed into a controller that prepares the nanoliter reactions. The plate

was then transferred into the BiomarkTM machine which includes a thermocycler for real-

time qPCR. A total of 40 PCR cycles were performed using the 96.96 dynamic array

IFC developed by the manufacturer. Cycle threshold (Ct) values were calculated by the

Fluidigm Real-Time PCR analysis software. The cutoff for detectable genes was those

Ct >27. The geometric mean of the two housekeeping genes was calculated and used

to obtain the delta Ct (dCt) values of the other 94 genes of interest. Fold changes were

calculated as 2-dCt relative to housekeeping genes. The parameters for the validation

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and qPCR system were the same and are described below. For primer validation using

the BiomarkTM HD system, two pools of embryos, 40-Day 5 morulae and 40-Day 7 and

Day 8 blastocysts, from a different IVF procedures were used. RNA was extracted from

both pools of embryos using the RNeasy micro kit (Qiagen, Valencia, CA, USA); a

DNase treatment was included as part of the protocol. Reverse transcription was

performed using the High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems, Carlsbad, CA, USA) following manufacturer’s instructions. The cDNA was

pre-amplified following the guidelines for the Ambion® Single Cell-to-CTTM kit

(ThermoFisher Scientific) and diluted-serially in 2 fold dilutions to a single cell

equivalent. Primer-probe sets and samples were loaded on an IFC plate and placed into

a controller that prepares the nanoliter reactions. The plate was then transferred into the

BiomarkTM machine for real-time qPCR. Standard curves and cycle threshold (Ct)

values were calculated by the Fluidigm Real-Time PCR analysis software.

Statistical Analysis

The Gene Cluster 3.0 clustering software (de Hoon et al. 2004) was used to

generate the heatmap and unsupervised cluster analysis; complete linkage and

Euclidean distance were used for classifying the cell types. Java treeview (Keil et al.

2016) was used to visualize and group the clustered data. The 9.4 version of SAS

software package (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis.

The effect of cell population on gene expression was performed by analysis of variance

using the generalized linear models procedure (PROC GLM) of SAS, v. 9.4 (SAS

Institute Inc., Cary, NC, USA). Data analyzed were values for fold change relative to

housekeeping genes; results shown are least-squares means ± SEM. When the main

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effect of treatment was significant (P<0.05), differences between individual populations

were determined using the pdiff statement of PROC GLM.

Results

Identification of Cell Populations Using Cluster Analysis

Expression of 94 genes was assessed for 68 individual cells. However, one of

the cells was excluded from further analysis because none of the 96 genes were

detectable by qPCR.

The complete dataset after normalizing to housekeeping genes, ACTB and

GAPDH, was subjected to unsupervised clustering to sort the cells on the basis of gene

expression. A total of 6 cell populations was identified (Figure 2-1) in two large clades

(A and B) that were subdivided into smaller subclades. Clade A contained two

subclades termed A1 (n=8 cells) and A2 (4 cells). Clade B was subdivided into two

subclades [B1 (13 cells) and B2 (43 cells), with clade B2 consisting of three smaller

subpopulations termed B2.1 (8 cells), B2.2 (17 cells) and B2.3 (18 cells).

Identification of Cell Subpopulations as Epiblast, Hypoblast, and TE

Expression patterns of genes known to be markers for specific cell lineages in

the bovine or other species was used to identify each subclade of cells. Expression

patterns of nine genes considered as markers of epiblast in bovine (Kuijk et al. 2012;

Denicol et al. 2014)], mouse (Guo et al. 2010; Boroviak et al. 2015), or human (Yan et

al. 2013; Blakeley et al. 2015) are shown in Figure 2-2. Three of these genes, FGF4,

NANOG, and POUF51, were more highly expressed in subclade A2 than other

subclades. In addition, another marker, HNF4A, was more highly expressed in

subclades A1 and A2 than for cells in clade B; FN1 was upregulated in clade A1 as

compared to the other five subclades. Based on these patterns of gene expression,

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subclade A2 was considered to represent epiblast. Nonetheless, several markers of

epiblast in the mouse (Guo et al. 2010; Boroviak et al. 2015) were not preferentially

expressed in this clade including BMP4, ESRRB, KLF2, and SOX2.

Expression of nine genes that are characteristic of hypoblast in either bovine

(Kuijk et al. 2012; Denicol et al. 2014), mouse (Chazaud et al. 2006; Guo et al. 2010;

Boroviak et al. 2015) or human (Yan et al. 2013; Blakeley et al. 2015) are shown in

Figure 2-3. Four of these genes (FGFR2, GATA6, PDGFRA and SOX17) were more

expressed in population A1 than other populations. Accordingly subclade A1 was

labeled as hypoblast. Note that this subclade, like the epiblast clade, also expresses

high amounts of HNF4A (Figure 2-2) and RUNX1, which is a hypoblast marker in

mouse (Guo et al. 2010). Other markers of hypoblast in mouse, including CREB3L2,

GRB2, SNAI1, and TCF23 (Chazaud et al. 2006; Guo et al. 2010) were not

overexpressed in the hypoblast clade.

Results for expression of 11 genes considered as TE markers in bovine (Ozawa

et al. 2012; Nagatomo et al. 2013; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al.

2016), mouse (Guo et al. 2010; Boroviak et al. 2015), or human (Yan et al. 2013;

Blakeley et al. 2015) are shown in Figure 2-4. The genes CDX2, GATA3, IFNT and

KRT8 exhibited higher expression in the subpopulations of subclade B than for cells of

subclade A. Accordingly, clade B was considered to represent TE and cell populations

were renamed as follows: B1=TE1, B2.1=TE2; B2.2=T3 and B2.4=T4. Expression of

CDX2 and GATA3 tended to be higher for TE1 and TE2 than TE3 and TE4, IFNT was

highest in TE3 and lowest in TE1, and KRT8 was lowest for TE4. In addition to these

four genes, another TE marker in mouse, ELF5 was more expressed in TE3 than for

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cells in other subclades (significant as compared to TE4). Also, the mouse TE marker,

EOMES, was more highly expressed in TE4 than in other TE subpopulations or in

epiblast or hypoblast. The bovine TE marker, PECAM1, was most highly expressed in

TE3, although the only significant difference was with TE4. There was no difference in

gene expression between subpopulations for DAB2, MBNL3, TEAD4 or TFAP2C. Also,

the mouse TE marker, MBNL3 was, most highly expressed in hypoblast (significantly

different from epiblast and TE2 and TE4).

Note that expression of GATA6, which is a marker of hypoblast in bovine, but

which also shows moderate immunolabeling in TE and low immunolabeling in epiblast

(Kuijk et al. 2012; Denicol et al. 2014) exhibited a similar pattern of transcript

abundance – high in hypoblast, low in epiblast and moderate in TE populations (Figure

2-3).

Other Genes Overexpressed in Epiblast and Hypoblast

In addition to genes used to define epiblast (Figure 2-2) and hypoblast (Figure 2-

3), there were six additional genes that were most highly expressed in epiblast (AJAP1,

DNMT3A, H2AFZ, KDM2B, SAV1, and SLIT2), two genes most highly expressed in

hypoblast (ALPL and GJA1) and three genes (CDH2, HDAC1, and HDAC8) that were

more expressed in both epiblast and hypoblast than the TE populations (Figure 2-5). Of

these latter three genes, CDH2 was expressed equally in epiblast and hypoblast,

HDAC1 was more expressed in hypoblast than epiblast, and HDAC8 was more

expressed in epiblast than hypoblast) (Figure 2-5). One gene, ID2, was expressed more

in hypoblast than epiblast, with amounts in TE being intermediate (Figure 2-5).

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Other Genes Overexpressed in All Subpopulations of TE

Besides the genes used to define TE populations, there were an additional five

genes that were expressed in all four TE subpopulations (Figure 2-6). Of these,

expression of GATA2, RAC1 and SFN was relatively uniform between TE

subpopulations, AMOT was higher for TE1 than TE4 (with amounts for TE2 and TE3

being intermediate), and expression of CYP11A1 tended to be higher for TE3 than other

TE populations.

Other Genes Overexpressed in Some TE Subpopulations

There were several genes that were differentially expressed in one or more TE

subpopulations as compared to other cell subpopulations. Cells in TE1 expressed

higher amounts of transcripts for CCL26, LATS1 and LATS2 than for all or some

subpopulations of cells (Figure 2-7). Expression of LATS1 was lowest for TE3 and TE4.

Expression of TET2 was also higher than for other subpopulations except TE2 (Figure

2-7). Cells in TE2 expressed higher amounts of transcript for ACKR4, CCL11, CCR2,

CCR3, CRB2, DNMT1, EPHA4, HPRT1, ID1, MST1, PPBP, TAZ and TJAP1 than other

TE subpopulations and, except for HPRT1 (high expression also in hypoblast), higher

than epiblast or hypoblast (Figure 2-7). Expression of INADL was higher for TE2 than

all cell subpopulations than TE1, KAT6B was higher for TE2 than TE4, STAT3 was

higher for TE2 than all populations except hypoblast, and STAT4 (Figure 2-7) was

higher for TE2 than all populations except epiblast (Figure 2-7). SOX2 was highly

expressed in epiblast, hypoblast, TE2 and TE4 but lowly expressed in TE1 and TE3

(Figure 2-2).

As compared to other cell populations, expression of ACTA2 was higher for TE3

than other TE populations and cells in TE4 expressed higher amounts of ASGR1,

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CCL24, CCR5, CDC42EP4 (difference with epiblast not significant), IL4, ITK, PLCB1

and SOX30 than other cells; cells in TE3 expressed lower amounts of transcripts for

EZH2, cells in TE3 and TE4 expressed lower amounts of LATS1 than TE1 and cells in

TE3 and TE4 expressed lower amounts of SDHA than for other populations (Figure 2-

8). KLF2 was expressed highest in TE4, moderately expressed in epiblast, TE2 and

TE3 and least expressed in hypoblast and TE1 (Figure 2-2).

Genes Whose Expression Did Not Differ Between Subpopulations

There were 16 genes in which there was no significant difference in expression

between cell populations (Figure 2-9).

Discussion

Knockout studies in mice illustrate that a successful pregnancy depends upon

acquisition and appropriate development of the first three cell lineages of the developing

blastocyst for a successful pregnancy (Avilion et al. 2003; Strumpf et al. 2005; Morin-

kensicki et al. 2006; Yagi et al. 2007; Jedrusik et al. 2015). Here we identify a variety of

molecular markers for epiblast, hypoblast and TE lineages in the bovine blastocyst,

including some markers that allow discrimination of 4 subpopulations of TE. In addition,

differential patterns of gene expression between cell subpopulations provide insights

into the differentiation and function of each cell lineage in the bovine blastocyst.

Shown in Figure 2-10 are sets of genes that are preferentially expressed in

epiblast, hypoblast, or TE as well as sets of genes that are either upregulated or

downregulated in specific subpopulations of TE. It is proposed that mRNA for these

genes, or the protein products encoded by those mRNA, can be used to identify and

study the function of specific lineages in the bovine blastocyst. Some of the molecular

markers for epiblast, hypoblast and TE were expressed almost exclusively by one cell

65

lineage. Examples include AJAP1, FGF4 and NANOG in epiblast, FGFR2, FN1, and

SOX17 in hypoblast, and CDX2, GATA2, GATA3, IFNT, KRT8 and SFN in TE. Others,

while more expressed in one cell subpopulation than others, were expressed to variable

degrees by more than one cell subpopulation. Examples include HDAC1 and POU5F1,

which were highest in epiblast but which were also expressed in hypoblast and TE cells,

GATA6, in which transcript abundance was highest for hypoblast but which was also

expressed in TE cell subpopulations, and SLIT2, most expressed in epiblast but also

moderately expressed in the TE4 subpopulation. Gene markers identified in Figure 2-10

that allow discrimination between the four subpopulations of TE also varied in the

degree to which expression was limited to one subpopulation. Thus, some genes, like

ACKR4, CCL11, CCL26, CCR2, CCR3, CRB2, DNMT1, ELF5, EOMES, EPHA4, ID1,

MST1, PECAM1, PPBP, STAT3, STAT4, TAZ, and TJAP1, were much more expressed

for one TE subpopulation than for other subpopulations whereas others, like AMOT,

CYP11A1 LATS1, and TET2, while being more expressed in one or more cell

subpopulations than others, were expressed more uniformly among the subpopulations.

Many of the epiblast, hypoblast and TE markers identified in Figure 2-10 have

been previously identified as cell lineage markers. Examples include Cdx2, Fgf4, Fgfr2,

Gata3, Gata6, Hnf4a, Krt8, Nanog, Pdgfra, Pou5f1, Runx1, and Sox17 for mouse (Guo

et al. 2010; Boroviak et al. 2015), HNF4A, NANOG, PDGFRA, POU5F1, and SOX17 for

human (Yan et al. 2013; Blakeley et al. 2015) and CDX2, GATA3, GATA6, IFNT,

KRT8, and NANOG for bovine (Kuijk et al. 2012; Ozawa et al. 2012; Nagatomo et al.

2013; Denicol et al. 2014; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al. 2016).

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Others have not been previously described as cell lineage markers, for example,

GATA2, MST1, SFN and TJAP1 for TE.

Many of the cell lineage markers in the mouse or human embryo displayed an

expression pattern in the bovine embryo distinct from the expected pattern. Examples

include BMP4, CREB3L2, ESRRB, FN1, GRB2, KLF2, MBNL3, SNA1, TCF23, and

TEAD4. Also, some TE markers in mice were only expressed in a subset of TE cells,

most notably ELF5 and EOMES. Differences between the bovine and other species is

not surprising because evolutionary analysis indicates that there is a large degree of

heterogeneity in the control of gene expression in the early embryo among species (Xie

et al. 2010; Berg et al. 2011; Niakan and Eggan 2013; Hosseini et al. 2015).

It remains to be determined whether differences in protein expression

recapitulate differences in transcript abundance and whether markers that are useful for

distinguishing between cell lineages at the mRNA level will also prove useful at the

protein level. It is notable, however, that for the proteins previously examined, there is

concordance with results on mRNA determined here. CDX2 is a widely-used protein

marker for TE (Ross et al. 2009; Kuijk et al. 2012; Schiffmacher and Keefer 2013;

Denicol et al. 2014) and was found to be highly expressed in all four TE populations in

the present study. In addition, IFNT, whose transcript was overexpressed in TE than

epiblast and hypoblast, was immunolocalized to TE but not ICM (Johnson et al. 2006).

Similarly, like for present findings regarding gene expression, GATA6 can be localized

to both hypoblast and TE, with amounts of immunoreactive protein greater in hypoblast

than TE (Kuijk et al. 2012; Denicol et al. 2014). The present finding that POU5F1 was

most highly expressed in the epiblast but was also detected in populations of hypoblast

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and TE is consistent with earlier results that immunoreactive POU5F1 was present in

ICM and TE, with more intense labeling in the former (Berg et al. 2011).

There was one cases, however, where there is a lack of concordance between

gene expression and protein accumulation. Immunoreactive YAP1 was greater in TE

compared to ICM in Day 7 bovine blastocysts (Tribulo et al. 2017 submitted) even

though YAP1 mRNA was not different between cell lineages in the current study.

Indeed, at least in the mouse, YAP1 is regulated post-translationally through control of

phosphorylation and degradation (Basu et al. 2003; Verghese et al. 2012). It is also

possible that differences in gene expression between cell lineages, seen here with

blastocysts at Day 8.75 of development, may be different at other stages in

development. One example is CCL24, whose expression in the blastocyst peaks at

about Day 7 of development (Chapter 4). At Days 7-8, CCL24 is more expressed in ICM

than TE (Ozawa et al. 2012; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al.

2016). In the current experiment, however, CCL24 expression was largely limited to one

TE subpopulation (TE4), suggesting a developmental switch in the site of expression of

this chemokine gene.

Patterns of gene expression revealed in this study provides some insight into the

processes leading to the formation and subsequent development of each cell lineage.

As expected, the epiblast, which remains in a pluripotent state after formation of the TE

and hypoblast (Nichols et al. 1998; Kirchhof et al. 2000; Avilion et al. 2003; Silva et al.

2009), exhibited upregulation of the pluripotency genes NANOG and POU5F1. The

epiblast also overexpressed FGF4, which has been associated with high levels of SOX2

expression in the epiblast of mouse species (Yuan et al. 1995). Expression of two

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genes involved in epigenetic remodeling in the epiblast, DNMT3A and KDMB2, are also

consistent with maintenance of epiblast cells. Studies using embryonic stem cells

deficient for Dnmt1/Dnmt3a/Dnmt3b indicated that methyltransferases are important for

allowing embryonic stem cells to contribute to embryonic lineages at Day E10.5 but is

not required for formation of extraembryonic tissues (Sakaue et al. 2010). KDM2B has

also been implicated in maintenance of pluripotency by driving noncanonical Polycomb

repressive complex 1 (ncPRC1) to CpG islands (Morey et al. 2015; Schoenfelder et al.

2015) and subsequent repression of differentiation promoting genes (Smith et al. 2016).

Another gene upregulated in epiblast, H2AFZ, encodes for a variant of the histone

octamer member H2A. The specific function of H2AFZ during early embryonic

development is unknown but H2afz deficient mouse embryos die by E5.5 and have an

abnormal ICM (Faast et al. 2001). A histone deacetylase, HDAC8, was upregulated in

both epiblast and hypoblast, in agreement with a previous finding that the gene was

upregulated in the ICM of the bovine embryo (Hosseini et al. 2015). The significance of

this finding is unclear since HDAC8 is involved in silencing pluripotency genes (Saha et

al. 2013). Another two genes upregulated in the epiblast may be involved in the Hippo

signaling pathway that controls formation of the ICM. AJAP1 is an adherens junction

associated protein. During formation of the ICM and TE, adherens junctions play a

crucial role in promoting ICM formation by interacting with E-cadherin and Amot (Sasaki

2015). Another gene upregulated in epiblast, SAV1, is an upstream member of the

phosphorylation cascade of the Hippo signaling pathway (Lee et al. 2008).

Differentiation of the hypoblast in mice involves actions of Ffg4 from neighboring

cells on hypoblast precursors (Morris et al. 2010, 2013). The situation with respect to

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the bovine is unclear. Treatment of bovine embryos with FGF4 and heparin can lead to

blastocysts where the ICM is composed entirely of hypoblast cells (Kuijk et al. 2012). In

this same paper, inhibition of FGF4 signaling caused by addition of sodium chlorate

caused no effect on number of hypoblast cells (Kuijk et al. 2012). Addition of FGF2 to

bovine blastocysts increased the outgrowth of hypoblast cells expressing GATA4 and

GATA6 (Yang et al. 2011). Present results evaluating gene expression are consistent

with a role of FGF4 in the formation of the hypoblast. In particular, the epiblast

subpopulation preferentially expressed FGF4 while cells of the hypoblast subpopulation

preferentially expressed FGFR2. One downstream gene for FGF signaling in human

pluripotent stem cells is HNF4A (Twaroski et al. 2015), which is also a hypoblast marker

in the mouse (Hermitte and Chazaud 2014) and human (Yan et al. 2013). This gene

was overexpressed in the epiblast and hypoblast subpopulations and mRNA was

localized to ICM, particularly those lining the blastocoele, in the bovine blastocyst

(Nagatomo et al. 2013). Another gene that is regulated by FGF4 in mouse embryo is the

transcription factor Sox17 (Frankenberg et al. 2011), which in turn can act as an

enhancer for Fn1 (Shirai et al. 2005). Both of these hypoblast markers were found to be

upregulated in the hypoblast subpopulation in the present experiment. FN1 encodes

fibronectin 1, a component of extracellular matrix. In the bovine, FN1 is primarily

localized to ICM although a small fraction of immunoreactive FN1 is associated with

what could be hypoblast (Goossens et al. 2009).

Another growth factor important for hypoblast survival and expansion in the

mouse is Pdgf (Artus et al. 2010, 2013). The receptor for this growth factor, PDGFRA,

was overexpressed in the hypoblast subpopulation. Hypoblast cells also overexpressed

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two genes involved in gap junction formation - GJA1, and CDH2, which was also highly

expressed in epiblast. Gap junctions play and important role in Hippo signaling in the

ICM by regulating localization of E-cadherin which is a direct regulator of the Hippo

signaling (Sasaki 2015). Perhaps these specialized junctional complexes are important

for differentiation or maintenance of the hypoblast.

Although hypoblast cells represent a more differentiated cell type than epiblast,

gene expression patterns suggest that this cell line retains some degree of an

undifferentiated estate, at least at this stage of development. Thus, hypoblast cells

overexpressed ALPL, which is highly expressed in embryonic stem cells in mouse

(Tielens et al. 2006) and pluripotent stem cells derived from cloned bovine embryos

(Wang et al. 2005). The hypoblast is also apparently undergoing epigenetic regulation

as indicated by overexpression of HDAC1 and HDAC8. Hdac1 is a histone deacetylase

that, when, is embryonic lethal (Montgomery et al. 2007) and, in mouse embryonic stem

cells, is involved in recruiting silencing complexes to maintain pluripotency (Dovey et al.

2010). In addition, Hdac1 cooperates with Brg1 to silence Nanog in the TE (Carey et al.

2015). Perhaps the same phenomenon occurs in hypoblast. Inhibition of Nanog

expression is sufficient to induce hypoblast formation in mouse embryos (Frankenberg

et al. 2011).

In the current experiment, a large number of genes involved in chemokine

signaling were selected for analysis because of a recent finding that suggests CCL24

participates in positioning of hypoblast precursors to the outside of the ICM (Chapter 4).

This chemokine gene reaches maximal expression at Day 7 of development (Chapter 4)

and is more highly expressed in the ICM than in the TE at Day 7-9 (Ozawa et al. 2012;

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Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al. 2016). Moreover, inhibition of

CCL24 signaling by addition of receptor antagonists or knockdown of CCL24 mRNA

reduces the proportion of GATA6+ cells in the outer portion of the ICM (Chapter 4).

Based on these results, it was expected to observe higher expression of CCL24 in

epiblast and of various chemokine receptor genes in hypoblast. In fact, however, CCL24

was overexpressed in one population of TE cells and there was no evidence for

overexpression of chemokine receptors in hypoblast. One possibility is that the apparent

expression of CCL24 in ICM reflects contamination with TE. This could be possible

because, as discussed below, the TE4 population may represent polar TE that is

adjacent to ICM. However, Immunolocalization of CCL24 indicated that the protein was

most commonly localized to the ICM (Chapter 4). It may be more likely, therefore, that

the site of CCL24 expression changes as the embryo advances in development so that

by Day 8.75 of development, the time examined here, CCL24 expression by cells of the

ICM becomes inhibited and the TE begins expressing CCL24.

CDX2 is the transcription factor upregulated in TE as compared to epiblast or

hypoblast that has been shown to be necessary for development of the trophoblast in

the bovine. Zygotic deletion of CDX2 does not prevent blastocyst formation (Berg et al.

2011) but is important for upregulation of GATA3 (Sakurai et al. 2016a), and

maintenance of tight junctions in the TE (Goissis and Cibelli 2014). Transgenic CDX2-

deficent embryos were unable to undergo trophoblast elongation at Day 14 of gestation

after transfer to females but expression of IFNT at this stage was not blocked (Berg et

al. 2011). A recent paper from the mouse indicates that maternally-derived Cdx2 is

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important for blastocyst formation (Jedrusik et al. 2015) and it remains to be determined

whether a similar phenomenon occurs in the bovine.

In the mouse, transcription of Cdx2 depends upon formation of a complex of

Yap1 and Tead4 after inactivation of Hippo signaling in the outer cells of the embryo

[reviewed by (Lorthongpanich and Issaragrisil 2015)]. It is not known whether a similar

signaling system is involved in the bovine. Expression of TEAD4 and YAP1 were not

different between cell types of the bovine blastocyst and other components of Hippo

signaling were increased in epiblast (SAV1), all four TE subpopulations (SFN), TE1

(AMOT, LATS1, or LATS2), or TE2 (MST1 or TAZ). Bovine blastocysts can form,

appear to show no differences of ICM and TE and have normal CDX2, GATA6, IFNT

and POU5F1 levels in the presence of reduced amounts of mRNA for TEAD4 (Sakurai

et al. 2016b).

Use of single cell analysis of blastocyst gene expression revealed that the cells

of the TE are heterogeneous. There are at least four subpopulations of TE cells at Day

8.75 of development with TE1 forming one subclade and TE2, TE3 and TE4 forming a

second subclade. The existence of so many populations of TE could reflect variation

among individual blastocysts or the presence of more than one population of cells in an

individual blastocyst. Blastocysts used for the experiment included a mixture of early,

expanding, hatching and hatched blastocysts and some of the heterogeneity in TE

populations could reflect variation among blastocysts in extent of development.

Moreover, there are differences between male and female blastocysts in gene

expression (Bermejo-Alvarez et al. 2010) and such differences could lead to differences

in gene expression among TE cells. However, it is also likely that the TE is not

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functionally uniform within an individual blastocyst. Indeed, there is evidence that gene

expression differs between mural and polar TE cells in the bovine (Nagatomo et al.

2013) and immunolocalization of IFNT in the TE is not uniform (Johnson et al. 2006).

Additional studies will be required to identify temporal, spatial and functional

differences between the TE subpopulations. However, there are indications that TE1,

which forms a separate subclade from TE2, TE3 and TE4, may represent immature TE

cells. This population of cells had low expression of the maternal recognition of

pregnancy signal IFNT as well as the lowest amounts of the transcription factor

EOMES. Secretion of IFNT was higher for expanded blastocysts than for non-expanded

blastocysts (Kubisch et al. 2001) and increased as blastocysts were cultured for 24 or

48 h (Kubisch et al. 2004). Eomes is a late TE marker in the mouse blastocyst and its

transcription depends on actions of Tead4 and Cdx2 (Probst and Arnold 2017). TE1

cells also exhibited elevated expression of three molecules involved in Hippo signaling –

AMOT, LATS1 and LATS2. Moreover, TE1 cells expressed high amounts of TET2

which is involved in inhibition of differentiation of mouse embryonic stem cells to

trophoblast (Koh et al. 2011). The fact that TE1 cells highly express CCL26, a marker of

polar TE in the bovine (Nagatomo et al. 2013), could mean that these cells are

preferentially localized in this region of the TE. While trophoblast stem cells (TSC) are

also located in polar TE, at least in the mouse (Probst and Arnold 2017), it is unlikely

that TE1 cells represent TSC because of the low expression of EOMES, which is highly

expressed in TSC of the mouse (Probst and Arnold 2017).

A better candidate for TSC are cells in the TE4 subpopulation. These cells highly

express EOMES and two markers of polar TE (Nagatomo et al. 2013) – ASGR1 and

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(non-significantly) TMEM232. TE4 cells also express high amounts of SOX2, which has

been implicated in maintenance of TSC in mouse (Adachi et al. 2013) as well as the

transcription factors KLF2, involved in maintenance of pluripotency in ESC (Qiu et al.

2015) and SOX30, a member of the SRY family whose function is not well understood.

Perhaps the most differentiated of the TE subpopulations is the TE2 subclade.

This subpopulation had the highest number of genes that were upregulated in

expression compared to other TE subpopulations. Among the 17 genes upregulated in

TE2 are three implicated in placental function in other species: the Hippo signaling

molecule MST1, the transcriptional regulator ID1 and the ephrin receptor EPHA4. Mst1

is important for placental differentiation in the mouse (Du et al. 2014), ID2, which is

closely related to ID1, participates in regulation of basic helix-loop-helix transcription

factors in human placenta (Liu et al. 2004), and interactions between ephrin A and

ephrin receptors have been proposed to block premature binding of blastocysts to

endometrium (Fujii et al. 2006). Another two genes that were upregulated were STAT3

and TAZ. STAT3 is an intracellular signal transduction molecule for various growth

factors affecting embryonic development including, in the mouse, Lif and Egf (Cheng et

al. 2016). TAZ is another Hippo signaling molecule and is directly related to YAP1

(Kanai et al. 2000). In mouse embryos, Taz works in conjunction with Yap1 to induce

Cdx2 and an increase of Taz is enough to upregulate CDX2 (Nishioka et al. 2009).

Analysis of single cells also revealed that some genes previously reported as not

being expressed by the bovine blastocyst, in particular the chemokine receptors CCR3

and CCR5 (Chapter 4) and EOMES (Berg et al. 2011) are in fact expressed by specific

cell populations in the TE.

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We conclude that the bovine late-blastocyst cell population is heterogeneous and

composed of epiblast, hypoblast and four TE cell types. Each cell population may be

identified with specific markers previously described and with novel markers identified

here. Further studies are needed to confirm whether subpopulations of TE cells

represent different maturity stages in development of a committed TE phenotype.

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Table 2-1. Information about genes selected for gene expression analysis

Gene Symbol RefSeq ID Reason for selection/previously reported effects

ACKR4 NM_174265.2 Chemokine receptor

ACTA2 NM_001034502.1 Involved in tight junction formation| cell polarity regulator

ACTB NM_173979.3 Housekeeping

AJAP1 NM_001206815.1 Cell polarity regulator

ALPL NM_176858.2 Alkaline phosphatase activity upregulated in bovine ICM

AMOT NM_001206309.1 Hippo signaling pathway

ASGR1 NM_001037590.2 Polar TE in bovine

ASH1L NM_001192743.1 Histone methyltransferase

BMP4 NM_001045877.1 Epiblast marker in mice

CCL11 NM_205773.2 Chemokine

CCL24 NM_001046596.2 Chemokine upregulated in bovine ICM

CCL26 NM_001205635.1 Chemokine upregulated in bovine polar TE

CCR2 NM_001194959.1 Chemokine receptor

CCR3 NM_001194960.1 Chemokine receptor|CCL24 chemokine receptor

CCR4 NM_001100293.2 Chemokine receptor

CCR5 NM_001011672.2 Chemokine receptor

CCR7 NM_001024930.3 Chemokine receptor

CDC42EP4 NM_001046471.1 Cell polarity regulator upregulated in bovine ICM

CDH1 NM_001002763.1 Involved in tight junction formation

CDH2 NM_001166492.1 Involved in tight junction formation

CDX2 NM_001206299.1 TE marker in mice, bovine and human

CRB2 XM_003586670.3 Cell polarity regulator

CREB3L2 NM_001102533.1 Hypoblast marker in mice

CYP11A1 NM_176644.2 Steroidogenesis regulator upregulated in bovine TE

DAB2 NM_001193246.1 TE marker in bovine and human

DNMT1 NM_182651.2 DNA methyltransferase

DNMT3A NM_001206502.1 DNA methyltransferase

ELF5 NM_001024569.1 TE marker in bovine

EOMES NM_001191188.1 TE marker in mice and bovine

EPHA4 NM_001083441.1 Axon guidance regulator upregulated in bovine ICM

ESRRB XM_010809669.1 Epiblast marker in mice

EZH2 NM_001193024.1 Methyltransferase

FGF4 NM_001040605.2 Epiblast marker in mice

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Table 2-1. Continued

Gene Symbol RefSeq ID Reason for selection/previously reported effects

FGFR2 NM_001205310.1 Hypoblast marker in bovine

FN1 NM_001163778.1 Epiblast marker in mice and upregulated in bovine ICM

GAPDH NM_001034034.2 Housekeeping

GATA2 NM_001192114.1 GATA binding protein gene upregulated in bovine TE

GATA3 NM_001076804.1 TE marker in bovine and mice

GATA6 XM_001253596.3 Hypoblast marker in bovine and mice

GJA1 NM_174068.2 Cell polarity regulator upregulated in bovine ICM

GNAT2 NM_174326.2 Histone acetyltransferase

GRB2 NM_001034630.1 Hypoblast marker in bovine

H2AFZ NM_174809.2 Housekeeping

HDAC1 NM_001037444.2 Histone deacetylase

HDAC8 NM_001076231.2 Histone deacetylase

HNF4A NM_001015557.1 Epiblast marker in mouse

HPRT1 NM_001034035.2 Housekeeping

HSD3B1 NM_174343.2 Steroidogenesis regulator upregulated in bovine TE

ID1 NM_001097568.2 Inhibitor of DNA binding upregulated in bovine ICM and mouse TE

ID2 NM_001034231.2 inhibitor of DNA binding upregulated in bovine ICM and mouse TE

IFNT NM_001168279.1 TE marker in bovine

IL4 NM_173921.2 Chemokine

INADL NM_001191501.1 Cell polarity regulator

ITK NM_001105388.1 Chemokine differentially expressed in bovine ICM

KAT6B XM_002698885.4 Histone acetyltransferase

KAT8 NM_001105483.2 Histone acetyltransferase

KDM2B XM_010814033.1 Histone demethylase

KLF2 XM_001787366.4 Epiblast marker in mice

KRT8 NM_001033610.1 TE marker in mice and bovine

LATS1 NM_001192866.1 Hippo signaling pathway kinase

LATS2 XM_002691865.3 Hippo signaling pathway kinase

MAPK13 NM_001014947.1 Regulator of MAP kinase activity upregulated in bovine TE

MBNL3 NM_001192740.1 TE marker in mice

MST1 NM_001075677.2 Hippo signaling pathway

NANOG NM_001025344.1 Epi marker in mice, bovine and human

NF2 XM_002694607.4 Hippo signaling pathway

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Table 2-1. Continued

Gene Symbol RefSeq ID Reason for selection/previously reported effects

PARD3 XM_010811174.1 Cell polarity regulator

PDGFRA NM_001192345.1 Hypoblast marker in human and mice

PECAM1 NM_174571.3 TE marker in bovine

PLCB1 NM_174817.1 Chemokine differentially expressed in bovine ICM

POU5F1 NM_174580.2 Epiblast marker in mice and human

PPBP XM_002688351.3 Chemokine differentially expressed in bovine ICM

RAC1 NM_174163.2 Cell polarity regulator|Rho GTPase family

ROBO1 NM_001192888.2 Axon guidance regulator upregulated in bovine ICM

RUNX1 NM_001256578.1 Hypoblast marker in mice

SAV1 NM_001191362.1 Hippo signaling pathway

SDHA NM_174178.2 Housekeeping

SFN NM_001075912.1 Hippo signaling

SLIT2 NM_001191516.2 Axon guidance regulator upregulated in bovine ICM

SMYD3 NM_001076406.2 H3 and H4 methyltransferase

SNAI1 NM_001112708.1 Hypoblast marker in mice and human

SOX17 NM_001206251.1 Hypoblast marker in mice and human

SOX2 NM_001105463.2 Epiblast marker in mice and human

SOX30 NM_001046429.2 Transcription factor of unknown function

STAT1 NM_001077900.1 Chemokine differentially expressed in bovine ICM

STAT3 NM_001012671.2 Chemokine differentially expressed in bovine ICM

STAT4 NM_001083692.2 Chemokine differentially expressed in bovine ICM

TAZ XM_002699714.3 Hippo signaling pathway

TCF23 NM_001038216.2 Hypoblast marker in mice

TEAD4 XM_002687882.1 TE marker in mice

TET1 XM_010820679.1 DNA demethylase

TET2 XM_005198583.2 DNA demethylase

TFAP2C NM_001075509.1 TE marker in mice

TJAP1 NM_001192419.1 Involved in tight junction formation

TMEM232 XM_010798106.1 pTE dominant

YAP1 XM_003586931.3 Hippo signaling pathway

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Figure 2-1. Identification of embryonic cell populations. An unsupervised clustering and

heat map generation software were used to show expression levels of 91 genes (HPRT1, H2AFZ and SDHA were excluded from the clustering analysis) from 67 individual blastomeres. The output yielded two mayor clades (A and B) that were subdivided into six smaller clusters (highlighted with orange lines): A1, A2, B1, B2.1, B2.2 and B2.3. The numbers in parenthesis are the number of cells in each group. Color key at the bottom right (-3=bright green, 0=black, 3=bright red).

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Figure 2-2. Suggested epiblast markers used to identify blastocyst cell populations.

Individual cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Effects of cell type on expression of genes whose expression was higher in cells designated as epiblast (clade A2). Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05). Species symbols are used to denominate upregulation of the gene in epiblast of mouse (Guo et al. 2010; Boroviak et al. 2015), human (Yan et al. 2013; Blakeley et al. 2015) or bovine (Denicol et al. 2014).

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Figure 2-3. Suggested hypoblast markers used to identify blastocyst cell populations.

Individual cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Effects of cell type on expression of genes whose expression was higher in cells designated as hypoblast (clade A1). Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05). Species symbols are used to denominate upregulation of the gene in hypoblast of mouse (Chazaud et al. 2006; Guo et al. 2010; Boroviak et al. 2015), human (Yan et al. 2013; Blakeley et al. 2015) or bovine (Kuijk et al. 2008; Denicol et al. 2014).

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Figure 2-4. Suggested trophectoderm markers used to identify blastocyst cell

populations. Individual cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Effects of cell type on expression of genes whose expression was higher in cells designated as trophectoderm (clades B). Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05). Species symbols are used to denominate upregulation of the gene in TE of mouse (Guo et al. 2010; Boroviak et al. 2015), human (Yan et al. 2013; Blakeley et al. 2015) or bovine (Ozawa et al. 2012; Nagatomo et al. 2013; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al. 2016).

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Figure 2-5. Additional epiblast and hypoblast differentially expressed genes. Individual

cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05).

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Figure 2-6. Additional trophectoderm genes equally expressed in all four TE. Individual

cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05).

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Figure 2-7. Trophectoderm-1 and trophectoderm-2 differentially expressed genes.

Individual cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05).

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Figure 2-8. Trophectoderm-3 and trophectoderm-4 differentially expressed genes. Individual cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18. Bars labeled with unequal lettering are different from each other (P<0.05).

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Figure 2-9. Transcripts whose expression was not significantly different among cell

populations. Individual cells were captured by C1 Single-Cell Auto Prep integrated fluidic circuit. The cDNA was obtained and used for real time PCR using the Fluidigm® qPCR microfluidic device BiomarkTM HD system. Data was normalized to the geometric mean of ACTB and GAPDH, and is presented as least-squares means ± SEM of the number of cells within each subgroup: epiblast (Epi)=4, hypoblast (Hypo)=7, TE1=13, TE2=8, TE3=17 and TE4=18.

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Figure 2-10. Model for cell specific expression amongst cell populations. The six

populations identified were defined according to differential gene expression specific to each cell type: epiblast (4 cells), hypoblast (7 cells), TE1 (13 cells), TE2 (8 cells), TE3 (17 cells) and TE4 (18 cells). The total of differentially expressed genes were: 6 in epiblast, 6 in hypoblast, 11 in all four TE, 7 in TE1, 17 in TE2, 5 in TE3, 18 in TE4, 8 in epiblast & hypoblast and 5 in hypoblast and TE. Differentially expressed genes in TE include upregulated (middle column) and downregulated (right column) genes compared to other TE genes.

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CHAPTER 3 ROLE OF YES-ASSOCIATED PROTEIN 1, ANGIOMOTIN AND MAP KINASE IN

BLASTOCYST DEVELOPMENT IN THE PREIMPLANTATION EMBRYO

Introduction

The first differentiation event in the mammalian embryo occurs when the

totipotent morula goes through processes of compaction and changes in cell polarity

that lead to formation of the blastocyst which contains a pluripotent ICM, a differentiated

TE and a blastocoel (Arnold and Robertson 2009; Pfeffer 2014). The second

differentiation event results in the partition of cells in the ICM into pluripotent epiblast

and differentiated hypoblast (also known as primitive endoderm). Together, the first

three cell types of the blastocyst are precursors of fetal tissues (epiblast), yolk sac and

extraembryonic endoderm (hypoblast), and placental tissues (TE) (Arnold and

Robertson 2009; Morris and Zernicka-Goetz 2012). In the bovine embryo, these events

occur between Days 6 and 7 (first differentiation), and Days 7 and 9 (second

differentiation).

The mouse is the best studied model for understanding the mechanisms by

which the first two differentiation events are achieved. In this species, differential

activation of the Hippo signaling pathway is an important determinant of cell fate with

respect to TE or ICM (Paramasivam et al. 2011; Hirate et al. 2013; Lorthongpanich and

Issaragrisil 2015). Activation of the Hippo pathway in the inner cells of the embryo lead

to the downstream regulator Yap1 being phosphorylated so that it is targeted for either

degradation or retention in the cytoplasm. As a result, Yap1 does not accumulate in the

nucleus, and transcription of genes such as Pou5f1 and Sox2 is favored to maintain

pluripotency (Lian et al. 2010; Lorthongpanich et al. 2012). In the outer cells of the

embryo, the Hippo pathway is inactivated so that Yap1 remains non-phosphorylated

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and is translocated into the nucleus where it associates with Tead4 and activates

transcription of genes promoting differentiation of trophectoderm including Cdx2 and

Gata3 (Nishioka et al. 2008, 2009; Ralston et al. 2010).

Activation of Hippo signaling in cells destined to become ICM is achieved by gap

junctions that allow cell to cell communication to activate KIBRA and NF2 which

synergistically act to phosphorylate LATS1/2. Simultaneously, MST1/2 are activated by

an unknown signal and further reinforce phosphorylation of LATS1/2 (Justice et al.

1995; Xu et al. 1995; Li et al. 2013). Phosphorylated LATS1/2 is functionally active and

phosphorylates YAP1. Amot is another protein that participates in inactivation of Yap1.

Amot, which is localized at the basolateral membrane of inner embryonic cells through

interactions with Cdh1, can activate LATS1/2 (Paramasivam et al. 2011; Hirate et al.

2013; Hirate and Sasaki 2014), directly phosphorylate Yap1 (Paramasivam et al. 2011;

Hirate et al. 2013) and bind to and retain YAP1 at the plasma membrane so that it

cannot enter the nucleus (Leung and Zernicka-Goetz 2013). Inactivation of Yap1 does

not occur in the outer cells of the compact morula which will become TE cells. The outer

cells become polarized and connected through tight junctions that inhibit cell to cell

communication. Lack of gap junctions prevent MST1/2 activation. In addition, Amot is

phosphorylated at S176 in inner cells to interact with LATS1/2 or Yap1 to reinforce

inactivation of Yap1. In the outer cells, Amot is unphosphorylated (Hirate and Sasaki

2014; Kono et al. 2014) and inactivation of Yap1 is relieved.

In the mouse, formation of hypoblast cells involves actions of Fgf4 from future

epiblast cells acting on future hypoblast cells via Fgfr2 to activate MAPK signaling,

transcription of Gata6 and differentiation into hypoblast cells (Yamanaka et al. 2010;

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Kuijk et al. 2012; Morris et al. 2013). Cells that secrete Fgf4 have low numbers of Fgf2r

so that Gata6 expression is low, expression of the pluripotency factor Nanog is high and

cells become epiblast (Morris et al. 2010).

Involvement of the Hippo signaling pathway in formation of the blastocyst in the

cow is largely unknown but the role of two downstream effectors, CDX2 and TEAD4,

have been evaluated. Embryos that were deficient for CDX2 could develop into

blastocysts (Berg et al. 2011; Goissis and Cibelli 2014) but there was disruption in

regulation of GATA3 (Sakurai et al. 2016a) and TE tight junctions TE (Goissis and

Cibelli 2014). After transfer of CDX2-knockout embryos to recipient females, the embryo

was not able to elongate at Day 14 although production of IFNT was the same in control

and CDX2-mutant embryos (Berg et al. 2011). These findings suggests that CDX2 is

not required for blastocyst formation in the bovine but is necessary for proper

functioning of the TE including subsequent elongation. Similarly, bovine embryos were

able to develop to the blastocyst stage when treated with an RNA interference molecule

to downregulate TEAD4 from the zygote to the blastocyst stage (Sakurai et al. 2016b).

Even though treatment decreased TEAD4 mRNA abundance, there was no effect on

number of ICM and TE cells in blastocysts at Day 7 (Sakurai et al. 2016b).

There is also little information about processes controlling hypoblast

differentiation in the bovine embryo. Embryos that were treated with FGF4 and heparin

developed an ICM that was rich in hypoblast cells (Kuijk et al. 2012). Treatment of

bovine blastocysts with FGF2 also increased the outgrowths of hypoblast-like cells and

expression of GATA4 and GATA6 (Yang et al. 2011). However, inhibition of FGF4 did

not result in a decrease in hypoblast cells in bovine blastocysts (Kuijk et al. 2012). Thus,

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it may be that the fibroblast growth factor signaling pathway is involved in differentiation

of the hypoblast but that FGF4 is not essential for that process.

In the current series of experiments, we evaluated the role of Hippo signaling

regulators, YAP1 and AMOT, as well as the MAPK pathway in formation and function of

TE, hypoblast and epiblast in the cow. The first objective was to characterize

developmental patterns in localization of YAP1 and CDX2 during early preimplantation

stages of development by immunolocalization and fluorescent imaging. The second

objective was to analyze the consequences of disruption of YAP1 activity by either

chemical interference with interaction with TEAD4 using the inhibitor verteporfin (Liu-

chittenden et al. 2012) or knockdown of the mRNA for YAP1. It was hypothesized that

these treatments would prevent CDX2 transcription, formation of TE and a blastocyst

with a normal blastocoele and capability of hatching. A third objective was to determine

whether interference with AMOT biosynthesis through mRNA knockdown of AMOT

would increase TE formation. It was hypothesized that hypoblast differentiation would

also be promoted as a result of AMOT inhibition. A fourth objective was to study the

effects of disruption of the MAPK pathway by inhibiting MAP2K1/2 (also known as MEK)

with PD0325901. It was hypothesized that the ratio of epiblast cells would increase and

hypoblast cells would decrease because of an essential role for the MAPK pathway in

formation of epiblast and hypoblast.

Materials and Methods

In Vitro Production of Bovine Embryos

All experiments were performed with embryos produced in vitro using the

protocol previously described (Ortega et al. 2016, 2017). Sperm and oocytes were from

a mixture of animals of various Bos taurus breeds as well as cattle containing and

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admixture of B. taurus and B. indicus. The procedures were done following the protocol

previously described (Ortega et al. 2016, 2017). Presumptive zygotes were collected

and exposed to hyaluronidase (1000 U/mL in approximately 0.5 mL HEPES-TALP) to

remove the cumulus cells, washed three times in HEPES-TALP and placed in groups of

25-30 zygotes per 50 µL drop of SOF-BE2 (Kannampuzha Francis et al. 2017) covered

with mineral oil in a humidified gas atmosphere of 5% (v/v) CO2, 5% (v/v) O2 and

balanced nitrogen, at 38.5°C. The proportion of embryos that cleaved was assessed at

Day 3 after fertilization and the proportion of embryos that became blastocysts was

verified at Day 7.5 (182±2 h) post fertilization.

A replicate was defined as a single in vitro fertilization procedure involving 200-

300 COCs and a pool from three bulls for fertilization. A total of 19 bulls were used for

the different replicates throughout all the experiments. Only replicates with a cleavage

rate > 65% in control embryos were used.

Immunofluorescent Analysis of Embryos

Procedures for immunolabeling were performed at room temperature unless

otherwise stated. For dual labeling of immunoreactive YAP1 and CDX2, oocytes and

embryos were incubated in permeabilization solution [DPBS-PVP containing 0.25%

(v/v) Triton X-100] for 30 min and then in blocking buffer [5% (w/v) bovine serum

albumin (BSA) in DPBS] for 1 h. Oocytes and embryos were then transferred to rabbit

monoclonal antibody against endogenous YAP1 (1 µg/mL; Cell Signaling Technology,

Danvers, MA, USA), and incubated overnight at 4°C. Afterwards, the embryos were

washed three times in washing buffer [DPBS + 0.1% bovine serum albumin (w/v) and

0.1 % (v/v) Tween-20] and incubated for 1 h in the first secondary antibody: Alexa Fluor

555 conjugated goat polyclonal anti-rabbit IgG (1 µg/mL; ThermoFisher Scientific,

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Waltham, MA, USA). The oocytes and embryos were washed another three times in

washing buffer and incubated for 1 h in the second primary antibody, which was mouse

monoclonal antibody against CDX2 (Biogenex, Fremont, CA, USA) at the 1 µg/mL

working concentration provided. Oocytes and embryos were washed again and

incubated for 1 h in the second secondary antibody [1 µg/mL fluorescein isothiocyanate

(FITC) conjugated goat polyclonal anti-mouse IgG; Abcam, Cambridge, MA, USA].

Then, the samples were washed three times and counterstained with 1 µg/mL Hoescht

33342 in DPBS-PVP for 15 min, washed once in DPBS-PVP and transferred to a 10 µL

drop of SlowFade Gold antifade reagent (ThermoFisher Scientific) on a glass

microscope slide and covered with a coverslip. To determine non-specific labeling,

primary antibodies were replaced with rabbit and mouse IgG (1 µg/mL).

The same procedures were used for dual labeling of NANOG (epiblast marker)

and GATA6 (hypoblast marker) except that the primary antibodies were substituted with

rabbit polyclonal antibody against human GATA6 (Santa Cruz Biotechnology, Dallas,

TX, USA) and mouse polyclonal antibody against human NANOG (eBioscience, San

Diego, CA, USA); both were used at 1 µg/mL.

Images were observed with a 40X objective using a Zeiss Axioplan 2

epifluorescence microscope (Zeiss, Göttingen, Germany) and Zeiss filter sets 02 [4’,6-

Diamidino-2-phenylindole (DAPI)], 03 (FITC), and 04 (rhodamine). Digital images were

acquired using AxioVision software (Zeiss) and a high-resolution black and white Zeiss

AxioCam MRm digital camera. Image J V. 1.48 (National Institutes of Health, Bethesda,

MD, USA) was used to visualize images, count the number of cells using the cell

counter tool and measure protein labeling intensity using the selection of area and

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measure options. The protein labeling intensity was adjusted by selecting a region in the

background, measuring the intensity, and subtracting this number from the measured

protein labeling intensity. Depending on the experiment, immunofluorescent intensity

was determined for the entire area of the embryo, all nuclei in the embryo or all nuclei in

the subset of nuclei that were positive for a specific marker (for example, CDX2 or

YAP1).

RNA Isolation

Embryos for analysis of reverse transcription (RT) PCR were washed three times

in DPBS-PVP, incubated in 0.1% (w/v) protease from Streptococcus griseus (Sigma-

Aldrich) in DPBS for ~3 minutes or until the zonae dissolved, washed three times in

fresh DPBS-PVP, snap frozen in liquid nitrogen and stored at -80°C until RNA isolation.

For RNA extraction, the PicoPure RNA isolation kit (Applied Biosystems,

Carlsbad, CA, USA) was used following the manufacturer’s instructions. Isolated RNA

(15 µL) was treated with 1 µL (2 U) of DNAseI (New England Biolabs, Ipswich, MA,

USA) for removal of DNA contamination prior to reverse transcription. The High

Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used for reverse

transcription following the manufacturer’s instructions. For each sample, there was a

negative control in which reverse transcriptase was omitted. The cDNA was stored at -

20°C until further gene expression analysis.

Quantitative Real-time PCR (qPCR)

The procedure was performed using the CFX96 Real-Time PCR Detection

System and SsoFast EvaGreen Supermix with Low ROX (Bio-Rad, Hercules, CA, USA)

or Fluidigm® qPCR microfluidic device BiomarkTM HD system. Primers for AMOT,

CDX2, GAPDH, GATA6, NANOG, SOX2 and YWHAZ were used as previously

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described (Ozawa et al. 2012; Denicol et al. 2015). Primers for YAP1

(XM_003586931.4; Fwd 5’-TTTGAGATCCCTGACGATGTG-3’; Rv 5’-

GCCAGGTTGTTGTCTGATCTA-3’) were designed using PrimerQuest (IDT DNA,

Coralville, IA, USA). Primers were validated using cDNA from a pool of 30 Day 7

blastocysts and qPCR. The protocol included the generation of a standard curve of at

least three points with a two-factor dilution between two subsequent points. The slope

was -3.2 and primer efficiency was 102%. Identification of amplicons was confirmed by

agarose gel electrophoresis, Sanger sequencing and alignment of the sequence using

the Basic Local Alignment Search Tool feature of the National Center for Biotechnology

Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The conditions for qPCR were as follows: an initial denaturation at 95°C for 30

sec followed by 40 cycles at 95°C for 5 sec, 60°C for 5 sec and 1 cycle for a melting

curve analysis at 65°C to 95°C in increments of 0.5°C every 2 sec. The cycle threshold

(Ct) for each gene of interest was normalized to the geometric mean of GAPDH or

YWHAZ reference genes to generate delta Ct (dCt) values that were used for statistical

analysis. Then, the dCt value of the treated samples were normalized to the control

sample to calculate the delta-delta Ct (ddCt) and fold changes were calculated as 2-ddCt

relative to vehicle treated samples that were used for graphical representation.

Gene Expression Analysis Using High Throughput RT-PCR

The Fluidigm® qPCR microfluidic device BiomarkTM HD system was used to

analyze the effect of YAP1 knockdown on 96 selected genes. Primers for 96 genes

(Chapter 2) were designed by Fluidigm® Delta GeneTM assays (Fluidigm Co., San

Francisco, CA, USA) and optimized by Miami Center for AIDS Research (CFAR) at the

University of Miami Miller School of Medicine (funded by NIH grant P30AI073961).

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Amongst the genes analyzed were 5 housekeeping genes, 9 epiblast potential specific

markers, 10 trophectoderm potential specific markers, 11 hypoblast potential specific

markers, 16 chemokine signaling pathway genes, 9 Hippo signaling pathway genes, 13

epigenetic modification gene markers, 14 tight junctions, cell polarity and axon

guidance, and another 12 genes of interest. The amplification process was also carried

out by Miami Center for AIDS Research (CFAR) at the University of Miami Miller School

of Medicine (funded by NIH grant P30AI073961). The cDNA of all the pools of embryos

was pre-amplified following the guidelines for the Ambion® Single Cell-to-CTTM kit

(ThermoFisher Scientific) and diluted in 2 fold down to a single cell equivalent.

The procedures for gene amplification was the same as previously described

(Siqueira and Hansen 2016), (Chapter 2). A total of 40 PCR cycles were performed

using the 96.96 dynamic array IFC developed by the manufacturer. The cutoff for

detectable genes was those Ct >27. Two housekeeping genes, ACTB and GAPDH,

showed failed readings in a few of the samples; thus, they were not included in the

analysis. The geometric mean of the three housekeeping genes (HPRT1, H2AFZ and

SDHA) was calculated and used to obtain the delta Ct (dCt) values of the other 91

genes of interest. Then, the dCt of treated samples was normalized to the dCt of control

(vehicle treated) samples to calculate the delta-delta Ct (ddCt). Then fold changes were

calculated as 2-ddCt relative to control samples. The dCt was used for statistical analysis

and the fold change was used to represent the data.

Experiment 1: Developmental Changes in Immunoreactive YAP1 and CDX2

All oocytes and embryos were produced in vitro and collected at specific times.

MII oocytes (n=5) were harvested at 22-24 h after maturation and denuded of the

cumulus cells using hyaluronidase as elsewhere (Ortega et al. 2016, 2017). Embryos

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were collected from culture drops at specific hours post insemination (hpi): 28-32 hpi (2-

cell, n=2), 44-48 hpi (3-4 cell, n=3), 50-55 hpi (5-8 cell, n=4), 72 hpi (9-16 cell, n=1), 120

hpi (Day 5 morula, n=5), 144 hpi (Day 6 morula), 168 hpi (Day 7 blastocyst), 192 hpi

(Day 8 blastocyst) and 216 hpi (Day 9 blastocyst). Oocytes and embryos were washed

three times in DBPS containing 0.1% (w/w) PVP (Kodak, Rochester, NY, USA), fixed in

4% (w/v) paraformaldehyde diluted in DPBS-PVP for 15 min, washed another three

times in DPBS-PVP and stored at 4ºC until protein immunolocalization procedures for

YAP1 and CDX2 as described earlier.

Experiment 2: Inhibition of Interactions between YAP1 and TEAD4

Verteporfin (VP) is a molecule that inhibits interactions between YAP1 and

TEAD4 (Liu-chittenden et al. 2012). For Experiment 2, it was tested whether VP would

alter the proportion of embryos becoming blastocysts, block blastocoel formation or

expansion, affect cell allocation to various lineages and alter blastocyst hatching.

Bovine embryos were randomly divided into groups and cultured in 45 µL SOF-BE2.

Embryos were treated at Day 5 after insemination with 5 µL VP (Sigma-Aldrich, St.

Louis, MO, USA) or vehicle [SOF-BE2 containing 1% (v/v) dimethyl sulfoxide (DMSO)]

to produce a final concentration of 10 µM or equivalent amount of vehicle [0.1% (v/v)

DMSO].

Embryos were evaluated for formation of the blastocoel at Day 7.5 post-

insemination. Some blastocysts were harvested at this point. The remaining blastocysts

were placed in one of the drops containing the same treatments as original, and

cultured until 8.5 or Day 9.5. Harvested blastocysts were analyzed for

immunolocalization of YAP1 and CDX2 at Days 7.5, 8.5 and 9.5 post-insemination or for

immunolocalization of NANOG and GATA6 at Day 9.5.

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A total of four replicates were used to produce embryos for the experiment with

the total number of blastocysts examined being: n=18 (Day 7.5 for CDX2 and YAP1),

n=18 (Day 8.5 for CDX2 and YAP1), n=11 (Day 9.5 for CDX2 and YAP1) and n=9 (Day

9.5 for GATA6 and NANOG) for embryos treated with VP and n=28 (Day 7.5 for CDX2

and YAP1), n=23 (Day 8.5 for CDX2 and YAP1), n=11 (Day 9.5 for CDX2 and YAP1)

and n=7 (Day 9.5 for GATA6 and NANOG) for embryos treated with vehicle.

Experiment 3: Knockdown of YAP1

After fertilization, putative zygotes were randomly divided in groups and placed in

culture medium containing either 3 µM antisense GapmeR (LNA GapmeRs in vivo

Ready, Exiqon, Inc., Woburn, MA, USA) directed against YAP1 (5’-

GAGCACTTTGACTGAT-3’), 3 µM of a standard negative control GapmeR designed by

the company (5’-AACACGTCTATACGC-3’), or vehicle [1.6% (v/v) diethylpyrocarbonate

(DEPC) treated double-distilled water). Embryos were evaluated for blastocyst

formation at Day 7.5 after insemination and were collected at either Day 7.5, 8.5 or 9.5.

For five replicates, blastocysts were processed for immunolocalization of YAP1 and

CDX2 on Days 7.5 and 8.5 and for immunolocalization of GATA6 and NANOG on Day

9.5. The number of embryos used for the immunolocalization analysis were n=6 (Day

7.5 for CDX2 and YAP1), n=10 (Day 8.5 for CDX2 and YAP1) and n=3 (Day 9.5 for

GATA6 and NANOG) for the YAP1 targeting GapmeR, n=23 (Day 7.5 for CDX2 and

YAP1), n=15 (Day 8.5 for CDX2 and YAP1) and n=3 (Day 9.5 for GATA6 and NANOG)

for the standard negative control GapmeR and n=11 (Day 7.5 for CDX2 and YAP1),

n=19 (Day 8.5 for CDX2 and YAP1) and n=6 (Day 9.5 for GATA6 and NANOG) for

embryos treated with vehicle. For five replicates, pools of Day 8.5 blastocysts,

representing n=6, 9, 7, 10 and 12 YAP1 targeting GapmeR treated blastocysts, n=19,

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12, 9, 16 and 13 standard negative control GapmeR treated blastocysts, and n=17, 15,

11, 10 and 14 vehicle-treated blastocysts, were used for analysis of mRNA for 96 genes

corresponding to various pathways involved in embryo differentiation and development.

Experiment 4: Knockdown of AMOT

This experiment was conducted as for Experiment 3 except that the GapmeR

against AMOT was 5’-GAACGCTGCTGGAGTA-3’. For five replicates, blastocysts were

collected at Day 7.5 and 8.5 and used for immunolabeling for YAP1 and CDX2 or

GATA6 and NANOG (Day 8.5 only). The number of blastocysts were n=23 (Day 7.5 for

CDX2 and YAP1), n=18 (Day 8.5 for CDX2 and YAP1) and n=19 (Day 8.5 for GATA6

and NANOG) for blastocysts treated with AMOT targeting GapmeR, n=21 (Day 7.5

CDX2 and YAP1), n=24 (Day 8.5 for CDX2 and YAP1) and n=22 (Day 8.5 for GATA6

and NANOG) for blastocysts treated with standard negative control GapmeR, and n=14

(Day 7.5 for CDX2 and YAP1), n=20 (Day 8.5 for CDX2 and YAP1) and n=15 (Day 8.5

for GATA6 and NANOG) for embryos treated with vehicle. For three replicates,

blastocysts were collected at Day 7.5 to produce three pools each of 14, 30, 31 AMOT

targeting GapmeR treated blastocysts, n=26, 23 and 37 standard negative control

GapmeR treated blastocysts, and n=28, 29 and 27 vehicle treated blastocysts were

used for RNA analysis.

Experiment 5: Inhibition of MAP2K1/2

PD0325901 is a molecule that inhibits MAP2K1/2 and which has been reported

to increase number of epiblast cells in bovine blastocysts (Liu-chittenden et al. 2012).

To verify this observation, embryos were randomly divided into groups and cultured in

45 µL SOF-BE2. Embryos were treated at Day 6 after insemination with 5 µL MAP2K1/2

inhibitor (PD0325901; Sigma-Aldrich) or vehicle [SOF-BE2 containing 1% (v/v) DMSO]

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to produce a final concentration of 0.5 µM or equivalent amount of vehicle [0.1% (v/v)

DMSO]. Embryos were evaluated for formation of the blastocoel at Day 7.5 and 8.5

post-insemination. Blastocysts were collected at Day 8.5 post-insemination for

immunolocalization of NANOG and GATA6.

A total of seven replicates were used to produce embryos for the experiment with

the total number of blastocysts examined for NANOG and GATA6 labeling being: n=26

(Day 8.5 for GATA6 and NANOG) for MAP2K1/2 inhibitor and n=25 (Day 8.5 for GATA6

and NANOG) for vehicle.

Statistical Analysis

The SAS v 9.4 software package (SAS Institute Inc., Cary, NC, USA) was used

for statistical analysis. The generalized linear mixed models procedures (Proc

GLIMMIX) was used to evaluate the effects of treatment on the percent of putative

zygotes to become blastocysts and the percent of blastocysts that were hatching or

hatched from the zona pellucida. Each embryo was considered as an individual

observation and development and hatching considered as a binary variable (0=did not

occur; 1=occurred). Treatment was considered as a fixed effect and replicate was

considered random.

Treatment effect on other variables was analyzed by analysis of variance using

the generalized linear models procedure (Proc GLM) of SAS. Main effects of treatment

and replicate were considered fixed. For knockdown experiments, differences between

treatments were separated into two individual orthogonal contrasts as follows: YAP1

targeting GapmeR or AMOT targeting GapmeR vs two controls (standard negative

GapmeR and vehicle control) and standard negative control vs vehicle. For analysis of

gene expression in the knockdown studies, treatment effects were determined by

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analysis of dCt data but results are shown as fold change relative to untreated control

(vehicle).

Unless otherwise stated, data shown are least-squares means + SEM.

Results

Experiment 1: Developmental Changes in YAP1 and CDX2

The presence of YAP1 and CDX2 in the nucleus and cytoplasm of MII oocyte

and embryos throughout development to the blastocyst stage was evaluated by dual

immunofluorescence labeling. Representative images are shown in Figure 3-1. In the

MII oocyte YAP1 was localized in the cytoplasm. From the 2-cell stage to the 9-16 cell

stage, YAP1 remained in the cytoplasm but also was present in nuclei. From the Day 5

morula onwards, YAP1 was present primarily in the nuclear compartments of cells.

Moreover, localization was brighter in the nuclei of the outer cells. In early stages of

development (through the 9-16 cell stage), CDX2 was localized exclusively in the

cytoplasm. By the morula stage, the pattern of localization changed so that CDX2 was

located in the nuclei. At the blastocyst stages (Days 7-9), intensity of nuclear CDX2 was

greater than at earlier stages.

Experiment 2: Inhibition of YAP1-TEAD Interactions by Treatment with Verteporfin

Representative images for labeling of blastocysts with antibodies against YAP1,

CDX2, NANOG and GATA6 are shown in Figure 3-2 while results from quantitative

analyses are summarized in Table 1. The proportion of putative zygotes that became

blastocysts at Day 7.5 post-insemination was decreased (P=0.04) by treatment with VP

(22.2±1.5% for vehicle vs. 16.7±1.5% for VP). The total number of nuclei in blastocysts

was decreased by VP at Days 7.5 and 8.5; P<0.001) but not at Day 9.5 (Table 1).

Verteporfin decreased (P≤0.0001) the number of nuclei that were CDX2+ at all three

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points examined (Days 7.5, 8.5 and 9.5) and decreased (P<0.05 or less) the number of

YAP1+ cells at the three time points examined (Days 7.5, 8.5 and 9.5). Verteporfin also

caused a significant decrease in numbers of NANOG+ and GATA6+ nuclei (Table 1).

Note that the reduction in percent of nuclei positive for CDX2, YAP1, GATA6 and

NANOG does not reflect absence of the proteins; cytoplasmic labeling was noticeable

for all four proteins (Figure 3-2).

A subset of blastocysts that formed at Day 7.5 were cultured for a further two

Days to evaluate whether VP would affect hatching from the zona pellucida.

Representative images of hatching are shown in Figure 3-2. The percentage of

blastocysts that either hatched or were undergoing hatching at Day 8.5 was 12.2±1.5%

for vehicle vs. 1.7±1.5% for VP (P=0.05). For a separate set of blastocysts, the percent

that hatched or were hatching at Day 9.5 was 21±1.5% for vehicle vs. 0±1.5% for VP

(P=0.02).

Experiment 3: YAP1 knockdown

The effectiveness of knockdown was confirmed with qPCR (Figure 3-3) and

immunoreactive YAP1 labeling at Day 8.5 (Figure 3-3). Addition of the YAP1 targeting

GapmeR did not affect cleavage rate (74.2±2.7% for vehicle, 72.3±3.5% for standard

negative control GapmeR, and 71.5±3.1% for YAP1 targeting GapmeR) but significantly

reduced the percent of putative zygotes becoming a blastocyst at Days 7.5 and 8.5 and

the percent of blastocysts that underwent hatching from the zona pellucida at Days 7.5

and 8.5 (Table 2).

Treatment with YAP1 targeting GapmeR did not affect total cell number of

blastocysts at either Days 7.5 or 8.5 (Table 2). However, the number of cells that were

CDX2+ cells were decreased at Days 7.5 (P=0.004) and 8.5 (P<0.0001) when

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compared to control groups. YAP1 targeting GapmeR did not reduce the number of

cells that had YAP1+ nuclei at Day 7.5 but did reduce these measurements at Day 8.5

(P<0.05). The majority of the Day 9.5 embryos showed poor labeling for GATA6 and

NANOG. As a result, there were few GATA6+ or NANOG+ nuclei in the YAP1

knockdown group at Day 9.5 but the difference was not significant.

The fluorescent intensity of each immunoreactive protein was measured in nuclei

that were positive for this protein (Figure 3-3). Intensity for CDX2+ in CDX2+ nuclei was

lower for the YAP1 targeting GapmeR group than for controls at Day 7.5 (P≤0.05) but

not at Day 8.5. The intensity for YAP1 in YAP1+ nuclei was also reduced by YAP1

targeting GapmeR at Day 7.5 (P=0.004) and Day 8.5 (P=0.07). Treatment with YAP1

targeting GapmeR did not affect intensity of labeling for GATA6+ or NANOG+ nuclei.

Effects of treatment on transcript abundance for 90 genes at Day 8.5 was also

examined. (Figure 3-4 and Figure 3-6A-C). Treatment of embryos with YAP1 targeting

GapmeR significantly reduced transcript abundance for 12 genes (AJAP1, ALPL,

CCR7, ELF5, FGF4, FGFR2, HSD3B1, ID1, IFNT, NANOG, SOX17 and TEAD4) and

tended to reduce transcript abundance for another 5 genes [(CDX2 (P=0.09), CRB2

(P=0.09), DNMT3A (P=0.08), HNF4A (P=0.09), and, KAT8 (P=0.08)]. In addition YAP1,

targeting GapmeR increased transcript abundance for 3 genes (CCL26, CDH1 and

KRT8) and tended to increase transcript abundance for another 3 genes [CCR5

(P=0.08), ITK (P=0.08), and MAPK13 (P=0.07)].

Experiment 4: AMOT Knockdown

As shown in Figure 3-5, AMOT targeting GapmeR was successful at knocking

down AMOT mRNA in blastocysts at Day 7.5 post-insemination (P<0.001). Treatment

with AMOT targeting GapmeR had no negative effects on the proportion of putative

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zygotes that cleaved (64.7±4.8% for vehicle, 70.8±4.3% for standard negative control

GapmeR, and 70.7±4.3% for AMOT targeting GapmeR) but the treatment decreased

the percent of putative zygotes that developed to the blastocyst stage at Day 7.5 but not

at Day 8.5 (Table 3). There was no effect of AMOT targeting GapmeR on the percent of

blastocysts that underwent hatching (Table 3).

Results for cell number on blastocysts are shown in Table 3. Total cell number at

Day 7.5 or Day 8.5 blastocysts was not different between treatments. Similarly, there

was no effect of treatment on the number of cells that were CDX2+ or YAP1+ cells. At

Day 8.5, in contrast, blastocysts produced with the AMOT targeting GapmeR had

significantly lower number (P<0.01) of cells that were CDX2+ or YAP1+. Treatment with

the AMOT targeting GapmeR increased the number of ICM nuclei that were GATA6+

as compared to both controls (P<0.05). However, numerically, the difference only

existed for the comparison of the two GapmeR treatments. There was no effect of

treatment on the number of ICM nuclei that were NANOG+.

Effects of treatment on transcript abundance for CDX2, YAP1, GATA6, NANOG

and SOX2 was examined in blastocysts at Day 7.5. All but SOX2 (P=0.7) were

decreased (P<0.05) by downregulation of AMOT (Figure 3-5).

Experiment 5: Inhibition of the MAP2K1/2

Results are shown in Table 4. Treatment with 0.5 µM MAP2K1/2 inhibitor did not

affect the proportion of putative zygotes becoming blastocysts. However, blastocysts in

the MAP2K1/2 inhibitor group had more cells than control blastocysts (P≤0.01) and this

increase resulted from an increase in number of CDX2- cells (i.e., cells in the ICM)

rather than from an increase in number of cells that were CDX2+. Moreover, the

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increase in number of cells in the ICM was ascribed to an increased number of

NANOG+ cells (i.e., epiblast) (P<0.001) rather than GATA6+ cells (i.e., hypoblast).

Discussion

Results reported here demonstrate for the first time that YAP1 and AMOT play a

role in the regulation of TE differentiation in the bovine embryo. Moreover, results

confirm that activation of MAPK signaling is important for differentiation of the hypoblast

(Kuijk et al. 2012) and indicate that YAP1 and AMOT are not required for development

of hypoblast (GATA6+) or epiblast (NANOG+) cells in the inner cell mass.

A central regulator of differentiation of the TE is the transcription factor CDX2.

Transcription of Cdx2 in the mouse is dependent upon interactions of YAP1 and

TEAD4. Embryonic transcription of Cdx2 is not required for blastocyst formation in mice

(Strumpf et al. 2005) but a recent study showed that the Cdx2 derived from the oocyte

is important for formation of the blastocyst (Jedrusik et al. 2015). In the cow, too,

formation of the TE is not dependent on embryonic-derived CDX2 because embryos

develop to the blastocyst stage when the gene is deleted (Berg et al. 2011) or knocked

down using siRNA technology (Goissis and Cibelli 2014; Sakurai et al. 2016a).

Similarly, blastocyst formation is not compromised in embryos with downregulated

TEAD4, which is required for transcription of CDX2 (Sakurai et al. 2016b). However,

blastocysts formed when CDX2 is low or absent experience abnormalities in GATA3

expression (Sakurai et al., 2016), maintenance of tight junctions (Goissis and Cibelli

2014) and trophoblast elongation after transfer to recipient females (Berg et al. 2011).

Present results suggest that, like in the mouse, accumulation of nuclear CDX2 is

dependent upon YAP1. In particular, both VP, which blocks the interaction of YAP1 and

TEAD4 (Liu-chittenden et al. 2012), and a YAP1 targeting GapmeR, which greatly

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reduced amount of YAP1 mRNA and protein, reduced the percent of embryos that

became blastocysts as well as the percent of blastomeres that had nuclei that were

positive for CDX2 as well as those that were positive for YAP1. Like for blastocysts

formed in the presence of inadequate CDX2, blastocysts produced in the presence of

both VP and the YAP1 targeting GapmeR were dysfunctional as indicated by a reduced

capacity for undergoing hatching from the zona pellucida. Effects of VP were more

exaggerated than for the YAP1 targeting GapmeR, possibly because VP can also affect

autophagy (Donohue et al. 2011; Liang et al. 2014) and RAS-signaling (Garcia-

Rendueles et al. 2015). However, most effects of VP paralleled those of the YAP1

targeting GapmeR.

It is likely that interactions between YAP1 and CDX2 to regulate differentiation of

the TE involve more than regulation of CDX2 transcription by YAP1-TEAD4. Indeed,

immunoreactive CDX2 was present in embryos after treatment with VP and the YAP1

targeting GapmeR. A similar phenomenon was observed when examining

developmental changes in immunoreactive YAP1 and CDX2. In the early stages of

development, CDX2 was localized primarily to the cytoplasm and did not become

primarily nuclear in location until the morula stage of development. Thus, it is likely that

development of the TE involves regulation of CDX2 accumulation in the nucleus

(Strumpf et al. 2005; Jedrusik et al. 2008) and that YAP1 is involved in this process

(Nishioka et al. 2009; Ralston et al. 2010). This pattern of protein expression is not

surprising as it has been observed in the mouse embryo (Deb et al. 2006). Like the

mouse (Jedrusik et al. 2015), transcription factors of maternal origin may be important

for differentiation of the embryo. In bovine embryos, transcript for CDX2 is low or absent

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prior to the morula stage and then increases up to the blastocyst stage (Jiang et al.

2014; Denicol et al. 2015). In contrast to transcript abundance, it was observed here

that CDX2 protein was present in all stages of development examined including the MII

oocyte.

The YAP1 targeting GapmeR also disrupted gene expression in the blastocyst in

a way consistent with reduced differentiation of TE. Thus, treatment with YAP1 targeting

GapmeR tended to reduce expression of CDX2 and CRB2, and significantly reduced

other genes characteristically expressed in TE including ID1, ELF5, HSD3B1, IFNT and

TEAD4. The exception was for KRT8, a TE marker that was upregulated by treatment

with YAP1 targeting GapmeR. Also, while the YAP1 targeting GapmeR did not have a

clear effect on numbers of epiblast and hypoblast cells, it disrupted expression of genes

characteristically expressed by those cell types. In particular, treatment decreased

expression of four hypoblast markers (ALPL, HNF4A, FGFR2 and SOX17) and four

markers of epiblast (AJAP1, DNMT3A, FGF4, and NANOG) (Chapter 2). It can be

inferred, therefore, that YAP1 is important for function of hypoblast and epiblast, either

directly because of actions on cells of the ICM, or indirectly because TE function is

compromised.

While results from experiments in which actions of YAP1 were disrupted were

mostly similar to what would be expected based of results in the mouse, this was not

the case for AMOT. Based on experiments in mice (Hirate et al. 2013), it was

hypothesized that an AMOT targeting GapmeR would alleviate inhibition of YAP1 by

AMOT and increase the number of cells designated as TE (i.e., nuclei labeled with

CDX2 or YAP1). Instead, treatment with the AMOT targeting GapmeR decreased the

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number of cells in the Day 8.5 blastocyst that were positive for CDX2 and YAP1 and

also reduced the proportion of blastocysts that underwent hatching. There was no effect

of the AMOT targeting GapmeR on number of cells positive for CDX2 or YAP1 at Day

7.5 of development but treatment did reduce expression of markers for TE (CDX2, and

YAP1) as well as markers of hypoblast (GATA6) and epiblast (NANOG). A role for

AMOT in development of the TE is also indirectly supported by the observation that

expression of AMOT is greater in TE than ICM at Days 7.5 and 8 (Ozawa et al. 2012;

Hosseini et al. 2015) (Chapter 2). Further work is needed to define how AMOT

participates in function of the TE.

It is not clear whether the effect of AMOT targeting GapmeR on expression of

NANOG and GATA6 represents a direct role in differentiation of the ICM into epiblast

and hypoblast or indirect effects caused by disruption of TE function. Except for SOX2,

the AMOT targeting GapmeR reduced expression of all genes examined including those

characteristics of epiblast (NANOG), hypoblast (GATA6) and TE (CDX2 and YAP1).

While the AMOT targeting GapmeR had no effect on the number of NANOG+ cells,

numbers of CDX2+ cells were decreased and numbers of GATA6+ cells were increased.

Treatment of embryos with the MAP2K1/2 inhibitor did not affect competence of

embryos to become blastocysts but the number of ICM cells increased as a result of an

increase in NANOG+ epiblast cells. This result was expected because the MAPK

pathway has been shown to be important for development of the hypoblast in cattle

(Kuijk et al. 2012). In mice, MAPK is activated by Fgf4 from epiblast cells activating the

Fgfr2 in the hypoblast to trigger Gata6 expression and subsequent downregulation of

Nanog transcription. In cattle, too, addition of FGF4 (Kuijk et al. 2012) and FGF2 (Yang

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et al. 2011) can promote hypoblast formation. However, it is not clear whether the

endogenous ligand for MAPK signaling is FGF4 since inhibition of activity of this

molecule had no effect on hypoblast numbers (Kuijk et al. 2012). That the MAP2K1/2

inhibitor affected epiblast numbers and not numbers of hypoblast suggests that the

main role of the MAPK pathway is to suppress epiblast formation.

Altogether, these results are indicative of a role of the Hippo pathway members,

YAP1, AMOT in the formation of ICM and TE in the bovine blastocyst and indicate that

both proteins can also affect function of the epiblast and hypoblast. Results also confirm

the importance of MAPK signaling for differentiation of the ICM into epiblast and

hypoblast. More studies are required to expand these findings and explore the role of

AMOT in the hypoblast and TE formation. This information may be relevant to

understanding how blastomeres communicate to regulate cell fate. In addition, this

information is valuable to improve in vitro production of embryos and promote culture

conditions more similar to in vivo. In conclusion, YAP1 and AMOT are regulators of cell

differentiation in the cow embryo but, the role is not as conserved as the mouse

embryo. YAP1 promotes differentiation of TE and hypoblast while AMOT downregulates

hypoblast differentiation.

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Table 3-1. Effects of verteporfin on characteristics of blastocyst development in the bovine embryo

Vehicle a Verteporfin a,b

Proportion of putative zygotes that reached the blastocyst stage, Day 7.5 (N=8) a

22.2±1.5% (n=923) 16.7±1.5% (n=887) *

Proportion of blastocysts that were undergoing hatching from the zona pellucida, Day 8.5 (N=5) a,c

12.2±1.5% (n=82) 1.7±1.5% (n=58) *

Proportion of blastocysts that hatched from the zona pellucida, Day 9.5 (N=5) a,c

21±1.5% (n=38) 0±1.5% (n=31) *

Total number of nuclei (Hoescht+), Day 7.5 (N=4) a

131.8±6.3 (n=28) 103.2±7.5 (n=18) ***

Total number of nuclei (Hoescht+), Day 8.5 (N=4) a

150.6±10.2 (n=23) 107.1±9.5 (n=18) ***

Total number of nuclei (Hoescht+), Day 9.5 (N=4) a

103.7±6.9 (n=11) 108.9±6.6 (n=11)

Number of CDX2- nuclei (ICM), Day 7.5 (N=4) a

53.5±3.9 (n=28) 65.5±4.7 (n=18) *

Number of CDX2- nuclei (ICM), Day 8.5 (N=4) a

30.3±6.2 (n=23) 108±5.8 (n=18) ***

Number of CDX2- nuclei (ICM), Day 9.5 (N=4) a

30.3±8.2 (n=11) 106.8±8.6 (n=11) ***

Number of CDX2+ nuclei (TE), Day 7.5 (N=4) a

78.3±5.8 (n=28) 37.7±6.9 (n=18) ***

Number of CDX2+ nuclei (TE), Day 8.5 (N=4) a

120.3±7.3 (n=23) -0.9±6.8 (n=18) ***

Number of CDX2+ nuclei (TE), Day 9.5 (N=4) a

81.3±5.3 (n=11) 6.7±5.5 (n=11) ***

Number of YAP1+ nuclei, Day 7.5 (N=4) a 64±6.4 (n=28) 35.4±8.6 (n=18) *

Number of YAP1+ nuclei, Day 8.5 (N=4) a 59.8±7.6 (n=23) 4.8±4.9 (n=18) ***

Number of YAP1+ nuclei, Day 9.5 (N=4) a 85±6.9 (n=11) 11.8±8.3 (n=11) ***

Number of GATA6+ nuclei (hypoblast), Day 9.5 (N=4) a

39.7±6 (n=7) -3.3±5.4 (n=9) ***

Number of NANOG+ nuclei (epiblast), Day 9.5 (N=4) a

20±4.2 (n=7) -2±3.8 (n=9) **

a N=Number of replicates; n = total number of embryos per treatment. b *P<0.05; **P<0.01; ***P<0.001 c A blastocyst was considered to be undergoing hatching it is was hatching from the zona pellucida or had completed hatching.

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Table 3-2. Consequences of YAP1 knockdown in the bovine embryo

Vehicle a Negative control

GapmeR a,c YAP1 targeting

GapmeR a,b

Proportion of putative zygotes that reached the blastocyst stage, Day 7.5 (N=13) a

15±1.6% (n=724) 16.2±2% (n=751) 9.1±1.2% (n=746) ***

Proportion of blastocysts that were undergoing hatching from the zona pellucida, Day 7.5 (N=5) a,d

21±6.6% (n=54) 20.3±7% (n=52) 2.6±2.6% (n=37) *

Proportion of blastocysts that were undergoing hatching from the zona pellucida, Day 8.5 (N=5) a,d

37±7.2% (n=68) 25.7±6% (n=65) 11.2±5% (n=43) **

Total number of nuclei (Hoescht+), Day 7.5 (N=6) a

94±8.6 (n=11) 103.7±6.6 (n=23) 99.9±11.7 (n=6)

Total number of nuclei (Hoescht+), Day 8.5 (N=6) a

112±7.4 (n=19) 95.2±8.6 (n=15) 101.3±5.1 (n=10)

Number of CDX2- nuclei (ICM), Day 7.5 (N=6) a

55.4±6.5 (n=11) 70.6±5.1 (n=23) 87.5±7.1 (n=6) *

Number of CDX2- nuclei (ICM), Day 8.5 (N=6) a

57±5.1 (n=19) 56.5±5.8 (n=15) 89.3±7.1 (n=10) ***

Number of CDX2+ nuclei (TE), Day 7.5 (N=6) a

38.7±5.2 (n=11) 33.1±4 (n=23) * 12.5±7.1 (n=6) **

Number of CDX2+ nuclei (TE), Day 8.5 (N=6) a

54.8±5.1 (n=19) 38.7±5.8 (n=15) 12±7.1 (n=10) ***

Number of YAP1+ nuclei, Day 7.5 (N=6) a

44.7±5.2 (n=11) 48±4 (n=23) 41.1±7.1 (n=6)

Number of YAP1+ nuclei, Day 8.5 (N=6) a

56.8±5.8 (n=19) 43.3±6.6 (n=15) 27.8±7.1 (n=10) *

Number of GATA6+ nuclei (hypoblast), Day 9.5 (N=4) a

28.2±5.5 (n=6) 16±8.1 (n=3) 2.5±10.4 (n=3)

Number of NANOG+ nuclei (epiblast), Day 9.5 (N=4) a

15.8±5.1 (n=6) 2.1±7.5 (n=3) 2.8±9.6 (n=3)

a N=Number of replicates; n = total number of embryos per treatment. b Controls vs treatment orthogonal contrast. *P<0.05; **P<0.01; ***P<0.001 † Statistical tendency, P≤0.1 c Vehicle vs Negative Control GapmeR orthogonal contrast. *P<0.05; **P<0.01; ***P<0.001 d A blastocyst was considered to be undergoing hatching it is was hatching from the zona pellucida or had completed hatching.

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Table 3-3. Effects of AMOT knockdown on the bovine embryo

Vehicle a Negative control

GapmeR a AMOT targeting

GapmeR a,c

Proportion of putative zygotes that reached the blastocyst stage, Day 7.5 (N=8) a

26±2.5% (n=562) 31±2.6% (n=590) 24±2.3% (n=613) *

Proportion of putative zygotes that reached the blastocyst stage, Day 8.5 (N=5) a

28.1±3% (n=344) 30.8±3% (n=380) 26±2.8% (n=368)

Proportion of blastocysts that were undergoing hatching from the zona pellucida, Day 7.5 (N=8) a

12±2.6% (n=153) 13±2.5% (n=187) 8.5±2.3% (n=153)

Proportion of blastocysts that were undergoing hatching from the zona pellucida, Day 8.5 (N=5) a

29±6.4% (n=51) 34±5.9% (n=64) 19±5.7% (n=49)

Total number of nuclei (Hoescht+), Day 7.5 (N=5) a

104±8.5 (n=14) 108.4±7.2 (n=21) 124.4±6.9 (n=23)

Total number of nuclei (Hoescht+), Day 8.5 (N=5) a,b

125±6.2 (n=35) 115.6±5.5 (n=46) 107±5.2 (n=37) †

Number of CDX2- nuclei (ICM), Day 7.5 (N=5) a

43.8±6.3 (n=14) 52.8±5.3 (n=21) 62.8±5.1 (n=23) †

Number of CDX2- nuclei (ICM), Day 8.5 (N=5) a

44.1±4.3 (n=20) 47.7±3.8 (n=24) 50±3.8 (n=18)

Number of CDX2+ nuclei (TE), Day 7.5 (N=5) a

59.7±4.9 (n=14) 55.6±4.1 (n=21) 61.6±4 (n=23)

Number of CDX2+ nuclei (TE), Day 8.5 (N=5) a

79±5.9 (n=20) 78.1±5.2 (n=24) 61.5±5.1 (n=18) **

Number of YAP1+ nuclei, Day 7.5 (N=5) a

56±5.8 (n=14) 54.1±4.9 (n=21) 60.2±4.7 (n=23)

Number of YAP1+ nuclei, Day 8.5 (N=5) a

78.4±5.9 (n=20) 79.2±5.2 (n=24) 56.4±5.1 (n=18) ***

Number of GATA6+ nuclei (hypoblast), Day 8.5 (N=5) a

25±2.1 (n=15) 18.7±1.9 (n=22) 24.7±1.7 (n=19) *

Number of NANOG+ nuclei (epiblast), Day 8.5 (N=5) a

12.4±1.5 (n=15) 8.4±1.4 (n=22) 8.2±1.2 (n=19)

a N=Number of replicates; n = total number of embryos per treatment. b n= all Day 8.5 embryos including the n for CDX2+, YAP1+, GATA6+, NANOG+ c Controls vs treatment orthogonal contrast. *P<0.05; **P<0.01; ***P<0.001 † Statistical tendency, P≤0.1

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Table 3-4. Effects of treatment with MAP2K1/2 inhibitor on development of bovine embryos to the blastocyst stage a

Vehicle PD0325901 b

Proportion of putative zygotes that reached the blastocyst stage, Day 7.5 (N=7) a

23.1±1.6% (n=471)

25.3±1.6% (n=503)

Proportion of putative zygotes that reached the blastocyst stage, Day 8.5 (N=7) a

22.8±1.5% (n=395) 23.6±1.5% (n=415)

Total number of nuclei, Day 8.5 (N=7) a 104.±4.4 (n=25) 119.9±4.2 (n=26) **

Number of CDX2- nuclei (ICM), Day 8.5 (N=7) a

48.2±3.5 (n=25) 62.5±3.3 (n=26) **

Number of CDX2+ nuclei (TE), Day 8.5 (N=7) a

56.7±3.6 (n=25) 57.4±3.5 (n=26)

Number of GATA6+ nuclei (hypoblast), Day 8.5 (N=7) a

36±2.4 (n=25) 37.2±2.3 (n=26)

Number of NANOG+ nuclei (epiblast), Day 8.5 (N=7) a

12.2±2 (n=25) 25.3±1.9 (n=26) ***

a N=Number of replicates; n = total number of embryos per treatment. b *P<0.05; **P<0.01; ***P<0.001

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Figure 3-1. Immunolocalization of CDX2 and YAP1 in the bovine oocyte and early embryo. MII oocytes and embryos were labeled with antibody against CDX2 (green) and YAP1 (red) or with nuclear labeling Hoescht 33342 (blue). Merged panel contains all three fluorochrome channels. Scale bar=20 µM.

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Figure 3-2. Representative images of blastocysts in absence (vehicle) or presence of

verteporfin (VP; YAP1-TEAD4 inhibitor) from Days 5-9.5 of development. Top row represents bright field images that are shown to indicate the negative effect of VP on morphology and hatching. While the control group (left panels) underwent blastocyst formation, expansion and hatching, the treated group (right panels) experienced blastocyst formation (indicated by presence of blastocoel) but blastocyst expansion and hatching from the zona pellucida was interrupted. Color panels show that VP led to loss of YAP1 (red) labeling, absence of CDX2 (green) nuclear labeling (but not cytoplasmic labeling) and reduced labeling of GATA6+ (red) and NANOG+ (green) in ICM nuclei (but not cytoplasm). All nuclei are labeled with Hoescht 33342 (blue). Scale bar on bright field images=50 µM; scale bar on fluorescent images=20 µM.

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Figure 3-3. Amounts of YAP1 mRNA, CDX2, YAP1, GATA6 and NANOG as affected

by treatment with YAP1 targeting GapmeR. (A and B) Analysis of YAP1 mRNA by quantitative real-time PCR for Day 8.5 blastocysts (5 pools per treatment). Data are presented as fold change relative to control (vehicle). Housekeeping genes were YWHAZ and GAPDH in panel A or HPRT1, H2AFZ and SHDA for panel B. (C-F) Intensity of immunoreactive YAP1 (C), CDX2 (D), GATA6 (E) and NANOG (F) in nuclei that were positive for the same protein. Immunoreactivity of GATA6 and NANOG were measured at Day 9.5 only. The number of embryos per group were n=6 (Day 7.5), n=10 (Day 8.5) and n=3 (Day 9.5) for those treated with YAP1 targeting GapmeR, n=23 (Day 7.5), n=15 (Day 8.5) and n=3 (Day 9.5) for those treated with standard negative control GapmeR and n=11 (Day 7.5), n=19 (Day 8.5) and n=6 (Day 9.5) for those treated with vehicle. Data are least-squares means ± SEM results. The P-values for effects of treatment are indicated by the value above the bars.

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Figure 3-4. Knockdown of YAP1 alters gene expression of 23 transcripts in blastocysts at Day 8.5 of development as determined by quantitative real-time PCR data (n=5 pools of blastocysts per treatment). Data are represented as fold change relative to control (vehicle); housekeeping genes were HPRT1, H2AFZ and SDHA. Data are least-squares means ± SEM results. The P-values for effects of treatment are indicated by the value above the bars. Abbreviations are Std Neg = standard negative control GapmeR; YAP1kd = YAP1 targeting GapmeR.

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Figure 3-5. Effect of AMOT knockdown on expression of genes associated with

blastocyst differentiation of epiblast and hypoblast. Gene expression was determined using quantitative real-time PCR data of three pools of Day 7.5 blastocysts per group. Data are presented as fold change relative to control (vehicle); housekeeping genes were YWHAZ and GAPDH. Data are least-squares means ± SEM results. The P-values for effects of treatment are indicated by the asterisk above the bars (*P<0.05; **P<0.01). Abbreviations are Std Neg = standard negative control GapmeR; AMOTkd = AMOT targeting GapmeR.

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Figure 3-6. Expression of genes in blastocyst at Day 8.5 of development that were not

affected by YAP1 knockdown as determined by quantitative real-time PCR data (n=5 pools of blastocysts per treatment). A total of 67 genes were not affected by the knockdown (A-C). Data are represented as fold change relative to control (vehicle); housekeeping genes were HPRT1, H2AFZ and SDHA. Data are least-squares means ± SEM. Abbreviations are Std Neg = standard negative control GapmeR; YAP1kd = YAP1 targeting GapmeR.

A

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B

Figure 3-6. Continued

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C

Figure 3-6. Continued

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CHAPTER 4 ROLE OF CC CYTOKINES IN SPATIAL ARRANGEMENT OF THE INNER CELL

MASS OF THE BLASTOCYST

Introduction

Following, formation of the hypoblast from cells of the ICM represents the second

differentiation event in the mammalian embryo, being preceded by differentiation of cells

into the ICM and TE. In the mouse, hypoblast formation from cells within the ICM is

regulated by the FGF4/FGFR2 pathway (Rossant et al. 2003; Chazaud et al. 2006;

Kang et al. 2013; Morris et al. 2013). A group of cells within the ICM secrete FGF4 that

then acts on neighboring cells through FGFR2 to activate expression of Gata6 and drive

hypoblast formation. The remaining cells, which are negative for FGFR2, express

Nanog and become epiblast cells (Rossant et al. 2003; Chazaud et al. 2006; Kang et al.

2013; Morris et al. 2013). Initially, GATA6+ and NANOG+ cells are scattered in the ICM

in a salt-and-pepper pattern but subsequently GATA6+ cells form an epithelium at the

edge of the ICM lining the blastocoel (Rossant et al. 2003; Chazaud et al. 2006). There

is species variation in the mechanisms by which hypoblast cells are delineated. In

particular, FGF4 has little or no role in formation of hypoblast in the human and bovine

embryo although downstream effectors of FGF4 signaling participate in promotion of

hypoblast differentiation and inhibition of epiblast differentiation in cattle (Kuijk et al.

2012).

Three models have been described to explain the mechanism by which GATA6+

cells in the ICM become spatially reorganized into an epithelium lining the blastocoele

(Hogan and Tilly 1978). The first model, that the cells near the blastocoel cavity are the

most sensitive to signals triggering hypoblast differentiation (Dziadek 1979), is not

consistent with the initial localization of GATA6+ cells throughout the ICM although it is

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possible that there is heterogeneity among GATA6+ cells in signal responsiveness.

Another model posits that localization depends on apoptosis as well as switching of

gene expression from Gata6 to Nanog or vice versa depending on cell position (Plusa et

al. 2008; Zernicka-Goetz et al. 2009). Finally, it has been proposed that hypoblast

precursors move to their corresponding location (Chazaud et al. 2006). Time-lapse

photography has been used to demonstrate movement of GATA6+ cells across the ICM

to face the blastocoel cavity (Plusa et al. 2008).

Among the genes differentially expressed between cells of the ICM and TE in the

bovine blastocyst is CCL24, which encodes for a chemokine also known as eotaxin-2

and which was overexpressed in the ICM of the Day 8 blastocyst when compared to the

TE (Ozawa et al. 2012; Nagatomo et al. 2013; Brinkhof et al. 2015; Zhao et al. 2016).

Other genes involved in chemokine signaling are also upregulated in ICM compared

with the TE including PPBP (previously known as CXCL7), ITK, and STAT3 (Ozawa et

al. 2012; Nagatomo et al. 2013; Brinkhof et al. 2015; Zhao et al. 2016). Chemokines

exhibit chemotactic activity and are involved in cellular migration, polarization and

proliferation (Senior et al. 1983; Parkinson et al. 1993; Gupta et al. 1995; Bonecchi et al.

1998). CCL24 signals through the G-protein coupled seven transmembrane receptor

CCR3 (Forssmann et al. 1997).

In the present study, we tested the hypothesis that CCL24 plays a role in the

arrangement of cells of the epiblast and hypoblast of the bovine embryo. The

hypothesis was analyzed through a series of experiments to evaluate gene expression,

protein localization and consequences of inhibition of CCL24 signaling.

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Materials and Methods

In Vitro Production of Embryos

Embryos were produced by in vitro fertilization using procedures described

elsewhere (Fields et al. 2011). Oocytes were obtained from ovaries recovered from a

local abattoir. Both oocytes and sperm were from a mixture of animals of various breeds

including Bos taurus, B. indicus and admixture of the two genetic types. The surface of

the ovaries were scored with a scalpel and washed in oocyte collection medium

(BoviPRO ™, MOFA Global, Verona, WI, USA) to collect cumulus oocyte complexes

(COCs). Those COCs covered with at least one layer of cumulus cells and containing

homogeneous cytoplasm were selected for maturation. The selected COCs were

washed in fresh oocyte collection medium, pooled in groups of 10 and placed in 50 µL

drops of oocyte maturation medium (Tissue Culture Medium-199 with Earle’s salts

supplemented with 2% (v/v) bovine steer serum, 100 U/mL penicillin-G, 0.1 mg/mL

streptomycin, and 1 mM glutamine) that were overlaid with mineral oil (Sigma-Aldrich,

St. Louis, MO, USA). The COCs were matured for 18-22 h at 38.5°C in an atmosphere

of 5% (v/v) CO2 in humidified air. Up to 300 COCs were pooled and fertilized in plates

containing 1.7 mL of IVF-TALP (Caisson Labs, Smithfield, UT, USA) and 80 µL PHE

(0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 µM epinephrine) prepared as

previously described (Ortega et al. 2017). Semen from frozen-thawed straws from three

bulls were pooled, purified with Isolate® [Irvine Scientific, Santa Ana, CA, USA; 50%

(vol/vol and 90% (vol/vol)] and diluted to a final concentration in the fertilization dishes

of 1x106/mL. Fertilization proceeded for 8-9 h in a humidified environment at 38.5°C and

5% (v/v) CO2. After fertilization, putative zygotes were removed from the fertilization

dish, denuded of cumulus cells by vortexing in 100 µL hyaluronidase (1000 U/mL in

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approximately 0.5 mL HEPES-TALP), and cultured in groups of 25-30 in 50 µL oil-

covered microdrops of a serum-free culture medium called SOF-BE2 (Kannampuzha

Francis et al. 2017) at 38.5°C and a humidified environment consisting of 5% (v/v) O2,

5% (v/v) CO2 and the balance nitrogen. The proportion of putative zygotes that cleaved

was determined at Day 3 after insemination and the proportion that developed to

blastocyst was determined at Day 7 after insemination.

A replicate was defined as a single in vitro fertilization procedure; each replicate

consisted of at least 300 COCs and involved fertilization with a pool of spermatozoa

from three bulls. A total of 48 bulls were used for the different replicates throughout all

the experiments. Only replicates with a cleavage rate > 65% in control embryos were

used.

Developmental Changes in mRNA for CCL24, CCR3 and CCR5

Matured oocytes and embryos were produced in vitro. Oocytes were harvested

at 18-22 h after maturation and cumulus cells were removed using hyaluronidase as

described above. Embryos were collected from cultures at the following times: 28-32 h

post insemination (hpi), (2-cell), 44-48 hpi (3-4 cell), 50-55 hpi (5-8 cell), 72 hpi (9-16

cell), 120 hpi (Day 5 morula), 144 hpi (Day 6 morula and Day 6 blastocysts), 168 hpi

(Day 7 blastocyst), 192 hpi (Day 8 blastocyst) and 216 hpi (Day 9 blastocyst). Oocytes

and embryos were collected for detection of CCL24 transcripts in two experiments. In

the first, 4 pools of 10-30 oocytes or embryos of the following stages were collected:

matured oocyte, 2-cell, 3-4 cell, 5-8 cell, 9-16 cell, Day 5 morula, and blastocysts at Day

7, 8 and 9 (840 oocytes and embryos total). In the second experiment, 3 pools each of

Day 5 morula, Day 6 morula, Day 6 blastocysts and Day 7 blastocysts were collected

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(360 total embryos). For detection of CCR3 and CCR5, only one pool of Day 7

blastocysts was analyzed (30 embryos total).

Pools of oocytes and embryos were washed three times in DPBS containing

0.1% (w/w) PVP (Kodak, Rochester, NY, USA), incubated in 0.1% (w/v) protease from

Streptococcus griseus (Sigma-Aldrich) in DPBS until the zonae dissolved, washed

another three times in fresh DPBS/PVP and stored at -80°C until processing for RNA

extraction. RNA was extracted from each pool of embryos using the RNeasy micro kit

(Qiagen, Valencia, CA, USA). A DNase treatment was included in the RNA isolation

procedure. Reverse transcription was performed using the High Capacity cDNA

Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) following

manufacturer’s instructions. For each sample, a negative control in which reverse

transcriptase was omitted was performed. The cDNA was stored at -20°C until further

use.

Primers for GAPDH and YWHAZ housekeeping genes were used as previously

described (Denicol et al. 2014). Primers for CCL24 (Fwd: 5’-

TAGAGGGCTCTTGGTCACA-3’; Rv: 5’-GTCCTCCAGGTCCATTCATTAC-3’), CCR3

(Fwd: 5’-AGACTTTCTGCAGTCCTCTTTAC-3’; Rv: 5’-

AGAGCGAGACCCAGGATATTT-3’) and CCR5 (Fwd:

5’GTGTCGCAACGAGAAGAAGA-3’; Rv: 5’-CAGGAGAAGGACGATGTTGTAG-3’) were

designed using PrimerQuest (IDT DNA, Coralville, IA, USA). Primers were validated

using pools of cDNA from 40 blastocysts, lymphocytes or blood samples, and

quantitative real-time PCR. Validation included generation of a standard curve of at

least three points with a two-factor dilution between two subsequent points. Slopes were

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between -3.00 and -3.50 and primer efficiency was between 98% and 103%.

Identification of amplicons was confirmed by agarose gel electrophoresis, Sanger

sequencing and alignment of the sequence using the Basic Local Alignment Search

Tool feature of the National Center for Biotechnology Information

(https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Reverse transcription PCR (RT-PCR) was analyzed two ways, firstly by

qualitative PCR, where amplicons were examined by agarose gel electrophoresis and

secondly, by qPCR. The reactions were performed using the CFX96 Real-Time PCR

Detection System and SsoFast EvaGreen Supermix with Low ROX (Bio-Rad, Hercules,

CA, USA). The conditions for both qualitative and qPCR were as follows: an initial

denaturation at 95°C for 30 sec followed by 40 cycles at 95°C for 5 sec, 60°C for 5 sec

and 1 cycle for a melting curve analysis at 65°C to 95°C in increments of 0.5°C every 2

sec (Denicol et al. 2015). The amplicons examined by gel electrophoresis were

separated by electrophoresis using 1% (w/v) agarose gels in Tris acetate-EDTA buffer.

Bands were visualized with 0.1 µg/mL ethidium bromide (ThermoFisher). All samples

were run on a single gel at 60 V for 1 h. For qPCR, the cycle threshold (Ct) for each

gene of interest was normalized to the mean of GAPDH or YWHAZ reference genes to

generate ΔCt values that were used for statistical analysis.

Production of Antisera to CCL24

Mouse anti-bovine CCL24 polyclonal antiserum was developed by immunizing

three Balb/C mice with a mix of three peptides that were each conjugated to keyhole

limpet hemocyanin. The peptides were H2N-CQKKASARARAMSTT-OH, H2N-

AGVIFTTQKGQKFC-OH, and H2N-SKKIPESRVISYQLC-OH. They corresponded to

regions of bovine CCL24 (NP_001040061.1) predicted to be antigenic. Each mouse

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was immunized with 125 µL of a solution of 1.9-2.0 mg/mL of each peptide conjugate

that was mixed with 125 µL MPL® + TDM Emulsion oil (2X concentration; Sigma-

Aldrich) and 10 µL of 1 mg/mL CpG stock (Invivogen, San Diego, CA, USA). Each

mouse was immunized, s.c., at three sites (50 µL in each ventral groin site and 160 µL

on the back). Immunization was repeated three times at 21 d intervals. A final

intraperitoneal injection of 125 µL without adjuvant (i.e. peptide conjugates only) was

administered at 88 d after the first immunization.

Blood was collected from each mouse at various intervals and tested for antibody

titer by ELISA. Isotype of the antiserum was determined by using three alkaline-

phosphatase labeled second antibodies in the ELISA: rabbit anti-mouse IgG, whole

molecule; goat anti-mouse IgG, gamma chain specific; and goat anti-mouse IgM, mu

chain specific. The sample of serum producing the highest titer (collected on Day 92

after initial immunization) was used for experiments. This antiserum had an IgG titer that

was 16 fold higher than the IgM titer. Specificity of the antiserum was confirmed by

neutralization of the antiserum with the conjugated peptides mixture followed by ELISA

and immunofluorescence.

Immunolocalization of CCL24 and CDX2

Dual labeling for immunoreactive CCL24 and CDX2 was examined in Day 5

(n=16) and 6 (n=13) morulae and Day 7 (n=93) and 8 (n=96) blastocysts. All steps

proceeded at room temperature. Harvested embryos were washed in DPBS with 0.1%

(w/v) PVP (DPBS/PVP), fixed in 4% (w/v) paraformaldehyde diluted in DPBS/PVP for

15 min and washed three times in DPBS/PVP. Embryos were then incubated for 30

min in DPBS-PVP containing 0.25% (v/v) Triton X-100, followed by a 1 h incubation in

blocking buffer [5% (w/v) BSA in DPBS]. This was followed by incubation in primary

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antibody [mouse anti-bovine CCL24 polyclonal antiserum diluted in DPBS containing

1% (w/v) BSA and 0.1% (v/v) Tween-20] overnight, washing in washing buffer [DPBS

containing 0.1% bovine serum albumin (w/v) and 0.1% (v/v) Tween-20] and incubation

with secondary antibody (1 µg/mL FITC conjugated goat polyclonal anti-mouse IgG;

Abcam, Cambridge, MA, USA) for 1 h. Embryos were again washed, incubated for 1 h

in mouse monoclonal antibody against CDX2 (Biogenex, Fremont, CA, USA) at the 1

µg/mL working concentration provided by the manufacturer. Embryos were washed and

incubated with the secondary antibody consisting of 1 µg/mL Alexa555 conjugated

rabbit polyclonal anti-mouse IgG. Finally, embryos were washed and incubated in 1

µg/mL Hoescht 33342 in DPBS/PVP to label nuclei, washed in DPBS/PVP, placed on a

glass microscope slide in 10 µL drops of SlowFade Gold antifade reagent

(ThermoFisher Scientific, Waltham, MA, USA), covered with a coverslip and observed

with a 40X objective using a Zeiss Axioplan 2 epifluorescence microscope (Zeiss,

Göttingen, Germany) and Zeiss filter sets 02 (DAPI), 03 (FITC) and 04 (rhodamine).

Digital images were acquired using AxioVision software (Zeiss) and a high-resolution

black and white Zeiss AxioCam MRm digital camera. Image J V. 1.48 (National

Institutes of Health, Bethesda, MD, USA) was used to visualize images and measure

labeling intensity.

To determine non-specific labeling, primary antibodies were replaced with normal

mouse serum [1:100 (v/v)] and mouse IgG (1 µg/mL).

Consequences of Inhibition of CCR3 for Localization of GATA6+ Cells in Hypoblast

Two experiments were conducted to determine effects of inhibition of CCR3 on

localization of hypoblast cells. One experiment utilized (S)-methyl-2-naphthoylamino-3-

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(4-nitrophenyl) propionate (SB328437; Calbiochem, Billerica, MA, USA) (White et al.

2000; Provost et al. 2013) and the second utilized N-benzoyl-4-nitroaniline ethyl ester

(SB297006; R&D Systems, Minneapolis, MN, USA) (Provost et al. 2013). In each

experiment, embryos were cultured in 45 µL SOF-BE2. At Day 6 post-insemination,

each drop of embryos received 5 µL of inhibitor [SB328437 (final concentration 1 µM) or

SB297006 (final concentration, 5 µM)] or 5 µL of vehicle [SOF-BE2 containing 1% (v/v)

dimethyl sulfoxide]. Concentrations used were shown to be effective at inhibiting

eosinophil chemotaxis (White et al. 2000; Provost et al. 2013). Blastocysts were

collected at Day 8 after insemination for immunolocalization of GATA6 (hypoblast) and

NANOG (epiblast). The number of embryos subjected to analysis were 8-9 embryos per

group for the experiment with SB328437 (from two different replicates) and 68 embryos

per group for SB297006 (from six different replicates).

Procedures for immunofluorescent labeling were as described above except that

the primary antibodies (both used at 1 µg/mL) were mouse polyclonal antibody against

human NANOG (eBioscience, San Diego, CA, USA) and rabbit polyclonal antibody

against human GATA6 (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary

antibodies were 1 µg/mL FITC conjugated goat polyclonal anti-mouse IgG (Abcam) and

Alexa Fluor 555 conjugated goat polyclonal anti-rabbit IgG (ThermoFisher).

Embryos were examined for immunofluorescence using confocal microscopy.

Embryos were placed in 10 µL drops of ProLong® Gold Anti-Fade Mounting Medium

(ThermoFisher Scientific) on clean chamber slides and examined on a spinning disk

confocal scanner mounted on an Olympus DSU-IX81 inverted fluorescent microscope.

Images were captured with a 40X objective using DAPI, FITC and red fluorescent

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protein (RFP) filter sets. Digital images were taken every 2 µm using an attached

Hamamatsu C4742-80-12AG monochrome CCD camera. SlideBook 6 Reader

(Intelligent Imaging Innovations, Inc., Denver, CO, USA) was used to visualize images

and count total number of cells. Images visualized and captured following confocal

microscopy were counted by sections. Embryos contained a total of 23 sections on

average.

Number and position of NANOG+ and GATA6+ cells in the ICM were counted by

examining images while scrolling serially through the 2 µm sections. Most embryos at

this stage of development do not have a clearly-delineable hypoblast where an epithelial

layer of GATA6+ cells lines the blastocoele (Kuijk et al. 2012; Denicol et al. 2014).

Accordingly, position of cells in the ICM was based on whether the cell was located in

the outer or inner portion of the ICM. An example of how cell position was determined in

shown in Figure 4-1. Cells in the first 25% and last 25% of the serial sections of ICM

were considered cells on the outside of the ICM. Cells in the intervening 50% of

sections were considered to be located in the inside of the ICM if located in the interior

of the section and to be on the outside of the ICM if located on the periphery of the

section. Note that treatments that would alter the directed positioning of NANOG+ and

GATA6+ cells would not lead to an absence of cells in either the outer or inner regions

of the ICM because cells would be randomly located in both regions.

Consequences of Knockdown of CCL24 for Localization of GATA6+ Cells in Hypoblast

Presumptive zygotes were harvested at 18-22 hpi and microinjected with a

morpholino targeting bovine CCL24 exon2/intron2 (5’-

TAATAGTTACTCACATCACTCCTGC-3’ with 3’-lissamine red emitting fluorescent tag)

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or a standard negative morpholino (5’-CCTCTTACCTCAGTTACAATTTATA-3’ with 3’-

lissamine red emitting fluorescent tag) (Gene Tools, LLC, Philomath, OR, USA).

Microinjection was performed using an inverted microscope (Diaphot, Nikon Instruments

Inc., Melville, NY, USA) with attached Narishige micromanipulator system (Tritech

Research Inc., Los Angeles, CA, USA) and oil/air manual microinjector (Cell tram®

Vario, Eppendorf, Hauppauge, NY, USA). Presumptive zygotes were placed in a

medium of DPBS containing 1 µM morpholino and injected using a holding pipette (15-

20 µm internal diameter, 120 µm outer diameter, 0.65 mm tip to elbow length; Smiths

Medical, Dublin, OH, USA) and an injection pipette (spiked tip, 5 µm internal diameter,

0.55 mm tip to elbow length; Smiths Medical). The injection solution was loaded into the

tip of the pipette each time before microinjection until the formed meniscus reached the

curve of the pipette. Then the pipette was advanced into the zygote and, through

application of negative pressure, the plasma membrane was ruptured. Then, positive

pressure was applied to return the cytoplasm into the zygote along with the injection

solution. The volume was regulated by observing the meniscus reaching the tip of the

pipette. It was estimated that 10-11 pl were injected based on the internal diameter and

the length of the tip of the pipette.

Zygotes were visualized under a digital inverted fluorescent microscope (Evos®

FL, Thermo Fisher Scientific). Those zygotes in which fluorescence was detected

(indicating successful injection of the fluorescently tagged morpholino) were cultured in

microdrops of SOF-BE2 as described earlier until Day 7 or Day 8 after insemination for

PCR and immunohistochemical analysis respectively. A total of 60 zygotes for each

morpholino group were cultured for each replicate. An additional negative control for the

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microinjection procedure was a group of 60 uninjected embryos per replicate that were

exposed to the same manipulation conditions as the microinjected embryos but that

were placed in DPBS during micromanipulation and were not injected.

Randomly selected blastocysts at Day 7 of development were harvested to

measure CCL24 mRNA. A total of three pools of 8-25 blastocysts per pool from three

different replicates were processed for qPCR as described above. A total of 12-22

blastocysts per group from another five different replicates were collected at Day 8 and

processed for immunolabeling for NANOG and GATA6 as described above. Number of

NANOG+ and GATA6+ cells and location of GATA6+ cell in the inner and outer part of

the ICM were determined as described above.

Statistical Analysis

The SAS v 9.4 software package (SAS Institute Inc., Cary, NC, USA) was used

for statistical analysis. Treatment effects were evaluated by analysis of variance using

the generalized linear models procedure (Proc GLM) of SAS; data shown are least-

squares means ± SEM. For main effects in which there were more than two levels,

differences between means were determined using the pdiff statement of PROC GLM

or by separating variation due to treatment into individual degree-of-freedom

comparisons using orthogonal contrasts. For example, differences in localization of

GATA6+ cells in the morpholino experiment were determined by separating variation

due to treatment into single degree-of-freedom orthogonal contrasts to determine the

difference between two controls (uninjected and standard negative morpholino) to the

targeting morpholino and the difference between the uninjected control to the standard

negative morpholino.

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Results

Developmental Changes in Expression of CCL24 in the Bovine Embryo

Expression of CCL24 was assessed in two experiments. In the first, presence of

CCL24 mRNA was evaluated by electrophoresis of PCR products (Figure 4-2A).

Expression of CCL24 was not detectable in the matured oocyte or in embryos at any

stage of development from the 2-cell stage through 16-cell stage. Transcript for CCL24

was detected in the morula at Day 6 of development and in blastocysts at Day 7, 8 and

9 of development. Amounts of mRNA were greater at Day 7 than at Day 8 or 9. In the

second experiment, amounts of CCL24 mRNA were quantified for embryos collected

from Day 5 to 7 of development (Figure 4-2B). There was no detectable CCL24 in Day 5

morulae but CCL24 mRNA was detectable in blastocysts at Day 6 and 7. Amounts of

mRNA was greater (P=0.01) at Day 7 than for other stages.

Immunolocalization of CCL24

Immunoreactive CCL24 was not detected in morulae collected at Day 5 or 6

(results not shown). There was also a fraction of embryos at both Days 7 (18% of the 93

embryos examined) and Day 8 (14% of the 96 embryos examined) that did not exhibit

immunoreactive CCL24. The remaining blastocysts exhibited immunoreactive CCL24 in

the cytoplasm but the pattern varied between embryos (Figure 4-3). The most common

pattern, exhibited in 43% of the Day 7 blastocysts examined and 48% of the Day 8

blastocysts examined, was for labeling in the ICM (defined as cells not labeled with

CDX2) to be greater than for cells in the TE (cells positive for CDX2) (Figure 4-3A). For

other blastocysts (28% at Day 7 and 32% at Day 8), labeling was similar for ICM and TE

(Figure 4-3B). In a minority of cases, labeling for CCL24 was greater for TE than ICM

(11% of Day 7 blastocysts and 6% of Day 8 blastocysts). Overall, intensity of labeling

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for CCL24, scored from 0 to 4, was greater for ICM than TE at both Days 7 and 8

(P<0.0001; Figure 4-4). In the negative control embryos, there was no specific labeling

(Figure 4-3C).

Consequences of Inhibition of CCR3 for Localization of GATA6+ Cells in Hypoblast

In two separate experiments, activation of CCR3, the receptor for CCL24, was

inhibited at Day 6 with one of two CCR3 antagonists, SB328437 and SB297006.

Neither inhibitor affected the proportion of embryos becoming a blastocyst at Day 7

(19.3% for control vs. 18.6% for SB328437, and 29.0% for control vs. 28.0% for

SB297006) or Day 8 (24.8% for control vs. 23.0% for SB328437, and 34.9% for control

vs. 34.1% for SB297006).

Labeling with anti-NANOG (epiblast) and GATA6 (hypoblast) was used to define

position of hypoblast cells within the ICM. Note that GATA6+ cells are present in both

TE and ICM, with the former being fluorescently dim and the latter being fluorescently

bright. Two examples of localization of GATA6+ cells in the ICM are shown in Figure 4-

5A (vehicle) and 5B (SB297006). For the experiment with SB328437, treatment did not

cause a change in number of NANOG+ (Figure 4-6A), GATA6+ (Figure 4-6B) or total

cells in the ICM (Figure 4-6C). However, the inhibitor did reduce (P=0.03) the percent

of GATA6+ cells located on the outside of the ICM (Figure 4-6D). Similarly, SB297006

had no effect on number of cells in the ICM (Figure 4-6E, 6F and 6G) but reduced

(P=0.03) the percent of GATA6+ cells localized on the outside of the ICM (Figure 4-6H).

Consequences of Knockdown of CCL24 for Localization of GATA6+ Cells in Hypoblast

Microinjection decreased the percent of putative zygotes that cleaved and that

became blastocysts (P<0.01) regardless of the solution injected but there was no

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difference between embryos injected with the standard negative or CCL24 morpholino

in cleavage rate or blastocyst development. Values for cleavage were 76.3% for

uninjected, 67.6% for the standard negative control and 64.1% for the CCL24

morpholino. Values for percent of putative zygotes developing to the blastocyst stage at

Day 7 were 18.7% for uninjected (from 529 zygotes), 11.1% for standard negative

control (from 477 zygotes) and 10.2% for CCL24 morpholino (from 495 zygotes). For

development of blastocysts at Day 8, values were 27.0% for uninjected, 16.5% for

standard negative control and 13.3% for CCL24 morpholino. Amounts of mRNA for

CCL24 in blastocysts at Day 7 were reduced for the targeting morpholino as compared

to the two control groups (P=0.08) (Figure 4-8A).

Labeling with anti-NANOG (epiblast) and GATA6 (hypoblast) was used to define

position of hypoblast cells within the ICM. Representative examples of labeling are

shown in Figure 4-7A, 7B and 7C. Treatment did not cause a change in number of

NANOG+ (Figure 4-8B), or GATA6+ (Figure 4-8C) in the ICM or total cells (Figure 4-8D).

However, the targeting morpholino reduced (P=0.02) the percent of GATA6+ cells

located on the outside of the ICM (Figure 4-8E).

Expression of CCR3 and CCR5

There was no detectable expression of CCR3 or CCR5 in the blastocyst at Day 7

or 8 of development even though primers were successful in amplifying both receptor

genes from lymphocyte cDNA (results not shown).

Discussion

One of the poorly understood phenomena involved in hypoblast formation is the

reorganization of the ICM by which GATA6+ cells change from being distributed

throughout the ICM to being limited to an epithelial layer lining the blastocoele (Hogan

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and Tilly 1978). Cell movement has been proposed to be involved in this process

(Chazaud et al. 2006) and Gata6+ cells have been shown to move across the ICM

(Plusa et al. 2008). The observation that 10 genes involved in the chemokine signaling

pathway are overexpressed in ICM of bovine embryos as compared to TE (Ozawa et al.

2012) suggests that chemokines could be involved in directed movement of GATA6+

cells in the ICM. Here we show that one of the genes overexpressed in the ICM of

bovine embryos, CCL24, does play a role in localization of GATA6+ cells in the bovine

embryo. This is so because the percent of GATA6+ cells localized to the outside portion

of the ICM was reduced by two antagonists of CCR3, the receptor for CCL24

(Daugherty et al. 1996; Gao et al. 1996; Kitaura et al. 1996) as well as by knockdown of

mRNA for CCL24.

The temporal pattern of expression of CCL24 in the bovine embryo is also

consistent with it playing a specific role in blastocyst formation. The gene does not

become expressed until Day 6, peaks in the blastocyst at Day 7 and then declines. In

vivo, as well, CCL24 transcript was not detected until the blastocyst stage (Jiang et al.

2014). Moreover, by the blastocyst stage, CCL24 is more expressed in ICM than TE

(Ozawa et al. 2012; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al. 2016).

Present results using an antibody raised to a CCL24 peptide indicated that the protein

can be found in both ICM and TE but that intensity of labeling is generally greater for

ICM. The variation in the pattern of localization could mean that either the protein can

be expressed by cells of the TE or that CCL24 secreted by ICM can move to the TE.

If CCL24 in the ICM is playing a role in chemotactic movement of GATA6+ cells

within the ICM, one would expect that a gradient of CCL24 would be set up within the

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blastocyst. It is not known whether cellular movement involved in positioning of GATA6+

and NANOG+ cells in the ICM in the bovine involves movement of GATA6+ cells,

NANOG+ cells, or both cell types. Thus, it is possible that CCL24 becomes preferentially

sequestered near or opposite to the site of hypoblast formation. Whether this is the

case could not be resolved immunochemically in this study.

One paradox of the present results is that two separate CCR3 antagonists

reduced the percent of GATA6+ cells located in the outer region of the ICM even though

mRNA for CCR3 and mRNA for a related receptor gene, CCR5, were not detectable in

embryos at the blastocyst stage. Similarly, CCR3 was not expressed in bovine embryos

produced in vivo from the 2-cell through blastocyst stage and CCR5 transcript was

absent in 2-cell, 8-cell, and morula stage embryos, weakly expressed in 4-cell embryos

and observed in only one of two pools of 16-cell embryos and one of two pools of

blastocysts examined (Jiang et al. 2014). There are several possible explanations for

the paradox. First, it is possible that only a small subset of cells in the ICM express

CCR3. Indeed, results from our laboratory (Chapter 2) using single cell RT-PCR is

indicative that a fraction of cells in the Day 8.75 blastocyst express CCR3. Secondly, it

is possible that receptors were synthesized in the oocyte or early embryo and persist

through the blastocyst stage despite low transcript abundance. Finally, it is possible that

CCL24 uses an alternate receptor other than CCR3 or CCR5 in the ICM. Chemokine

receptors share ligands and CCR3 can be activated by CCL5, CCL7, CCL11, CCL13,

CCL24, and CCL26 (Daugherty et al. 1996; Uguccioni et al. 1996, 1997). There are

other chemokine receptor genes expressed in the ICM of the bovine blastocyst

including ACKR4 (previously known as CCRL1), CCR5, CCR7 and CXCR4 (Ozawa et

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al. 2012; Jiang et al. 2014; Brinkhof et al. 2015; Hosseini et al. 2015). It is also possible

that the antagonists are blocking receptors distinct from those involved in chemokine

signaling although the fact that the knockdown of CCL24 caused similar effects makes

this less likely.

Typically, knockdown of mRNA in embryos is achieved by injection of siRNA into

the zygote (Wang et al. 2012; Driver et al. 2013; Goissis and Cibelli 2014). However,

efficiency of knockdown declines after several Days (Wang et al. 2012; GE Healthcare

Dharmacon Inc. 2016). Given that CCL24 does not become highly expressed until the

blastocyst stage, use of siRNA was deemed to be unlikely to reduce transcript

abundance for CCL24 in the blastocyst. However, microinjection of morpholinos

complementary towards CCL24 was effective at reducing mRNA for CCL24.

Morpholinos are oligonucleotides that are complementary to the sequence of the

targeting gene and where ribose is substituted with a morpholino ring to increase

stability inside the cell (Gene Tools LLC 2016). The selected targeting CCL24

morpholino interrupted the splicing region downstream of exon 2 (i.e. intron 2) to

produce a non-functional CCL24 mRNA.

The fact that large numbers of GATA6+ cells remained in the outer portion of the

ICM after treatment with CCR3 inhibitors or the CCL24 morpholino was to be expected

even if CCL24 participates in cell positioning. GATA6+ cells are originally dispersed

throughout the ICM and then GATA6+ cells become restricted to the hypoblastic

epithelium (Denicol et al. 2014). If a treatment (inhibitor or morpholino) completely

prevented the change in position, one would still find GATA6+ cells in the outer part of

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the ICM although, as observed here, the percent of cells in the outer portion would be

reduced.

Although evidence was obtained that CCL24 is involved in hypoblast formation in

cattle, the chemokine is not important in blastocyst differentiation in the mouse because

the gene is not expressed in the preimplantation embryo and because matings of Ccl24

null females with null males results in live offspring. Expression of Ccl24 in the

preimplantation mouse embryo was analyzed in silico by reviewing previous datasets

from studies of global gene expression. In none of the studies, which encompassed

analysis of oocytes, cleavage-stage embryos, and blastocysts up to E4.5, were

detectable amounts of Ccl24 consistently identified. This was true whether analysis was

performed using microarray analysis or RNA-Seq. Similarly, personal communication

from Marc Rothenberg, University of Cincinnati, indicates that female mice homozygous

for a deletion in a 4.0 kb fragment of mouse chromosome 5 containing exons 1-3 of

Ccl24 (Pope et al. 2005) bred to homozygous null males for the same deletion produce

live young with an average litter size of 4.9±0.5 pups at weaning (3-4 wk of age).

It is likely that the chemokine landscape is different between bovine and mouse

embryos not only for CCL24 but also for other chemokines. Besides CCL24 (Ozawa et

al. 2012; Jiang et al. 2014; Brinkhof et al. 2015; Hosseini et al. 2015; Zhao et al. 2016),

other chemokine genes expressed in the bovine blastocyst include CCL17, CCL25,

CCL26, CXCL17, and PPBP (Nagatomo et al. 2013; Jiang et al. 2014; Hosseini et al.

2015). CCL26 is expressed in the polar TE of the bovine embryo (Nagatomo et al. 2013;

Hosseini et al. 2015) and such a location for synthesis of CCL26 could set up a

chemokine gradient that facilitates directed movement of GATA6+ cells to the

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blastocoele. In the mouse, in contrast, Ccl17, Ccl25, Cxcl14 and Ppbp were very low or

non-detectable in blastocysts in most (Zeng et al. 2004; Maekawa et al. 2005; Tang et

al. 2009; Xie et al. 2010; Boroviak et al. 2015) but not all (Xie et al. 2010) transcriptome

databases. Moreover, Ccl26 is not expressed in the blastocyst (Zeng et al. 2004;

Maekawa et al. 2005; Tang et al. 2009; Xie et al. 2010; Boroviak et al. 2015) or any

other tissue as it is considered a pseudogene (Pope et al. 2005).

The fact that CCL24 has different roles in the bovine and mouse embryo is not

surprising because of species divergence in preimplantation development among

mammals including for the mechanisms controlling the first differentiation events in the

blastocyst. Following embryonic genome activation, transcription occurs in a species-

specific pattern, with that of the bovine and human being more similar to each other

than to the mouse (Zeng et al. 2004). Mutations in the regulatory elements for Pou5f1

(i.e., Oct4) occurring in the mouse mean that expression of this transcription factor is

limited to the ICM in the mouse but not in the cow and human (Wang et al. 2012).

Formation of the hypoblast involves actions of FGF4 in the mouse (Rossant et al. 2003;

Chazaud et al. 2006; Kang et al. 2013; Morris et al. 2013) but not in the bovine and

human (Kuijk et al. 2012). Greater similarity between cattle and humans as compared to

the mouse despite the fact that humans diverged from mice more recently than from

cattle may reflect the high rate of evolutionary change in mice [see discussion in ref.

(Hansen 2010)].

In conclusion, findings of the present experiments indicate that the chemokine

CCL24 participates in reorganization of the ICM of the bovine blastocyst to facilitate

localization of GATA6+ cells to the outside of the ICM.

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Figure 4-1. Schematic representation of methodology used for determination of number

of cells located in inner and outer regions of the ICM. Blastocysts were collected at Day 8 and subjected to differential immunolocalization of GATA6 (hypoblast marker; red) and NANOG (epiblast marker; green). (I) Z-stack image projection of sections A-H. Following confocal microscopy, cells in each embryo section were counted sequentially. Panels A, B, G and H are representative of the sections to denominate that the cells in the first and last 25% of the sections are outer cells. Panels C-F are representative of the sections to denominate that the inner 50% are inner cells of the inner cell mass. The exception in the inner sections were the cells on the outside that were denominated outer cells. Closed arrows indicate outer cells=o. Closed diamond arrows indicate inner cells=i. Scale bar=10 µM.

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Figure 4-2. Developmental changes in CCL24 expression. (A) Representative results for amplification of CCL24 by RT-PCR from cDNA of oocytes and embryos at various stages of development. Amplicons were separated by electrophoresis using 1% (w/v) agarose gels. DNA was labeled with ethidium bromide. The size of the CCL24 amplicon is 120 bp. The image is representative of results from 4 biological replicates per stage. (B) Results of a separate experiment in which expression of CCL24 relative to housekeeping genes (GAPDH and YWHAZ; n=3 biological replicates) was determined by qPCR. Transcript for CCL24 was non-detectable for morulae at Day 5 or Day 6 but was detected for blastocysts at Day 6 and 7. Using statistical analysis, expression was higher for Day 7 blastocysts (P=0.01) than for embryos at other stages. Data are least-squares means + SEM.

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Figure 4-3. Representative examples of patterns of immunoreactive CCL24 in the Day

7 and Day 8 blastocyst as determined by epiflourescent microscopy. Shown are two representative embryos. Note that immunoreactivity was greater for ICM than TE for one embryo (A) whereas immunoreactive CCL24 (green) was present in both ICM and TE for another embryo (B). The ICM, which was identified by lack of immunolabeling of CDX2 (red), is outlined in white. Panel (C) shows an example of a negative control embryo labeled with normal mouse serum (NMS) and mouse IgG (mIgG) instead of CCL24 and CDX2. All nuclei were labeled with Hoescht (blue). Scale bar=10 µM.

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Figure 4-4. Differential immunolocalization of CCL24 in the ICM and TE of Day 7 and Day 8 blastocysts. CCL24 intensity was arbitrarily scored from 0-4 (0=no expression, 4=very bright) in the ICM and TE. Cell lineage was determined by labeling with anti-CDX2 (TE marker). Data are least-squares means ± SEM of results from 93 Day 7 and 96 Day 8 embryos. The P values for effects of cell type are indicated by the value above the bars.

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Figure 4-5. Confocal z-stack projections of representative Day 8 blastocysts after

inhibition of CCR3. Embryos were treated with vehicle (A) or SB297006 (B) from Day 6 to Day 8. Labeling with anti-GATA6 (hypoblast) and anti-NANOG (epiblast) was used to define position of hypoblast cells within the ICM. Note that GATA6+ cells are present in both TE and ICM, with the former being fluorescently dim and the latter being fluorescently bright. Panel A shows two embryos and Panel B a single embryo. Open arrowheads point to outer cells and closed arrowheads point to inner cells. All nuclei were labeled with Hoescht (blue). Red=GATA6+ and green=NANOG+. Scale bar=10 µM.

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Figure 4-6. Inhibition of CCR3 affects the location of GATA6+ cells at Day 8 of

development. Data from the experiment with SB328437 are in panels A-D while data from the experiment with SB297006 are in panels E-H. The number of embryos per treatment for panels A-D were 8 for vehicle and 9 for SB328437. The number of embryos per treatment for panels D-H were 68 for vehicle and 68 for SB297006. Data are least-squares means ± SEM. The P values for significant treatment effects are indicated by the value above the bar for treated embryos.

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Figure 4-7. Confocal z-stack projections of representative Day 8 blastocysts as affected

by morpholino treatment. Selected zygotes were randomly injected with standard negative or CCL24 targeting morpholino and returned to culture until Day 8; uninjected control embryos were exposed to same conditions expect microinjection. Labeling with anti-GATA6 (hypoblast) and anti-NANOG (epiblast) was used to define position of hypoblast cells within the ICM. Panels A and B are representative images showing an uninjected embryo (A), an embryo injected with the standard negative control (B) and an embryo injected with targeting morpholino (C). Open arrowheads point to outer cells and closed arrowheads point to inner cells. All nuclei were labeled with Hoescht (blue). Red=GATA6+ and green=NANOG+. Scale bar=10 µM.

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Figure 4-8. Injection of a morpholino against CCL24 affects the location of GATA6+

cells. (A) Real-time RT-PCR CCL24 mRNA data from three pools of Day 7 blastocysts. Data are presented as fold change relative to uninjected control; housekeeping genes were YWHAZ and GAPDH. (B-D) Number of NANOG+ (B) and GATA6+ cells (C) in the ICM as well as total number of cells in the ICM (D). Treatment did not cause a change in number of any cell type. E) Percent of GATA6+ cells in the ICM that were located on the outside of the ICM. The percent of cells on the outside was reduced by the morpholino against CCL24 as compared to the two control treatments (P=0.02). The number of embryos analyzed in panels B-E was 22 for uninjected, 13 for the standard negative morpholino and 12 for the morpholino against CCL24.

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CHAPTER 5 THE BOVINE EMBRYO HATCHES FROM THE ZONA PELLUCIDA THROUGH

EITHER THE EMBRYONIC OR ABEMBRYONIC POLE

Introduction

Hatching from the zona pellucida is a prerequisite for the preimplantation embryo

to attach to the uterus and initiate placentation. Failure of the process could conceivably

lead to pregnancy loss, as indicated by some studies where assisted hatching improved

clinical pregnancy rate in women (Carney et al. 2012) and cattle (Taniyama et al. 2011).

Three mechanisms are known to be involved in the process of blastocyst hatching:

mechanical forces exerted on the zona pellucida by blastocyst expansion, (Cole 1967;

Massip and Mulnard 1980; Massip et al. 1982), weakening of the zona pellucida by

enzymatic degradation (Sawada et al. 1990; Berg and Menino, Jr. 1992; Mishra and

Seshagiri 2000), and penetration of the zona pellucida by projections of

trophectodermal cells (Gonzales et al. 1996; Seshagiri et al. 2009). The relative

importance of these mechanisms varies between species. In the hamster, for example,

the blastocoel cavity shrinks in size prior to hatching (Seshagiri et al. 2009). There is

also variation between species in the nature of the proteinases implicated in dissolution

of the zona pellucida including a trypsin-like enzyme in the mouse (Perona and

Wassarman 1986; O’Sullivan et al. 2001), cathepsins in the hamster (Sireesha et al.

2008) and a urokinase-type plasminogen activator (PLAU) in cattle (Berg and Menino,

Jr. 1992; Coates and Menino 1994).

There is evidence that the blastocyst preferentially hatches from the abembryonic

pole (i.e., opposite the ICM and involving mural TE) regardless of whether attachment of

the blastocyst to the endometrium occurs at the abembryonic (guinea pig, hamster,

mouse) or embryonic pole (human). Hatching is more frequent from the abembryonic

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pole in mice (Perona and Wassarman 1986) and humans (Sathananthan et al. 2003).

Also, trophectodermal projections predominate in this part of the TE for guinea pig

(Spee 1883), hamster (Gonzales et al. 1996) and human, (Sathananthan et al. 2003),

and trypsin-like proteinase is limited to mural TE in mice (Perona and Wassarman

1986).

All of the species mentioned in the previous paragraph undergo implantation in

the uterine endometrium shortly after hatching. Whether species that undergo a

prolonged period of time after hatching before attachment to the endometrium are

similarly polarized with respect to the site of hatching has not been established. The

cow is one such species. While hatching occurs about 7 to 10 Days after fertilization

(Betteridge and Fléchon 1988), the first attachments between TE and endometrium do

not occur for about 10 Days, at Day 20 of gestation (King et al. 1981). In the only study

conducted to date, it was found that 48% of bovine embryos hatched through an

opening in the zona pellucida near the embryonic pole while the remainder hatched

from either the TE near the side of the ICM (embryonic mural TE; 36%) or from the

abembryonic polar TE (16%) (Niimura et al. 2010). Here we reexamined the question of

the location of hatching through the zona pellucida in the bovine using a combination of

light microscopy and epifluorescence microscopy of embryos labeled with various

markers of cell lineage. Among the markers used were CDX2 and YAP1, both markers

of TE (Strumpf et al. 2005; Chen et al. 2010), NANOG, which is specific to epiblast cells

of the ICM (Denicol et al. 2014) and GATA6, which is most abundant in cells of the

hypoblast (Denicol et al. 2014).

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Materials and Methods

In Vitro Production of Embryos

Production of embryos was performed as described earlier (Ortega et al. 2017),

using sperm and oocytes from a mixture of animals of various breeds including B.

taurus, B. indicus and admixture of the two genetic groups. Embryos were cultured in

groups of 30 in 50 µL oil-covered microdrops of a serum-free culture medium, SOF-BE2

(Kannampuzha Francis et al. 2017) at 38.5°C in a humidified environment consisting of

5% (v/v) O2, 5% (v/v) CO2 and the balance nitrogen.

A total of 110 hatching blastocysts at Day 7 or 8 after insemination were

collected for analysis. Each of these blastocysts had an ICM that could be clearly

identified using a digital inverted microscope (Evos® FL, Thermo Fisher Scientific,

Waltham, MA, USA). A subset of these blastocysts (n=26) were also subjected to

analysis by immunofluorescence.

Immunolocalization of Cells Labeled with Epiblast, Hypoblast and TE Markers

A set of hatching blastocysts were labeled using Hoescht 33342 (to label all

nuclei) and a combination of two antibodies against GATA6 and CDX2 (n=4), CDX2 and

YAP1 (n=2), or GATA6 and NANOG (n=8). In addition, another set of hatching

blastocysts were labeled using Hoescht 33342 and either CDX2 (n=8), β-catenin (n=3)

or non-phospho (active) β-catenin (n=1). Primary antibodies used were mouse

monoclonal antibody against CDX2 (Biogenex, Fremont, CA, USA), rabbit polyclonal

antibody against human GATA6 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit

monoclonal anti-YAP1 (Cell Signaling Technology, Danver, MA, USA), rabbit polyclonal

anti-β-catenin (Abcam, Cambridge, MA, USA), rabbit monoclonal anti non-phospho

(active) β-catenin (Cell Signaling Technology) and mouse polyclonal antibody against

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human NANOG (eBioscience, San Diego, CA, USA). The secondary antibodies were

FITC conjugated goat polyclonal anti-mouse IgG (Abcam) and Alexa Fluor 555

conjugated goat polyclonal anti-rabbit IgG (ThermoFisher). All antibodies were used at 1

µg/mL except for mouse monoclonal antibody against CDX2 (Biogenex, Fremont, CA,

USA), which was used at the working concentration provided by the manufacturer.

Non-specific binding was evaluated by substituting IgG for the primary antibody.

All steps for immunolocalization proceeded at room temperature unless

otherwise stated. Briefly, blastocysts were collected, washed 3 times in DPBS

containing 0.1% (w/v) PVP (DPBS/PVP), fixed for 15 min in 4% (w/v) paraformaldehyde

diluted in DPBS/PVP, washed 3 times in PBS/PVP, incubated for 30 min in

permeabilization buffer [DPBS/PVP containing 0.25% (v/v) Triton X-100], and then

incubated for 1 h in blocking buffer [5% (w/v) bovine serum albumin (BSA) in DPBS].

Blastocysts were then incubated overnight with the first primary antibody at 4°C,

washed 3 times in washing buffer [DPBS containing 0.1% (w/v) BSA and 0.1% (v/v)

Tween-20], and for 1 h in secondary antibody. The immunolabeling procedure was then

repeated with a second primary antibody for those blastocysts labeled with two primary

antibodies. Following labeling with antibodies, blastocysts were washed 3 times in

washing buffer, incubated with 1 µg/mL Hoescht 33342 in DPBS/PVP for 15 min to label

nuclei, washed once in DPBS/PVP and mounted on glass slides in 5-10 µL of SlowFade

Gold antifade reagent (ThermoFisher Scientific). Blastocysts were visualized at 40X

objective using a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Göttingen,

Germany) and Zeiss filter sets 02 (DAPI), 03 (FITC) and 04 (rhodamine). Digital images

were acquired using AxioVision software (Zeiss) and a high-resolution black and white

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Zeiss AxioCam MRm digital camera. Image J V. 1.48 (National Institutes of Health,

Bethesda, MD, USA) was used to visualize images, count the number of cells, and

measure the embryo diameter and length of the hatching opening.

One hatching blastocyst was subjected to confocal microscopy after

immunolocalization of GATA6 and NANOG. The blastocyst was examined on a spinning

disk confocal scanner mounted on an Olympus DSU-IX81 inverted fluorescent

microscope. Images were captured with a 40X objective using DAPI, FITC and red

fluorescent protein (RFP) filter sets. Digital images were taken using an attached

Hamamatsu C4742-80-12AG monochrome CCD camera. SlideBook 6 Reader

(Intelligent Imaging Innovations, Inc., Denver, CO, USA) was used to visualize images

and count total number of cells.

Identification of Cell Types and Embryonic Poles

Hatching embryos were separated in two categories based on the location of the

hatching opening: 1- embryonic pole, if hatching occurred ipsilateral to the ICM, or 2-

abembryonic pole, if hatching occurred from the opposite end to the ICM. Because of

occasional difficulties in assigning exact location of the initial site of penetration of the

zona pellucida, the abembryonic group included blastocysts in which hatching occurred

from the lateral side of the embryo. The orientation of the hatching site was determined

by locating the ICM by light microscopy, and for embryos that were immunolabeled, by

examining the cell type present in the hatched portion of the blastocyst. Nuclei that were

either NANOG+, bright GATA6+, YAP1- or CDX2- were considered to be ICM. Nuclei

that were CDX2+ cells, dim GATA6+ or YAP1+ were considered TE. Immunoreactive β-

catenin was detected on the membrane of all cells but was more intense for cells of the

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ICM. Accordingly, cells with bright β-catenin labeling were considered ICM and cells

with less intense labeling were considered TE.

Statistical Analysis

Data were analyzed using the SAS v 9.4 software package (SAS Institute Inc.,

Cary, NC, USA); embryo was considered the experimental unit. The frequency

procedure (Proc FREQ) was used to calculate the proportion of blastocysts that hatched

from the embryonic and abembryonic pole. Differences between the two types of

embryos in terms of proportion of ICM and TE cells in the hatched portion of the embryo

was determined by analysis of variance using the generalized linear models procedure

(Proc GLM) of SAS. Data shown are least-squares means ± SEM.

Results

Examples of blastocysts hatching from the embryonic and abembryonic poles as

determined by light microscopy are shown in Figure 5-1. A total of 55% (60/110) of

blastocysts hatched through the embryonic pole and 45% (50/110) through the

abembryonic pole. Of these 50 embryos, 31 hatched from the lateral TE (i.e., to the side

of the ICM) and 19 from the contralateral TE (i.e., opposite from the ICM). Note that, in

many cases, blastocysts were examined when hatching was extensive and

classification as to lateral vs contralateral locations is tentative. There was no difference

in frequency between Days 7 (55% embryonic pole vs 45% abembryonic pole) and 8

(54% embryonic pole vs 46% abembryonic pole) (Table 1).

Use of immunofluorescence to examine the cells that had passed through the

zona pellucida demonstrated how the site of hatching affects the composition of the

hatched portion of the blastocyst. For blastocysts hatching through the embryonic pole,

the hatched portion of the blastocyst contained cells of TE and/or ICM origin. For

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example, the embryo in Figure 5-2A, none of the cells in the hatched area expressed

the TE markers CDX2 or YAP1. For the blastocyst in Figure 5-2B, the hatched area

contained numerous cells that were positive for the ICM marker NANOG but also cells

negative for ICM markers. In contrast, when hatching was through the abembryonic

pole, all or most cells in the hatched region were TE. For example, in Figure 5-2C, the

hatched region was devoid of NANOG+ and bright GATA6+ cells. Overall, the proportion

of cells in the hatched portion of the blastocyst that was ICM was higher (P<0.0001) for

blastocysts experiencing hatching through the embryonic pole than for blastocysts

hatching through the abembryonic pole (68.3 vs 13.0%) (Table 2). In addition, 49.3% of

the cells of the ICM were in the hatched portion of the blastocyst for those hatching

through the embryonic pole vs 8.1% for those hatching through the abembryonic pole

(P<0.0001; Table 2).

One hatching blastocyst labeled with antibodies to NANOG and GATA6 was

examined by confocal microscopy (Figure 5-3). This embryo was hatching through the

embryonic pole. Sections taken through the plane of focus where the zona pellucida

had been penetrated by cells of the blastocyst show clearly that the ICM has been

stretched across the opening in zona pellucida with the hatching portion outside the

zona pellucida, the larger inner portion still within the zona, and with two NANOG+ cells

on either side of the opening – one that is passing through the zona and another that

appears to be following behind the first cell.

Discussion

Present results confirm earlier results using light microscopy (Niimura et al. 2010)

that the in vitro developed bovine blastocyst can hatch from either the embryonic or

abembryonic pole, with about 50% of blastocysts experiencing hatching through the

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embryonic pole. Given that less than 50% of the surface area of the zona pellucida is

adjacent to the ICM, a preference for hatching through the embryonic pole is indicated.

Results also confirm earlier results (Niimura et al. 2010) that, when hatching does not

occur through the embryonic pole, it is more likely to commence in a region of the TE

lateral to the ICM than directly opposite to it. The present results extend earlier findings

(Niimura et al. 2010) by demonstrating how the site of hatching affects the composition

of the hatched portion of the blastocyst. When hatching is from the embryonic pole, a

majority of the cells in the hatched region of the blastocyst are ICM whereas, TE cells

predominate in the hatched region of blastocysts hatching from the abembryonic pole.

Thus, the nature of the first physical contact of the cells of the embryo with the female

reproductive tract is different for blastocysts hatching from the embryonic vs

abembryonic pole. In the cow, hatching takes place in the uterus (Betteridge and

Fléchon 1988) and it remains to be seen whether the endometrium responds differently

to a blastocyst hatching from the embryonal vs abembryonal poles. This is a possibility

because gene expression varies between ICM and TE (Nagatomo et al. 2013) and

recent experiments in cattle indicate that the cleavage-stage embryo can interact with

the oviduct to change gene expression (Lonergan and Forde 2014; Gómez and Muñoz

2015).

The cow is distinct from other species studied because abembryonal hatching

predominates in the mouse (Perona and Wassarman 1986), human (Sathananthan et

al. 2003), guinea pig (Spee 1883) and hamster (Gonzales and Bavister 1995). The

reason for the difference is not known. Except for the cow, all of the above-named

species attach to the endometrium soon after hatching whereas the bovine blastocyst

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resides in the uterus for 10 or more Days before attaching to the endometrium. Perhaps

orientation of hatching is less critical in species where the embryo spends a prolonged

period free of permanent attachment to the endometrium. Examination of the location of

hatching in species that share this characteristic with the cow (sheep, pig, and horse)

could provide illumination on this point.

It also remains to be determined why some bovine blastocysts hatch from one

location whereas others hatch from another location. In species in which hatching is

biased towards the abembryonal pole, trophectodermal projections and proteinase

activity is localized to this region (Spee 1883; Perona and Wassarman 1986; Gonzales

and Bavister 1995; Sathananthan et al. 2003). One possibility is that trophectodermal

projections or proteinase activity develops uniformly in the bovine blastocyst and that

the site of hatching depends on physical characteristics of the zona pellucida.

Alternatively the specific location of trophectodermal projections or proteinase activity

could vary between embryos.

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Table 5-1. Percent and frequency of embryos hatching from the embryonic or abembryonic pole at Days 7 and 8a

Hatching pole related to the inner cell mass (ICM)

Embryonic Abembryonic

Adjacent to the ICM Lateral to the ICM Opposite to the ICM

Day 7 55% (32/58) 28% (16/58) 17% (10/58) Day 8 54% (28/52) 29% (15/52) 17% (9/52)

a A total of 110 embryos were evaluated after bright field and epifluorescence microscopy imaging; 58 at Day 7 and 52 at Day 8

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Table 5-2. Proportion of hatched cells that were inner cell mass (ICM) and trophectoderm (TE) as affected by hatching pole a

Hatching pole Embryonic Abembryonic

Percent of hatched cells that were ICM

68.3±5.6*** 13.0±10.2

Percent of ICM cells that hatched

49.3±5.2*** 8.1±9.6

a A total of 26 embryos (7 at Day 7 and 19 at Day 8) were evaluated by epifluorescence microscopy ***Values with asterisks indicate significant difference between groups (P<0.001)

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Figure 5-1. Representative images of embryos hatching through the embryonic or

abembryonic pole. Panels A-B show embryos escaping the zona through the embryonic pole. Panels C-D represent embryos hatching through the abembryonic pole completely opposite to the ICM (C, D). The area encircled with the dotted line represents the inner cell mass. Scale bar=50 µM.

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Figure 5-2. Examples of immunolocalization of inner cell mass (ICM) and trophectoderm (TE) in blastocyst experiencing hatching through the embryonic pole (A, B) and abembryonic pole (C). Immunofluorescence was evaluated using epifluorescence microscopy. Panel A is a blastocyst labeled with anti-YAP1 (red) and anti-CDX2 (green) while panel B and C are blastocysts labeled with anti-GATA6 (red) and anti-NANOG (green). Nuclei were labeled with Hoescht 33342 (blue). For each blastocyst, immunofluorescence is shown separately for the red, green and blue channels. For panel A, ICM cells were identified as those that the nuclei were YAP1- and CDX2- while TE cells had nuclei that were YAP1+ and CDX2+. For panels B and C, cells of the ICM that are epiblast are those with nuclei that are NANOG+; cells of the ICM that are hypoblast are those with nuclei that have bright GATA6+. Cells of the TE are those with nuclei that are NANOG- and have dim GATA6+. Panel B shows 50% of the ICM cells on the hatched area and 50% remaining inside. Panel C shows the hatched area being devoid of NANOG+ and bright GATA6+ nuclei. The area encircled with the dotted line represents the ICM. The white arrowheads indicate the opening through which the embryo is exiting. Scale bar=20 µM.

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Figure 5-3. Analysis of a blastocyst hatching through the embryonic pole using confocal

microscopy. The blastocyst was labeled using antibodies against NANOG (green) and GATA6 (red). Nuclei were labeled with Hoescht 33342 (blue). Cells of the inner cell mass that are epiblast are NANOG+ and GATA6- while hypoblast cells are NANOG- and GATA6+. The white arrowhead points to the hatching opening. Note the pair of NANOG+ epiblast cells (pointed by the arrowhead) exiting the zona pellucida. Scale bar=20 µM.

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CHAPTER 6 GENERAL DISCUSSION

As stated throughout the literature review, the process by which the newly

fertilized embryo develops into a blastocyst has been well mapped out in the mouse. In

contrast, the cellular differentiation processes involved in preimplantation development

have not been studied in detail in the cow. There is a compelling need to understand

these processes in cattle because many mechanisms involved in early development

have not been conserved during evolution (Kuijk et al. 2008; Xie et al. 2010; Berg et al.

2011). Moreover, proper embryo differentiation and preimplantation development is

crucial for establishment of pregnancy, fetal development throughout gestation and birth

of a normal offspring (Fischer-Brown et al. 2004; Hansen 2011; Wiltbank et al. 2016).

Making study of preimplantation development in the cow has been more difficult in cattle

than in the mouse not only because it is more difficult to utilize gene knockout models

but also because there are few molecular markers described in the cow to allow

discrimination between epiblast, hypoblast and TE. The aims of the research described

in this dissertation were to use the cow as a model to develop markers for

characterization of cell lineages in the blastocyst, understand the role of key molecules

in the first and second lineage determination events of the blastocyst, and gain

understanding of the spatial orientation of the processes for formation of the hypoblast

and hatching from the zona pellucida. As will be highlighted in this chapter, the research

has resulted not only in a large number of markers that define epiblast, hypoblast, and

four populations of TE but also has provided key details in our understanding of the

mechanisms controlling blastocyst formation in the cow.

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By combining results of the research presented here and what has been

previously described in the literature, we can propose a new model for how the bovine

blastocyst forms, differentiates into the three cell layers, and hatches from the zona

pellucida (Figure 6-1).

First, experiments in this dissertation are indicative that development to the

blastocyst stage of development can occur independent of mRNA for AMOT (Chapter

3), CCL24 (Chapter 4), CDX2 (Berg et al. 2011; Goissis and Cibelli 2014), FGFR2

(Akizawa et al. 2016), or YAP1 (Chapter 3). Results from Chapter 3 indicate that a

decrease of either AMOT or YAP1, or inhibition of the YAP1-TEAD4 interaction,

decreases the proportion of embryos becoming blastocysts. However, the overall ability

of the embryo to form a blastocoel, was not affected by knockdown of either AMOT or

YAP1. The same was true for CCL24 knockdown (Chapter 4) which did not affect the

proportion of embryos becoming blastocysts. Nonetheless, function of the resultant

blastocyst is compromised by reduction in mRNA for AMOT, CCL24 and YAP1. In

particular, the role of the Hippo pathway member, YAP1, is reflected in the mid-late

blastocyst because when the YAP1-TEAD4 interaction is inhibited, formation of the

epiblast and hypoblast is inhibited and, the embryo is not able to hatch from the zona

pellucida (Chapter 3). Similarly, when YAP1 is decreased, the proportion of embryos

that hatched from the zona pellucida was also lower. The membrane bound protein,

AMOT, which is another regulator of the Hippo pathway (Paramasivam et al. 2011;

Hirate et al. 2013), is dispensable for maintenance of the blastocyst because the ability

of the embryo to hatch was not compromised after the decrease of AMOT but, the

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amount of TE cells was lower indicating that it is playing a role in TE proliferation

(Chapter 3).

Results of experiments in the dissertation also revealed new insights into

regulation of the second differentiation event in the blastocyst – formation of epiblast or

hypoblast from ICM precursors (Figure 6-1). In particular, results are indicative that

YAP1 can promote hypoblast differentiation or function while AMOT inhibits hypoblast

differentiation (Chapter 3). Treatment of embryos with YAP1 targeting GapmeR not only

decreased genes that are markers for TE but several genes that were markers of

epiblast and hypoblast. It is not clear from these results whether YAP1 acts directly in

epiblast or hypoblast, as they both express YAP1 mRNA (Chapter 2) or whether the

disruption in TE function caused by reduction in YAP1 mRNA affects other cell types in

the blastocyst. The same is true for AMOT knockdown embryos (Chapter 3). The mRNA

for TE marker, CDX2, was lower at Day 7.5 but, the number of TE cells was unaffected

then. The number of TE cells was reduced in the Day 8.5 blastocysts with lower AMOT.

Moreover, the number of epiblast and hypoblast cells was not decreased as a result of

AMOT knockdown but the epiblast and hypoblast markers, NANOG and GATA6,

respectively, were lower in embryos with lower AMOT.

Previous results indicate that FGFs, FGFR2 and MAPK are involved in

differentiation of the epiblast and hypoblast (Yang et al. 2011; Kuijk et al. 2012; Akizawa

et al. 2016). The experiment using the MAP2K1/2 inhibitor in Chapter 3 confirmed the

importance of the MAPK pathway for inhibition of epiblast formation. The MAPK

pathway is activated as a result of the FGFR2 activation by one of the FGF ligands. In

the bovine, these ligands are probably FGF2 and FGF4. FGF2 is highly detected in the

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embryo throughout the preimplantation stages (Jiang et al. 2014) and when blastocysts

are supplemented with FGF2, the outgrowths of the ICM are mainly composed of

hypoblast cells (Yang et al. 2011). Although FGF4 was not detected in in vivo produced

embryos, single cell gene expression analysis in Chapter 2 revealed that epiblast cells

have high expression of FGF4 when compared to other cell populations. Furthermore,

addition of FGF4 in the presence of heparin increased number of hypoblast cells (Kuijk

et al. 2012). In vivo embryos express a decrease in FGFR2 from the zygote to the 8-16

cell stage embryo coinciding with EGA and, after EGA, FGFR2 increases from the

morula to the blastocyst stage (Jiang et al. 2014). FGFR2 has been detected to be

differentially expressed between ICM and TE (Nagatomo et al. 2013; Hosseini et al.

2015) and, our results indicate that FGFR2 is expressed only in hypoblast cells

(Chapter 2). When FGFR2 is knocked down, expression of GATA6 and NANOG is

unchanged but another hypoblast marker, HNF4A, appeared to be decreased (Akizawa

et al. 2016). Thus it is likely that FGFR2 is involved in hypoblast formation.

As mentioned before, experiments in Chapter 4 are indicative that CCL24 is

involved in the formation of the epiblast and hypoblast but it is important for correct

cellular spatial distribution of hypoblast cells (Figure 6-1). The first evidence for this

suggestion is that CCL24 is more expressed in the ICM when compared to the TE

(Ozawa et al. 2012; Nagatomo et al. 2013; Brinkhof et al. 2015; Hosseini et al. 2015;

Zhao et al. 2016) and that CCL24 is involved in cell migration (White et al. 2000;

Provost et al. 2013). Our results indicate that CCL24 is not required for blastocyst

formation as the proportion of embryos becoming blastocysts was not affected after

CCL24 knockdown (Chapter 4). However, deregulation of CCL24 resulted in a decrease

169

proportion of GATA6+ cells on the periphery of the ICM. The fact that a large proportion

of cells remained on the outside of the ICM after CCL24 knockdown could suggest that

other chemokines are working along with CCL24. However, it is more likely that,

because epiblast and hypoblast cells begin to form scattered in the ICM, these outer

cells were already on the outside and thus did not need to be moved. Nevertheless, our

findings present a new role for chemokines, and for CCL24, in the organization of the

ICM.

Lastly, we confirmed earlier observations (Niimura et al. 2010) that the hatching

process by which the blastocyst escapes the zona pellucida can occur with equal

frequency through the embryonic or abembryonic poles of the embryo (Chapter 5).

Because the surface area on the embryonic pole is smaller, there is probably some bias

towards hatching through this pole. It is not known whether the preference for an

individual blastocyst to hatch from the embryonic or abembryonic poles resides in

differences in mechanisms embryos use for hatching or whether the zona pellucida has

different physical characteristics that causes hatching to be favored from one pole or the

other. The bovine embryo can communicate with the female reproductive tract as early

as the cleavage-stage (Lonergan and Forde 2014; Gómez and Muñoz 2015). Thus, it is

possible that the orientation of hatching could affect the nature of the first blastomeres

that come in contact with the endometrial epithelium. When hatching was through the

embryonic pole, 68% of cells in the hatching portion of the blastocyst were ICM vs only

13% when hatching was through the abembryonic pole. Analysis of gene expression by

single cells from the blastocyst (Chapter 2) has led to the identity of a large number of

markers of epiblast, hypoblast and TE that can be useful for further studies on the

170

function of the bovine blastocyst. The identity of these markers is summarized in Figure

6-2. Examination of the function of these markers is consistent with what is known about

the function of specific cell types in the blastocyst. The epiblast remains pluripotent

through the mid-late phases of blastocyst development (Nichols et al. 1998; Kirchhof et

al. 2000; Avilion et al. 2003; Silva et al. 2009). Consistent with this fact was upregulation

of genes involved in pluripotency (NANOG and POU5F1). Moreover, gene expression

differences between cell types is consistent with the idea that it is FGF4 from epiblast

precursors acting on FGFR2 expressed by hypoblast precursors that causes hypoblast

and epiblast differentiation (Kujik et al., 2012). Our results agree with the hypoblast

involving FGF4 from the epiblast and FGFR2 from itself to differentiate. Downstream

genes of the FGF pathway, SOX17 (Frankenberg et al. 2011) and FN1 (Shirai et al.

2005), which are also markers for hypoblast in the mouse, were also upregulated in the

bovine hypoblast cells. The hypoblast also seems to be under epigenetic regulation as

depicted by the detection of HDAC1 which in mouse TE is involved in silencing Nanog

(Carey et al. 2015) and could possibly be acting in a similar way in the bovine

hypoblast.

The TE was characterized by upregulation of well-known differentiation markers

such as CDX2, GATA3, IFNT and KRT8. More importantly, it was found that the TE is

heterogeneous with respect to gene expression. Four subpopulations could be identified

(Chapter 2). One of the populations belonged to a single subclade, TE1, while the other

three were, TE2, TE3, TE4 formed a second subclade. Based on the low expression of

EOMES and IFNT, which can be considered markers for mature TE, it is proposed that

TE1 is the least differentiated TE subpopulation. Based on high expression of EOMES

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and KLF2 [an ESC pluripotency marker (Qiu et al. 2015)], it is proposed that TE4 could

be a TSC. Moreover, TE2, which contains 17 genes that are upregulated compared to

other subpopulations, is the most differentiated TE subpopulation and TE3 is more

mature than TE1 but not fully differentiated and not a TSC.

It is uncertain if the heterogeneity of the TE is related to variation in the

differentiation status of TE cells within the same blastocyst or to variation among

blastocysts. It is proposed that an individual blastocyst contains more than one type of

TE subpopulation and that the TE goes through several levels of differentiation as the

embryo develops. Supporting this idea are observations that there is heterogeneity of

expression of IFNT with TE cells (Johnson et al. 2006) and that gene expression differs

between polar and mural TE (Nagatomo et al. 2013).

A pending question is whether the translated protein from these cell-specific

transcripts follow the same pattern of expression. Previous studies show that this is the

case for CDX2, GATA6, IFNT and POU5F1 (Johnson et al. 2006; Ross et al. 2009;

Berg et al. 2011; Kuijk et al. 2012; Schiffmacher and Keefer 2013; Denicol et al. 2014).

If other proteins recapitulate the gene expression, antibodies for these proteins can be

used to distinguish between cell populations in an experimental setting.

Another unresolved question is whether some of the differences in gene

expression between cell types change as the embryo goes through development. This

was the case observed for the chemokine, CCL24, which is more highly expressed in

the ICM than TE at Day 7-8 (Ozawa et al. 2012; Hosseini et al. 2015) but was

upregulated in TE4 as compared to other cell populations at Day 8.75 (Chapter 2) . A

variety of questions arise from this dissertation. Among these are the role of AMOT in

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TE differentiation and whether YAP1-TEAD4 interactions are important for TE

differentiation in the cow. As discussed previously, Amot plays an important role in

preventing TE formation in the mouse (Paramasivam et al. 2011; Hirate et al. 2013).

However, results in Chapter 3 indicate that, AMOT promotes TE formation in the cow.

However, it is not known how AMOT functions to promote TE. One possibility is that

AMOT interacts with YAP1 while another possibility is that AMOT acts through a

pathway independent of YAP1. YAP1 and YAP1-TEAD4 interaction are also promoting

TE formation and are important for the embryo to hatch from the zona pellucida

(Chapter 3).

In the mouse, Yap1 and Tead4 act together to activate transcription of Cdx2

(Nishioka et al. 2008; Stephenson et al. 2012; Lorthongpanich and Issaragrisil 2015).

Results from Chapter 3 confirm the importance of YAP1 for TE function. However, an

earlier study found no effect of knocking down TEAD4 on blastocyst formation or

function (Sakurai et al. 2016). Thus, it is possible that TEAD4 is dispensable for

formation of TE. It is possible that TEAD4 is involved but that proteins in redundant

pathways compensate for the decreased amounts of TEAD4 in gene knockdown

studies. Alternatively, in the cow, YAP1 does not require TEAD4 to activate transcription

of CDX2.

Overall, the insight to bovine embryo differentiation and development obtained in

this dissertation is of particular importance for future experiments. Understating the

preimplantation embryo development and the mechanisms used by the embryo gives us

an opportunity to improve in vitro production conditions for developing embryos to make

them more similar to their in vivo derived counterparts. It may also be that infertility in

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cattle is the result in part of failure of the mechanisms studied in this dissertation.

Perhaps, treatments can be identified that can enhance embryonic development to

reduce these errors. Perhaps embryokines such as IGF1 and CSF2 (de Moraes and

Hansen 1997; Block et al. 2007; Loureiro et al. 2011; Denicol et al. 2014) improve

fertility by directing preimplantation development in a way that ensures proper

differentiation of the blastocyst.

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Figure 6-1. Schematic representation of a new model for development of the bovine preimplantation embryo to the blastocyst stage of development. The model is explained in the text. Light-gold cells=undifferentiated cells, blue-gray cells= TE, orange cells= undifferentiated ICM, green cells= epiblast cells, red cells= hypoblast cells.

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Figure 6-2. Gene expression specific for epiblast, hypoblast and specific trophectoderm (TE) cell populations in the cow. Left panel - genes that were upregulated in the epiblast, hypoblast and TE cells when compared to the other cell populations. Right panel- transcripts that are upregulated (↑) and downregulated (↓) in the four subpopulations of trophectoderm as compared to other cell populations. All differences were significant except for TMEM232.

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BIOGRAPHICAL SKETCH

Verónica M. Negrón-Pérez was born and raised in Puerto Rico as the child of

Nerybelle Pérez Rosas and Mariano Negrón. She is the youngest sister of Efraín,

Mariano, Martín and Cecilia Negrón-Pérez. From a young age she learned from her

grandparents and family to love agriculture, science and education. Verónica graduated

from the Animal Industry, Program at the University of Puerto Rico at Mayagüez with a

BS degree in 2011. Upon receiving her undergraduate degree, she pursued a master’s

degree at the University of Missouri in reproductive physiology under the supervision of

Dr. Rocío M. Rivera. Her research was on the epigenetics of the mammalian embryo. In

2013 she moved to Gainesville, FL to begin her doctoral studies in the Animal Molecular

and Cellular Biology Graduate Program at the University of Florida under the

supervision of Dr. Peter J. Hansen. She was supported in her studies through award of

a McKnight Doctoral Fellowship of the Florida Education Funds.

Upon completion of her Doctor of Philosophy degree, Verónica will start working

as a postdoctoral research assistant in the Department of Animal and Poultry Sciences

at Virginia Tech. Her long-term goals are to develop an independent career as a

research scientist and educator, and to encourage underrepresented students to

consider a career in science.