characterlzatton of the function of the otx-2 …iii functional analysis of otx-2 was performed by a...
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CHARACTERlZATtON OF THE FUNCTION OF THE OTX-2
GENE IN EARLY MAMMALIAN DEVELOPMENT
Ou Jin
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Molecular and Medical Genetics University of Toronto
@ Copyright by Ou Jin, 1997
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Dedication
to my father
CHARACTERlZATlON OF THE FUNCTION OF OTX-2 GENE IN
EARLY MAMMALIAN DEVELOPMENT
by Ou Jin
A thesis submitted in conformrty with the requirements for the degree of Doctor
of Philosophy in the Graduate Department of Molecular and Medical Genetics at
the University of Toronto, 1997
ABSTRACT
How the anterior region of the vertebrate embryo is specified is still an
unsolved question in early pattern formation. In this thesis, I have undertaken a
study of the roles of a homeobox gene, Otx-2, in the early murine development,
with particular reference to its role in anterior patterning.
Otw-2, one of the two mammalian Otx genes, related to the Drosophila otd
gene, has been cloned and characterized. It encodes a protein containing a 60
amino acid homeodomain motif with only two amino acid differences from the
sequence of Drasophila Otd. 00r-2 is one of the earliest genes showing
restricted anterior expression domains. During early embryogenesis, OW2 is
first expressed in the entire epiblast which gives rise to the embryo proper, then
is gradually restricted to the anterior region of the embryo and finally to the
presumptive forebrain and midbrain. This expression profile suggests that Ok-
2 may have multiple roles in the early development and anterior patterning of
the embryo. These studies provide fundamental background information for
further functional analysis.
iii
Functional analysis of Otx-2 was performed by a gene targeting
approach. A loss-of-f unction mutation of Otx-2 leads to loss of forebrain,
midbrain and anterior hindbrain, suggesting the critical role of Otx.2 in head
formation. In addition, homozygous Ok-2 mutant embryos also show early
gastrulation defects and defects in the prechordal mesoderm and notochord
precursors. These defects are consistent with multiple roles for Otx-2 in
gastrulation and the patterning of the rostra1 brain in mice.
To further dissect the function of 0tx-2 in anterior patterning, a double
heterozygous mutant was made with HNF-313, a member of the winged-helix
transcription factor family. Generation of Otx-2+/-; HNF-3&/- double
heterozygous mutants demonstrates a new lethal phenotype in mice. The
phenotype is characterized by varying degrees of holoprosencephaly, cyclopia
with proboscis-like structures, suggesting the genetic interaction of the two
genes in anterior midline patterning. Co-expression of Otx-2 and HNF-3B in the
anterior midline of the embryo further supported such interactions. Furthermore,
the loss of Shh expression, a secreted protein, in the anterior region of 0k-2+'
;HNF-3B+/- embryos indicates that both Ob(-2 and HNF-3B are involved in
regulating the Shh signalling pathway in the anterior midline. This study also
illustrates the way in which dosage-dependent effects of transcription factors
can reveal novel genetic interactions and help define genetic pathways.
iv
ACKNOWLEDGMENTS
First, I sincerely thank my supewisor, Dr. Janet Rossant, for her
consistent support, encouragement and advice throughout the program. I also
would like to take this opportunity to thank Drs. Martin Breitman, Rod Mclnnes
and Andrew Spence for their suggestions and comments during yearly
romm ittee meetings.
I thank people in our lab, old and new, not only for sharing friendships
but also for sharing their expertise. In particular, I would like to thank Lois
Schwartz for aggregation experiments, Ken Harpal for preparing wax sections
and Celine Champigny for some sequencing.
Five years of a life time is short, but this period was an extremely
important time, not only for my career but also for my life. Three years ago, I
lost my father, the most important person in my Me. It was under my father's
influence that I decided to go to medical school. Later, when I became
interested in medical research, he gave me great encouragement. Five years
ago, it was my father who strengthened my decision to pursue a Ph.D. degree. I
wish he could see the day I receive my degree. I would also like to thank my
mother for all her support, especially since she moved to Toronto. Finally, I
would like to thank my wife, Evie, for her understanding and support.
CONTENTS
ABSTRACT .......................................................................................................... ...... ....... .ii ACKNOWLEDGMENTS .......................... ...- ............... .. ................................ iv UST OF FIGURES ............. .., ..................................................................................... vii ... LIST OF TABLES ....................................................................................................... VIII UST OF ABBREVIATIONS ............................................................................................. ix
CHAPTER 1 : INTRODUCTION ....................................................................................... I
Early mouse development ............................................................................................ 2 Gastrulation ....... .... .............................................................................................. 2 Neurulation .............. ., ........................................................................................ 8
Hox genes and vertebrate A-P patterning ................................................................ 14 Otd and ems in Drosophila anterior head development ........................... 4
........................................................................................................................ otd 20 ................................................................................ ems ............................... ...,.. 21
Mammalian homologues of otd and ems and their expression patterns during mouse embryogenesis .................... .. ............................................................ 21
Mouse Otx- 1 and Otx-2 ................................................................................... -22 Mouse Emx- I and Emx-2 ................................................................................. 23
Outline uf the thesis ....................................................................................................... 26
CHAPTER 2: IDENTIFICATiON AND CHARACTERIZATION OF 07X-2 GENE .. 27
...... ........*........................................................*.........-....*.......-..... Introduction ........ ........ .. 28 Materials and Methods .................................................................................................. 29 Resuks .............................................................................................................................. 31
Isolation of Otx-2 cDNAs ................................................................................... 31 Northern blot analysis and identification of 5'-OW-2 cDNA ........................ 33 Exon-intron structure of Otx-2 gene ................................................................ 34
.................................................. Early expression pattern of the OW2 gene -35 Discussion ...................... ....., ......................................................................................... 35
CHAPTER 3: TARGETlNG OF OX-2 ......................................................................... 55
Introduction ......... ,., .......................................................................................................... 56 Materials and Methods ................................................................................................ 58 Resu ks .............................................................................................................................. 60
A targeted disruption of Otx-2 in ES cells by homologous . . recomb~nat~on .................................................................................................... 60 The Obr-2 mutation leads to embryonic lethality ............... ...... ................ 63 Morphological and histological analysis of the Otx-2 homozygous phenotype ........................................................................................................... 64 Deletion of anterior brain regions rostra1 to hornbornere 3 ..................... 36 Defects in gastrulation in Otw-2 homozygous embryos ............................ 66
vii
LIST OF FIGURES
Chapter 1 Figure 1.1 :
Figure 1 2: Figure 1.3: Figure 1.4: Figure 1.5:
Figure 1.6:
Chapter 2 Figure 2.1 :
Figure 2.2:
Figure 2.3: Figure 2.4:
Figure 2.5: Figure 2.6: Figure 2.7:
Figure 2.8:
Chapter 3 Figure 3.1 : Figure 3.2:
Figure 3.3: Figure 3.4: Figure 3.5:
Figure 3.6:
Schematic representation of mouse development from E6.0 to E8.5 ............................................................................................................. 3 Morphological features of the node ...................................................... 6 Neural induction in Xenopus ............................................................... 1 0 Early mouse brain development ......................................................... 12 Genomic organization and collinear expression patterns
............. of Drosophiia HOM-C genes and mammalian Hox genes 17 Comparison of expression of Otx and Emx genes in the mouse
......................................................................................... embryo at El0 24
The nucleotide sequence of the 1.6kb cDNA clone and the amino ........ ............. acid sequence of the putative Otw-2 gene product .. 39
Comparison of Otx-2 homeodomain with Otx-1 , Xotx.2, SpOtx. Otd homeodomains ............................................................................. -41 Transcription of the O&-2 gene ........................................................ 43 Diagram of the 5' RACE and primer extension experiments
......................................... (A) and sequence of 5' RACE product (B) 45 .............................. Result of primer extension experiment 4
Exon-intron structure of Otx-2 gene ................................................ -49 Expression of Otx-2 from prestreak to early somite stages
....................... revealed by whole mount RNA in situ hybridization 51 ................................ Tissue sections analyzed for Otx-2 expression 53
........ Targeted disruption of the Otx-2 gene ................................ .. 75 (A) Southern bolt analysis of DNA from wild-type and
... targeted ES cell lines. 5-1 2. 5-23 and C- 1 2 .................. ....... -37 (B) PCR genotyping of yolk sac biopsies isolated from E8.5
embryos of intercross between Otx-% mice ........................ 79 Phenotype of Otx-2 homozygous ...................................................... -81 Histological analysis of Otx-2 homozygous embryo ....................... 82 Whole mount RNA in situ hybridization of Krox-20 in E8.5 wild-type or heterozygous embryo and homozygous mutant embryo ..................................................................................................... 84 Whole-mount analysis of mesoderm markers in wild-type or heterozygous embryos and homozygous mutant
............. embryos at E7.25E8.5 .. .................................................... -86
Chapter 4 Figure 4.1 : Phenotype of Otx-2 +/- ; HNF-30 +/- embryos at El 2.5 ................. 1 05 Figure 4.2. Phenotype of 0&-2 +/- ; HNF-3B +/- embryos at E9.5 ................... 1 07 Figure 4.3: Histological analysis of Otx-2 +/- ; HNF-38 +/- embryos at 12.5 .. 1 09
viii
Figure 4.4: Comparison expression of OtK-2 and HNF-3B +/- in wildtype mouse embryos between E7.5 and €9.5 ................ ... ............. 1 1 1
Figure 4.5: Expression of Otw-2, Hoxb- 1, Six-3 and Mox- 1 in Otx-2 +/- ; HNF- .......................................................... ..................... 38 +/- embryos .. 1 13
Figure 4.6: Whole-mount in situ hybridization and cross section analysis of Otx-2 +/- ; H W 3 B +I- mutant with BF1, Krm-20 and Shh ....... 115
.............................................. Figure4.7: Phenotypeof Otx-24-embryos 1 1 7 Figure 4.8: Schematic diagram showing that OtK-2 and HNF-3B are
involved in regulating Shh expression in the anterior region ..... 1 1 9
Chapter 5 Figure 5.1 : Topologiocal equivalence between the Xenopus fate map
and mouse fate map at the early gastrula stage ............................ 1 28
LIST OF TABLES
Chapter 3 Table 3.1:
Table 3.2: Table 3.3:
Chapter 4 Table 4.1:
Table 4.2:
Table 4.3:
Results of screens for homologous recombination into OW2 locus .......... ........,. .................................................................................... 62 Frequency of germ-line transmission of targeted ES cells lines ... 62 Genotype of mice resulting from Otw-2 heterozygous intercrosses.. ...... .... .....*.. ...,,. ..,,.. ..-....-........*.... 64
Frequency of genotypes resulting from Otx-2 +/- and HNF-3B 4- intercrosses at weaning stage .......................................................... 102 Phenotypes resulting from OW+/- and HNF-3B +/- intercrosses at E12.5 ............................... .,., ........................................................... 103 Frequency of genotypes resulting from Obr-2 and HNF-38 intercrosses at E9.5 .......................................................................... 1 04
ABBREVIATtONS
A-P Anterior-Posterior
CNS Central Nervous System
D-V Dorsal -Ventral
E Embryonic Day
ES Embryonic Stem cells
RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
RACE Rapid Amplification of cDNA End
Chapter I
INTRODUCTION
The mouse (Mus musculus) is a widely used system for studying
vertebrate development. Not only is it closer to humans than non-mammalian
species, but it also has many valuable genetic resources available such as a
large collection of mutants for investigators to characterize phenotypic variants
and explore the molecular definition of these mutants. However, other systems
have many advantages for experimental embryology. For example, in Xenopus,
chick and zebrafish, embryos are more plentiful and develop outside the
mother. Thus, they are often easier to manipulate and observe. In recent years,
it has become clear that the mechanisms governing early development are
highly conserved between species. Thus, information from other species is
certainly very useful for understanding mouse development. In this section, I will
briefly outline early mouse development with emphasis on those processes
which are relevant to studies described in this thesis.
Gastrulation
Cleavage, gastrulation and organogenesis are three major stages during
mouse embryogenesis. After the stage of cleavage and the formation of a
blastocyst, the embryo enters one of the most critical stages in its development:
the stage of gastrulation. Before gastrulation starts, the embryonic compartment
of the embryo has two components: the primitive ectoderrn or epiblast and
primitive endoderm (Fig.l.1 A). Gastrulation begins at embryonic day 6.5 (E6.5).
It is a process of profound but well-ordered rearrangements of the cells in the
embryo, resulting in the formation of two new layers, mesoderm and definitive
endoderm. Cells at the junction between the primitive ectoderrn and the
Figure 1 -1. Schematic representation of mouse development from E6.0 to E8.5.
(adapted from the following sources with slight modifications: Hogan et al.,
1986, 1994).
xtracmbryontc
Mesodermal cells ingrcsslng into primltlvc streak
E6.0 E7.0
extraembryonic ectoderm undergo an epithelial to mesenchymal transition and
start to migrate and come to lie between the primitive ectoderm and the
endoderm, resulting in the formation of the primitive streak (Fig. 1.1 8, C, D, E).
As mesoderm migration proceeds, the primitive streak gradually elongates and
reaches the distal tip of the egg cylinder. The lateral migration of the mesoderm
through the primitive streak results in a wing-shaped mesodermal layer that
extends around the embryonic ectoderm (Fig. 1.1 C). A specialized structure,
called the node, appears at mid-late streak stages in the anterior end of the
primitive streak (Fig. 1.1 E and Fig. 1.2). It is a localized two-layered area and
there is no visceral endoderm underlying this area (Fig. 12C) (reviewed in
Sulik et al., 1994). Cells from the node migrate fonnrard in the midline, resulting
in the formation of the head process. The cells of the anterior head process
form the socalled prechordal region or prechordal plate which is much more
readily recognizable in the chick. At late streak stage, head process cells meet
mesoderm wings at the anterior end and complete the mesoderm layer. In the
midline, cells displace the overlying primitive endoderm to form definitive
endoderm or gut endoderm (Fig.1 .l E). However, the origin of gut endoderm is
still not clear. HNF4B homozygous mutants lack node formation, and foregut
morphogenesis is severely affected in those mutants (Ang and Rossant, 1994;
Weinstein et al., 1994), suggesting that the node might be one of the cell
sources involved in gut endoderm formation.
The basic body plan along the anteriorposterior (A-P) axis and the future
dorsal-ventral (D-V) axis are established during gastrulation. In addition, the
formation of three germ layers also provides the appropriate tissue interactions
required for further differentiation during organogenesis. The primitive streak
initiates the A-P axis, representing the future posterior portion of the embryo.
Fig. 1.2. Morphological features of the node. (A) Midsagittal section showing the
position of the node. (B) Cross section through a similar stage embryo
immediately anterior of the node. Note that in the midline, there are only two cell
layers. (C) Ventral view of the surface of the node and notochord in €7.5
embryo. (D) Higher magnification view shows cilia-like projections from these
cells (arrows). Abbreviations: .A: allantois; Am: amnion; E: embryonic ectoderm;
En: endoderm; ExEn: extraembryonic endoderm; ExM: extraembryonic
mesoderm of the yolk sac; FG: foregut diverticulum; H: presumptive heart; M:
mesoderm; N: node; NC: notochord; NF: neural folds; PS: primitive streak.
(adapted from Hogan et al., 1994)
The node, which is equivalent to Hensen's node in the chick (Waddington,
1933), the dorsal blastopore lip in Xenopus and the embryonic shield in the
zebrafish (Ha, 1992). plays a prime role in organizing and patterning the
midline axis of the embryo. The organizer function of the dorsal lip was first
suggested by Spemann and Mangold (1 924), who demonstrated the induction
of a secondary axis by grafting a dorsal blastopore lip to the ventral side of a
host embryo. Spemann (1931) also found that the early dorsal lip could induce
a complete axis including a head, suggesting that the early dorsal lip has a
head organizer activity. However, the later stage dorsal lip could only induce a
trunk-tail structure, suggesting that at later stages, it only has trunk organizer
function. In mouse, Beddington (1 994) provided the first direct evidence for the
organizing function of the node, if it is transplanted heterotopically. When the
mid-gastrulation node was grafted to a posterolateral location in a host embryo
at the same developmental stage, a second neural axis was induced. However,
the most anterior end of the second axis is always at hindbrain level. Therefore,
whether the node can induce the most anterior structures such as forebrain still
remains open.
Neurula tion
When mesoderm extends to the anterior end of the embryo, the overlying
anterior ectoderm forms the neural plate which is first recognizable soon after
€7.5. The neural plate gradually folds up to form a neural tube. At E9.0, the
anterior neuropore is completely closed. The formation of the neural plate and
its transformation to neural tube is one of the most dramatic events following
gastrulation. Induction and axial patterning of the neural plate, which has been
mainly investigated in amphibian and chick embryos, is a complex process
involving in planar signals from the organizer region and vertical signals from
underlying axial mesoderm (reviewed by Slack and Tannahill, 1992; Ruiz i
Altaba, 1994; Kessler and Melton. 1994; Kelly and Melton, 1995). A two-step
model of neural induction has been proposed based on a series of
transplantation and recombination experiments in amphibian embryos using
morphological and molecular markers. It suggests that the ectoderm is first
induced to anterior neural fate by the signals present in the mesoderm, followed
by a transformation to a more posterior neural fate by a graded posterior signal
coming from posterior mesoderm (Fig. 1.3). It also has been found that different
regions of the underlying mesoderm have different inducing potential. In mouse,
similar observations have been achieved by explant-recombination assays
(Ang and Rossant, 1993; Ang et al., 1994). These studies suggest the
importance of tissue interactions in neural induction and regional specification
along the A-P axis of the neural plate and neural tube.
Regionalization of the neural tube becomes morphologically apparent as
a result of changes in the shape of the tube. In the anterior region of the neural
tube (the future brain), there are three primary brain compartments: forebrain,
midbrain and hindbrain (Fig. 1.4). The forebrain further subdivides into
telencephalon and diencephalon. The telencephalon will form the cerebral
hemispheres while the diencephalon will form the thalamus, hypothalamus and
retina, each of which has distinct morphologies and histologies (reviewed by
Puelles and Rubenstein, 1993). The midbrain does not undergo further
transverse subdivision. However, the hindbrain further evolves into a series of
repeated undulations or segments. These segments are so-called
rhombomeres(r), rl-r7, each of which has a unique developmental fate
(Lumsden and Keynes, 1989; Lumsden et al.. 1991).
Fig. 1.3. Neural induction in Xenopus. During gastrulation. dorsal mesoderm
involutes and migrates underneath the ectodermal surface of the gastrula. As
dorsoanterior mesoderm involutes, the adjacent ectoderm is initially induced to
form anterior neural tissues. The mesoderm migrates toward the former animal
pole, and newly contacted ectoderrn is also induced to form anterior neural
tissue. Ectoderm is progressively contacted by more posterior mesoderm
resulting in posterior neural elements (darker shades of purple). As a result of
this process, the anteroposterior pattern of induced neural tissue reflects the
anteroposterior character of underlying dorsal mesoderm. Both vertical and
planar signals emanating from dorsal mesoderm have been implicated in the
transformation of overlying embryonic ectoderm to neural ectoderm. 0,
organizer mesoderm. (Adapted from Kessler and Melton, 1994)
Figure 1.4. Early mouse brain development. The three primary brain vesicles
are subdivided as development continues. (adapted from Gilbert. 1994)
In recent years, a number of candidate molecules implicated in axial
patterning have been identified. Signaling molecules such as chordin (Sasai et
al., 1 994), follistatin (reviewed in Kessler and Melton, 1 994) and noggin (Smith
and Harland, 1992; Smith et al., 1993) are candidates for neural inducers.
Nuclear transcription factors such as Otx-2, gsc, HNF-3R and Lim-7 (reviewed
by De Robertis, 1994; BallyGuif and Boncinelli, 1997) and secreted molecules
such as wnt-I (reviewed in Joyner, 1996) and Shh (reviewed by
Hammerschmidt, 1997) have been implicated in the patterning of the neural
tube in vertebrates. The roles of Drosophila horneotic complex (HOW) , and
their vertebrate homologues, Hox (homeobox) genes, in the axial pattern have
been extensively studied. In the following section, l will briefly discuss the role
of Hox genes in the axial patterning of vertebrate embryo body.
ox aenesmd verteb~te A-P oattemmg
One of the most fascinating findings in recent years in developmental
biology is that the molecular mechanisms of axial patterning have been highly
conserved during evolution. H O W genes (reviewed by Akam, 1989) were
discovered in the Drosophila due to the dramatic phenotypes that result from
mutations in these genes. Pioneering work by Lewis (1 978) demonstrated that
the segments of Drosophila differ from each other because of the action of a
small set of homeotic genes along the A-P axis of the body. For example,
Ultrabithorax (Ubx) mutations result in homeotic transformations of the third
thoracic (T3) segment carrying halteres to a second thoracic (T2) segment with
wings (Lewis, 1978, 1981, 1982). As figure I .5 illustrates, by genetic criteria, the
labial (lab), Deformed (Dfd) and proboscipedia @b) genes are involved in
specify the identity of anterior segments, the Sexcombs reduced (Scr) and
Antennapedia (Antp) genes contribute thoracic identity functions, and the
bithorax complex genes (Ubx, abdominal-A and Abdominal-@ are required for
the specification of segmental identities in the posterior thorax and in all
abdominal segments (reviewed in McGinnis and Krumlauf, 1 992). Loss-of-
function mutations and gain-of-function mutations of HOM-C genes lead to
homeotic transformations of the embryonic and /or the adult Drosophila body
plan, suggesting that these genes are involved in the genetic control of A-P
patterning in Drosophila (reviewed by McGinnis and Krumlauf, 1 992; Lawrence
and Morata. 1994). McGinnis and his colleagues (1 984a). and Scott and
Wiener (1984) first discovered that Drosophila H O W genes contain a
conserved sequence called the Homeobox motif. The homeobox motif is not
only present in flies but also found in all vertebrates, such as Xenopus, chick,
mice and human (McGinnis et al., 1984b; Carrasco et al., 1984, ). The
homeobox encodes a 60 amino acid motif (homeodomain). These
homeodomains have sequence-specific DNA binding activities in vifro and
have subsequently been found in many transcription factors (reviewed by
Levine and Hoey, 1988; Scott et al., 1989; Laughon, 1991; Gehring et al.,
1994). However, how these homeobox gene products achieve their in vivo
specificities in axial patterning is still an important and unresolved problem.
The mammalian Hox genes are aefined an the basis of their homology to
the genes of the Drosophila HOMC. Analysis of mouse and human Hox genes
reveals that there are at least 39 genes which are organized in four clusters,
HoxA. HoxB, HoxC, and HoxD. Based on sequence similarities and relative
genomic arrangements, the individual Hox genes with in different clusters can
be aligned with each other and with genes of the Drosophila HomC cluster
(Fig. 1.5). In mammals, Hox genes are expressed in overlapping domains along
A-P axis of the embryo with spatial colinearity according to the order of genes
along the chromosomes. For example, the more 3' located genes are
expressed in more anterior regions whereas the more 5' located genes are
expressed in more posterior regions (Fig. 1.58).
Genetic approaches provide a critical test for the postulated role of
candidate genes in development (Rossant, 1990; Joyner and Guillemont,
1994). The importance of Hox genes in vertebrate axial patterning has been
further demonstrated by loss-of-function mutations and gain-of-function
mutation studies (reviewed by McGinnis and Krumlauf, 1 992; Krumlauf, 1 994).
In Xenopus, these functional studies were usually performed through either
microinjection of mRNA into early embryos. resulting in ectopic production of the
protein, or m icroinjection of antibody into eggs, resulting in interference with the
normal function of the gene. In mouse, most functional studies used either the
approach of generating transgenic mice or the gene targeting approach to
generate null mutant mice (reviewed in McGinnis and Krumlauf, 1992).
Targeted disruption of members of the Hox gene families have demonstrated a
wide spectrum of phenotypes which include structural defects such as in the
formation of the vertebral column and limbs, and the formation of branchial
archderived tissues. For example, loss-of-function mutations of Hoxa-1 and
Hoxa-3 lead to defects in hindbrain and branchial regions of the mouse, but do
not appear to cause homeotic transformations of the affected regions (Chisaka
and Capecchi, 1991 ; Lufkin et al., 1991 ; Carpenter et al., 1993). However, some
phenotypes can be considered as anterior or posterior homeotic
transformations. For example, loss-of-function mutation of Hoxb-4 causes partial
transformation of the axis to atlas identity by the formation of a ventral arch at
Fig. 1.5. Genomic organization and colinear expression patterns of Drosophila
H O W genes and mammalian Hox genes. (A) Example of evolutionary
conservation of horneotic gene organization and expression in Drosophila
embryo and mouse embryo. (B) Schematic representation of the Drosophila
homeotic complex (HOM-C), the four human Hox complexes and a hypothetical
ancestral homeotic complex are displayed showing their possible phylogenetic
relationships. Each gene is represented by a colored box. For the simplicity, the
partial overlap between HOM gene transcripts in thoracic and abdominal
segments of Drosophila and overlapping expression domains of mammalian
Hox genes along the body axis are not represented; therefore, each color
represents the anteriormost expression domain of a given subfamily. (C)
Illustration of the transcription direction of these homeobox genes and
responses to retinoic acid. All these genes are transcribed in the same
direction. Those that are expressed more anteriorly are expressed earlier and
can be induced by low doses of retinoic acid. HOM gene abbreviations are: lab.
labial; pb, proboscipedia; Md, Deformed; Scr, Sex combs reduced; Antp,
Antennapedia; Ubx, Ultrabithoraw; abd-a, abdominaM; Abd-B, Abdominal-8.
(Adapted from the following sources with slight modifications: Gilbert, 1994;
Mark et al., 1 997).
the level of the second cervical vertebra (Ramirez-Solis et al., 1993).
Overexpression of Hoxa-7, analogous to gain-of-function homeotic mutations in
Drosophila. converts the basioccipital bone into a proatlas structure (Kessel, et
al., 1990). Hox mutations in mice often exhibit variably penetrant phenotypes,
suggesting functional redundancy among Hox genes. Consistent with this
hypothesis. mice with combinations of Hox mutations (i.e. double mutations),
show more severe defects than mice with the single mutations (Davis et al.,
1995; Davis and Capecchi, 1996; Fromental-Ramain et al., 1996; Chen and
Capecchi, 1997), suggesting quantitative genetic interactions among Hox
genes in regional pattern formation. For example, Hoxa-9 and Hoxd-9
compound mutants demonstrate synergistically altered phenotypes consisting
of an increase of penetrance and expressivity of malformations present in the
single mutants. The compound mutants also show novel alterations in the
forelimb stylopod (humerus) and additional vertebral transformations. These
observations suggest that Hoxa-9 and Hoxd-9 genetically interacted in forelimb
and axial skeleton patterning (FromentaCRamain et al., 1 996).
The Hox genes are involved in the genetic control of the identity of
specific regions in the hindbrain and spinal cord but they do not specify the
identity of the rostra1 brain region. Therefore, how the anterior region of the
brain is specified becomes one of the most interesting questions in early pattern
formation. However, until recently little was known about this field either in flies
or in vertebrates. A breakthrough has come with the identification of other
homeobox genes in Drosophila, namely orthodenticle (of@ (Finkelstein et al.,
1990) and empty spiracles (ems) (Dalton et al., 1989). In the following section, I
will discuss the roles of atd and ems in Drosophila anterior head development.
development
otd
otd was originally found in a large screen for loci that affect development
of the larval cuticle (Wieschaus et al., 1984). R encodes a homeodomain-
containing protein (Finkelstein et al., 1990). suggesting that it may function as a
transcriptional factor during development. The expression of ofd was detected
as early as 2.5 hr, the stage of blastoderm, by in situ hybridization to wild-type
embryos (Finkelstein et a!.. 1990). It is expressed in a large circumferential
stripe at the anterior end of the embryo. This expression domain includes cells
which will give rise to many of the future head structures (Jurgens et al.. 1986).
According to the fate map, several head structures derived from the anterior
expression domain of otd are deleted in mutant embryos (Cohen and Jiirgens,
1990; Finkelstein and Perrimon, 1990). The early otd expression pattern and
the head defects in mutant embryos suggest that otd plays an important role in
anterior patterning of the Dt-osophila embryo. At about 5-6 hr, shortly after
gastrulation, a second domain of otd expression was detected in a longitudinal
strip of cells along the ventral midline of the embryos. This expression domain
includes cells which are neuronal, glial and epidermal precursors (Jacobs and
Goodman, 1989a.b). In otd mutant embryos, neural and epidermal defects have
been found in the ventral medial region (Finkelstein et al., 1990; Klambt et al.,
1991; Wieschaus et al., 1992), suggesting that otd is also required for the
specification of ventral midline cells in the CNS and epidermis.
ems
ems was found in a search for zygotic patterning mutations (Jurgens et
al., 1984). ems mutant embryos demonstrate severe patterning defects in the
anterior head structures (Jiirgens et al.. 1 984; Dalton et al.. 1 989). suggesting a
role for ems in the patterning of the developing head in Drosaphila. The ems
gene was isolated later using the even-skipped homeo box as a probe (Dalton
et al., 1989). Like the otd gene, ems also encodes a homeodomaincontaining
protein and thus probably functions as a transcription factor during
development. ems is expressed in a stripe at the anterior of the embryo as early
as the blastoderrn stage (Dalton et al., 1989). These ems expressing cells are
fated to give rise to a variety of head stmctures, such as the antenna! sense
organs, the dorsal arms, and the vertical plates and mandibular segments
(Jiirgens et al., 1986). In ems mutant embryos, all of these structures are
missing or disrupted (Jurgens et al., 1984; Dalton et al., 1989), suggesting that
ems plays a role in the patterning of the anterior head. A second expression
domain of ems appears at the beginning of germ-band extension stage in the
anterior lateral ectoderrn and seems to be required for the morphogenesis of
the posterior tracheal tubes (Dalton et al., 1989).
The cloning of the otd and ems genes provided the first molecular
evidence for homeobox-containing genes being involved in the most anterior
patterning of the embryo. Many labs including our lab started to search for their
vertebrate homologues and to explore the molecular mechanisms underlying
patterning of the most anterior regions of the embryo.
an homologlbes of bid and ems and the r exoresslon ~merns durinq
ouse e m b r y ~ e n e s ~ ~
In recent years, vertebrate otd homologues. O k - 1 and &-2, have been
cloned from Xenopus (Bally-Cuif et al.. 1995; Pannese et al., 1995), zebrafish
(Li et al., 1994; Mercier et al.. 1995). chick (Bally-Cuif et al., 1995), mouse and
human (Simeone et al., 1992a. 1993; Ang et al.. 1994). Vertebrate ems
homologues, Emx-1 and Emx-2, have also been isolated and characterized in
the mouse (Simeone et a!., 1992b). In this section, I will summarize the
expression patterns of mammalian homologues of otd and ems during mouse
em bryogenesis.
Mouse Otx-7 and Otx-2
Otx-2 is one of the earliest genes showing restricted anterior expression
domains. At E5.56.0, Otx-2 is expressed throughout the embryonic ectoderm or
epiblast, which gives rise to the embryo proper, but not in the extraembryonic
tissues (Simeone et al., 1993; Ang et al., 1994; Acampora et al., 1995). At E6.0-
6.5, the expression pattern of 00~-2 in the epiblast appears to be the same.
Between E6.5-7.75, Otx-2 expression becomes progressively restricted to the
anterior half of the embryo and later becomes further restricted to the
anterionost third of the embryo. The expression domain includes the anterior
three germ layers. At €8.0-8.5, Obr-2 is expressed in forebrain, midbrain. and
optic eminence of the central nervous system (CNS). Weaker expression is also
found in the notochord, head mesenchyme and foregut at the same axial levels.
as well as in the ectoderm and endoderm cells of the first branchial arch. At
E9.5, the sharp posterior boundary of OtK-2 expression corresponds precisely to
the border between the midbrain and hindbrain. The domain of Otx-2
expression covers almost the entire forebrain and midbrain except the regions
of the optic chiasrna and optic recess. Expression of Otu-1 starts later than Otx-2
and is first observed at approximately at E8.0. R is expressed in similar regions
with Otu-2 except some presumptive ventral forebrain regions and at E8.5, both
genes have the same posterior boundary of expression between the midbrain
and hindbrain. The two genes are also expressed in the developing eyes, ears
and the epithelia cells in the nasal cavities (Simeone et al., 1993).
Mouse Emx-1 and Emx-2
E m - 2 is first expressed in the anterior dorsal neuroectodermal regions
of the embryo at E8.5 (Simeone et al., 1992b). At E9.5, Emx-7 is expressed in
similar regions but the expression domain is smaller than Emx-2. The posterior
boundary of the Emx-1 expression domain coincides with the region between
presumptive diencephalon and telecephalon. Besides dorsal telencephalon,
Emx-2 is also expressed in restricted regions of the diencephalon: the anterior
dorsal and posterior ventral diencephalon. At E9.75, the expression of Emx-2 is
detected in some mesencephalic regions (Simeone. et al., 1992b) and at €13.5,
both genes are expressed in the developing lens, but Emx-2 is also expressed
in the olfactory epithelia in nasal cavities and posterior hypothalamus (Simeone
et al., 1992b).
In summary, the expression domains of Otx and Emx genes are
contained within each other, in the sequence E m x l < E m x - 0 - 2 Figure
1.6 schematically summarizes the expression patterns of the four genes in the
forebrain and midbrain at E10. These expression studies suggest that OW-I and
Otw-2 may cooperate to determine the territory of forebrain and midbrain while
Emx-1 and Emx-2 are involved in the patterning of dorsal telecephalon. The
Fig. 1.6. Comparison of expression of Otx and Emx genes in the mouse embryo
at E10. (modified from Finkelstein and Boncinelli, 1994). For details see text.
Abbreviations: Di: diencephalon; Mes: mesencephalon; Met: metencephalon;
Tel: telencephalon; My: myencephalon.
early expression pattern of Otx-2 may suggest that Obr-2 is also involved in
specifying the developing brain in early embryogenesis.
ne of the thesis
The major goal of the research described in this thesis is to understand
the role of Otx-2 gene, one of the mouse homologues of Drosophila otd, in
mouse embryogenesis. Chapter 2 describes the identification and
characterization of the mouse Otx-2 gene. Chapter 3 describes the functional
analysis of Otx-2 gene by gene targeting. Chapter 4 describes the further
analysis of the role of Mx-2 in anterior midline patterning by making double
heterozygous mutants with HNF-38 , a member of the winged-helix transcription
factor family. Chapter 5 will discuss the significance of the thesis work, the
future experiments and recent important progress in the field of anterior
patterning.
Chapter 2
Identification and Characterization Of the 0txm2 Gene
A portion of this chapter appeared in the following publication:
Ang, S.-L.. Conlon, R.A., Jin, 0. and Rossant, J. (1 994) Positive and negative
signals from mesoderm regulate the expression of mouse Otx-2 in ectoderm
explants. Development 120, 2979-2989.
I performed all work described here, with exception of isolation of the 1 kb Otx-2
cDNA, sectioned in situ hybridization (Fig. 2.8) and most whole-mount in situ
hybridization experiments (Fig. 2.7A. B, C, 0).
INTRODUCTION
How the anterior region of the vertebrate embryo is specified is still an
unresolved question in early pattern formation. Studies on the Drosophila
homeotic complex ( H O W ) genes and their vertebrate homologues Hox
(homeobox) genes, reveal that molecular mechanisms involved in axial
patterning are remarkably conserved during evolution (reviewed by McGinnis
and Krumlauf, 1992). In particular, specific Hox genes are involved in the
regional specification of the hindbrain and spinal cord. However, until recently,
little was known about the development of the most anterior region of the animal
embryo. A breakthrough has come with the identification of other homeobox
genes in Drosophila, namely orthodenticle (atd) (Finkelstein et al., 1990) and
empty spiracles (ems) (Dalton et al., 1989). These two genes are expressed in
overlapping domains in the anterior pole of the blastoderm stage of embryo.
Absence of either of the two genes leads to loss of specific head structures,
suggesting that both otd and ems are involved in the establishment of different
head structures. Recently, mammalian homologues of otd and ems have been
cloned, namely Otu-I, Otx-2, Emx-1 and Emx-2 (Simeone et al., 1992a, 1992b).
Expression analysis at E9.5 showed that these genes are expressed in nested
A-P domains in developing brain (Simeone et al., 1992a).
We cloned Otw-2 independently and analyzed the early expression
pattern of Otx-2 during embryogenesis. At pre- and early streak stages of
embryogenesis (E6.0-E6.5), Otw-2 is expressed throughout the epiblast which
gives rise to the embryo proper. As development proceeds, the expression of
Otx-2 is gradually restricted to the anterior half of the embryo including the
anterior three germ layers. At the somite stage of embryogenesis, the
expression of the Otx-2 is in the presumptive forebrain and midbrain. The
expression profile of Otr-2 suggests multiple roles of Otx-2 gene during early
em bryogenesis.
Methods and Materials
clones
Using a human otd-related cDNA clone EST01828 (Adam et al., 1992).
an 8.5 day embryonic mouse cDNA library (a gift of Brigid Hogan; Frohman et
al., 1987) was screened under low stringency hybridization conditions (5x
Denhart's, 5xSSC. 0.1 % SDS at 42'C). Three independent phage clones were
isolated and inserts were subcloned into pKS plasmid vector. One clone,
pkSotd9, showed specific expression in the anterior part of the embryo by
whole-mount RNA in situ hybridization and, when sequenced, contained a
partial horneodomain with homology to otd. The insert from this clone was used
to screen a 129Sv genomic library in Dash2 (gift of A. Reaume and R Zirngibl)
using high stringency hybridization conditions (5x Denhart's, 5x SSC,
O.l%SDS at 65'C). Two overlaping genomic clones, spanning about 25kb
were isolated, and a detailed restriction map was constructed. To obtain large
cDNA clones, three cDNA libraries, one 11.5 day (a gift of C.C Hui), and two
12.5 day cDNA libraries(gifts of M. Hanks and J. McGlade) were screened with
the 1 kb cDNA probe or with a 170 bp Acc I-Kpn / fragment under high
stringency hybridization conditions (5x Denhart's, 5x SSC, 0.1 %SDS at 65°C).
In total six cDNA clones were isolated and double strand DNA was prepared for
sequencing. Sequence was carried out using Sequenase according to the
method described by United States Biochemical Corporation (USB) .
In situ hybridizations were carried out on whole-mount and sectioned
material as described (Conlon and Rossant, 1992). Single-stranded RNA
probes labeled with digoxigenin-or 3%-labeled UTP were synthesized from
linearized template DNA as directed by the manufacturer (Boehringer
Mann heim Biochemicals). The OfK-2 cDNA containing plasmid, pOtd9, was
linearized with Xbal and transcribed in vitro using T3 polymerase to obtain an
antisense transcript.
Total RNA was isolated from ES cells according to the procedure
described (Chomczynski and Sacchi, 1987). BRACE was performed following
the instructions provided with the B'RAGE system (BRL). Briefly. total RNA
prepared from ES cells was used as template for reverse transcription primed
by Exten 1 (5'CCGCCITACGCAGTCAATGGGCTG-3'). The first strand cDNA
was treated with RNase H for 10 rnin to degrade the RNA template, and it was
then purified using a Glassmax spin cartridge column (BRL). A poly (C) tail was
added to the 5'end of the cDNA using terminal transferase. Second strand
cDNA synthesis and first round PCR were accomplished by an anchor primer
(BRL:5'-CUACUACUACUAGGCCACGCGTCGACTACGGGGGGGGGG-3')
and a Exten 1 primer. A second round PCR was performed using UAS primer
(BRL:5'-CUACUACUACUAGGCCACCGCGTCGACTAGTAC-) and Exten2
primer (5'-CCCTGACCCllTCCATTTCCAGTCGAATCGAGA-3'). DNA
fragments amplified by PCR were cloned and then sequenced to identify 5' 0tx-
2 cDNA sequence.
Northern and orimer extension a n d v s i ~
Total RNA was prepared from ES cells and embryos as described above.
Poly(A) RNA was purified using an mRNA purification kit (Pharmacia). lOpg
total RNA and 1pg mRNA were denatured and electrophoresed on a 1 %
formaldehyde agarose gel and then transferred to a Genescreen Plus
membrane (DuPont). The 1 kb Eco Rl fragment was used as a probe with the
random priming method. Hybridization was carried overnight at 42'C in 50%
formamide, 5x Denhart's, 5x SSC, O.l%SDS, 100rng/ml sheared salmon sperm
DNA. The filter was washed at room temperature twice and then exposed to
Kodak X A R 5 film for two days. After that, the filter was washed again at 55%
twice, and then reexposed for three days. Then the filter was stripped by boiling
and reprobed with a mouse O-actin probe.
Primer extension was carried out as follows. A single stranded
oligonucleotide (Exten 3: 5'-GATAGCTGGCTATITGGAATTTGAAGG-3').
which is complementary to 00r-2 mRNA beginning 194 upstream of the ATG
start codon, was end-labeled using T4 polynucleotide kinase and # 2 ~ ] - ~ ~ ~ .
The labeled oligonucleotides were then hybridized to 5pg and 10pg of poly (A)
RNA or 25pg total RNA derived from ES cells and were extended with reverse
transcriptase at 42°C for 2 hr. The extension products were visualized by
autoradiography after fractionation on a 6 % sequencing gel.
Results
Using a partial human cDNA with homology to the Drosophila Oid gene
(Adam et al.. 1992), S.L. Ang screened a mouse 8.5 day cDNA library and
isolated a single I kb cDNA clone under low stringency hybridization conditions.
I sequenced this clone and found that it contains only a partial 3' homeodomain
(1 1 amino acids) with 100% identity to that of Otd (Finkelstein and Perrimon,
1990). Later, I isolated a 1.6 kb cDNA from a 12.5 day cDNA library.
Analysis of possible reading frames reveals only one open reading frame
(ORF) of significant length (Fig.2.1). The open reading frame is 866
nucleotides long. It is proceeded by a 5' untranslated region of 228 bp and
followed by a 3' untranslated region of 535 bp. At the dend, no polyadenylation
signal (Proudfoot and Brownlee, 1976) is present. However, there is a 13bp
poly(A) tract at the 3' end of cDNA. Sequence analysis from the corresponding
exon (exon 5) also reveals a 15bp poly(A) tract in the genomic region. So it is
likely that the cDNA is the result of internal priming within the 3WTR of the
mRNA. While we were isolating this cDNA, Boncinelli's group published that
there were two mammalian CWs (Simeone et al., 1992a). Sequence
comparison of the horneodomain showed that the cDNA we isolated
corresponded to the gene they called Otx-2. Fig. 2.2 shows a comparison of
a x - 2 with other Obc related homeodomain sequences in different species. The
horneodomains of the predicted mouse Otx-1 and Oh-2 proteins are extremely
similar to those of the Xenopus, sea urchin and Drosophila Otd proteins. In
addition, they all share the lysine residue near to C-terminal end of the
homeodomain. This residue is also found in the products ofDrosophila bicoid
(bcd) and goosecoid (gsc) genes (Frigerio et al., 1986. Blumberg et ai., 1991) . This particular lysine has been shown to play a key role in in vitro DNA binding
specificity (Hanes and Brent, 1989; Treisman et al., 1989).
orthern blot an on of 5'-0tx - 2 cDNq
Fig. 2.3 illustrates a Northern blot analysis using the 1 kb Otx-2 cDNA as a
probe. The probe detected a 2.5kb transcript in ES cells, 10.5 and 12.5d
embryos. However, another 6.6kb band was detected in 10.5d and 12.5d
embryos but not in ES cells, although the signal is weaker than that of the 2.5kb
band. The longest cDNA I have is 1.6kb in length and 900bp shorter than the
mRNA detected in ES cells and El 0.5/€11.5 embryos by Northern blot analysis.
In order to identify regulatory elements upstream of the Otx-2 gene, it is
necessary to identify the transcription initiation site. Two additional 1 1.5 and
12.5 day cDNA libraries were screened in order to obtain more 5' sequence of
Otx-2 cDNA. However, partial sequence analysis of all clones showed that
none extended beyond the 5' end of the 1.6 kb cDNA. In order to get more 5'
sequence of Otx-2, the 5'-RACE technique was employed with ES cell RNA.
Two RACE products were cloned and sequenced. One of the products
contains 240bp 5'-sequence of the Obr-2 gene (Fig. 2.4). Comparing with the
1.6kb cDNA, the product contains an addiiional 70 nucleotides but is still not full
length. Primer extension was used to identify the transcription initiation site(Fig.
2.5). It demonstrated that there are possibly two or three transcription initiation
start sites which are about 523bp upstream of the ATG start codon. Thus, the
largest cDNA and RACE product we isolated suggested that it may be still
missing about 225 bp of 5'-UTR and 576 bp of 3' WR. 6RACE cloning will be
needed to clone the remaining 5'-sequence.
on-lntran strucme of the Otx - 2 gene
After screening the 129Sv genomic library using the 1 kb EcoRl cDNA
fragment as a probe, two overlapping genomic clones, G3 and G9, spanning
about 25kb were isolated. These two 15kb genomic fragments were then
subcloned into plasrnid vectors (pKS), and characterized by restriction
mapping, subcloning, and partial sequencing. The exon-intron organization of
Otr-2 gene was determined by comparison of the corresponding regions of
cDNA and genornic clones and is illustrated in Fig. 2.6. Comparison of the 3'
end of the Ofw-2 cDNA with Exon 5 reveals no consensus 3' splice site,
indicating that the 1.6 kb cDNA is incomplete at 3' end. An intron is present in
Otx-2 immediately upstream from the homeodomain, as is often the case for
homeobox genes (Boncinelli et al. 1991). An additional intron is present in the
Otr-2 gene within the homeobox. The results from 5' RACE and primer
extension experiments (Fig. 2.5) suggest that about 225 bp of 5' UTR of Otx-2
cDNA is missing. Whether the exon(s) encoding this region are present within
the G3 genomic clone is not clear. However, the missing exons may be quite far
away from the following exons, as is the case of SpOtx(B) in sea urchin (Li et
al., 1997).
Fig. 3.7 illustrates the early expression pattern of the OW-2 gene. At €6.0-
6.5, Otr-2 appears to be already expressed throughout the embryonic
ectoderm, or epiblast which gives rise to the embryo proper, but not in the
extraembryonic tissues. Between E6.5-7.75. Otr-2 expression becomes
progressively restricted to the anterior half of the embryo and later becomes
further restricted to the anteriormost third of the embryo. The expression domain
includes the anterior three germ layers (Fig2.8C). At 8.0-8.5 dpc, &-2 is
expressed in forebrain, midbrain, and optic eminence of the central nervous
system (CNS) (Rgs2.7D; 2.8D). Weaker expression is also found in the
notochord, head mesenchyme and foregut at the same axial levels. as well as
in the ectoderm and endoderm cells of the first branchial arch (Fig.2.80 and
data not shown). At E9.5, the sharp posterior boundary of Otx-2 expression
corresponds precisely to the border between the midbrain and hindbrain (Fig.
2.7E).
Discussion
I cloned OtK-2, a gene related to the Drosophila otd homeobox gene, and
analyzed the expression pattern of Otx-2 during early embryogenesis. In my
Northern blot analysis I detected a 2.5kb transcript in the ES cells, E l 0.5 and
12.5 embryos. However, l also found a 6.6 band from €1 0.5 and El25 of
embryos but not in ES cells (Fig. 2.3). Southern blot analysis with the same
probe yield a single or two bands when genornic DNA was digested with three
different restriction enzymes (data not shown). These bands correspond to the
genomic clone. tt suggests that Ofx-2 is a single copy gene, although more
distantly related genes may exist. The finding of two transcripts raises a
question whether different promoter utilization and alternative splicing of Otw-2
gene might be important in mouse embryogenesis. Recently, differential
promoter utilization and alternative splicing of Otx have been found in sea
urchin (Li et al., 1997). Four different SpOtx mRNAs were found during sea
urchin embryogenesis. All of these mRNA were products of a single SpOtx gene
resulting from differential promoter utilization and alternative splicing. They
code for two different SpOtx proteins that differ only in their N-terminal regions.
To further investigate the origin of the 6.6 band, the following experiments will
be performed. First, mRNA from early to late stages of embryogenesis will be
used for Northern blot analysis to get the whole profile of this band. Since five
exons so far have been identified and the previous probe used for Northern blot
was the fifth exon, all the other four exons, especially the third exon which
contains most of the homeodomain sequence, will be used individually for
further Northern blot analysis to search whether these four exons are
responsible for this 6.6 band. If all or the third exon can hybridize to this 6.6
band, it may suggest that this 6.6 band is alternative form of Otx-2. If not. it
would indicate that the 6.6 band represents part of another gene which has
sequence homology to the fifth exon.
Unlike hindbrain and spinal cord, little was known about the molecular
mechanism involved in patterning the anterior regions (forebrain and midbrain)
of the embryo. However, identification of the homeobox genes otd and ems in
Drosophila (Dalton et al.. 1989; Finkelstein et al.. 1990) and their mammalian
otdrelated genes, Otw-1 and Otx-2, along with the ems-related genes, Emx-1
and Emx-2 (Simeone et al., 1992b) open a useful way to investigate how the
anterior of embryo is specified. The mammalian Obc and Emx genes are
expressed in nested anterior-posterior (A-P) domains in the anterior C N S
(Simeone et al., 1992a). The Otx-2 gene shows the earliest restricted anterior
expression domain. It is already expressed in epiblast as early as E6.0 of the
mouse embryo. Between 6.5 - 7.5 dpc, Otx-2 expression becomes progressively
restricted to the anterior half of the embryo and later becomes further restricted
to the anteriormost third of the embryo. At 8.0 - 8.5 dpc, Otx-2 is expressed in
the developing anterior neural tube with a posterior boundary corresponding to
the border between
expression domain
the presumptive midbrain and hindbrain. By 9.5 dpc, the
spans the developing forebrain and midbrain. We also
detected Otr-2 expression in anterior mesoderm and endoderrn during early
gastrulation. The progressive restriction of Otx-2 expression to the anterior of
the embryo by headfold stage correlates with the anterior migration of
mesendoderm. Early expression of Ofx-2 raises the issue of whether the Otw-2
plays a direct role in specifying anterior structure.
The expression pattern of Otx-2 is largely consistent with the recently
reported results in Xenopus (Pannese et al., 1995; Blitz et al., 1995) and chick
(Bally-Cuif et al., 1995). In Xenopus, XOLx-2 is expressed throughout early
development from unfertilized egg to late blastula with increasing level of
expression. At stage 9.5, expression of XOtw-2 is localized in the dorsal region
of the marginal zone and at stage 10.25 it is in dorsal bottle cells and cells of the
dorsal deep zone fated to give rise to prechordal mesendoderm. At stage 10.5,
the expression of XOtx-2 extends to presumptive anterior neuroectoderm and it
persists in subsequent stages. Similarly, in the chick, expression of c-Otx-2 is
first detected in all epiblast and associated with cells with presumptive anterior
mesendoderm fate and it correlates with mesendoderm migration toward
anteriormost regions of the embryo. Subsequently it extends to anterior
neuroectoderm.
Early expression of OW-2 raises the issue of whether Otx-2 plays a direct
role in specifying anterior structure. The study of experimentally manipulated
Xenopus embryos suggests a role for XOtx-2 in development of anterior
structures (Pannese et al.. 1995; Blitz et al., 1995). Microinjection of XOtx-2
mRNA into I-, 2-, and 4cell stage embryos produces embryos with severely
reduced trunk and tail structures and the appearance of secondary cement
glands. The cement gland is one of the most anterior structures of the
developing Xenopus embryo. The induction of a secondary cement gland by
ectopic expression of XOtx-2 indicates a role of XOtx-2 in specifying developing
anterior head structures.
conservation of the expression pattern of Otd-related gene OW-2 in
mouse, Xenopus and chick suggests that the underlying molecular mechanisms
of head patterning may have been conserved throughout evolution.
Overexpression of XObr-2 in Xenopus already suggests a role for this gene in
specifying anterior head structures. Loss-function-mutation of Otx-2 in mouse
will be a powerful way to address its role in anterior patterning of embryo.
Fig. 2.1. The nucleotide sequence of the 1.6kb cDNA clone and the amino acid
sequence of the putative Otx-2 gene product. The underlined sequence
corresponds to the homeodomain, and the asterisk marks the translational
termination codon.
tcgctagaggagctgagtcgccacctctactttgatagctggctctttggaattttgaag gataatttgattttttttttcttttctaacgtccaatgcggctgtaagttccgtcactcc aaatctacccaccaaggaccctgatcctgtccactccaggcgaatcgagaccgtccggct gggtccccccaatttgggccgactttgcgcctaaaaacaaccttagcatgatgtcttatc
M M S Y L taaagcaaccgccttacgcagtcaatgggctgagtctgaccacttcgggtatggacttgc K Q P P Y A V N G L S L T T S G M D L L
tgcatccctccgtgggctaccc~g~~acc~~~~ggaaacagcgaagggagaggacgactt H P S V G Y P A T P R K O R R E R T T F
ttactagggcacagctcgacgttctggaagctctgtttgccaagacccggtacccagaca T R A O T , D V T , E A L F A K T R Y p D L
tcttcatgagggaagaggtggcactgaaaatcaacttgccagaatccagggtgcaggtat F M R E E V A ~ , K I N I , P E S R V O V X
ggtttaagaatcgaagagctaagtgccgccaacagcagcagcagcagcagaatggaggtc F R N R R A K C R O O O G Q ~ Q N G G Q
agaacaaagtgaggcctgccaagaagaagagctctccagctcgggaagtgagttcagaga N K V R P A K K K S S P A R E V S S E S
gtggaacaagtggccagtt~agtcccccctctagtacctcagtcccaaccattgccagca G T S G Q F S P P S S T S V P T I A S S
gcagtgctccagtgtctatctggagccccagcgtccatctccccactgtctgaccccttgt S A P V S I W S P A S I S P L S G P L S
ccacttcctcctcctgcatgcagaggtcctatcccatgacctatactcaggcttcaggtt T S S S C M Q R S Y P M T Y T Q A S G Y
atagtcaaggctatgctggctcaacttcctactttgggggcatggactgtggatcttatt S Q G Y A G S T S Y F G G M D C G S Y L
tgacccctatgcatcacca~cttcctggaccaggggccacactcagtcccatgggtacca T P M H H Q L P G P G A T L S P M G T N
atgctgttaccagccatctcaatcagtccccagcttctctttccacccagggatatggag A V T S H L N Q S P A S L S T Q G Y G A
cttcaagcttgggttttaactcaaccactgattgcttggattataaggaccaaactgcct S S L G F N S T T D C L D Y K D Q T A S
cttggaagcttaacttcaatgctgact9cttggatcataaagatcagacgtcctcatgga W K L N F N A D C L D Y K D Q T S S W K
aattccaggttttgtgaagacctgtagaagctctttttgtgggtgatttttaaatatgct F Q V L *
cggctgaacattccagttttagccaggcattggttaaaaaagttagatggaacgatgctc tcagactcctgatcaaagttaccgagaggcatagaaggaanaaggaaggggccttagaag ggtccatcaaccagcaacctgaaatggacaaaccaatctacttaagattctgttatagtt ctagatcattggtttcctgatttgcaaatgattgatcaaanatattctagcgacatgcaa ccaaataccactcaaaacaaaaatccagcaaaactgagttgtgagggaagggagggaagg tcatggccttcaaagcagaggtgatccggtgttttagccaatctttggttgaatgttagg aatggacaatgtcccaggctcattcacgtttcatgaccaacaggtagttggcactgaaaa acttttcagggctgtgtggttgtgcgactgattgtcctagatgcagtactttatttaaaa aaaaaaaaa 16 2 9
Fig. 2.2. Comparison of Otx-2 homeodomain with Otx-I , Xotx-2(Xenopus),
SpOtx(sea urchin) and Otd (Drosophila) homeodomains. Red amino acids
indicate differences with Otx-2. The lysine residue at position 9 of the third helix
is marked with pink arrow.
Fig. 2.3. Transcription of the OW-2 gene. Lanes 1 and 2 contain 1 pg poly(A)+
RNA derived from El 0.5 and E12.5 mouse embryos, respectively. Lane 3
contains 10 pg of total RNA derived from ES cells. The probe is 1 kb Eco FU
fragment from pKSotd9. The blot first (A) was washed at room temperature
(details see methods and materials) and then (B) at 55°C. The same Northern
blot that had been stripped and rehybridized with an mB-actin probe((;).
Fig. 2.4. Diagram of the 5' RACE and primer extension experiments (A) and
sequence of 5' RACE product (8). (A) Primer Exten 1 was used to synthesize
first strand cDNA. Anchor Primer was added later for first round PCR (details
see method and materials). Second round PCR was performed using primer
Exten 2b (with Eco RI cloning site) and UAP (Universal Amplification Primer with
Sal I cloning site). Primer Exten 3 was used for primer extension experiments.
(6) The nucleotide sequence of 5' RACE product. The underlined sequence
highlighted in red is primer Exten 2b.
Anchor Primer - UAP -
\\ Otx-2 mRNA - Exten 3 (for primer extension)
240bp from RACE
(3 2 5 bp estimated by Primer Extension)
TGCAAATCTCCCTGAGAGCGGAACCTCCTCAGCTCCAACTAAGCCnCCACKTTACTAAAAAAT AAAAATCGCTAGAGGAGCTCAGTCGCCACCTCTACTlTGATAGCTGGCTAmGGAAmGAAGG ATGGmGAmC'TmCTAACGTCCAATGCGGCTGTAAGTCCGTCACTCCAAATCTACC CACCAAGGT CCCTGACCCTTTCCAICCAGTCGAATCGAGA
Exten 2b
Fig. 2.5. Result of primer extension experiment. Lanes 1 and 2 contain 5 and 10
pg of poly(A) RNA derived from ES cells, respectively. Lane 3 contains 25 pg
total RNA derived from ES cells. Primer Exten 3 was used in this experiment.
Fig. 2.6. Exon-intron structure of Ofx-2 gene. (A) The overlapping two genomic
clones are represented by the thin lines above the gene. (B) Lightly shaded
rectangles represent exons, darkly shaded rectangles denote the
homeodomain, striped ban represent open reading frame (ORF), pKSotd9
represents the cDNA clone containing a 1 kb Otx-2 cDNA, pEXloxOl.1
represents the clone containing a 1.6 kb Otr-2 cDNA. pKS-5-RACE2 obtained
from 5'RACE cloning contains 240bp Otw-2 cDNA which was used to identify the
two additional exons upstream of the ATG-startcodon-exon .
Fig. 2.7 Expression of Otw-2 from prestreak to early somite stages revealed by
whole mount RNA in situ hybridization. (A) Pre- to early streak stage (E6.0-E6.5)
embryos showing widespread expression of Otx-2. (B) Mid- to late streak stage
(E7.0-€7.3). Expression became progressively restricted to the anterior half of
the embryo. (C) Headfold stage (E7.5E7.7). Further restriction of Otx-2
expression to the anterior third of the embryo. (D) At the somite stage (E8.0-
E8.5), Otx-2 expression in the neuroectoderm was found in the forebrain and
midbrain regions. (E) At E9.5 the sharp boundary of Otx-2 expression
corresponds precisely to the boundary between the midbrain and hindbrain
(arrows). Scale bars in D and E represent 100 pm, while the scale bar in A
represents I 00 p n for the other embryos.
Fig. 2.8 Tissue sections analyzed for Otx-2 expression. (A,B) Dark-field and
bright-field views of a sagittal section of an early streak stage embryo analyzed
by radioactive RNA in siiu hybridization showing widespread expression ofOtx-
2 in the ectoderm and delaminating mesoderm in the posterior end. (C)
Parasagittal section of a headfold stage embryo previously stained by whole
mount in situ hybridization showing Otx-2 expression in all three germ layers at
the anterior end.(D) Frontal section of a 10- to 15-somite stage embryo assayed
by whole mount RNA in situ hybridization showing Obr-2 expression in optic
eminence (oe) , diencephalon (di), notochord (No), foregut (fg), ectoderm cells of
the first branchial arch (ba) and endoderm cells surrounding the first branchial
pouch (arrowed). Scale bar represent 20 pm. abbreviations: A: anterior; P:
posterior; ect: ectoderm; mes: mesoderm; en: endoderm; ne: neuroectoderrn;
am: amnion.
Chapter 3
Targeting of the Ofx-2 Gene
This chapter is a modified version of the following publication:
In. 0.. Rhinn. M.. Daigle, N., Stevenson, L. and Rossant, J. (1996). A
targeted mouse Otx-2 mutation leads to severe defects in gastrulation and
formation of axial mesoderm and to deletion of rostra1 brain.Development 1 22:
243-252
The first two authors contributed equally to this work.
I am responsible for the following work: generating two targeting vectors (Fig.
3.1), most of the work in the table 3.1, a portion of the work in the table 3.3, the
work in figures 3.2,3.3,3.4 (with exception of the preparing wax sections) and
fig. 3.5.
INTRODUCTION
The patterning and development of the vertebrate neural tube is a
complex process involving both cell extrinsic and cell intrinsic events. Among
the cell extrinsic events, the mesoderm in the organizer region, namely the
dorsal blastopore lip in amphibians, Hensen's node in birds and the node in the
mouse, is able to induce neural differentiation in the surrounding ectoderm
tissue (Spemann, 1938; Waddington, 1933; Beddington, 1994). Furthermore, in
vim experiments have demonstrated that the prechordal mesoderm and
notochord, descendants of the organizer, can also induce and pattern the
neural tube along the anteroposterior(A-P) axis in Xenopus embryos (reviewed
in Slack and Tannahill, 1992; Doniach, 1993; Ruiz i Altaba, 1994). Support for
a role of the prechordal mesoderm in the induction of forebrain and midbrain
has also come from the analysis of the phenotype of hornozygous Lim-1 mutant
embryos: a defect in prechordal mesoderm cells in these mutant mice is
suggested to be responsible for the subsequent loss of anterior brain tissues
(Shawlot and Behringer, 1995). However, the role of the notochord in A-P (A-P)
patterning of the neural tube is still in question, since embryos homozygous for
a mutation in the gene H W B lack an organized node and notochord but show
relatively normal A-P patterning of the central nervous system (CNS) (Ang and
Rossant, 1994; Weinstein et al., 1 994).
A major contribution to the identification of cell intrinsic molecules
responsible for neural tube regionalization has come from cloning of genes
homologous to homeobox-containing genes within the HOM-C complex in
Drosophila (reviewed in Lawrence and Morata, 1994), namely the Hox genes in
mice. The function of these genes in the regional specification of hindbrain and
vertebrae has been investigated using both loss-of-function and gain-of-
function studies (reviewed by Krumlauf, 1994). Hox genes are not expressed in
the forebrain and midbrain, suggesting that some other classes of horneobox
genes are involved in the development of these rostral regions. Recently, two
new classes of homeobox genes, related to the Drosophila orthoden tide (otd)
(Finkelstein and Perrimon, 1990) and empty spiracles (ems) gene (Dalton et al.,
1989), have been cloned. These genes, Otx-I, Obr-2,, Emx-1 and Emx-2? are
expressed in nested domains in the forebrain and midbrain regions (Simeone
et al., IW2a). Since otd and ems have been shown to participate in a
regulatory network required for head formation in flies, it has been suggested
that the conserved murine genes serve similar roles in the patterning of rostral
brain in mice (Finkelstein and Boncinelli, 1 994).
The mouse owrelated genes. Otx-1 and Otx-2, belong to the bicoid-class
of homeobox genes. The amino acid sequences of the homeodomains of their
protein products differ by two and three amino acids from that of the otd gene
product respectively (Simeone et al., 1993). These genes are expressed in
overlapping domains in the anterior GNS with the domain of Otx-1 expression
contained within the Otu-2 domain. Otx-2 is already expressed by embryonic
day 5.5 (E5.5). while the expression of Otr-I mRNA is not detected until early
E8.0. Otx-2 expression at E5.5 is widespread in the epiblast, which gives rise to
the embryo proper. From the early primitive streak to headfold stages, Otr-2
expression in the ectoderrn becomes restricted to the anterior end of the embryo
(Simeone et al., 1993, Ang et al., 1994). We and others have previously shown
that this Otx-2 expression in the anterior ectoderm depends on interactions with
the underlying mesoderm at the anterior end of the embryo in mice and
Xenopus (Ang et al.. 1994; Pannese et al., 1995; Blitz and Cho. 1 995). Obr-2
expression was also found in anterior endomesoderm tissues, including axial
mesoderm tissues, such as notochord and prechordal mesoderm (Ang et al.,
1994; Simeone et al.. 1995). The earlier expression of 0tx-2, compared toOtx-
1, and its expression in axial mesoderm tissues that possess neural patterning
capabilities suggests that this gene could be involved in the patterning of
anterior neural tissues.
To begin to dissect the roles of OW2 in vivo, I have generated a
homeobox deletion in the gene using homologous recombination in ES cells.
This mutation results in early gastrulation defects. Homozygous Otw-2 mutant
embryos also show defects in the prechordal mesoderm and notochord
precursors by the headfold stage. By E8.25, rostral deletion of the neural tube
to rhombornere 3 was clearly apparent. These defects are consistent with
multiple roles for 0tx-2 in gastrulation and the patterning of rostral brain in mice.
MATERIALS AND METHODS
A l kb mouse 00r-2 partial cDNA probe was used to isolate two
overlapping genomic clones, containing the entire coding region of the Otu-2
gene from a 129SVlJ genomic library. To construct the first targeting vector,
pPNT02. a 1.7 kb Bgl 11-Xba I fragment that maps 5' to the OtK-2 homeobox was
subcloned into the BamH I-Xba I site of pPKT (Tybulewicz et al., 1991). A 4.4 kb
Stu I fragment located 3' to the homeobox was subsequently cloned into the
Xho I site of the above vector. The PGKneo and PGKtk cassettes were in the
opposite transcriptional orientation compared to the endogenous Otx-2 gene
(Fig. 3.1). To construct the second targeting vector, pPNTKSN, a 6.5 kb Sma I
fragment from the 5' region of Utx-2 was first subcloned into the Xho I site of
pPNT. A 2.5 kb Kpn I fragment from the 3' end of the gene was then subcloned
into Kpn I site of the same vector. in pPNTKSN, the PGKneo and PGKtk
cassettes were in the same transcriptional orientation with respect to the OtK-2
fragments as the endogenous Otx-2 gene (Fig. 3.1).
Generation of the mutation
The R1 ES cells (Nagy et al., 1993) were cultured and electroporated
with Nod-linearized pPNTO2 and pPNTKSN as described (Wurst and Joyner,
1 993). Doubly resistant cells were selected in a concentration of 1 50 pg/ml of
active G418 and 2 pM gancyclovir for 10-1 1 days before picking. Colonies were
picked onto gelatinized 96-well plates and grown to near confluency before
splitting into two 96-well plates. The master plate was frozen down, and ES cell
genornic DNA from the other plate was analyzed by Southern blotting for
homologous recombination events.
Genomic DNA from these cell lines was digested with EwR I and Xho I
and probed with a 2.0 kb Hindl 11-Xbal 3' flanking probe and a 1.7 kb 8gl 11-Xba I
5' internal probe. Hybridization was carried out overnight at 42OC in 50%
formamide, 5x Denhart's, 5x SSC, O.l%SDS, 100 pg/ml sheared salmon sperm
DNA.
Chimeras were generated by ES-morula aggregation and blastocyst
injection with targeted ES lines. Chimeric males were bred to CD1 females to
establish F1 heterozygotes. Embryos from intercrosses of F1 heterozygotes
were typed either by Southern analysis or by PCR of yolk sac DNA. To
genotype €7.5 embryos, ectoplacentai cones were isolated and cultured for 1
week in 96 well dishes using Dulbeccots modified Eaglets medium plus 15%
fetal calf serum. Cells were lysed in 35 pi of proteinase K buffer (50 mM KCI,
1 OmM Tris.HCI (pH 8.3). 2.0mM MgC12, 0.1 mg/mL gelatin. 0.45% Nonidet p40,
and 0.45% Tween-20). To detect a 1.3 kb wildtype band, the following primers
were used: sense strand (5'-ATGATGTCTTATCTAAAGCAACCGCCTTACG-3')
and an tisen se strand (5'-TCATTGGGTCATCAGTATAAACCA-3'). The OW-2
mutant allele was detected by amplifying a 650 bp neo fragment using a set of
primers corresponding to the sense strand of the neo gene (5'-
ATCTCCTGTCATCTCACClTGC-3') and antisense PGK poly (A) sequence (5'-
ACCCCACCCCCACCCCCGTAGC-3'). Samples were amplified for 35 cycles
(94°C 40 seconds; 55°C 1 minute; 72°C 1.5 minutes) for the wild-type allele and
for 40 cycles (94°C for 1 minute; 65OC for 1 minute; 72°C for 2 minutes) for the
mutant allele. Amplified bands were visualized by agarose gel electrophoresis
and ethidium bromide staining.
Wholemount in situ hybridization was performed as described
previously (Conlon and Herrmann, 1993). For histology and in sifu
hybridization to sections, embryos were fixed overnight in 4%
paraformaldehyde in PBS. They were then processed and embedded in wax
and sectioned at 5-6pm. Slides were then dewaxed, rehydrated and stained
with hematoxylin and eosin. Probes used for in situ hybridization of sections
were: gsc (Blum et al., 1 W2), Lim-1 (Barnes et al., 1 994), Brachyury (Hermann,
1991). Mox-7 (Candia et al., 1992). HNF-3B (Ang et al., 1993). Krox-20
(Wilkinson et al., 1989b).
RESULTS
tarwted disr~ption of Otx 3 in ES cells bv homolo~pus recambtnat~on . . -
Two positivdnegative targeting vectors were made (Fig.3.1). One
targeting vector (pPNTO2). containing 1.7 kb of 5' and 4.4 kb of 3' genomic
sequence, was designed to delete the homeodomain region of Otx-2, and
replace it with the PGKneo cassette from the pPNT vector (Tybulewicz et al..
1991). Therefore the PGKneo insertion should truncate the Otx-2 coding
sequence immediately after the first exon which encodes 32 amino acids. The
other targeting vector, designated pPNTKSN. consisted of 6.5 kb of 5' and 2.5
kb of 3' genomic sequence and was to designed to delete the exon containing
the predicted initiation codon.
The linearized targeting vectors were electroporated into ES cells (Nagy
et al., 1 993) and clones were selected for resistance to 641 8 and gancyclovir.
A total of 11 94 double resistant ES cell colonies were analyzed by Southern
blot using a 5' internal and a 3' external probe, six cell lines, including 5-23 and
C12, were isolated in which the Ob-2 locus was correctly targeted. Table 3.1
summarizes the screening results. All targeted cell lines are from pPNTO2
vector. Using a genornic probe spanning exon 2, the deletion of the homeobox
region was confirmed in the the genomic DNA of homozygous Ofx-2 mutants by
Southern blot analysis (data not shown).
The 5-23 and C-12 targeted ES cell lines were used to generate
chimeras by ES cell-morula aggregation (Nagy et al., 1993) or by blastocyst
injection. These chimeras transmitted the mutation to their progeny. Mice
generated from both lines showed identical phenotypes. All analyses were
carried out on a mixed CD1/129 background.
Table 3.1. Results of Screens for Homologous Recombination into
Otx-2 Locus
Targeting Vectors G418 and Gancyclovir Recombinants
ES Colonies
DPNTO~ 648 6
fable 3.2. Frequency of Germ-Line Transmission of Targeted E S
Cell tines
Cell
Lines
Total Embryos Number of Number of Germ tine
Transmission
* Cell line 5-23, contributed to germ line later in S.L.Angls lab.
he Obr - 2 m w n leads to e w o n i c lethality
When heterozygous animals were crossed with wild-type CD1 females
and their progeny were genotyped at 3 weeks of age, heterozygous mice were
obtained with at a frequency of 142/311, less than the expected 50% ratio.
These heterozygous mice appeared normal and were fertile. However, when
offspring from heterozygous intercrosses were harvested at E9.5 and E10.5, a
small fraction (81149) of normal size embryos showed an open neural tube
defect at the forebrain and midbrain levels. Caudal to the midbrain region, these
embryos looked identical to wild-type embryos (data not shown). All these
embryos have been genotyped to be heterozygous animals. The Otw-2
mutation thus results in a dominant phenotype that is weakly penetrant on the
CD11129 background, and this dominant phenotype may explain the slightly
lower number of heterozygous animals obtained at 3 weeks of age. This
phenotype will be analyzed in more detail elsewhere.
When the progeny of intercrossed of O W heterozygous animals were
analyzed at birth, no homozygous newborn animals were found, indicating that
Oh-2 is required for embryonic development. To characterize the embryonic
lethality, we analyzed litters from heterozygous intercrosses from E7.25 to
E l 0.5. Homozygous mutant embryos were present between E7.25 and E9.5 at
roughly the expected frequency of 2596, however, at €10.5, the proportion of
mutants obtained declined to 17% and these mutant embryos were either
severely growth retarded or being resorbed. Thus, the Otw-2 mutation leads to
embryonic lethality around E10.5 (Table 3.3). The results from a typical
genotyping analysis of yolk sacs from E8.5 embryos generated from
intercrosses of Obr-2 heterozygotes are illustrated in Fig. 3-28.
s of the Obr - m o 7 y o o u s he no
By E7.25-€7.5 (mid- to late-streak stage), homozygous embryos were
morphologically distinguishable from normal embryos (Fig. 3.38). The
Table 3.3. Genotype of mice resulting from 0-2 heterozygous
*Embryos were either severely growth retarded or being resorbed.
i ntercrosses
abnormal looking embryos were smaller than their littermates, and these
embryos were confirmed to be homozygous mutant by PCR analysis. Sections
through mutant embryos at this stage demonstrated clearly that these embryos
had initiated gastrulation and mesoderm had formed all around the embryo
(Fig.3.4B,C,D). Embryonic ectoderm, mesoderm and endoderm cells are
present in mutant embryos. However, the accumulation of mesoderm cells has
been observed in these homozygous embryos (Fig.3.4B,C9D). In most of the
Stage
7.5
8.5
9.5
10.5
Postpartum
+/+
12
24
16
15
18
OtK-2+/-
27
43
32
23
36
Otx-&/- (%)
1 3(25)
22(25)
1 4(23)
8(17)*
0
mutants, the allantois was poorly developed and formed a round ball of cells, in
contrast to its fairly long and extended appearance in normal embryos at this
stage. It was also often disconnected from the embryonic portion of the
conceptus.
At E7.75 (headfold stage), the headfolds forming at the rostra1 end of
wild-type embryos were not apparent in homozygous mutant embryos. At €8.5
(somite stage), variations in the phenotype of the mutants were found (Fig.
3.3D,E,F). We have divided the mutants at this stage into a less severe class,
which includes embryos enclosed in the yolk sac, and a more severe class in
which the embryos are completely excluded from the yolk sac. The less
severely affected class of mutants exhibited fairly good development of
posterior somites and a rudimentary heart. At the anterior end, the neural tube
looked extremely abnormal with either numerous folds or a single fused
structure instead of open neural folds. Compared to wild-type or heterozygous
embryos, no forebrain or midbrain tissue could be identified suggesting that
deletion of neural tissue had occurred anteriorly. The sornites were not always
normal; in some embryos they were fused at the midline or were irregularly
shaped. The more severely affected homozygous mutant embryos were
smaller than the first class of mutants, wrapped up anteriorly in definitive
endoderm and showed improper segmentation of mesoderm into somites.
Some of the mutants in this class were very thin and spiralshaped and showed
no sign of organogenesis and segmentation of mesoderm into somites
(Fig.3.3F). The two different classes of mutants occurred at approximately the
same frequency.
To determine precisely how much rostra1 brain tissue was deleted in
mutant embryos, several A-P regionspecific neural markers such as Krox-20
and En were used to characterize Otx-2 homozygous embryos (Fig. 3.5 and
data not shown). For these studies, we only used E8.25-8.5 mutant embryos
from the less severe class. Krox-20 was expressed in two bands across the
neural tube (rhombomere 3 and 5) in wild-type or heterozygous embryos.
However, in mutants the first band of expression either completely abolished or
weakly detectable at the anterior most end of the embryos (Fig. 3.5 and data not
show).These results demonstrate that in homozygous mutants. neural tissues
anterior to rhombomere 3 have been deleted.
ous embrvo~
The small size and abnormal morphology of E7.25 embryos
demonstrated that defects had already occurred in homozygous mutants at this
early stage. To determine if defects occurred in the primitive streak, we
analyzed the expression of the genes Goosecoid (gsc) and Brachyury.
Goosecoid is a homeobox gene expressed in the anterior primitive streak
region in mid- to latestreak stage embryos (Fig.3.6A and Blum et al., 1992).
Goosecoidexpressing cells were found ectopically located in the proximal
region of homozygous mutants in one case and were absent in three other
embryos analyzed (Fig. 3.68 and data not shown). Previous studies have
demonstrated that gsc is expressed transiently in the anterior primitive streak in
wild-type embryos (Hum et al., 1992). Since it is difficult to accurately stage the
homozygous mutant embryos due to their abnormal morphology, we cannot
distinguish whether the mutant embryos that fail to express gsc do so because
they are older than the late-streak stage or because Otx-2 is required for the
maintenance of gsc expression at this early stage.
The Brachyurygene is expressed along the entire proximaldistal extent
of the primitive streak at the mid40 late-streak stage (Hermann, 1991). In some
homozygous Otu-2 mutants, Brachyury expression was only found in the
proximal region in a position similar to the gsc-expressing cells in Otu-2
homozygous mutants (data not shown). Other Otx-2 mutant embryos showed
almost complete extension but a thickening of the region of the primitive streak
(Fig.3.6F). Together, these results demonstrate that Otw-2 homozygous mutants
show severe early gastrulation defects, characterized by a lack of proper
primitive streak organization.
oderm and n o w o r d are severelv affected tant
embrvos
To determine if the different populations of embryonic mesoderm tissues
were present in mutants at E8.5, the markers Brachyury, Mox-1 (Candia et al.,
1992) and Lim-1 (Barnes et al., 1994) were used to identify axial, paraxial and
lateral mesoderm cells, respectively. Both Mox-1 and Lim-1 expression could
be detected in mesoderm cells of mutant E8.5 embryos, indicating that the
homozygous mutants contain paraxial and lateral mesoderm cells (Fig.3.6 J, L).
Max-1 expression in the somites in posterior regions was similar in
homozygous mutants and their littermates. Anteriorly however, Mox-1
expression spread across the midline, as expected from the observed fusion of
somites. In eight out of ten embryos analyzed, Brachyury expression in
homozygous mutants was absent in the anterior midline, at the normal position
of the notochord. In one case, Brachyuyexpressing cells were present
anteriorly but appeared to bud off and diverge from the axial notochord (Fig.
3.6N). However, in another case, expression of Brachyury seemed normal in
axial notochord (data not shown). Thus axial mesoderm cells are severely
affected in Otx-2 homozygous embryos, while paraxial and lateral mesoderm
cells do develop more normally. The appearance of fused somites is consistent
with axial mesoderm defects since it has also been seen in other notochordless
mouse mutants and notochordless chick embryos (Dietrich et al., 1993; Ang and
Rossant, 1 994; Teillet and le Douarin, 1983; Rong et al., 1992).
We next tested whether axial mesoderm defects occurred at earlier
stages than those examined above. Lim-1, Brachyury, and HNFGl3 (Ang et at.,
1993; Monaghan et al., 1993; Sasaki and Hogan, 1993) are all expressed in the
node and head process in wild-type embryos at E7.5 (Fig.3.6C.E.G). H W 3 B
and Lim-1 are also expressed in midline cells anterior to the notochord known
as prechordal mesoderm cells (Fig.3.6C.G-arrows). In homozygous Otw-2
mutants, all three genes were expressed in the node and in a few ceils
extending anteriorly at a short distance from the node. This was in sharp
contrast to wild-type embryos in which labeled headprocess cells had migrated
much further anteriorly (Ag.C,E.G). In particular, the anterior-most midline
expression of Lim-1 and HNF3B in the prechordal mesoderm cells was
missing in the mutants (arrows in 40 and ti). These results suggest that the
defects in axial mesoderm observed at E8.5 are due to a failure in the
generation of these cells at earlier stages. Prechordal mesoderm fails to
develop properly and notochord development is incomplete in €7.5 OtK-2
homozygous embryos.
DISCUSSION
Deletion of the homeobox region of the Otx-2 gene produced an
embryonic lethal phenotype in mice. The phenotype was characterized by
severe gastrulation and prechordal mesoderm defects, absence or reduction of
the notochord, and severe anterior truncations. All mutant embryos were
severely growth retarded or resorbed by E10.5, presumably because the
separation of embryonic and extraembryonic regions resulted in defective yolk
sac circulation. Given that Otx-2 is broadly expressed at the pre-streak and
early streak stage embryos, and is later restricted to anterior structures, this
phenotype implicates Otx-2 in several different aspects of early postimplantation
patterning.
Earlv ~ t r u l a t i o n defects in Otx-2 homqy~gpus m u m
In the mid- to latestreak stage mutant embryos, the incomplete
elongation of the primitive streak, the accumulation of mesoderm cells between
embryonic and extraembryonic region of the embryo and the ectopic location of
gsc-expressing cells in the proximal region, indicate that OtK-2 is required in
some manner for the normal organization of the streak.
The smaller size of the embryonic portion of mutant embryos at the mid-
streak stage suggests that proliferation of the epiblast tissue could also be
affected or delayed in the absence of Obr-2. To examine whether cell
proliferation in the epiblast is affected at this early stage, BrdU incorporation
experiments will be performed. In addition, the severe constriction observed
between embryonic and extraembryonic regions in Otw-2 mutants suggest that
other processes besides proliferation are also affected in the mutants. A very
similar extraembryonic-embryonic constriction was also observed in HNF-38
mutants (Ang and Rossant, 1994; Weinstein et al.. 1994). to a lesser extent in
Lim-1 mutants (Shawlot and Behringer, 1995), and in nodaldeficient embryos
(Varlet, et al., 1997). All of these three genes are expressed in the visceral
endoderm and recent evidence from mosaic analysis of nodal suggests the
involvement of the endoderrn in the constriction (Varlet et al., 1 997).
regy~red for Drooer orechordal mesoderm and notochord develooment
We have demonstrated that the prechordal mesoderm is severely
affected in Otx-2 homozygous mutant embryos at the headfold stage using Lim-
7 and HNF-3B genes as markers for this tissue. These results demonstrate that
Otx-2 is an essential regulator of prechordal mesoderm development. Recent
phenotypic studies on Lim-7 hornozygous mutants have also demonstrated an
essential role for the Lim-1 gene in prechordal mesoderm development. Since
Oh-2 and Lim-1 are both expressed in prechordal mesoderm in headfold stage
embryos and are required for its development, it will be interesting to study
whether these two genes might function in the same genetic pathway.
Defects in notochord development were also observed in Otx-2
homozygous embryos. In the late streak and headfold stage embryos, there
was limited midline extension of the head process. In later embryos, the
notochord formed only at the most posterior end in some cases, while in other
cases, notochord cells were present but misplaced lateral to the midline.
Anterior notochord normally expresses Otx-2 at the midbrain level (Ang et al.,
1 9941, perhaps explaining the anterior notochord defects observed in mutant
embryos. However abnormalities in the development of notochord cells, which
normally do not express Otx-2 at the hindbrain and trunk levels, cannot be
readily explained. It seems likely that the failure of later notochord development
reflects earlier defects in headprocess development. Interestingly, studies of
Otx-2 in Xenopus and chick have demonstrated Otx-2 expression in the
organizer tissue of these species, namely dorsal blastopore lip and Hensen's
node respectively (Pannese et al., 1995; Blitz and Cho, 1995; Bailly-Cuif et al..
1995). Thus, by analogy to the situation seen in these species, Otr-2 might be
expressed in the presumptive node at the anterior end of the primitive streak in
early streak stage mouse embryos (Lawson et al., 1 991 ), this expression being
obscured by the simultaneous widespread expression of Otr-2 in the epiblast at
this stage. Loss of Otx-2 in this structure may be responsible for the notochord
defects at later stages.
brain develq~rnent during mouse s m a m
Otx-2 homozygous mutants that failed to become enclosed by visceral
yolk sac by E8.5 because of the severe constriction between embryonic and
extraembryonic regions, were extremely abnormal and in some cases seemed
to lack any axial organization. However, specific anterior defects could be
observed in the less severely affected embryos that were still enclosed in the
yolk sac.
By E8.25, deletions in the anterior neural tube rostra1 to rhombomere 3
were clearly apparent in these hornozygous mutants. Loss of forebrain,
midbrain and anterior hindbrain was demonstrated using early molecular
markers for these tissues such as BF1, Emx-2 and En (data not shown). This
phenotype could have been predicted. on the basis of the anterior expression of
Otx-2 and its relationship to the otd gene in Drosophila. Loss of function
mutations in otd in flies lead to deletion of anterior head structures (Finkelstein
et al.. 1990). These results support evolutionary conservation of the function of
these genes in head development in flies and mice.
This interpretation of the phenotype assumes that 00r-2 acts
autonomously in the anterior CNS and is required for the specification of these
regions. However, this hypothesis cannot readily explain why structures
posterior to the expression domain of Otw-2 are also deleted. The caudal
boundary of Otx-2 expression marks the mid-hindbrain boundary. However,
deletions of the hindbrain region, containing the Errexpression domain of the
metencephalon and close to the anterior border of rhomobomere 3, occur in
mutant embryos. This could either be due to early expression of OW-2 in cells
fated to become hindbrain at the late-streak stage or to a dependence of
anterior hindbrain development on more rostra1 neural tube. Comparison of the
fate-map studies of late streak stage embryos with the domain of Otr-2
expression at this stage suggest that this domain does not include hindbrain
territories (Tam. 1989). However a more extensive study is necessary to
exclude this possibility.
Alternatively, loss of anterior neural tissue could be an indirect
consequence of the loss of notochord and prechordal mesoderm in the Otx-2
homozygous mutants. Classical embryological studies have demonstrated a
role for both these tissues in the induction and patterning of the neural tube
(reviewed by Slack and Tannahill, 1992; Doniach, 1993; Ruiz i Altaba, 1994). A
role for the prechordal mesoderm was supported by the phenotype cf Lim-1 null
homozygous mutants (Shawlot and Behringer, 1995). In contrast, a role for
notochord tissue in A-P neural tube patterning has not been confirmed by
mutant studies in mice. Mouse embryos lacking the HNF38 gene do not
develop a notochord, yet they showed expression of rostral brain markers (Ang
and Rossant, 1 994; Weinstein et al., 1 994). Together, these results suggest an
essential role for the prechordal mesoderm, but not the notochord in patterning
of the anterior neural tube in mice. Furthermore, the Lim-1 mutant phenotype is
remarkably similar to that of the Otw-2 homozygous mutants, in that deletions of
the anterior neural tube occur at about the same anteroposterior level (Shawlot
and Behringer, 1995). Thus, loss of rostral brain tissues observed in Otx-2
homozygous mutants could be a consequence of the lack of prechordal
mesoderm development.
To distinguish between these hypotheses, I am planning to recombine
mesoderm tissue from homozygous mutant Otx-2 embryos with ectoderm tissue
from wild-type embryos to determine if the mutant mesoderm tissue can induce
expression of anterior neural markers such as En genes. The reverse
experiment of recombining ectoderm tissue from OW2 homozygous embryos
and mesoderm from wild-type embryos will be performed to determine if the
OW-2 negative ectoderm tissue can respond to inducing signals from the
mesoderm. The use of the in vitro tissue recombination assay (Ang and
Rossant, 1993, 1994) to analyze Otx-2 mutants should allow us to dissect the
roles of 00r-2 in the ectoderm and mesoderm tissues at the latestreak stage.
In conclusion, Otx-2 homozygous mutants show complex defects in
gastrulation, axial mesoderm and rostral brain development that implicate Otx-2
in the pathways of primitive streak organization, axial mesoderm development
as well as anterior head development. Further experiments will be required to
dissect out these different roles.
Figure 3.1. Targeted disruption of the OW2 gene. The first targeting vector,
pPNTO2, contains 7.1 kb of the Otx-2 genomic locus while the second one,
pPNTKSN, contains 10 kb. The open boxes represent the coding region and
the solid boxes indicate the homeodomain. The 5' and 3' probes used for
Southern blot analysis are indicated. The sizes of the expected restriction
fragments from the wild-type and mutated Otu-2 alleles with specific probes are
indicated in the following table. The sequences amplified by PCR to identify the
wild-type and Otx-2-1- alleles are indicated as red boxes.
Expected Restriction
Fragrnents(kb)
5'- Xho I Eco RI
Expected Restriction
Fragments(kb)
5'- Xho I Em RI
Abbreviations: 6: Bglll; E: Eco RI; H: Hind Ill; K: Kpn I; S: Stu I; Sm: Sma I; Xb:
Xba I; Xh: Xho I. m: mutant; wt: wild-type.
Figure 3.2. (A)Southern blot analysis of DNA from wild-type and targeted ES
cell lines, 5-12, 5-23 and C-12. The sizes of the DNA bands are indicated in
kilobases (kb). Both 5' and 3' probes detected predicted restriction fragments for
the wild-type (wt) and mutated (m) allele. (B) PCR gemtyping of yolk sac
biopsies isolated from E8.5 embryos of intercross between Otx-2+/- mice.
Embryos were scored phenotypically as either normal (N) or mutant (M).
3' probe Eco RI
5' probe Xhol
' 8.0 kb (m)
2.6 kb (wt)
I ) C 8 . O kb (m)
5' probe Xhol
genow Pe +I- +/- +I+ +I+ 4 4- +I- phenotype N N N N M M N
Figure 3.3. Phenotype of Otx-2 homozygotes. (A,B) Lateral views of E7.5
embryos. In mutant embryos (B), a constriction is seen between embryonic
regions and the extraembryonic portion, and the embryonic portion is smaller
than that of wild-type or heterozygous embryos (A). (C,D,E,F) Views of E8.5
embryos. (C) A phenotypically wildtype embryo (either +/+ or +/-). (D,E) are
examples of less severely affected homozygous mutants and (F) is an example
of more severely affected ones. (G,H) are examples of some homozygous
mutants which are either completely outside of the yolk sac (G) or partially
outside of the yolk sac (H). Scale bar, 100 pm.
Figure 3.4. Histological analysis of OtK-2 homozygous embryo. Saggittal
sections of E7.5 wild-type embryo (A) and homozygous mutant embryo (B,C,D
serial sections). In the mutant embryo, embryonic ectoderm, mesoderm and
endoderm cells can be seen. Note the accumulation of mesoderm cells. Scale
bar, 100 urn. ect: ectoderm; me: mesoderm; en: endoderm.
Figure 3.5. Whole mount RNA in situ hybridization of Krox-20 in €8.5 wild-type
or heterozygous embryo and homozygous mutant embryo. Krox-20 is normally
expressed in both rhombomeres 3 (r3) and 5 (r5) in this stage. However, in the
homozygous Otr-2 mutant, the expression of Krox-20 in rhombomere 3 is not
detected.
Figure 3.6. Whole-mount analysis of mesoderm markers in wild-type or heterozygous embryos (A,C,E,G,I,K,M) and homozygous mutant embryos (B,D,F,H J,L,N.O) at E7.25E8.5 (anterior is to the left in A-H, and MO or to the top in I-L). (A,@ gsc expression in E7.25 embryos. (A) gsc expression in anterior primitive streak cells in normal embryos. (B) gsc-expressing cells were present but only in the proximal region of the mutant embryo. (C,D) Lim-7 expression in €7.25 embryos. (C) In normal embryos, Lim-7 is expressed in the mesodermal wings, node, head process and prechordal mesoderm cells (arrow in C). In mutants, Lim-1 is expressed in the same areas except there was no expression in the prechordal mesoderm region (arrow in D). (E, F) Brachyury expression in E7.75 embryos at headfold stage. (E) Brachyury is expressed in the primitive streak, node and head process in normal embryos. (F) In mutant embryos, Brachyury expression is expanded in the primitive streak (open arrowhead), in the node (arrow) and in a few headprocess cells extending anteriorly from the node (arrowhead). (G,H) HNF-3B expression in E7.75 embryos. (G) In €7.75 wild-type embryos, HNFGB is expressed in the node, head process and prechordal mesoderm (arrow). (H). In mutant embryos, HNF- 38 is expressed in the node, in a shortened headprocess but not in the prechordal mesoderm area (arrow). (1,J)Mox-1 expression in €8.5 embryos. (1)Mox-1 is expressed in the somitic mesoderm of wild-type embryos. (J) In mutants,Mox-1 expression anteriorly is spread across the midline (arrow). (K,L) Lim-1 expression in E8.5 embryos. Lim-1 is expressed in the lateral mesoderm of normal embryos (K) and mutant embryos (L). (M.N.0) Brachyuryexpression in E8.5 embryos. In normal embryos, Brachyury is expressed in the notochord and in the posterior primitive streak region. In mutant embryos, Brachyury expressing cells are displaced from the midline anteriorly (arrowheads in N), or missing from the anterior and trunk regions (0). However, Brachyury was expressed in the primitive streak of mutants (N,O). Scale bar, 100pm.
Chapter 4
0 t . 2 and HNF-38 Genetically Interact in Anterior Midline Patterning
This chapter will be submitted as the following manuscript:
Jin, O., Harpal, K., Ang, S-L. and Rossant, J. (1997) Otw-2 and HNF-3B
Genetically lnteract in Anterior Midline Patterning. (in preparation)
I performed all work described here, with the exception of preparing the wax
sections.
INTRODUCTION
Genetic and experimental evidence points to the importance of the
midline axial mesoderm and the ventral midline of the neural tube as critical
sources of signals far dorsal-ventral (D-V) patterning of the neural tube and
head structures. Sonic hedgehog (Shh), a vertebrate homologue of the
Drosophila hedgehog (hh) gene, is an important molecule involved in D-V
patterning. Shh encodes a secreted protein. In the mouse embryo, it is
expressed in the node, notochord, floor plate, ventral forebrain and midbrain
and other patterning centres such as the posterior margin of the limb bud
(Riddle et al., 1993; Echelard et al., 1993; Chang et al., 1994; Marti et al., 1995).
Ectopic expression of Shh in the central neural system (CNS) leads to a
ventralization of large regions in the midbrain and hindbrain of the mouse and
zebrafish (Echelard et al., 1993; Krauss et al., 1993). In early neural plate or
intermediate neuroectoderm explants, Shh protein also induces ventral cell
fates in a dose-dependent fashion (reviewed by Placzek, 1995; Tanabe and
Jessell, 1996).
The phenotype that results from lossof-function mutation of Shh in mice
further demonstrates its critical role in D-V patterning (Chiang et al., 1996).
Homozygous embryos show a single fused optic vesicle (cyclopia), proboscis
and other severe P V patterning defects in the neural tube. The phenotype of
cyclopia was also observed when midline signaling was antagonized by
overexpression of protein kinase A (PKA), a negative regulator of Shh
(Hammerschmidt et aI., 1996). In zebrafish, embryos homozygous for the
cyclops mutation also exhibit cyclopia or partial cyclopia, and abnormal
development of forebrain (Hatta et al., 1991, 1994; Macdonald et al., 1995). In
these mutant embryos, no Shh expression is detected in the midline structures
of forebrain and midbrain (Krauss et al., 1993; Barth and Wilson, 1995).
consistently supporting the role of Shh in D-V patterning of head structures.
HNF-30, a transcription factor of the winged-helix family, has been
proposed as a candidate upstream regulator of Shh. It has been suggested that
HNF3B may activate Shh expression in the midline and that they
subsequently regulate each other's expression by a positive feedback
mechanism (Echelard et a(., 1 993; Sasaki and Hogan, 1 994; Hynes et al., 1 995;
Filosa et al., 1997). HNF3B is expressed in the visceral endoderm, node,
notochord, floor plate, and gut in the mouse embryos (Sasaki and Hogan, 1993;
Monaghan et al., 1993; Ang et al., 1993; Weinstein et al., 1994). Ectopic
expression of HNF-30 in mice leads to induction of ventral structures in the
dorsal region of the brain (Sasaki and Hogan, 1994). Similarly, misexpression
of another member of the winged-helix family, pintallavis, in Xenopus embryos
also induces expression of markers of ventral structures in the dorsal hindbrain
region (Ruiz i Altaba and Jessell, 1992). These studies suggest that HNF3a
has roles in D-V patterning.
The phenotypic consequences of loss-of-function mutation of HNF3B in
mice further demonstrate its key role in D-V patterning (Ang and Rossant, 1994;
Weinstein et al., 1994). Homozygous embryos lack node and notochord
structures and show severe abnormalities in D-V patterning of the neural tube.
In addition, homozygous embryos also show some anterior deletions, although
anterior-posterior patterning (A-P) is relatively normal. Mice heterozygous for
this mutation are viable but some show an incompletely penetrant phenotype
affecting development of the lower jaw. This heterozygous phenotype suggests
that H N W is present in limiting amounts. In such a situation, exacerbation of
the HNF-3B heterozygous phenotype might be expected in mutant mice doubly
heterozygous for the HNF-3B mutation and for mutations in genetically
interacting loci. Otx-2, anoMrelated homeobox gene, is normally expressed in
epiblast of pre-and early streak. Its expression is then gradually restricted to the
anterior of embryo and, at somite stages, to the presumptive forebrain and
midbrain (Simeone et al, 1992a. 1993; Ang et al, 1994). Loss-of-function
mutation of Otx-2 leads to deletion of anterior CNS structures up to rhombomere
3 (Acampora et al., 1995; Matsuo et al.. 1 995; Ang et al., 1 W6), suggesting that
Otw-2 is in involved in head organizer function in patterning of the anterior
embryo, but no specific role of Otx-2 in D V patterning of the CNS has been
proposed.
Comparison of the expression patterns of Obr-2 and HNF30 between
E7.5 and E9.5 revealed that they are co-expressed in the anterior midline of
embryos as early as E7.5. To investigate the potential genetic interactions
between the 00-2 and HNF3B genes, we have generated double
heterozygous mutant Otr-2 and HNF-30 embryos. We found that at €12.5,
double heterozygous embryos show varying degrees of holoprosencephaly,
and cyclopia with proboscis-like structures, suggesting genetic interaction of the
two genes in the anterior midline. Further analysis of the double heterozygous
mutant phenotype demonstrates that Shh expression is severely affected in the
anterior of the embryos, suggesting that both 0tx-2 and H H are involved in
regulating anterior midline signaling during embryogenesis.
MATERIALS AND METHODS
Otx-2 heterozygous mice of 129ISvxCDI background (Ang et al., 1 996)
were crossed with HNF-3B heterozygous mice of 129ISVxCD1 background
(Ang and Rossant. 1994) to generate double heterozygous mice. Genotyping of
newborn mice and embryos was performed by Southern blot analysis with
genomic DNA prepared from biopsies of tails and yolk sacs. Each DNA sample
is divided into two parts. One aliquot was analyzed with an Ok-2 probe and the
other with an HNF* probe in two separate blots. Probes used for Southern
blot analysis were described previously (Ang and Rossant, 1994; Ang et al.,
1996). Hybridizations were camed out at 42'C overnight in 50% formamide,
5xDenhart9s, 5xSSC, 1 %SDS. 100pg/ml sheared salmon sperm DNA. The
filters were finally washed in 0.2xSSC at 63'C. The filters were then exposed to
phosphor imager screens overnight.
. . . . . stoloav. wholemount RNA In sriu hvhr~dmt~on and
Mid-day of the day of the vaginal plug was considered as E0.5 in the
timing of embryo collection. Embryos were dissected and staged according to
morphological criteria (Kaufman, 1992). Embryos were photographed on a
Leitz Wild MI0 microscope. For histological analysis, embryos were fixed
overnight in 4% paraformaldehyde at 4'C, processed, embedded in wax and
sectioned. 5-6 pm sections were dewaxed in xylene, rehydrated through an
ethanol series into PBS and stained with hernatoxylin and eosin.
Whole-mount RNA in situ hybridization was performed as described
previously (Conlon and Hermann, 1 993). Single-strand RNA probes were
labeled with digoxigenin as directed by the manufacturer (Boehringer
Mannheim Biochemicals). The probes used for the whole-mount in situ
hybridization studies were as follows: Shh (Echelard et al., 1993); Of%-2 (Ang et
al.. 1994); BF-l (Tao and Lai. 1992); Hoxb-7 (Wilkinson et al.. 1989a); Six-3
(Oliver et al., 1 995); Mox-7 (Candia et al.. 1 992). After RNA in situ hybridization,
embryos were pastfiixed in 4% paraformaldehyde at 4OC overnight and then
followed by whole-mount antibody staining. Whole-mount antibody staining was
performed as described (Davis et al., 1991) using an anti-HNF3R antibody at a
dilution of 1 :I000 (Sasaki et al., 1993). For sectioning of whole-mount stained
specimens, embryos were postfixed in 4% paraformaldehyde at 4OC overnight.
Sections were cut at 5-6pm and some sections were counterstained lightly with
eosin, and photographed using a Leitz Orthoplan compound microscope and
Nomarski optics.
RESULTS
s of Otx - 3 and HNF-3B double heterozyp~us mutant
To investigate the possible interaction between 00~-2 and HNF-313, Otw-2
heterozygous mice of 129ISvxCDl background were crossed with HNF-30
heterozygous mice of 129/SvxCD1 background and offspring were genotyped 3
weeks after birth. Southern blot analysis revealed that the number of double
heterozygous weanlings was significantly reduced below the expected n urn ber
(Table 4.1). suggesting embryonic or post-natal lethality of double heterozygous
mutants. I observed that several double heterozygous pups died the day of
birth and showed mandible defects. Among the survivors, two died after eight
months and two died after eleven months. All these had incisor overgrowth and
jaw defects. The remaining four appeared normal and healthy, although one of
them had overgrowth of the incisors.
To characterize the embryonic lethality, I dissected litters at €18.5 and
found that some embryos had already begun to be resorbed (data not shown).
Taken together, my observations suggests that the doubly heterozygous
condition causes a variable penetrant lethal phenotype. These double
heterozygous mutants may be lost at late gestation or after birth.
I then dissected and genotyped litters at E12.5 and €9.5. Genotyping
results from E12.5 and €9.5 yolk sacs produced genotypes at roughly the
expected Mendelian frequencies (Table 4.2. Table 4.3). At El 2.5, about 56% of
double heterozygous mutants displayed an obvious phenotype which is
characterized by cyclopic or partial cyclopic eye, reduced distance between the
eyes, and proboscis and other defects (Table 4.2, Fig. 4.1 and data not shown).
At €9.5, this phenotype was already apparent and varied in its severity (Fig.
4.2). Some double heterozygotes showed a clear phenotype, demonstrating a
dramatic reduction in the size of the forebrain. In the most extreme case, the
forebrain was lost (Fig. 4.28). However, in most of these mutants, the forebrain
is always present but shows a reduction in size (Fig. 4.4C, D). In those embryos
with an obviously reduced forebrain, the floor and roof of the neural tube were
almost in contact with each other at the diencephalic and mesencephalic
junction (Fig. 4.2C). Although the overall size of most Otw-Z+/-;HNF33R+/- mutant
embryos was smaller than wild-type or heterozygous embryos, the posterior
part of each embryo appeared normal.
vsis of the Otx2+/:HNF3R+/ - - - ohenohrne
To characterize theOtx-Z+';HNF3B+/- phenotype in more detail, El 2.5
and E9.5 mutant embryos were sectioned for histological analysis (Fig. 4.3, 4.6
and data not shown). In wild-type or singly heterozygous embryos there are two
telencephalic vesicles (future lateral ventn'cles Fig. 4.3A.B). However, in Otx-
24-;HNF30+/- mutants these two vesicles were fused into a single
telencephalic vesicle (Fig. 4.3D, E). The size of the telencephalon and
diencephalon was reduced in Ofx-Z+/-;HNF3B+/- mutants (Fig. 4.3D,E). The
more striking phenotype was a fused single eye (cyclopia) and a proboscis-like
structure (Fig. 4.3F,H). In vertebrate, optic vesicles are formed from evagination
of the lateral walls of forebrain (Carlson, 1996). The optic vesicle invaginates
and forms a double-layered optic cup. The outer layer gives rise to the pigment
layer of the retina and the inner layer gives rise to the neural layer of the retina
which is much thickened than the outer layer. The two cups are connected to
the diencephalon by the optic stalks. However, in those Otx-2+/-;HNF-3B+/-
mutants which showed the cyclopia phenotype, the optic vesicles were fused at
midline and the lateral optic stalks were absent (data not shown). Although
some of Otx-2+/-;HNF38+/- mutants had smaller eyes (data not shown), most
of them seem morphologically normal. The lens, neural layer of the retina and
corneal ectoderrn were apparently normal (Fig. 4.3F. H). In addition, the
mandible of Otx-Z+/-;HNF-3B+/- embryos was smaller than wild-type or
heterozygotes (Fig. 4.3H). However, the morphology of the floor plate,
notochord and the other body structures were normal (Fig. 4.6M.N). These
results suggest that defects in Ob-Z+/-;HNF-38+/-= mutants were confined to
anterior structures.
on of T)fx 2 and H N F 3 B exDrssron In W.5-E9.5 mouse embrvos . . -
To investigate further how Otx-2 and HNF-3B could be interacting in the
anterior regions of the embryo, I compared the expression of Obi-2 RNA and
HNF3O protein in E7.5-€9.5 mouse embryos by double-labelling to examine
regions of overlap. In these studies, embryos were first stained for Otx-2 RNA
by whole-mount in situ hybridization and then for HNF38 protein by whole-
mount antibody staining. Further analysis was performed on sections. At E7.5,
I found that Otx-2 and HNF3B coexpressing cells overlapped in the foregut
pocket and anterior midline (Fig. 4.40). Sections through embryos double-
labelled for Otx-2 and HNF3B revealed coexpression of these two genes in
prechordal mesoderm (Fig. 4.4C), anterior ventral neural fold and ventral
endomesoderm (Fig. 4.4E).
At €8.5, I found that sections through forebrain, midbrain and hindbrain
regions of embryos showed Otx-2 and HNF-3B co-expressing cells in the
ventral forebrain and midbrain region (Fig. 4.4G,H). However, by €9.5, the
expression of these two genes only overlapped in the ventral midbrain regions
(Fig. 4-44. My double-labelling studies clearly suggests that Ofx-2 and HNF-
are co-expressed in the anterior midline of mouse embryo.
* . . ~ t ~ e s rn Ofx2r-/:HNFI3R+/ - - - - mutant emhrvo~
To characterize the anterior abnormality of 0&-2+/-;HNF3B+/- mutant
embryos further, the expression of several genes normally transcribed along the
A-P axis of the embryos was examined at €9.5 (Fig. 4.5). Otr-2, Hoxb- I and
Krm-20 were all expressed at the correct level of A-P axis, suggesting that A-P
patterning in the neural tube of OW-2+/-;HNF-33+/- mutants was not affected.
I also analyzed BF-I and S i x 3 expressions in Otx-2+/-;HNF3B+/-
embryos. BF-I, a winged-helix transcription factor, is normally expressed in the
telencephalon of brain. In homozygous mutants of BF-I, the dorsal
telencephalon is reduced in size while the ventral telencephalon is almost
completely absent (Xuan et al.. 1994), suggesting a role for BF-7 in the
development of telencephalon, especially ventral telencephalon. The
expression domain of BF-1 was missing in Otx-2+/-;HNFF-30+/- mutant embryos
(Fig. 4.58 and data not shown). I then checked the expression of Six-3 which is
normally expressed in the ventral forebrain and optic vesicles at this stage. It
has been demonstrated that Six3 can induce ectopic lens in fish embryos
(reviewed in Oliver and Gruss, 1997). suggesting that it plays a key role inlens
induction. In OtK-2+/-;HNF38+/- embryos, the expression domain of Six-3 in
ventral forebrain was missing but expression was still detectable in the reduced
single optical vesicle (Fig.4.5E.F). Those observations suggest that the ventral
forebrain is severely affected.
h h expression in anterior of Otx - 2+/ - : H N F + a n t - em bwos is severelv
ilmax!
Loss of ventral forebrain expression of Six4 and expression of BF-1,
fused telencephalon, cyclopia and mandible defects in Otx-2+/-;HNF-3&/-
mutants suggest that the chief defect is loss of ventral midline signaling in these
mutants, reminiscent of the phenotype of Shh homozygous mutants in the
anterior region (Chiang et al., 1996).Therefore, I analyzed Otx-24-;HNF-3B+/-
mutant embryos at E9.5 for the expression of Shh. At E9.5, Shh is normally
expressed in the ventral forebrain, midbrain, notochord, floor plate and gut. I
found expression of Shh was almost completely lost at the anterior end (Fig. 4.6
and data not shown). Sections through these mutants showed that the
expression of Shh was lost in ventral forebrain and floor plate, although the
floor plate was present morphologically (Fig.. 4.5). The loss of Shh expression
in the floor plate could be explained by lower level of Shh expression in the
underlying notochord. Very weak expression of Shh could still be detected at
ventral mid brain level (Fig. 4.5E, arrows) and anterior notochord (Fig.4.5M.
black arrowhead). The expression of Shh in the rest of the notochord, foregut
and hindgut is still detectable although the level of the expression is reduced,
perhaps due to reduced dosage of HNF-3B (Fig.4.5 M, N and data not shown).
DISCUSSION
Otw-2+/-; HNF-30+/- doubly heterozygous mutant mice exhibited a
variably penetrant lethal phenotype. The major phenotype is characterized by
varying degrees of holoprosencephaly, cyclopia and proboscis with normal
posterior structure. Shh expression in the anterior of the embryo was severely
affected. This new phenotype was observed in &-2+l-;HNF-3B+/- embryos but
not in Otr-2+/- or HNF-3B+/- single mutant embryos.
In previous single-mutant studies, haploinsufficiency of H N F a or Otx-2
has been demonstrated in HNF3B +/- or Otw-2 +/- animals (Ang and Rossant,
1994; Weinstein, et al. 1994; Acampora et al., 1995; Matsuo et al., 1995; Ang et
al., 1996). About 20% of HNF4B heterozygous adults exhibit malocclusion of
jaws and overgrowth of the incisors. Some of them died later although special
care such as providing soft food and cutting the overgrowth of incisors
periodically can significantly reduce mortality of these mice. 5% of Otx-2+/-
heterozygotes exhibit open neural tube defects at €9.5 (Fig. 4.7A) and
subsequently at €12.5 (Fig. 4-78). showing exposure of neural tissue with
characteristic exencep haly . This phenotype contributed to about 5% loss of
heterozygotes. Variation in the threshold levels of factors or modifiers which
may act synergistically with either HNF3B or Otx-2 may contribute to the
variable penetrance of these haploinsufficiencies.
In the double heterozygotes, the phenotype discovered may be
considered as a regionspecific exacerbation of the HNF-3B mutant phenotype.
H N F a is essential for the formation of midline structures, such as notochord
and floor plate. Shh is a prime signalling molecule in the midline for 0-V
patterning and in HNF-3B homozygous mutants, Shh expression is absent in
the midline of the HNF4B homozygotes. However, HNF3B homozygous
embryos died much earlier than Shh homozygous mutants. The early lethality
probably results from circulation defects, since HNF-36 has a role in endoderm
development. HNF-38 homozygous mutants demonstrate severe D-V patterning
defects along the entire body axis including the head. Recent study of
overexpression of HNF-38 in the entire midline exhibited anterior defects with
normal posterior (Dufort and Rossant, unpublished data). It suggests that the
anterior region is more sensitive for the dosage of HN-8 than that of the
posterior. The phenotype observed in HNF3B heterozygous mice is probably
due to mild D-V defects in mandible development. When HNF3B heterozygous
mice are crossed with Otx-2 heterozygous mice, double heterozygotes show
enhanced anterior midline patterning defects.
It is as yet unclear whether OtK-2 and HNF-3B directly interact to regulate
midline signalling. However, they do overlap in expression in the anterior
midline, suggesting a possibility of directly interacting. At E7.5, Otx-2 and HNF-
38 are coexpressed in the cells of the foregut pocket and anterior midline but
not in the posterior of the embryo. At €8.5, the two genes continue to be co-
expressed in the cells of ventral forebrain and midbrain. At E9.5, the expression
domains of the two genes no longer overlap in the ventral diencephalon but still
overlap in ventral midbrain. These studies support the potential interactions
between the two genes in these tissues.
The phenotype of Otx-2 and HNF38 double heterozygotes again
supports the critical role for Shh signalling in the forebrain where patterning is
the most complex and the exact source of signals is not well understood. The
hypothesis that ventral forebrain serves as an important source of signals for D-
V patterning in the anterior GNS is suggested by the phenotypes of zebrafish
embryos homozygous for the cyclops mutation (Hatta et al., 1991, 1994) and
mouse embryos homozygous for the Shh lossof-function mutation (Chiang et
al., 1996).
The molecular identity of cyclops is unknown. However, mutant embryos
show the fusion of two lateral eyes into a single median vsntral cyclopic one,
reduction in size of the diencephalon and loss of the floor plate in posterior
CNS. Cell transplantation experiments suggest that the cyclops gene product is
involved in regulating a signalling pathway in the forebrain midline cells (Hatta
et al., 1994). Consistent with this, there is no Shh expression in the ventral
forebrain or the posterior of ventral midline (Krauss et al., 1993; Barth and
Wilson, 1995).
In mouse, the most striking phenotype of Shh homozygous mutant
embryo is a single fused telencephalic vesicle with a single fused optic vesicle
and proboscis-like structure (Chiang et al., 1996). The phenotype demonstrated
from Otx-Z+/-;HNF4&/- mutants is not only reminiscent of Shh homozygous
mutant mouse embryos but also reminiscent of a variable spectrum in facial
findings in patients with Shh mutations (Erich et al., 1996). The most severe
group of these patients exhibits cyclopia, synophthalmia, proboscis and
microcephaly with normal development of the rest of the body, suggesting the
role of Shh in the anterior midline patterning in humans. In Otx-2+/-;HNF-33a+/-
mutants, the expression of Shh is lost or almost lost in ventral forebrain and
midbrain, suggesting that both Ofx-2 and HNF-3B are involved in regulating this
pathway in the anterior midline (Fig. 4.8).
These studies have revealed an expected role for Otx-2 in regulating
anterior D-V patterning, which is independent of its role in A-P patterining.
Since Otx-2 and HNF-3R are coexpressed in the Shh expressing tissues, the
ventral forebrain in early embryogenesis, it is possible that the two genes
interact directly by binding to the Shh promoter. Recently. three enhancer
elements have been identified in the regulatory region of Shh by transgenic
approaches (Epstein and Joyner, 1997). One enhancer element drives the
expression of a reporter gene in the caudal diencephalon and ventral midbrain.
The other two enhancer elements drive the expression of a reporter gene in the
floor plate from the mid-hindbrain boundary to a posterior level at the hindlim bs.
Weak expression of the reporter gene was also detected in the notochord.
Further analysis demonstrated that several HNF3 binding sites and a
consensus binding site for a bicoid type homeodomain protein have been found
in these enhancer elements. Altering or deleting these sites resulted in either
restriction or the abolishment of reporter gene activity (Epstein and Joyner,
1997). Their studies suggest that HNF3B family members may be involved in
regulating Shh expression in cooperation with other regulatory proteins in a
regionalized fashion. Whether Otx-2 binds to the ventral forebrain enhancer
remains to be determined. The genetic interaction we report here is also
consistent with other possible mechanisms. For example, it is possible that two
different pathways controlled by Otx-2 and HNF-3B may be separately involved
in regulating Shh signalling pathway.
In conclusion, the phenotype of Otx-2+/-;HNF3B+/- mutants
demonstrates genetic interaction of Otx-2 and HNF-38 in the anterior midline of
the embryo. Coexpression of Obr-2 and HNF4B in anterior midline of the
embryo supports a relatively direct interaction of the two genes in the
development of the anterior midline structures in the embryo. The loss of Sh h
expression in anterior of Otx-2+/-;HNF3B+/- embryos suggests that both Otw-2
and HNF-38 are involved in regulating the anterior Shh signalling pathway.
Fig.4.1 Phenotype of Obr-Z+/-;HNF38+/- embryos at €1 2.5. (A) Lateral view of
normal El 2.5 embryo. (B, C, D) lateral views of Otx-2+/-;HNFF-3B+/- mutants,
demonstrating closely spaced or fused eyes with proboscis.
Fig. 4.2 Phenotype of Otx-24-;HNF-38+/- embryos at E9.5. Lateral views of
wildtype embryo (A) and Otx-24-;HNFdB+/- embryos (6, C. D). (8) Otx-2+/-
;HNF-3hk embryo shows forebrain deletion. (C, D) Otw-2+/-;HNF38+/-
embryos display forebrain defects. It is noted that the floor and roof of the neural
tube of the mutants are in contact each other at the caudal diencephalic and
mesencephalic junction (arrowhead). Abbreviations: fb: forebrain; te:
telencephalon; di: diencephalon; mb: midbrain; hb: hindbrain; ov: otic vesicle.
Fig. 4.3 Histological analysis of Otx-2+/-;HNFaB+/- embryos at Ef2.5. Cross-
sections of wild-type (A, B, C) and Ofx-2+/-;HNF38+/- mutants (D, E, F). The
wild-type embryo has two telencephalic vesicles (Mure lateral ventricles) (A, B)
while in double mutant these two lateral ventricles are fused to form a single
ventricle(D, E). (C, F) show cross-sections at level of eyes. The O~X-~+~;HNF-
3&/- mutant has a single eye (cyclopia) (F). Note the two ears are close to the
single eye in the double mutant (hypotelorism). (G, H) Middle sagittal sections
of embryos. (G) wildtype (H) double heterozygotes. Note a single eye in anterior
midline and the smaller mandible in the double mutant (H). Abbreviations: TeV:
telenceph alic vesicle; cyc: cyclopia; 3rdV: the third vertical; 4thV: the fourth
ventricle; md: mandible.
Fig. 4.4. Comparison of Otx-2 and HNF38 in wildtype mouse embryos between
E7.5 and E9.5. (A,D) Anteroventral views of E7.5 embryo, stained for Otx-2 RNA
(purple)(A) and then stained forHNF3B protein (brown) (D). (D) Co-expressing
OtK-2 and HNF4B cells were found in the foregut pocket (fg) and anterior
midline (ml) (B,C,E) are cross sections through different levels of the anterior
portion of an embryo: top of neural fold tissue (B), under neural fold (C) and
foregut pocket region (E), demonstrating the presence of coexpressing cells
Otw-2 and HNF3B in prechordal mesoderm (pm) (C, arrowed), anterior
endomesoderm (em) (E, white arrows), and anterior ventral neural fold (vn) (E,
black arrow). (F.1) Lateral views of E8.5 and €9.5 embryos. stained for the
expression of Otw-2 and HNF-38. (G,H) Cross sections show co-expressing
cells of Otr-2 (purple) and HNF-3B (brown) in the ventral midbrain (vrnb) and
diencephalon (vdi) at E8.5. (J) Cross section shows co-expressing cells of Otx-
2(purple) and HNF-3B(brown) in ventral midbrain (vmb) at E9.5. (K) Cross
section shows expression of Obr-2 in the ventral diencephalon (vdi) and HNF-
38 in the floor plate (white arrow) at E9.5. At this stage, the two genes are no
longer coexpressed in the ventral diencenphalon. Abbreviations: em:
endomesoderm; fg: foregut pocket; fi: floor plate; ml: midline; op: optical vesicle;
pm: prechordal mesoderm; vdi: ventral diencephalon; vmb: ventral midbrain; vn:
ventral neural fold.
Fig. 4.5 Expression of Obr-2, Hox& 1, Six-3 and Mox-7 in Otx-2+/-;HNFF3Bi/-
embryos. (A) Lateral view of wild-type embryo, showing Otw-2 expression is
forebrain and midbrain. Hoxb-l expression in rhomomere 4 (arrowed) and
posterior spinal cord. (B, C) Otu-2+/-;HNF-33Bt/- embryos show reduced Otw-2
expression domain. (D) Lateral view of wild-type embryo, showing Six-3
expression in the optic vesicle (white arrow) and ventral forebrain (black arrow),
and Mox-I expression in second, third, and fourth branchial arches
(arrowheads), and somites. (E, F) Otx-2+/-;HNF-3+/- embryos, showing loss of
Six-3 expression domain in ventral forebrain but resistent weak expression in
the reduced single optic vesicle(arrows) .
Fig. 4.6. Whole-mount in situ hybridization and cross section analysis of Otx-
2+/-;HNF3R+/- mutant with BFI, Krox-20 and Shh. (A, D) lateral view of E9.5
wildtype (A) OtK-2+/-;HNF-3&/- (D) embryos, respectively, stained for BF-7,
Krox-20 and Shh expression. O ~ X - ~ + ~ ; H N F ~ $ + / - embryo demonstrates lack of
BF1 expression in telencephalon. There is much reduced expression of Shh in
the posterior region of the embryo, and expression is absent from the anterior
ventral midline. (B, C) Cross sections of wildtype embryo show Shh expression
in the ventral neural tube, notochord and foregut. Note Krox-20 expression in
the rhombomere 5. (E, F, G) cross sections of 01%-24-;HNF-33B+/- embryo,
showing loss of Shh expression in anterior ventral neural tube. Note very weak
expression of Shh in ventral midbrain (E, white arrows), anterior notochord (G,
black arrowhead) and expression in the tip of Rathkes's pouch (G, white arrow).
Krox-20 is still normally expressed in the rhombomere 5. (H, I, J, K) cross
sections of wildtype embryo through foregut and hindgut level show Shh
expression in the floor plate, notochord, foregut and hindgut. (L, M, N) cross
sections of Otx-2+/-;HNF-30+/- embryo show reduced Sh h expression in the
notochord, foregut and hindgut. However, Shh expression in the floor plate
seems lost but the morphology of the floor plate appears normal. Abbreviations:
fg: foregut; fl: floor plate; hg: hindgut; no: notochord; ot: otic vesicle; r5:
rhombomere 5; vdi: ventral diencep halon; wt: wildtype; 0o:Hh: Otx-2+/-;HNF-
3R+/-.
Fig.4.7 Phenotype of O H +/- embryos. (A) lateral view of E9.5 embryo.
showing open anterior neural tube. (8) Lateral view of €12.5 embryo, showing
characteristic exencephaly phenotype.
Fig. 4.8. Schematic diagram showing that both Otw-2 and HNF-30 are involved
in regulating Shh expression in the anterior region. For details, see text.
Chapter 5
DISCUSSION
The primary aim of the research described in this thesis was to
investigate the molecular mechanisms involved in patterning the anterior region
of the mammalian embryo. Otx-2, a gene related to the Drosophila otd
homeobox gene, has been cloned (Chapter 2). Obr-2 is expressed first in the
epi blast, then gradually restricted to the anterior region of the embryo, incl udi ng
all three anterior germ layers, and later to the presumptive forebrain and
midbrain. The phenotypic analysis of OW-2 homozygous mutants demonstrated
a critical role for Otx-2 in head formation (Chapter 3). At late-streak and
headfold stages, &-2 is expressed in the anterior midline of the embryo. Its
expression overlaps with that of HNF-38 , another organizer gene involved
mainly in D-V patterning of the embryo. The phenotype of Otx-2 and HNF-3B
double heterozygous mutants reveals a role for 01%-2 in midline patterning in
conjunction with HNF-38 (chapter 4). My studies here thus revealed some of
the multiple roles of the Otx-2 gene during mouse embryogenesis. In the
following sections, I will discuss how my findings on Otx-2 function relate to the
current progress in understanding anterior patterning and suggest future
experiments which may further our understanding of the molecular basis and
cellular interactions for the roles of OW2 in different lineage development.
. . Ivat~on and pattern na of the rostra1 b r m
The loss-of-function mutation of Otx-2 leads to early embryonic lethality
and total loss of the rostral brain. These early defects hinder the analysis of the
role of &-2 in the regionalitation and patterning of the rostral brain. Recently,
Otx-1 (Acampora et al., 1 %6), Emx-7 and Emx-2 have been knocked-out
(Pellegrini et al., 1996; Yoshida et al., 1997). About 30% of OW-1 homozygotes
died in the first postnatal month and all homozygotes exhibited epileptic
behavior with the characteristics of focal and generalized seizures. Further
analysis revealed multiple subtle abnormalities affecting the dorsal
telencephalic cortex, mesencenphalon and cerebellum (Acampora et al., 1996).
For Emx-1 hornozygous mice, half of them died in the neonatal stage. The Emx-
I mutation caused no apparent defects at the embryonic stage but most of the
mutant embryos displayed subtle defects as adults. These defects are restricted
to some regions of dorsal forebrain (Yoshida et al., 1997). Emx-2 homozygous
mice died within a few hours after birth, presumably due to the absence of the
kidneys and other parts of the urogenital system (Pellegrini et al., 1996; Yoshida
et al., 1997). Emx-2 is expressed in the primordia of the urogenital system
during embryogenesis (Simeone et al., 1992b). The mutant phenotype and
expression pattern of Emx-2 strongly suggest that Emx-2 is required for the
development of urogenital system. Analysis of Emx-2 homozygous brains
revealed that the dorsal structures of cerebral hemisphere are affected,
especially the medial limbic cortex and hippocampal region. Dentate gyrus is
always absent (Pellegrini et al.. 1 996; Yoshida et al., 1997). The dentate gyrus
is involved in the formation of hippocampus and is a part of the limbic system.
Further analysis of the role of Emx-2 in this system might be achieved by
rescuing development of the urogenital system. Since the two Otx and two Emx
genes are expressed in nested A-P domains in the sequence Emx-IcEmx-
2=0tK-1<0tw-2, one way to analyze how these genes cooperate in the
regionalization and patterning of the rostra1 brain is to generate double
heterozygous mutants.
In fact, the analysis of phenotype in the Otx-I+/-;0tx-2+/- (Yoshida et al.,
1997) or Otx-I-l-;0&-24- (Pellegrini et al.. 1996) mutants have revealed that the
Otx genes play a role in the regionalization and patterning of the rostral brain.
These mutants exhibit variable defects in the forebrain and midbrain, which are
more severe in the dorsal parts than ventral parts of the brain. These mutants
lack mesencephalon, pretectal area, dorsal thymus and exhibit a large
reduction of Ammon's horn. The metencephalon is expanded and the isthrnic-
like structure is rostrally shifted in the presumptive caudal diencephalon. It has
been suggested that the isthmus at the mesencephalic-metencephalic (mes-
met) boundary might have organizer function since it can induce the
surrounding tissues to acquire a mes-met fate (Marin and Puelles, et al., 1994;
Joyner, 1996). The shift of isthmic-like structure rostrally in these compound
mutants suggests that the Obr gene dosage is also required for establishing the
correct position of the isthmic organizer.
Recent evidence from Drosophila suggests that different threshold levels
of Otd protein are required for the formation of specific subdomains of the head
(Royet and Finkelstein, 1995). Similarly in mammals, appropriate threshold
levels of Otx proteins might also be required for the regionalization and
patterning of the rostral brain. These studies reveal that appropriate threshold
levels of Otx proteins are required in the regionalization and patterning of the
rostral brain. It would be of interest to search for potential interactions of Otu-2
with the Emx genes in regional specification. Besides Otx-I, Ok-2, Emx-7 and
Emx-2, a number of genes are now known to be expressed in the different
regions of the rostral brain, such as BF-1, the Wnt family of genes, the parfamily
of genes and the Dlxgenes (Bulfone et al., 1993, 1995; Puelles and Rubenstein
1 993; Figdor and Stein 1993; Rubenstein et al 1994). Since 00r-2 is expressed
in almost the entire forebrain and midbrain, it would be of interest to investigate
the interactions of Ofx-2 with these genes in regionalization of the brain.
s of the late role of 0&? - brain bv condfi~onal gene tarae . .
ting
Since Otx-2% embryos lack the entire rostral brain and die at an early
stage of embryogenesis, one way to further address the role of Ofx-2 in the
patterning of the different regions of the brain is to take a tissue-specific gene
targeting or conditional targeted rnutagenesis approach. This approach may
allow to specifically address the later role of &-2 in the regionalization and
patterning of the rostral brain. The &-2 locus will be targeted such that the
genomic region containing the ATG start codon and most of the homeodomain
region (exon3 and 4) will be flanked by loxP sites. Another line will be
generated that expresses Cre recombinase is under the control of brain (tissue)
specific promoter, such as the nestin promoter (Lendahl et al., 1990;
Zimmerman et al., 1994). When these two lines of mice are crossed, animals
that lack 0tx-2 function specifically in the brain tissue will be generated. This
Cre-loxP system has been successfully used recently in the conditional
targeting of the DNA polymerase 8 gene in T cells (Gu et al., 1994) and the Apc
gene in colorectal epithelium (Shibata et al., 1997). This system may allow us
to perform a fine, detailed analysis of Obr-2 function in the regionalization and
patterning of the forebrain and midbrain.
Analvsis of the role of Otr-2 in the head omanizer formation
As I discussed in Chapter 1, Spemann and Mangold (1924) first
demonstrated the induction of a secondary axis including head by grafting a
dorsal blastopore lip to the ventral side of a host amphibian embryo. Spemann
(1 931 ) also found that the early dorsal lip could induce a complete axis
including a head but at a later stage, the dorsal lip could only induce trunk and
tail structures, suggesting that the early dorsal lip has a head and trunk
organizer activity but as development proceeds, it only has trunk organizer
function. In mouse, Beddington (1994) has provided direct evidence for the
organizing function of the node by performing heterotropic transplantation
experiments. When the midgastrulation of node was grafted to a posterolateral
location in a host embryo at the same developmental stage, a second neural
axis was induced. However, the second axis always lacked forebrain and
midbrain, suggesting that the mouse node has trunk organizer activity but lacks
head organizer function.
Recent evidence suggests the important role of visceral endoderm in the
most anterior patterning. Cerberus, a putative secreted protein, was isolated by
differential screening in Xenopus (Bouwmeester et al., 1996). It is expressed in
the anterior endoderm. Ectopic heads could be induced when cerberus mRNA
was injected into Xenopus embryos. The inductive interactions between the
visceral endoderm and underlying ectoderm have been further demonstrated
by physically removing the anterior endoderm at earlier stages (Thomas and
Beddington, 1 996). A homeobox gene, HesxVRpx (Hermesz et al., 1 996), has
been used as a marker in this study. Hesxl is first expressed in a small domain
of anterior endoderrn at the start of gastrulation but about 24 hours later, it
begins to be expressed in the underlying ectoderm. The ectoderm expression of
Hesxl is almost lost by physical removal of endoderm cells which express
Hesxl at earlier stages, suggesting the ectodermal expression of Hesxl is
mainly dependent on signals from the endoderm.
Furthermore, evidence from chimeric analysis of nodal also supports this
hypothesis (Varlet et al., 1997). Nodal is a member of the TGFB family of
secreted protein and is required for gastrulation (Zhou et al.. 1993; Conlon et
al., 1 994). Nodal is expressed throughout the embryonic ectoderm before streak
formation and then localized within the most proximal embryonic ectoderrn.
However, nodal is also transiently expressed in the visceral endoderm at the
onset of gastrulation. To distinguish nodal signaling in the primitive ectoderm
and endoderm of the embryo. chimeric embryos in which the visceral endoderrn
is exclusively composed of nodakieficient cells have been generated by
injecting wild-type ES cells into nodakdeficient blastocysts. These chimeric
embryos demonstrate the lack of the most anterior structures, suggesting nodal
signaling in the endoderm is required for anterior patterning during mouse
gastrulation (Varlet et al., 1 997).
Taken together, a model for the presence of two organizers in the
Xenopus and mouse embryos has been proposed recently (Fig.5.2)
(Bouwmeester and Leyns, 1997). In Xenopus, the head and trunk organizers
are overlapping or directly adjacent at early stages. However, during
gastrulation, they become physically separated. In the mouse, the topography is
different. The anterior visceral endoderm , which is topological equivalent to the
anterior-dorsal endoderm in the Xenopus, is not adjacent to the node (Fig.5.1).
The spatial separation of the node and anterior visceral endoderm might
explain the absence of head structures in secondary axes induced by
heterotopical transplantation of mouse node.
Fig. 5.1. Topological equivalence between the Xenopus fate map and mouse
fate map at the early gastrula stage. The large arrows project the extremities of
the embryonic axis, A-P and D V , from the Xenopus onto the mouse fate map.
The smaller arrows indicate the movements of the mesoderm through the
blastopore lip (Xenopus) and the primitive streak (mouse). The different colors
represent the prospective embryonic regions as indicated. (Adapted from
Bouwrneester and Leyns, 1997)
Otx-2 is expressed in :he visceral endoderm as early as E5.5, raising the
issue of whether OW2 is required in the visceral endoderm. To address the role
of Otx-2 in endoderrn development, it is of interest to generate chimeric embryos
which have wild-type endoderm and homozygous Otx-2 embryonic ectoderm. If
ES cells are aggregated with eight cell stage embryos or injected into blastocyst
stage embryos, they can contribute to any tissue in the embryo. However, ES
cells contribute poorly to the extraem bryonic lineages including primitive
endoden (Beddington and Robertson, 1989). In contrast, tetraploid embryonic
cells contribute mainly to the extraembryonic tissues (Tarkowski, 1 977). In this
analysis, tetraploid wild-type embryos will be aggregated with O M - ES cell
lines to generate embryos in which the embryonic compartment is derived from
Otx-24- ES cells and the extraembryonic lineages including primitive endoderm
are derived from wildtype tissues. The isolation of Otx-2-1- ES cell lines would
be achieved by high G418 selection (Mortensen et al., 1992), and the method
for tetraploid aggregation was as previously described (Nagy et al., 1993). If the
Ok-2 homozygous phenotype is rescued or partially rescued, it would strongly
support the role of Otx-2 in the endoderm development. The reverse tetraploid
aggregation experiment (wild-type ES cells c-> Otx-24- tetraploid embryos)
would further address whether OW-2 in the endoderm is required for the anterior
patterning. In this case, we would expect that the chimeric embryos may
reproduce the Otw-2 homozygous head phenotype.
s of the role of Otx-2 n the mesoderm and ectoderm
As previously demonstrated (Chapter 2), the progressive restriction of
Otx-2 expression correlates with the anterior migration of mesoderm in the
embryo, suggesting that interactions with mesoderm might be involved in
setting up the anterior domain of Obc-2 expression in the overlying ectoderrn.
Our previous study demonstrated that a positive signal from anterior
mesendoderm is required to stabilize expression of Otx-2 in ectoderm explants
and a negative signal from the later-forming posterior mesendoderm represses
Otx-2 expression (Ang et al., I 994).
Since the anterior mesendoderm cells are involved in the induction of the
rostra1 brain (Ang et al., 1994) and Otx-2 is also expressed in the anterior
mesendoderrn, diploid chimera analysis (generation of mosaic embryos which
contain contributions from OW-2-1- ES cells and wild-type cells) will provide a
powerful tool for in vivo analysis of the role of OW2 in the anterior
mesendoderrn. For chimera analysis, it is important to have an independent cell
lineage marker to follow the distribution of mutant or wild-type cells in the
mosaic embryos. The mouse line carrying ROSA26 transgene, which
constitutively expresses lacZ in all tissues (Friedrich and Soriano, 1991), will be
crossed with Otx-2+/- mice on a l29Sv background. The Oh-2-/-;ROSA26 ES
cells will be isolated directly from cultured blastocysts which come from the
interbreeding of the offspring of ROSA26 and OW-2+/- mouse lines. Potential
chimeras will be recovered at E7.5E9.5 and processed for 8-galactosidase
staining to analysis the distribution of mutant ES cells and the phenotypic
consequences of Otr-2 mosaicism in the embryo. For example, the consistent
exclusion of Otx-2 mutant cells from anterior mesoderm will suggest that Otx-2
plays a cell autonomous role in this tissue. On the other hand, if chimeras with a
high percentage of Oa-U- cells in the anterior mesoderm reproduce the Otr-2
homozygous phenotype, whereas chimeras with a high percentage of wild-type
cells partially rescue this phenotype, it will suggest that Otx-2 plays a role in the
anterior mesoderm.
Alternatively, using the in v h tissue recombination assay (Ang and
Rossant, 1993, Ang et al., 1994) to analyze Otx-2 mutants may also allow us to
dissect the roles of O W in the ectoderm and mesoderm tissues at the late-
streak stage. In such an assay, mesoderm tissue from homozygous mutant Ob-
2 embryo will be recombined with ectoderm tissue from wild-type embryos to
determine if the mutant mesoderm tissue can induce expression of anterior
neural markers such as En genes. The reverse experiment of recombining
ectoderm tissue from Otx-2 homozygous embryos and mesoderm from wild-type
embryos will be performed to determine if the Oh-2 negative ectoderm tissue
can respond to inducing signals from the mesoderm.
Cloning the _aenesg,plated bv Otx-2fHNF-3B
As discussed in Chapter 4, Ofx-2 and HNF-30 synergistically interact in
anterior midline patterning. RNA in situ hybridization experiments suggest that
Shh, a secreted molecule, and Six3, a nuclear transcriptional factor, may be
regulated by Otx-2/HNF3B. It is of interest to delineate the regulatory elements
which may interact with Otx-2 and HNF3B in these genes. To better understand
the molecular basis of anterior midline patterning, it would be also very useful to
identify other potential target genes which are involved in anterior midline
patterning. One way to clone the downstream genes regulated by Otw-2WNF3R
is to combine the method for establishing single cell cDNA libraries (Brady and
Iscove, 1993) with suppression subtractive hybridization (Diatchenko et al.,
1996). The method of generating cDNA libraries from a small number of cells
overcomes the tissue limitation of the embryo and the suppression subtractive
hybridization enriches for rare sequences greater than 1 000-fold and
simdtaneously prevents non-target (not differentially expressed) DNA
amplification in a model system (Diatchenko et al., 1996). Therefore, it would
be reasonable to combine these two methods to clone genes regulated by Otw-
2WNF4B. In this experiment, the tester cDNA will be prepared from the anterior
midline region, where both genes are coexpressed, in double heterozygous
embryos at E7.5-7.7 and the driver cDNA will be prepared from the same region
of wild-type embryos. If the combined method is successful, it can be applied to
clone other genes involved in anterior patterning using anterior tissues
(endoderm or mesendoderm) as testers and posterior tissues as drivers. These
studies will contribute our insight to better understanding the genetic control of
anterior patterning .
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