Chapter 23 Developmental Genetics

Homeotic mutation

Central themes of developmental genetics - pattern formation, construction of complex form, operation through developmental program

Logic of building the body plan - Cell fate : differentiate into particular kinds of cells : positional information developmental field - Fate refinement : asymmetric division different regulatory instruction : decision by neighboring or paracrine signals - Specify fate option of cells of a given cell lineage in step by step manner : totipotent fate refinement cell lineage

Major decisions in building the embryo - simple binary decisions : separation of the germ line from the soma : establishment of the sex of the organism - specification decisions : establishment of the positional information : anterior-posterior axis segment dorsal-ventral axis germ layer : production of the various organs, tissues, systems

Applying regulatory mechanisms to developmental decisions

- Simple developmental pathway

: the concentration of some

key molecules determine

the “on” or “off” binary choice

- making a pathway decision and

subsequently remembering that

decision are both key to cell fate

commitment

Decision making in developmental pathway

Regulation of protein activity

- Simple developmental pathway

: During development,

regulatory mechanisms

at any of level can control

the production of active

protein products

Gene regulation at levels other than transcriptionInitiation: examples Tissue-specific regulation at thelevel of DNA structure

Tissue-specific amplificationof the number of copies of a gene leads to high levels of gene expression in that tissue

Structure of the two eggshell-gene clusters in Drosophila melanigaster

: Eggshell gene copy number is increased by somatic DNA rearrangement only in the follicle cell: each ACE region contains an amplification control element act as a special site for the initiation of tissue-specific DNA replication in follicle cells of the ovary

Transcript processing and tissue-specific regulation

The production of an active protein can be regulated by controllingthe pattern of splicing of an initial transcript into an mRNA

Germ line specific expression of Drosophila P elements: examples of tissue-specific regulation by RNA splicing

a) Somatic and germ line mRNA structure for the wild type P element

b) Modified P element transgene, P {2, 3}; in all tissues, transposase is active

Transcript processing and tissue-specific regulation

Somatic expression of P transposase in Drosophila containing P(w+) transposon

P transposase derived from a wild type P element: P element is inactive in soma (red eye)

P transposase derives from the P { 2,3} transgene: P transposase is active in soma (red and white)

Posttranscriptional regulation

* A certain sequences of 3’UTR of mRNA - regulate the degradation or the translation efficiency of mRNA - target sites for proteins that digest mRNA molecule or that block their translation

* A 3’UTR sequences interact with a regulatory RNA molecule ex) in C. elegans - premature adult development, or reiterated, producing delayed adulthood - RNA product of the lin-4 repress translation of lin-14 mRNA (complementarity between lin-4 RNA and 3’UTR of lin-14)

* A 3’UTR act as sites for anchoring an mRNA to particular structures within a cell

Regulatory instructions are also contained within noncoding regions of mRNAs

Posttranslational regulation

* Enzymatic modifications of proteins - phosphorylation - acetylation…..

* Multiprotein complexes - protein-protein interactions

Binary fate decisions: pathways of sex determination* In many species, sex determination is associated with the inheritance of a heteromorphic chromosome pair in one sex (XX-XY system) * The XX-XY system of flies and mammals arose independently

Drosophila sex determination: every cell for itself

Phenotypic consequences of different x-chromosome-to-autosome ratios

Drosophila n = 4one sex chromosomethree different autosomes

X : A > 1 or = 1 -> femaleX : A < 0.5 or = 0.5 -> male

(A : number of autosome set)

Every cell in Drosophila independently determines its sex

Basics of the regulatory pathway

The pathway of sex determination and differentiation in drosophila

Sexual phenotype is carried out by

a master regulatory switch and several

downstream sex-specific genes

Regulatory switch; the activity of Sxl (Sex lethal) protein1. Setting the switch in the “on” or “off” position

<The initiation & maintenance of the Sxl switch>- NUM (numerator gene in X ch.)- DEM (denominator gene in autosome)- NUM and DEM (both bHLH) forms dimers at random- Only NUM-NUM dimers forms active transcription factor

Regulatory switch

2. Maintaining the switch in a stable position

- The Sxl gene has two promoters :- PE is active only early in embryogenesis

:- PL is active in every cell for the remainder of the life cycle

- SXL protein is an RNA-binding protein that alters the splicing of the nascent Sxl transcript coming from late promoter

- Feedback or autoregulatory loop controlled at the level of RNA splicing

:- maintains SXL activity throughout development in flies with an X : A

ration of 1.0

Autoregulatory loop exemplifies how an early developmental decision can be “remembered” for the rest of development, even after the initial signals that established the decision have long disappeared

Regulatory switch

3. Propagating the decision Two forms of tra mRNA

Different dsx mRNA are produced in both sexes

Alternative splicing of tra and dsx transcripts

Mutational analysis of Drosophila sex determination

“Treasure your exceptions”

* The effect of null mutation - transform female into phenotypic male - Sis-b (sisterless-b); a numerator gene - Sxl (sex-lethal) and tra (transformer); the RNA splicing regulators - Must be active for female development

* Dsx (doublesex) - Dsx-/- leads to the production of flies that simultaneously have male and female attributes

Sex determination in mammals: coordinated control by the endocrine system;- the presence or absence of a Y chromosome

Mammalian reproductive development and endocrine organ control

The embryonic genital ridge consists of a medulla surrounded by a cortex- Female germ cells migrate into the cortex & become organized into a ovary- Male germ cells migrate into the medulla & become organized into a testis

In the initial urogenital organization at theindifferent gonad stage, precursors of both male (Wolffian) and female (Mullerian)ducts are present

Setting the switch in the “on” or “off” position

Testis-determining factor on Y chromosome (TDF in human, Tdy in mice), as same as SRY (human) –Sry (mice) gene

The wild type XY individual has SRY gene, which activates maleshunt pathway

Normal XX individual lacking SRY remains in the female default pathway

Mammalian reproductive development and endocrine organ control

* It is the presence or absence of a testis that determines the sexual phenotype, through the endocrine release of testosterone (in XY embryos lacking the androgen receptor, development proceeds along a completely female pathway even though the embryos have testes)

Mutational analysis of mammalian sex determination

Sex reversal- Sex reversed XX individuals are phenotypic males and carry a fragment of the Y chromosome in their genomes

sex reversal on Y gene (SRY)

A molecular map of the distal part of the short arm of the human Y chromosome

PCR of genomic DNA shows thatMouse 33.13 lacks a DNA marker(Zfy-1) for Y chromosomebut, contains the Sry transgene

The external genitalia of sex-reversedXX transgene mice 33.13 and normal XY male sib (33.17)

A transgenic mouse that proves Sry can cause the sex-reversal syndrome

Binary fate decision: the germ line versus the soma

In making the decision of germ line versus soma,

the embryo exploits its machinery for creating

asymmetries-the cytoskeleton, to localize a germ-line

determinant to a subset of early embryonic cells

Cytoskeleton of the cell

Different cytoskeletal system in the same cell

Intermediate filament (vimentin)

Microtubules (tubulin)Microfilament (actin)

Roles: control of the location of the mitotic cleavage plane control of the cell shape directed transport of molecules and organelles Cytoskeletal rods are polar structures

Intrinsic asymmetry of cytoskeletal filaments

The cytoskeleton serves as a highway system for the directedmovement of subcellular particles and organelles;- polarity is essential

Polarity of subunits in an actin microfilament

The distribution of tubulin in an interphase animal fibroblast

Movement of vesicles along microtubules

Electron micrograph of two smallvesicles attached to a microtubule

Kinesin attaches and moves the cargoes along the microtubules

A diagram of the kinesin protein

Localizing determinants through cytoskeletal asymmetries: the germ line

The early development of C. elegans, showing the early divisions of the zygote

The asymmetric distribution of P granules is microfilament dependent

Pole-cell formation at the syncitial stage of the early Drosophila embryo

Eggshell removed Drosophila embryo and longitudinal

section

In drosophila, microtubles provide the essential asymmetry of germ-line determinants

Forming complex pattern: establishing positional information

Mutational analysis of early Drosophila developmentGenetic assays for recessive zygotic and maternal effect mutations

The phenotypes of offspring are purely a manifestation of their own genotype

The phenotypes of offspring are purely a manifestation of their mother’s genotype

Cytoskeletal asymmetries and the Drosophila anterior-posterior axis

BCD protein (bicoid gene product) steeper gradient in the early embryos

HB-M protein (hunchback gene product) shallower, longer gradient

Positional information along the A-P axis of Drosophila embryo;creation of concentration gradients of two transcription factors (the BCD and HB-M proteins)

The expression of the localized A-P determinant

bcd mRNA BCD protein

nos mRNA NOS protein

Localization of mRNA within a cell is accomplished byanchoring the mRNAs to one end of polarized cytoskeleton chains

* Bicoid mRNA is tethered to the – ends of microtubules (anterior pole)* hb-m mRNA is uniformly distributed in the embryo - nos mRNA is localized at the posterior pole - NOS protein specifically block translation of hb-m mRNA - produce the shallow A-P gradient of HB-M protein

The effect of replacement of the 3’UTR of the nanos mRNA with the 3’UTR of the bicoid mRNA on mRNA localization and embryonic phenotype

* There are specific microtubule-association sequences located within the 3’UTRs - bcd mRNA 3’UTR localization sequences are bound a protein that can also bind the – ends of the microtubules

Studying the BCD gradient

Genetic changes in the bcd gene alter anterior fate

Exoskeletons of larvae derived from wild type and bcd maternal effect lethal mutant mothers

Left : wild type, normal phenotypeRight : bcd, anterior head and thoracic structures missing

Concentration of BCD proteinaffects A-P cell fates

The position of the cephalic furrow arises farther forward the posterior according to bcd+ gene dosage

Studying the BCD gradient

Bcd mRNA can completely substitute for the anterior determinantactivity of anterior cytoplasm

The bcd anteriorless mutant phenotype can be rescued by wildtype cytoplasm or purified bcd+ mRNA

Cell-cell signaling and the Drosophila dorsal-ventral axis

Positional information can be established through cell-cell signalingby means of a concentration gradient of a secreted molecule

The D-V positional information : DL protein activity

The distribution of DL in response to the SPZ signal (spaetzle gene product)

DL protein is in the nucleus ventrally, throughout the cell laterally and in the cytoplasm dorsally

A mature oocyte

Dl; dorsalCact; cactusSpz; spaetzle

The signaling pathway that leads to the gradient of nuclear versus

cytoplasmic localization of DL proteins

Cell-cell signaling and the Drosophila dorsal-ventral axis

The two classes of positional information

I. Localization of mRNAs within a cell II. Formation of a concentration gradient of an extracellular diffusible molecule

Unicellular fieldMulticellular fieldMorphogen; concentration-dependent determinants of form

Forming complex pattern: utilizing positional information to establish cell fates

Initial interpretation of positional information

Positional information ; a gradient of transcription factor activities(AP axis: BCD & HB-M, DV axis: DL) Activation of cardinal genes - the first genes to respond to the maternally supplied positional information - the zygotically expressed genes

Review of Drosophila embryology

A syncitium-stage embryo, common cytoplasm toward theperiphery and the central yolk-field region

A cellular blastoderm embryo, columnar cells

Changes during cellularization

Review of Drosophila embryology

3-hour Drosophila embryo

10-hour Drosophila embryo

Newly hatched larva

Segmentation identity of cells along the A-Paxis is already fated early in development

* A-P cardinal genes: gap genes* The gap genes can be expressed in a series of distinct domains by having promoters that are sensitive to the concentrations of A-P transcription factors

Early embryonic expression pattern of gap genes

Early blastoderm expression patterns of protein from three gap genes: hb-z, kr, and kni

Refining fate assignments through transcription-factor interactions

* The gap genes activate a set of secondary A-P patterning genes ; pair-rule genes in a repeating pattern of seven stripes the correct number of segments

Late blastoderm expression patterns of protein from two pair-rule genes:ftz (gray), and eve (brown)

Regulatory element complexity of the primary pair-rule genes turns

an asymmetric (gap gene) expression pattern into a repeating one

* There is a hierarchy within the pair-rule genes ; combination of pair-rule genes transcriptional regulation of the segment-polarity genes (the correct number of segments)

* Repeating expression pattern of primary pair-rule genes ?

Homeotic gene transformation of the third thoracic segment (T3) of Drosophila into an extra second thoracic segment (T2)

Wild type,one copy T2one copy T3

A bithorax triple mutant homozygote transforms T3 into a second copy of T2

* The establishment of segmental identity ; homeotic gene complexes - ANT-C (segmental identity in head and anterior thorax) - BX-C (segmental identity in posterior thorax and abdomen)* Homeosis : the conversion of one body part into another

Homeotic gene-encoded protein expression pattern in Drosophila

SCR ANTP

UBX ABD-B

Linear arrangement of the corresponding genes along chromosome 3

Segment identity is established through asymmetric gap-gene expression patternsthat deploy and asymmetric pattern of homeotic gene expression

A cascade of regulatory events

Hierarchical cascade that activates the elements forming the A-Psegmentation pattern in drosophila

Additional aspects of pattern formation

Memory systems for remembering cell fate

Two types of positive feedback loops in maintaining the levelof activity of transcription factors determining cell fate

The transcription factor binds to an enhancer of its own gene,maintaining its transcription

Each adjacent cell sends out a signal that activates receptors, signal transduction pathways, and transcription factors (TF) expression in the other cell (cell-cell interactions)

When the fate of a cell lineage has been established, it must be remembered

Ensuring that all fates are allocated: decisions by committee

Adult Caenorhabditis elegans

Cell-cell interactions:one type is the ability of one cell to induce a developmental commitmentin one neighbor of many, the other is the ability of one cell to inhibit its neighbors from adopting its fate.

The production of the C. elegans vulva from the equivalence groupby cell-cell interactions

Fate allocation can be

made through

a combination

of inductive and

lateral inhibitory

interactions

between cells

Developmental pathways are composed of plug and play modules

Different developmental decisions are made by using mixed and matched combinations of pathway components

Differences in the developmental context of different cell lineages -that is, the transcription factors active in these cells- permit different inputs to, and outputs from, a given developmental circuit

The many parallels in vertebrate and insect pattern formation

The similarity between the mammalian homeobox gene cluster (Hox complex) and insect ANT-C and BX-C homeotic gene cluster (HOM-C)

The comparative anatomy of the HOM-C and Hox gene clusters

The expression domains and regionsof the Drosophila and mouse embryos: the arrangements of the homeotic genes is colinear with their spatial pattern of expression

paralogous

The RNA expression pattern of three mouse Hox genes in thevertebral column of a sectioned 12.5-day-old mouse embryo:the anterior limit of each of the expression pattern is different

Each Hox gene is expressed in a continuous block beginning at a

Specific anterior limit and running posteriorly to the end of the

developing vertebral column

The phenotype of a homeotic mutant mouse

The phenotypes of the homozygous knockout mice are thematicallyparallel to the phenotypes of homozygous null HOM-C flies

Enlargement of the thoracic and lumbarvertebrae of a homozygous Hoxc-8- mouse: L1 in WT mice has no ribs

homozygous Hoxc-8- mouseright: clenched fingerLeft: normal finger

Evolutionary conserved pathways

The signaling pathway for activation of the drosophila DL morphogenparallels a mammalian signaling pathway for activation of NK-kB

Do the lessons of animal development apply to plants?

The general themes for establishing cell fates in animals are likely to be seen in plants as

well. But, the participating molecules in these developmental pathways are likely to be

considerably different from those encountered in animal development.

Flower development in Arabidopsis thaliana

A series of transcriptional regulator determine the fate of the four layers of the flower

Flower-identity gene expression and the establishment of whorl fate