Universal mechanism of animal development

Gene expression controls 4 essential process

Homologous proteins=functionally interchangeable

Eyeless =Pax-6

Share basic anatomical features

Epidermal cellGut cellsMuscle cellsNeuron & sensory cells

GastrulationEctoderm-epidermis, nervousEndoderm-gut, lung, liverMesoderm-muscle, connective

gastrulation

Multicellular animals are enriched in proteins mediating cell-cell interaction and gene regulation

Genome sequencingC. Elegans 19,000 genes Drosophila 14,000Homo sapiens 30,000

50% homologsNon-conserved---1) minor importance---free to mutation2) Gene duplication

Two classes of genes (same molecules for body construction) 1. TM (cell-cell interaction) cell adhesion and cell signaling2. Gene regulatory proteins (differential gene expression)

Regulatory DNA define the program of developmentDifferent arrangement of regulatory modules

Same cell types, different body structureAssembling the components in different combination—instruction in non-coding region

Descriptive embryology—track the individual cells (cell lineage)

Xenopus : cell division, growth, movement

Experimental embryology—remove, rearrange, transplantcell and tissue interaction

Chick & Xenopus

Developmental Genetics—action of genes

Developmental Genetics—action of genes

1.Isolation of mutant animals—genetic screen Mutations in their germ cells2.Interesting abnormality3.Discover genes4.Cloning and sequencing5.How does gene work6.Regulatory DNA that controls its expression

Fruit flyC. ElegansZebrafishMouseHuman—medical care system (abnormalities compatible with life)

Cell fate determinationtransplanting test—alter environments

Specified or committed—strong tendency

Positional values—reflect their location in the bodyregionally determined—switch on and maintain expression

Signaling system that controls the differencesbetween the parts of the limb is the same—tip to be toesdetermined as leg already

Markers of position

Gene regulatory proteins-T-box

Same genome to different cell fates

(Neuronblast)

Different environmentsadjacent similar cells –exchange signals

Cell-cell contact

Notch pathway—gain an advantage—stronger inhibitory signalSelf-reinforcing

Inductive interaction—signal is limited in time and space

Short-rang-----transmitted via cell-cell contactLong range----molecule diffuses through extracellular medium

Similar cells—equivalence group or morphogenetic field

Morphogen—finely graded

Localized sourceDifferent cell fatesHigh conc.Medium conc.low. Conc.

Sonic hedgehog protein (shh) –thumb to little finger axis

Mirror duplication of the pattern of digits

2,3,4 according to their distances from the source of shh

Extracellular inhibitors of signal molecules shape the response to the inducer

Chordin—neuron tissueInhibitor of BMP/TGFb—induces epidermal

Refined by sequential induction

Morphogen—1mmCell proliferation increase sizeLocal induction-more cell fates

C.elegans--anatomically simple

1000 somatic cells, 1000-2000 germ cells

Hermaphrodite –female + limited number of sperms—self-fertilized (homozygote progeny)Male-cross fertilized

Single fertilized egg-558 cells (egg shell)Growth (further division) and sex maturationThrough 4 larval stages and molts to adults in three days

Small and transparent—follow individual cells by direct observationGenome is small

Hard to do transplantationNo similar body structure as human

Lineage analysis

Clone-one founder cell-germ line and intestineCell-cell interaction

Maternal-effect genes in asymmetric division

Sperm entry point—posterior poleMother’s mRNA—to proteins (organized in relation to this point)Par-partitioning defectiveP granules—ribonucleoprotein particles to the posterior poleVasa homologs—RNA binding protein (germ cell determining material)

Complex pattern by cell-cell interaction

What point: decisive internal changes signals from other cellsMethod:1. Microsurgery a. laser microbeam microsurgery b. early embryo, cell pushed around and rearranged c. remove egg shell—in culture

2. Genetic screen (gene cloning, sequencing) p2-EMS interaction Screen for a. no gut cells –mom mutant : more mesoderm (Wnt, Frizzled) b. extra gut-pop mutant: plenty of pharynx (LEF-1/TCF)—E cells

Developmental Biology by Scott F. GilbertSinauer Associates, publishersSix edition, 2000

Cell change over time in their responsiveness to developmental signals

Age and past history

4 cell stage ABp –Notch signalABa

12 cell stageGrand daughter ABp (no response) Aba-Notch signal--Pharynx

Heterochronic genes

Loss of lin-14—prematureGain of lin-14—stay at 1st larva stage

Division and differentiation

Less cells Cell death

Green with lin-14 which disappears at larva feeding

Lin 4, Let 7 upstream of lin 14

short untranslated RNA (21-22nts)–complementary sequences in non-coding regionControl rate of translation or degradation

Lin 4 RNA increase—in stage 1 but like in stage 3Let 7 RNA increase– late larva to adult stages

Let7 homologs , and its target homologs in human, fly and zebrafishmicroRNA84 family-down regulate Ras (Let60)Low expression in lung cancer

Programmed cell death

Control cell numbers131 dieGenetic screen-Cell death abnormalced-3—caspaseced-4—Apaf-1ced-9—Bcl-2egl-1--Bad

Cell movementXenopus

Three daysBig-transplantation

The polarity of embryo depends on the polarity of the egg

VegT-T box familyVg1-TGF-Wnt-DishevelledVegetal pole—inner tissueAnimal pole—outer tissueD/V rotate—cortex Dishevelled

cleavage

Blastomeres—smaller cells 12 cleavages—synchronously without transcriptionAsymmetric—vegetal (fewer cells, but larger)

Three germ layers

Determinants distributed asymmetrically—different cell fatesEctodermMesodermEndoderm

Blastula—epithelial sheet

Na+ pump in water in

Only outer most cells

After gastrulation, the arrangement of three germ layers

Endoderm-digestive tractMesoderm-connective tissue, muscle (vascular system)Ectoderm-epidermisNervous system

Gastrulation

Gastrulation

Mesoderm: somite, notochordSegregate from the epithelium

Chemical signals trigger the mechanical processes

Organizer –gastrulation & pattern of specialization of tissues

Chick & Xenopus

Changes of cell packing provide force

Bottle cells-narrow necks-anchor them to the surface of epithelium

Convergent extension (main force)

Frizzled/Dishevelled polarity signaling pathway

Selective cell-cell adhesion

Sorting outReconstructionCadherins (Ca2+ dependent)Differentially expressed in the various tissuesInvolved in gastrulation, neurulation, somite formation

Red-epidermalGreen-mesodermBlue-neural plate

neurulation

Mesoderm-notochord-convergent extensionNotochord expresses Brachyury (T box family)

Neural tube formation

Gene expression oscillationmesoderm to somite segmentation

C-hairy-1: pair-rule genePeak-one set of genesTrough-another set of genes

Cadherin familyParaxial protocadeherinMark out the somite

somite- to muscle-cell precursors

Muscle precursor-myoblast (MyoD)

nucleoli

The main pathways of neural crest cell migration

From epidermis

Fibronectin-provide adhesive siteChondroitin sulfate proteoglycan-repel

Effect of mutations in the kit gene

Pigment cells depend on the kit product as a receptor for a survival factor

Albino-no pigment, megacolon-lack Endothelin-3

Drosophila melanogaster

From egg to adult

Body segments

Syncytial blastoderm

Extended germ bandGastrulation

Clear segment

Segments in different stages

Development of Drosophila egg

Fate map

Egg polarity genes

Maternal effect genes

Oocyte in its follicle

Egg polarity gradient system

Dorsal gradientNF-kB

Cactus-I-kB

Patterning the dorsoventral axis

Patterning the dorsoventral axis

Mesoderm from cells expressing twist

Ventral furrow formation--invagination

Dorsoventral inversion

Dpp=BMP4 (TGF-)Sog=Chordin (high-neurogenic tissue)

Three types of segmentation genes

The regulatory hierarchy

Modular organization of the regulatory DNA of the eve gene.

The formation of ftz and eve stripes in the Drosophila blastoderm

Interactions between Gap and pair-rule genesMaintained by segment polarity and homeotic selector genes

The expression of engrailed, a segment-polarity gene

Throughout the life!

A homeotic mutation

Antenaapedia mutantRegulatory region of the gene(expressed in the head)

The effect of deleting most of the genes of the bithorax complex

Molecular address labels(each segment)-segment identityDNA binding homeodomain(60 amino acids)DNA contains homeobox

All like ps5

The spatial pattern of expression of

genes of the bithorax complex

Bithorax—Ultrabithorax –5-12 Abdominal-A—7-13 Abdominal-B—10-13

Bithorax mutant –PS 4 default state+Ubx—5,6+Abd-A—7,8,9+Abd-B—10Combinatorial mannerLack Ubx—5,6 to 4 also 7-14 thorax structure in the abdomen

Hox—gap, pair-rule for the first 4 hours, then polycomb (repression), and Trithorax (activation)

The patterns of expression compared to the chromosomal locations of the genes of

the Hox complex

Dark-high levelMedium-lowerLight (not affect phenotype)

14 parasegment-at extended germ band stage

Action of genes of the polycomb group

Permanent record of positional information1. autoactivation-homeotic selector genes2. Polycomb-represses Trithorax-on

Affect histone H4 acetylationHyperacetylated H4-Hox exposed to activator

polycomb on chromosome

The Hox complex of an insect and the Hox complexes o

f a mammal

Expression domains of Hox genes in a mouse

Hox BTransgenic mouse withLacZ reporter

Segmentation and Hox gene expression in the hindbrain

Rhombomeres=segemnt