Universal mechanism of animal development

1. Universal mechanism of animal development Gene expression controls 4 essential process

2. Homologous proteins=functionally interchangeable Eyeless =Pax-6

3. Share basic anatomical features Epidermal cell Gut cells Muscle cells Neuron & sensory cells Gastrulation Ectoderm-epidermis, nervous Endoderm-gut, lung, liver Mesoderm-muscle, connective

4. gastrulation

5. Multicellularanimals are enriched in proteins mediating • cell-cell interaction and gene regulation • Genome sequencing • C. Elegans 19,000 genes • Drosophila 14,000 • Homo sapiens 30,000 • 50% homologs • Non-conserved--- • minor importance---free to mutation • Gene duplication • Two classes of genes (same molecules for body construction) • 1. TM (cell-cell interaction) cell adhesion and cell signaling • 2. Gene regulatory proteins (differential gene expression)

6. Regulatory DNA define the program of developmentDifferent arrangement of regulatory modules Same cell types, different body structure Assembling the components in different combination— instruction in non-coding region

7. Descriptive embryology—track the individual cells (cell lineage) Xenopus :cell division, growth, movement

8. Experimental embryology—remove, rearrange, transplantcell and tissue interaction Chick & Xenopus Developmental Genetics—action of genes

9. Developmental Genetics—action of genes 1.Isolation of mutant animals—genetic screen Mutations in their germ cells 2.Interesting abnormality 3.Discover genes 4.Cloning and sequencing 5.How does gene work 6.Regulatory DNA that controls its expression Fruit fly C. Elegans Zebrafish Mouse Human—medical care system (abnormalities compatible with life)

10. Cell fate determinationtransplanting test—alter environments Specified or committed—strong tendency

11. Positional values—reflect their location in the bodyregionally determined—switch on and maintain expression Markers of position Signaling system that controls the differences between the parts of the limb is the same— tip to be toes determined as leg already

12. Gene regulatory proteins-T-box

13. Same genome to different cell fates (Neuronblast)

14. Different environmentsadjacent similar cells –exchange signalsCell-cell contact Notch pathway—gain an advantage—stronger inhibitory signal Self-reinforcing

15. Inductive interaction—signal is limited in time and space Short-rang-----transmitted via cell-cell contact Long range----molecule diffuses through extracellular medium Similar cells—equivalence group or morphogenetic field

16. Morphogen—finely graded Localized source Different cell fates High conc. Medium conc. low. Conc. Sonic hedgehog protein (shh) – thumb to little finger axis

17. Mirror duplication of the pattern of digits 2,3,4 according to their distances from the source of shh

18. Extracellular inhibitors of signal molecules shape the response to the inducer Chordin—neuron tissue Inhibitor of BMP/TGFb— induces epidermal

19. Refined by sequential induction Morphogen—1mm Cell proliferation increase size Local induction-more cell fates

20. C.elegans--anatomically simple

21. 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 maturation Through 4 larval stages and molts to adults in three days Small and transparent—follow individual cells by direct observation Genome is small Hard to do transplantation No similar body structure as human

22. Lineage analysis Clone-one founder cell-germ line and intestine Cell-cell interaction

23. Maternal-effect genes in asymmetric division Sperm entry point—posterior pole Mother’s mRNA—to proteins (organized in relation to this point) Par-partitioning defective P granules—ribonucleoprotein particles to the posterior pole Vasa homologs—RNA binding protein (germ cell determining material)

24. Complex pattern by cell-cell interaction

25. What point: decisive internal changes • signals from other cells • Method: • 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

26. Developmental Biology by Scott F. Gilbert Sinauer Associates, publishers Six edition, 2000

27. Cell change over time in their responsiveness to developmental signals Age and past history 4 cell stage ABp –Notch signal ABa 12 cell stage Grand daughter ABp (no response) Aba-Notch signal--Pharynx

28. Heterochronic genes Cell death Less cells Division and differentiation Green with lin-14 which disappears at larva feeding Loss of lin-14—premature Gain of lin-14—stay at 1st larva stage

29. Lin 4, Let 7 upstream of lin 14 short untranslated RNA (21-22nts) –complementary sequences in non-coding region Control rate of translation or degradation Lin 4 RNA increase—in stage 1 but like in stage 3 Let 7 RNA increase– late larva to adult stages Let7 homologs , and its target homologs in human, fly and zebrafish microRNA84 family-down regulate Ras (Let60) Low expression in lung cancer

30. Programmed cell death Control cell numbers 131 die Genetic screen- Cell death abnormal ced-3—caspase ced-4—Apaf-1 ced-9—Bcl-2 egl-1--Bad

31. Cell movementXenopus Three days Big-transplantation

32. The polarity of embryo depends on the polarity of the egg VegT-T box family Vg1-TGF-b Wnt-Dishevelled Vegetal pole—inner tissue Animal pole—outer tissue D/V rotate—cortex Dishevelled

33. cleavage Blastomeres—smaller cells 12 cleavages—synchronously without transcription Asymmetric—vegetal (fewer cells, but larger)

34. Three germ layers Determinants distributed asymmetrically—different cell fates Ectoderm Mesoderm Endoderm

35. Blastula—epithelial sheet Na+ pump in water in Only outer most cells

36. After gastrulation, the arrangement of three germ layers Endoderm-digestive tract Mesoderm-connective tissue, muscle (vascular system) Ectoderm-epidermis Nervous system

37. Gastrulation

38. Gastrulation Mesoderm: somite, notochord Segregate from the epithelium

39. Chemical signals trigger the mechanical processes

40. Organizer –gastrulation & pattern of specialization of tissues Chick & Xenopus

41. Changes of cell packing provide force Bottle cells-narrow necks -anchor them to the surface of epithelium

42. Convergent extension (main force) Frizzled/Dishevelled polarity signaling pathway

43. Selective cell-cell adhesion Sorting out Reconstruction Cadherins (Ca2+ dependent) Differentially expressed in the various tissues Involved in gastrulation, neurulation, somite formation Red-epidermal Green-mesoderm Blue-neural plate

44. neurulation Mesoderm-notochord-convergent extension Notochord expresses Brachyury (T box family)

45. Neural tube formation

46. Gene expression oscillationmesoderm to somite segmentation Cadherin family Paraxial protocadeherin Mark out the somite C-hairy-1: pair-rule gene Peak-one set of genes Trough-another set of genes

47. somite- to muscle-cell precursors Muscle precursor-myoblast (MyoD) nucleoli

48. The main pathways of neural crest cell migration From epidermis Fibronectin- provide adhesive site Chondroitin sulfate proteoglycan- repel

49. Effect of mutations in the kit gene Albino-no pigment, megacolon-lack Endothelin-3 Pigment cells depend on the kit product as a receptor for a survival factor

50. Drosophila melanogaster