The early development of vertebrates: birds and mammals

1. The early development of vertebrates: birds and mammals 杨雪松 医学院 组胚系 & 生科院 再生医学教育部重点实验室 医学院 641， Tel：85228316， E-mail: yang_xuesong@126.com

2. The e-mail for my lecture: ppt or any notices Development_2012@126.com Password: development

3. 1984 1991 1997 1999 2007 now 暨南大学医学院 英国Dundee大学 哈尔滨医科大学生理学硕士博士 英国Manchester大学 哈尔滨医科大学医疗系 日本东京医科齿科大学博士 原肠胚形成期三胚层形成中细胞迁移以及血管发生中的基因调控 膜生理,神经科学 生殖内分泌 胰腺外 分泌 细胞生理学 细胞生物学 杨雪松 Scientific career

4. Human Embryo development movie

5. Developmental biology Definition: Field of biology that studies how a single cell (the fertilized egg) gives rise to a fully formed organism. Enjoyment: why do people get excited about the topic? Complexity, beauty, imaging, relevance (human congenital defects, cancer)

6. Developmental biology Description: Draw out embryonic development from 1-cell stage, cleavage, blastula, gastrula (germ layer formation), body plan development, neurulation, organogenesis So most of what you have studied so far has been intracellular biology, focusing on molecules and what happens inside individual cells; one important aspect of biology that has not yet been covered is how cells communicate with each other, how they form multicellular organisms, how cells form different tissues, how these different tissues form organs that in turn interact with each other.

7. Fundamentals questions in developmental biology Axis determination: AP, DV, LR. how do you ‘break’ the symmetry of the egg? Sperm entry, localized determinants Cell differentiation Cell proliferation Cell migration:mainly in gastrulation Cell polarization (symmetric vs asymmetric cell division), Cell shape (giving cells different morphologies, e.g. neurons, EMT), Cell death Morphogenesis (how cells come together to form tissues Organogenesis Germ cell development: fertilization Stem cells: multipotency, ability to self-renew, so ‘trendy’ Regeneration

8. Tools for developmental biology study Gene knock-out or knock down: mutation, RNAi, antisense oligos Gain-of-function experiment: mRNA injections, transgenics Biochemistry and molecular biology: Western blot, PCR… Imaging: live, gene and protein expression Experimental embryology: cell or tissue ablations, bead implantation, chick-quail chimeras

9. Model systems for developmental biology Worm (C. elegans) Fly (Drosophila melanogaster) Fish (zebrafish, medaka) Mouse Xenopus (laevis, tropicalis) Chick (Quail)

10. Chick: Gallus domesticus Fly: Drosophila melanogaster MODEL SYSTEMS for the experimental analysis of development Mouse: Mus musculis Xenopus laevis and tropicalis C elegans: Caenorhabditis elegans Fish: Danio rerio Model systems for developmental biology

11. The early development of chick embryo

12. The developmental stage of chick embryo

13. The five fundamental main steps Gametogenesis fertilization cleavage gastrulation organogenesis

14. Modulation of cell migration in chick embryo gastrulation

15. Is cell migration the only event happen during gastrulation? • Answer is No • Proliferation: divide 2-3 time during gastrulation • Apoptosis However, comparing those… no doubt that cell migration is major event, at least in chick gastrulation

16. The advantage of chick embryo 1. Good model for human being, short life-span 2. Very easy to accessible and be manipulated. 3. Combination with Classic and molecular biological technologies. • transplantation • cell labelling • Gene overexpression • Gene knock-down • Immunocytochemistry • chemical treatment • in situ hybridization

17. What is gastrulation? • Gastrulation – the stage of animal development where a ball of cells consisting of a single, undeveloped layer is converted into an embryo consisting of three separate tissue layers : • Ectoderm – outer layer • Endoderm – inner layer surrounding the gut • Mesoderm – middle tissue layer

18. The first structure – primitive streak

19. Hensen’s node primitive streak epiblast hypoblast migrating cells endoderm displacing hypoblst mesoderm Cell movement in chick embryo Gastrulation

20. When does gastrulation happen? • List of developmental events in order: • Fertilization) • Cleavage • Blastocyst • Grastrulation • Neuralization

21. Primitive Streak Formation (elongation to anterior)

22. The main axes of a developing embryos

23. Specification of the chick anterior-posterior axis by gravity Rotation in the shell gland (A) results in the lighter components of the yolk pushing up one side of the blastoderm (B). That more elevated region becomes the posterior of the embryo (C).

24. Discoidal meroblastic cleavage in a chick egg (A-D) Four stages viewed from the animal pole (the future dorsal side of the embryo). (E) An early-cleavage embryo viewed from the side

25. Formation of the two-layered blastoderm of the chick embryo (A, B) Primary hypoblast cells delaminate individually to form islands of cells beneath the epiblast. (C) Secondary hypoblast cells from the posterior margin (Koller's sickle and the posterior marginal cells behind it) migrate beneath the epiblast and incorporate the polyinvagination islands. (D) This sagittal section of an embryo near the posterior margin shows an upper layer consisting of a central epiblast that trails into the cells of Koller's sickle (ks) and the posterior marginal zone (mz).

26. Streak formation in chick

27. Regulation of the chick blastoderm posterior marginal zone (PMZ) (Only Vg1 expression PMZ Supplied by Xuesong Yang’s lab) The ability to initiate a primitive streak is found throughout the marginal zone. When the blastoderm is divided into four parts, each part can initiate gastrulation and give rise to an embryo.

28. New Culture EC culture Susan C etc. DEVELOPMENTAL DYNAMICS 220:284–289 (2001) The in vitro developmental methods for chick embryos

29. The microscopy for studying cell migration in gastrulation of chick embryos inverted Compound & Dissection Optical Systems Confocal Two-photon

30. Gene transfection …training required Just think of ….and keep trying…..

31. Cell migration in gastrulation • Cell movement pattern: • 1. Epiblast cells movement during primitive streak formation. • Mesoderm formation during gastrulation. • 3. Vasculogenesis • Cell movement mechanism: • 1. Cell-cell adhesive molecule. • Cell polarity. • 3. chemotaxis.

32. The correlation between morphogen and cells in pattern formation

33. Cell migration in gastrulation • Cell movement pattern: • 1.Epiblast cells movement during primitive streak formation. • Mesoderm formation during gastrulation. • 3. Vasculogenesis • Cell movement mechanism: • 1. Cell-cell adhesive molecule. • Cell polarity. • 3. chemotaxis.

34. The genes involved in streak formation in chick

35. Convergent extension in Keller explants

36. The Wnt planar polarity pathway in convergent extension

37. Cell movement patterns in gastrulation

38. The role of Wnt11 signalling

39. Streak formation as a result of intercalation and/or oriented cell division R Keller et al, 2003

40. Cell movements of the primitive streak of the chick embryo (A-C) Dorsal view of the formation and elongation of the primitive streak. The blastoderm is seen at (A) 3–4 hours, (B) 7–8 hours, and (C) 15–16 hours after fertilization. The early movements of the migrating epiblast cells are shown by arrows. (D-F) Formation of notochord and mesodermal somites as the primitive streak regresses, shown at (F) 19–22 hours, (E) 23–24 hours, and (F) the four-somite stage. Fate maps of the chick epiblast are shown for two stages, the definitive primitive streak stage (C) and neurulation (F). The endoderm has already ingressed beneath the epiblast, and convergent extension is seen in the midline.

41. Migration of endodermal and mesodermal cells through the primitive streak (A) Scanning electron micrograph shows epiblast cells passing into the blastocoel and extending their apical ends to become bottle cells. (B) Stereogram of a gastrulating chick embryo, showing the relationship of the primitive streak, the migrating cells, and the two original layers of the blastoderm. The lower layer becomes a mosaic of hypoblast and endodermal cells; the hypoblast cells eventually sort out to form a layer beneath the endoderm and contribute to the yolk sac.

42. 哈尔滨医科大学 –难忘的大学及研究生时代 Advertising for old school and hometown…

43. 哈尔滨–难忘的大学及硕士研究生时代 Advertising for old school and hometown…

44. Primitive Streak Regression (shorten to posterior)

45. Chick gastrulation roughly from 24 to 28 hours (A) The primitive streak at full extension (24 hours). The head process (anterior notochord) can be seen extending from Hensen's node. (B) Two-somite stage (25 hours). Pharyngeal endoderm is seen anteriorly, while the anterior notochord pushes up the head process beneath it. The primitive streak is regressing. (C) Four-somite stage (27 hours). (D) At 28 hours, the primitive streak has regressed to the caudal portion of the embryo. (E) Regression of the primitive streak, leaving the notochord in its wake. Various points of the streak (represented by letters) were followed after it achieved its maximum length. Time represents hours after achieving maximum length (the reference line, about 18 hours after incubation).

46. Formation of Hensen's node from Koller's sickle (A) Diagram of the posterior end of an early (pre-streak) embryo, showing the cells labeled with fluorescent dyes in the photographs. (B) Just before gastrulation, cells in the anterior end of Koller's sickle (the epiblast and middle layer) were labeled with green dye. Cells of the posterior portion of Koller's sickle were labeled with red dye. As the cells migrate, the anterior cells formed Hensen's node and its notochord derivatives.

47. Induction of a new embryo by transplantation of Hensen's node (A) A Hensen's node from a duck embryo is transplanted into the epiblast of a chick embryo. (B) A secondary embryo is induced (as is evident by the neural tube) from host tissues at the graft site.

48. Nobel prize was presented to the discover of Organizer

49. The change in cell shape in the neural plate during chick neurulation

50. The change in cell shape in the neural plate during chick neurulation