神经发生的分子机制

1. 神经发生的分子机制 景乃禾 中国科学院上海生命科学研究院 生物化学与细胞生物学研究所

2. 人脑的构成

3. 神经元的种类

4. 中枢神经系统 (脑＋脊髓) 细胞数量：1012 (1万亿) 其中： 神经细胞（神经元）1011 神经胶质细胞：9 X 1011 细胞种类：神经元 (Neuron，许多种类) 神经胶质细胞 (Glia) 星型胶质细胞 (Astrocyte) 少突神经胶质细胞 (Oligodendrocyte) 细胞联系：神经元－神经元 神经元－神经胶质细胞 1012 X (102-103)=1014-1015

5. 人脑的发育 (Gilbert, 1991)

6. 中枢神经系统发育的基本过程 一、神经系统的诱导 (Neural Induction) 主要研究：早期胚胎的神经外胚层(神经干细胞，Neural Stem Cell)是如何产生的？ 二、神经系统的发生 (Neurogenesis) 主要研究：神经干细胞是如何分化为各种神经元和神经胶质细胞的？ 三、神经联系的建立 (Axon Guidance, Synapse Formation) 主要研究：神经细胞是如何与其靶细胞建立神经联系的？其中包括：轴突的靶向生长和突触联系的建立。 四、神经系统的可塑性 (Neural Plasticity, Neural Stem Cells) 主要研究：成年动物神经系统的可塑性和神经系统损伤后的修复；神经干细胞和胚胎干细胞神经分化的分子机制。

7. 神经诱导 (Neural Induction) 多潜能干细胞 神经干细胞

8. Major Steps in Neural Differentiation Competence: Cells have the ability to become neural precursors if they are exposed to the right combination of signals. Specification: Cells have received the signals to become neural precursor cells but will still respond to signals that repress a neural character. Commitment: Cells have received the signals to become neural precursor cells and will progress to become neurons even in the presence of signals that repress a neural character. Differentiation:Neural precursor cells exit the cell cycle to become post-mitotic neurons.

9. History of Neural Induction Hypothesis • Spemann Organizer (1924-1990) • Default Model (1992-1997) • Neural Induction in Chick and Mouse (2001-)

10. Induction of Embryonic Primordia by Implantation of Organizers from a Different Species Hans Spemann and Hilde Mangold Arch. Mikr. Anat. Entw. Mech. 100, 599-638, 1924

11. Development of Xenopus embryo Early gastrula: Dorsal blastopore lip

12. Fate map of the Xenopus gastrula (Hemmati-Brivanlou & Melton, 1997)

13. Classical Transplantation Experiment by Spemann and Mangold The donor tissues could recruit the host cells to become the secondary neural tube. Dorsal blastopore lip (Hemmati-Brivanlou & Melton, 1997)

14. “Spemann Organizer” Spemann named the dorsal blastopore lip the “organizer”, and proposed that in normal development this region induces and organizes a correctly patterned nervous system in neighboring dorsal ectoderm. In the absence of this influence, as on the ventral side, the ectoderm differentiates as epidermis. Epidermis: “Default” fate for gastrula ectoderm Neural specification: needs a positive signal from neighboring cells (Neural Induction). “Default”: Cell autonomous.

15. Neural Induction Hypothesis Organizer Mesoderm (Notochord) Neural plate Kessler & Melton, 1994

16. This hypothesis dominated the developmental biology field for several decades. A considerable effort over several decades failed to identify the gene products responsible for neural induction in the embryo.

17. A Animal cap Markers expression (RT-PCR) : Mesoderm Neural ectoderm Epidermis Endoderm In vitro culture V Blastula (stage 8) Animal Cap Assay In early 1980, TGF-b and FGF family members have been found to have the mesodermal and neural induction activities. Questions: Posterior neural ectoderm? No mesoderm induction?

18. A Animal cap In vitro culture Epidermis V Neural Tissue Dissociated into signal cell, 4-5hr Reaggregation and culture Blastula (stage 8) Inconsistency with Neural Induction Hypothesis (Grunz & Tacke, 1989; Godsave & Slack, 1991) The idea of a positive signal involved in neural induction so dominated thinking in the field that the significance of results inconsistent with this idea were not widely appreciated.

19. Truncated activin receptor induces neural tisse Ali Hemmati-Brivanlou & Douglas Melton Nature, 1992, 359, 609-614 D1XAR1 In vitro culture Neural Tissue 2-cell stage Blastula • Questions raised: • Activin is a mesoderm inducer, not a neural inducer. • The block in activin signal transduction autoinduces cells of the animal cap to switch to a neuronal fate in the absence of any detectable mesoderm. XAR1 D1XAR1 ??? Organizer Mesoderm Neural tissue

20. Experiments in Xenopus that support the default model Stern, Development, 2005

21. “Default Model” BMP inhibitors: Noggin, Chordin, Follistatin Wilson & Edlund, 2001

22. “Default Model” (Hemmati-Brivanlou & Melton, 1997)

23. The “default model” in Xenopus Stern, Development, 2005 Question: How do these BMP inhibitors antagonize BMPs’ function?

24. By Eddy De Robertis Group in UCLA

25. Chordin protein is secreted by cultured cells and by Xenopus organizer tissues VMZ DMZ Chordin is a 120 kDa secreted protein.

26. How could Chordin antagonize BMPs’ signaling? • Interfere with BMP maturation or secretion; • Act via a parallel pathway to that of the BMP-receptor interaction; • Bind to the BMP receptor; • Bind directly to mature BMPs.

27. Chordin protein inhibits the osteogenic activity of mature BMP proteins AP activity can also be induced by RA, but this activity could not be inhibited by Chordin. (BMP specific inhibition)

28. Chordin inhibits BMP-4 binding to its receptor (100X) (100X)

29. Chordin binds to BMPs but not to Activin Chd-BMP4 interaction could not compete by other GFs. Chd-BMP4 interaction could compete by BMP2, but not by activin and TGFb1.

30. Cross-linking analysis confirms the direct bind of Chordin and BMP4

31. Chordin binds to BMP4 with high affinity Chd could bind to BMP4 with the same affinity as BMP4 binds to its receptor. Chd could bind to BMP4 homodimer and BMP4/BMP7 heterodimer with same affinity.

32. Neural induction and mesoderm dorsalization by Chordin protein and was inhibited by BMP-4 Neural induction (without mesoderm induction) Mesoderm dorsalization (ventral marginal zone)

33. Molecular mechanism of Chordin antagonizes BMP4 signaling

34. By Richard Harland Group at UC Berkeley

35. Default Model in Chick and Mouse Early Development • Questions unsolved: • In HNF3b KO mice, there is no node, but the embryos have the neural tissues (Node = Organizer in Xenopus). • Neural induction is initiated before gastulation. • BMP antagonists are not required for neural induction.

36. An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo Wilson et al., Curr. Biol., 2000, 10, 421-429

37. Specification of cells of the stage XII chick embryo Neural markers Epidermal In stage XII chick embryo, medial epiblast differentiate into neural cells, while lateral epiblast acquire the epidermal character.

38. FGF signaling and BMP expression in stage XII epiblast cells Medial Lateral Bef. Cul. 40h+ SU Bef. Cul. 40h+ SU+Nog Cul. 40h Cul. 40h BMP4 BMP7 FGF3 S17 FGFR2b Acquisition of neural character is accompanied by repression of BMP4 and BMP7 expression. S17

39. BMP4 induces epidermal character in prospective neural cells in stage XII BMP4 1.3nM + Medial

40. The FGFR tyrosine kinase inhibitor SU5402 inhibits the acquisition of neural character Medial + SU5402 Medial + SU5402 +Noggin Medial +SU5402 +IB/Fc +IA/Fc Noggin: BMP binding protein IB/Fc & IA/Fc: soluble dominant negative BMP receptors.

41. The status of Wnt signaling regulates neural and epidermal fates in the chick embryo Nature, 2001, 411, 325-330 Wilson et al.,

42. Wnts, FGF3 and BMP4 expression in medial and lateral epiblast of chick embryo M: Medial epiblast; L: Lateral epiblast. mFrz8CRD-IgG: Soluble Frizzle 8, Wnt receptor inhibitor.

43. Regulation of neural and epidermal fate in medial epiblast Neural Markers Epidermal Markers

44. Regulation of neural and epidermal fate in lateral epiblast Epidermal Markers Neural Markers

45. Summary of Experiments

46. An unifying mechanism of “neural induction” ? FGF, WNT and BMP play important roles in neuralization of amniote embryos (humans, rodents and birds) Wilson et al., Nature Neurosci., 2001

47. Neural induction in chick embryos ---Embryologist’s view Stage XIII-2 Stage XI-XII Stage 3+-4 End of Stage 4 ???

48. Neural induction in chick embryos ---Genetic cascade

49. Models of neural induction Xenopus Chick

50. Establishment of A-P axis in neural plate Two-inducer model:Anterior and posterior neural inducers Two-step model: Nieuwkoop's activation–transformation model