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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. 人脑的构成.

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Presentation on theme: "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. 人脑的构成."— Presentation transcript:

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: (1 万亿 ) 其中:神经细胞(神经元) 神经胶质细胞: 9 X 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 X ( )=

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?多潜能干细胞是如何分化为神经干细胞的? 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, Adult Neural Stem Cell) 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;成体神经 干细胞分化的机制。

7 多潜能干细胞 神经诱导 (Neural Induction) 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 (not fully committed). 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 1.Spemann Organizer ( ) 2.Default Model ( ) 3.Neural Induction in Chick (2001) 4.Neural induction in Mouse (2007)

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

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

12 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. “Spemann Organizer” Epidermis: “Default” fate for gastrula ectoderm Neural specification: needs a positive signal from neighboring cells (Neural Induction). “Default”: Cell autonomous.

13 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.

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

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

16 Stern, Development, 2005 The “default model” in Xenopus Question: How do these BMP inhibitors antagonize BMPs’ function? BMP4 Neural Ectoderm Chordin Noggin Follistatin

17 Default Model in Chick and Mouse Early Development Questions unsolved: 1.In Foxa2 (HNF3  KO mice, there is no node, but the embryos have the neural tissues (Node = Organizer in Xenopus). 2.Neural induction is initiated before gastulation. 3.BMP antagonists are not required for neural induction.

18 Gastrulation in Chicken Embryo

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

20 Summary of Experiments

21 FGF, WNT and BMP play important roles in neuralization of amniote embryos (humans, rodents and birds) Wilson et al., Nature Neurosci., 2001 An unifying mechanism of “neural induction” ? Question: 1.How does FGF induce neural? 2.What about BMP inhibitors?

22 Stage XI-XII Stage XIII-2 Stage End of Stage 4 ??? Neural induction in chick embryos ---Embryologist’s view

23 Neural induction in chick embryos ---Genetic cascade

24 Models of neural induction Xenopus Chick

25 Default Model in Chick and Mouse Early Development Questions unsolved: Why Xenopus and Chick or Mouse have used different mechanisms for neural induction?

26 Neural induction in mouse embryos

27 Early mouse development Preimplantation

28 Early mouse development E3.5 E4.5 ICM

29 Postimplantation Early mouse development (early) Epiblast (late) Epiblast (Primitive ectoderm) Anterior neuroectoderm VE: visceral endoderm AVE: anterior VE DVE: distal VE

30 Pluripotent cell lineages in mouse embryo Niwa, Development, 2007 E3.5 E4.5E5.5 E3.0

31 Neuroectodermal fate of epiblast cells in the distal region of the mouse egg cylinder: implication for body plan organization during early embryogenesis Development 121, (1995)

32 Fate-mapping of the distal cap epiblast by carbocyanine dye labeling E6.5Dye injection

33 The distal cap epiblast cells migrate to the anterior neuroectoderm

34 Distal VE to AVE Cell movements in early mouse embryos Epiblast to posterior Extraembryonic ectoderm to posterior epiblast Epiblast to posterior

35 Otx2 VEcis-lacZ 5.5 dpc 5.75 dpc 6.25 dpc 6.0 dpc Otx2 KI-lacZ Movement of Otx2-positive cells from DVE to AVE DVE: distal visceral endoderm; AVE: anterior visceral endoderm

36 Cell movements in early mouse embryos

37

38 Early post-implantation development in the mouse Nature Rev Gen, 8, 368, 2007

39 Mouse Gastrulation and Germ Layer Formation Cell 132, 661–680, 2008

40 Default Model in Chick and Mouse Early Development Why do Xenopus and Chick or Mouse use different mechanisms for neural induction??? Questions unsolved: 1.In Foxa2 (HNF3  KO mice, there is no node, but the embryos have the neural tissues (Node = Organizer in Xenopus). 2.Neural induction is initiated before gastulation. 3.BMP antagonists are not required for neural induction.

41 BMP signaling inhibits premature neural differentiation in the mouse embryo Development 134, (2007) New findings

42 BMP-Smads Signaling Pathway

43 BMPR1a is essential for BMP signaling in the early mouse embryo pSmad1/5/8: BMP pathway activated

44 WT Bmpr1a-/- WT Bmpr1a-/- Pluripotent markers: Oct4, Nanog and Fgf5 Neural stem cell markers: Six3, Hesx1 and Sox1 Premature neural differentiation of the epiblast occurs in BMPR1a -/- embryo

45 Suppression of mesoderm in BMPR1a -/- mouse embryo E6.5 Mesoderm markers and mesoderm-inducing signals: FGF8, Eomes, T, Nodal, Cripto, Wnt3 Note: Ectopic neural differentiation occurred in the same embryo

46 Bmpr1a epiblast- specific KO at E6.5 WT BMP signaling is required in the epiblast for mesoderm specification and to inhibit neural differentiation E7.5 E6.5 E5.5 Bmpr1a epiblast- specific KO at E6.5 E7.5 E6.5

47 Inhibition of FGF signaling does not block neural specification in BMPR1a -/- mouse embryo E5.5 E6.5 Hesx1 (neural marker) Control Epiblast KO ControlEpiblast KOBmpr1a-/- FGFs are not acting as direct neural inducers in the early post- implantation mouse embryo.

48 BMP signaling is required to inhibit epiblast neural differentiation BMP2/4 signal via Bmpr1a to maintain epiblast pluripotency Model for BMPs maintain epiblast pluripotency in mouse Node

49 Signaling centers and molecules implicated in neural induction AVE: Anterior visceral endoderm; MGO: Mid-gastrula organizer; EE: Extra-embryonic region; PS: Primitive streak

50 Tissues implicated in mouse neural induction NecessarySufficientCorrect time/place Signaling factors AVE Node GO No Yes No Yes Lefty, Cerberus Chordin, Noggin Chordin

51 AVE protects pre-specified anterior neural tissue from posteriorization

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

53 A model for mouse neural induction 1.The early mouse embryo exists in a pre-anterior neural state and that this cell fate must be blocked to allow the formation of other tissues. 2.The actual “Induction” of neural tissue during early gastrulation begins when the early/mid-gastrula organizer inhibits these posterior signals (a double negative) and thus protects a local region of the epiblast, allowing it to remain as prospective anterior neural tissue. BMP4 Neural Epiblast Chordin Noggin

54 3.The specified anterior neural cells move from the distal epiblast to the anterior epiblast, to be juxtaposed with the AVE that expresses inhibitors of posteriorizing factors to protect the pre-specified anterior neural tissue from acquiring posterior character. 4.More posterior types of neural tissue are subsequently induced by sequential derivatives of the gastrula organizer (Node). 5.The ultimate derivatives of the gastrula organizer and node form the anterior mesendoderm that stabilizes and maintains the overlying neural tissue. A model for mouse neural induction

55 Neural induction of the mouse embryo from E6.0 to 8.5 AVE: Anterior visceral endoderm; GO: Gastrula Organizer; AME: Anterior mesendoderm Yellow: AVE; Blue: Early neural markers; Orange: Primitive streak; Purple: AME

56 Spemann Organizer (Newt, 1924) Default model (Xenopus, 1996) FGF, WNT and BMP play important roles (Chick, 2001) Default model in mouse (Mouse, 2007) BMP4 NeuralEctoderm Chordin Noggin Follistatin Evolution of neural induction hypothesis

57 Function of BMP signaling in the epiblast of early embryo What are the downstream targets of BMP signaling? How does BMP signaling cross-talk with other pathways in its neural induction inhibition? Scientific questions: BMP signaling maintains epiblast pluripotency and prevents precocious neural differentiation of this tissue BMP

58 mESC Cell lineages in the early mouse embryo Morula Inner Cell Mass Trophectoderm Primitive endoderm Epiblast Parietal endoderm Visceral endoderm Definitive endoderm MesodermEctoderm liver pancreas lung blood heart skeletal muscle CNS skin

59 Pluripotent cell lineages in mouse embryo Development, 134, 2007 E3.5 Early blastocyst E4.5 Late blastocyst E5.5 Egg cylinder E3.0 Morula Late epiblast mES cells, 1981 hES cells, 1998

60 Derivation of pluripotent epiblast stem cells (EpiSCs) from mouse embryos Nature, 448, 2007 New cell lines from mouse epiblast share defining features with human embryonic stem cells Nature, 448, 2007 Derivation of pluripotent epiblast stem cells from mammalian embryos E , from late epiblast cells in egg cylinder stage

61 Mouse ES cells and EpiSCs have distinct gene expression and culture condition Gene names shown in red were detected in hES cell cultures Cell type mESCmEpiSChESC GFs LIF BMP4 FGF2 Activin FGF2 Activin GFs required to culture EpiSCs Nature 448, 2007

62 mESC Cell lineages in the early mouse embryo Morula Inner Cell Mass Trophectoderm Primitive endoderm Epiblast Parietal endoderm Visceral endoderm Definitive endoderm MesodermEctoderm liver pancreas lung blood heart skeletal muscle CNS skin EpiSC

63 Questions: 1.Do ES cells represent cell states in early embryos or are they only the artifact of culture condition? 2.Does ES cell in vitro differentiation recapitulate in vivo early embryo development?

64 Can ES cells recapitulate in vivo development? ICM mES EpiSCNSC Markers Oct4 Nanog Rex1 Oct4 Nanog Rex1 Fgf4 Sox2 Oct4 Nanog Fgf5 Sox1 Nestin Epiblast Late epiblast E3.5E4.5 E5.5E7.5 Anterior neuroectoderm

65 Rex1 + /Oct4 + and Rex1 - /Oct4 + subpopulations in undifferentiated ES cell culture Rex1-GFP (Rex1 + ) Rex1-GFP/Oct4-CFP (Rex1 + /Oct4 + ) Rex1-GFP/Oct4-CFP (Rex1 - /Oct4 + ) Development 135, 2008

66 Reversible phenotypes of mouse Rex1 + and Rex1 - populations GFP + /Rex1 + GFP - /Rex1 - Development 135, 2008

67 Reversible subpopulations of Rex1 + /Oct4 + and Rex1 – /Oct4 + cells Development 135, 2008

68 Heterogeneous expression of Stella in undifferentiated ESCs Stella-GFP ESCs Cell Stem Cell 3, 2008

69 ESCs display a state of dynamic equilibrium A. B. Cell Stem Cell 3, 2008

70 Model for the maintenance in ESCs composed of distinct cell types in a dynamic equilibrium Cell Stem Cell 3, 2008 E3.5 Early blastocyst E4.5 Late blastocyst E5.5 Egg cylinder

71 Origin, culture conditions, and functional properties of different pluripotent stem cell lines Cell 135, 2008 FGF-Ativin-Bio-Blastocyst-Derived Stem Cells (FAB-SC)

72 FAB-SCs share features with EpiSCs and mESCs, but are distinct from both A. B. C. Cell 135, 2008

73 Growth factor stimulation induces FAB-SC pluripotency A. B.C. Cell 135, 2008

74 Capturing Pluripotency Cell 132, 2008 Maintaining pluripotency A Metastable Coalition

75 Can neural induction of ES cells recapitulate in vivo development? ICMEarly epiblast Late epiblastNeural ectoderm Oct4 Nanog Fgf4 Sox2 Oct4 Nanog Nodal Fgf5 Sox1 Nestin EpiSCNSC ESC Stella+ Rex+ Stella- Rex- LIF/BMP4 FGF2/Activin In vivo In vitro ? ?

76 Epiblast-like stage is crucial for BMP inhibition of ES cell neural differentiation

77 Thank you!


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