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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.

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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."— 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:1012 (1万亿) 细胞种类:神经元 (Neuron,许多种类) 细胞联系:神经元-神经元
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 ( )=

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) 二、神经系统的发生 (Neurogenesis)
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 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 (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
Spemann Organizer ( ) Default Model ( ) Neural Induction in Chick (2001) Neural induction in Mouse (2007)

10 Hans Spemann and Hilde Mangold Arch. Mikr. Anat. Entw. Mech.
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
The donor tissues could recruit the host cells to become the secondary neural tube. Dorsal blastopore lip (Hemmati-Brivanlou & Melton, 1997)

12 “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.

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

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

17 Default Model in Chick and Mouse Early Development
Questions unsolved: In Foxa2 (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.

18 Gastrulation in Chicken Embryo

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

20 Summary of Experiments

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

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

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
ICM E4.5

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

30 Pluripotent cell lineages in mouse embryo
Niwa, Development, 2007

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
Dye injection

33 The distal cap epiblast cells migrate to the anterior neuroectoderm

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

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

36 Cell movements in early mouse embryos

37 Cell movements in early mouse embryos

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
Questions unsolved: In Foxa2 (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. Why do Xenopus and Chick or Mouse use different mechanisms for neural induction???

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

42 BMP-Smads Signaling Pathway

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

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

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

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

47 Inhibition of FGF signaling does not block neural specification in BMPR1a-/- mouse embryo
Hesx1 (neural marker) E6.5 E5.5 Control Epiblast KO Control Epiblast KO Bmpr1a-/- FGFs are not acting as direct neural inducers in the early post-implantation mouse embryo.

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

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
Necessary Sufficient Correct time/place Signaling factors AVE No No Yes Lefty, Cerberus Node No Yes No Chordin, Noggin GO Yes Yes Yes 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
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. 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 A model for mouse neural induction
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. More posterior types of neural tissue are subsequently induced by sequential derivatives of the gastrula organizer (Node). The ultimate derivatives of the gastrula organizer and node form the anterior mesendoderm that stabilizes and maintains the overlying neural tissue.

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

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

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

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

60 E5.5-5.75, from late epiblast cells in egg cylinder stage
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
GFs required to culture EpiSCs Cell type mESC mEpiSC hESC GFs LIF BMP4 FGF2 Activin Nature 448, 2007 Gene names shown in red were detected in hES cell cultures

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

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

64 Can ES cells recapitulate in vivo development?
Anterior neuroectoderm Epiblast Late epiblast ICM mES EpiSC NSC Oct4 Nanog Rex1 Oct4 Nanog Rex1 Fgf4 Sox2 Oct4 Nanog Fgf5 Sox1 Nestin Markers

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
Cell Stem Cell 3, 2008

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

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

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

73 Growth factor stimulation induces FAB-SC pluripotency
Cell 135, 2008

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

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

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

77 Thank you!


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