8Major 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.
9History of Neural Induction Hypothesis Spemann Organizer ( )Default Model ( )Neural Induction in Chick (2001)Neural induction in Mouse (2007)
10Hans Spemann and Hilde Mangold Arch. Mikr. Anat. Entw. Mech. Induction of Embryonic Primordia by Implantation of Organizers from a Different SpeciesHans Spemann and Hilde MangoldArch. Mikr. Anat. Entw. Mech.100, , 1924
11Classical 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 ectodermNeural specification: needs a positive signal from neighboring cells (Neural Induction).“Default”: Cell autonomous.
13This 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.
16The “default model” in Xenopus BMP4NeuralEctodermChordinNogginFollistatinQuestion: How do these BMP inhibitors antagonize BMPs’ function?Stern, Development, 2005
17Default 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.
21An 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
22Neural induction in chick embryos ---Embryologist’s view Stage XI-XIIStage XIII-2Stage 3+-4End of Stage 4???
23Neural induction in chick embryos ---Genetic cascade
38Early post-implantation development in the mouse Nature Rev Gen, 8, 368, 2007
39Mouse Gastrulation and Germ Layer Formation Cell 132, 661–680, 2008
40Default 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???
41New findingsBMP signaling inhibits premature neural differentiation in the mouse embryoDevelopment 134, (2007)
43pSmad1/5/8: BMP pathway activated BMPR1a is essential for BMP signaling in the early mouse embryopSmad1/5/8: BMP pathway activated
44Premature neural differentiation of the epiblast occurs in BMPR1a-/- embryo WTPluripotent markers: Oct4, Nanog and Fgf5Bmpr1a-/-Neural stem cell markers: Six3, Hesx1 and Sox1WTBmpr1a-/-
45Suppression of mesoderm in BMPR1a-/- mouse embryo Mesoderm markers and mesoderm-inducing signals: FGF8, Eomes, T, Nodal, Cripto, Wnt3Note: Ectopic neural differentiation occurred in the same embryo
46BMP signaling is required in the epiblast for mesoderm specification and to inhibit neural differentiationE5.5E6.5E7.5WTBmpr1a epiblast-specific KO at E6.5WTBmpr1a epiblast-specific KO at E6.5E6.5E7.5
47Inhibition of FGF signaling does not block neural specification in BMPR1a-/- mouse embryo Hesx1 (neural marker)E6.5E5.5ControlEpiblast KOControlEpiblast KOBmpr1a-/-FGFs are not acting as direct neural inducers in the early post-implantation mouse embryo.
48Model for BMPs maintain epiblast pluripotency in mouse NodeBMP signaling is required to inhibit epiblast neural differentiationBMP2/4 signal via Bmpr1a to maintain epiblast pluripotency
51AVE protects pre-specified anterior neural tissue from posteriorization
52Establishment of A-P axis in neural plate Two-inducer model: Anterior and posterior neural inducersTwo-step model: Nieuwkoop's activation–transformation model
53A 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.BMP4NeuralEpiblastChordinNoggin
54A 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.
55Neural induction of the mouse embryo from E6.0 to 8.5 AVE: Anterior visceral endoderm; GO: Gastrula Organizer; AME: Anterior mesendodermYellow: AVE; Blue: Early neural markers; Orange: Primitive streak; Purple: AME
56Evolution of neural induction hypothesis Default modelin mouse(Mouse, 2007)FGF, WNT and BMPplay important roles(Chick, 2001)Default model(Xenopus, 1996)ChordinNogginFollistatinSpemann Organizer(Newt, 1924)BMP4EctodermNeural
57Function of BMP signaling in the epiblast of early embryo BMP signaling maintains epiblast pluripotency and prevents precocious neural differentiation of this tissueScientific questions:What are the downstream targets of BMP signaling?How does BMP signaling cross-talk with other pathways in its neural induction inhibition?
58Cell lineages in the early mouse embryo MorulamESCInner Cell MassTrophectodermPrimitive endodermEpiblastParietal endodermVisceral endodermDefinitive endodermMesodermEctodermliver pancreas lungblood heart skeletal muscleCNSskin
60E5.5-5.75, from late epiblast cells in egg cylinder stage Derivation of pluripotent epiblast stem cells (EpiSCs) from mouse embryosNature, 448, 2007New cell lines from mouse epiblast share defining features with human embryonic stem cellsNature, 448, 2007Derivation of pluripotent epiblast stem cells from mammalian embryosE , from late epiblast cells in egg cylinder stage
61Mouse ES cells and EpiSCs have distinct gene expression and culture condition GFs required to culture EpiSCsCelltypemESCmEpiSChESCGFsLIF BMP4FGF2ActivinNature 448, 2007Gene names shown in red were detected in hES cell cultures
62Cell lineages in the early mouse embryo MorulamESCInner Cell MassTrophectodermEpiSCPrimitive endodermEpiblastParietal endodermVisceral endodermDefinitive endodermMesodermEctodermliver pancreas lungblood heart skeletal muscleCNSskin
63Questions: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?
64Can ES cells recapitulate in vivo development? Anterior neuroectodermEpiblastLate epiblastICMmESEpiSCNSCOct4NanogRex1Oct4NanogRex1Fgf4Sox2Oct4NanogFgf5Sox1NestinMarkers
65Rex1+/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
66Reversible phenotypes of mouse Rex1+ and Rex1- populations GFP+/Rex1+GFP-/Rex1-Development 135, 2008
67Reversible subpopulations of Rex1+/Oct4+ and Rex1–/Oct4+ cells Development 135, 2008
68Heterogeneous expression of Stella in undifferentiated ESCs Stella-GFP ESCsCell Stem Cell 3, 2008
69ESCs display a state of dynamic equilibrium Cell Stem Cell 3, 2008
70Model for the maintenance in ESCs composed of distinct cell types in a dynamic equilibrium Early blastocystE4.5Late blastocystE5.5Egg cylinderCell Stem Cell 3, 2008
71Origin, culture conditions, and functional properties of different pluripotent stem cell lines FGF-Ativin-Bio-Blastocyst-Derived Stem Cells (FAB-SC)Cell 135, 2008
72FAB-SCs share features with EpiSCs and mESCs, but are distinct from both Cell 135, 2008
75Can neural induction of ES cells recapitulate in vivo development? ICMEarly epiblastLate epiblastNeural ectodermLIF/BMP4FGF2/ActivinESCEpiSCNSC??In vitroStella-Rex-Stella+Rex+Oct4 Nanog Fgf4 Sox2Oct4NanogNodalFgf5Sox1Nestin
76Epiblast-like stage is crucial for BMP inhibition of ES cell neural differentiation