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.
History of Neural Induction Hypothesis 1.Spemann Organizer ( ) 2.Default Model ( ) 3.Neural Induction in Chick (2001) 4.Neural induction in Mouse (2007)
Induction of Embryonic Primordia by Implantation of Organizers from a Different Species Hans Spemann and Hilde Mangold Arch. Mikr. Anat. Entw. Mech. 100, , 1924
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)
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.
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.
Stern, Development, 2005 The “default model” in Xenopus Question: How do these BMP inhibitors antagonize BMPs’ function? BMP4 Neural Ectoderm Chordin Noggin Follistatin
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.
Gastrulation in Chicken Embryo
The status of Wnt signaling regulates neural and epidermal fates in the chick embryo Wilson et al., Nature, 2001, 411,
Summary of Experiments
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?
Stage XI-XII Stage XIII-2 Stage End of Stage 4 ??? Neural induction in chick embryos ---Embryologist’s view
Neural induction in chick embryos ---Genetic cascade
Models of neural induction Xenopus Chick
Default Model in Chick and Mouse Early Development Questions unsolved: Why Xenopus and Chick or Mouse have used different mechanisms for neural induction?
Neural induction in mouse embryos
Early mouse development Preimplantation
Early mouse development E3.5 E4.5 ICM
Postimplantation Early mouse development (early) Epiblast (late) Epiblast (Primitive ectoderm) Anterior neuroectoderm VE: visceral endoderm AVE: anterior VE DVE: distal VE
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.
BMP signaling inhibits premature neural differentiation in the mouse embryo Development 134, (2007) New findings
BMP-Smads Signaling Pathway
BMPR1a is essential for BMP signaling in the early mouse embryo pSmad1/5/8: BMP pathway activated
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
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
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
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.
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
Tissues implicated in mouse neural induction NecessarySufficientCorrect time/place Signaling factors AVE Node GO No Yes No Yes Lefty, Cerberus Chordin, Noggin Chordin
AVE protects pre-specified anterior neural tissue from posteriorization
Establishment of A-P axis in neural plate Two-inducer model: Anterior and posterior neural inducers Two-step model: Nieuwkoop's activation–transformation model
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
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
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
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
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
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
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
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
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
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
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?
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
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
Reversible phenotypes of mouse Rex1 + and Rex1 - populations GFP + /Rex1 + GFP - /Rex1 - Development 135, 2008
Reversible subpopulations of Rex1 + /Oct4 + and Rex1 – /Oct4 + cells Development 135, 2008
Heterogeneous expression of Stella in undifferentiated ESCs Stella-GFP ESCs Cell Stem Cell 3, 2008
ESCs display a state of dynamic equilibrium A. B. Cell Stem Cell 3, 2008
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
Origin, culture conditions, and functional properties of different pluripotent stem cell lines Cell 135, 2008 FGF-Ativin-Bio-Blastocyst-Derived Stem Cells (FAB-SC)
FAB-SCs share features with EpiSCs and mESCs, but are distinct from both A. B. C. Cell 135, 2008
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 ? ?
Epiblast-like stage is crucial for BMP inhibition of ES cell neural differentiation