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
神经元的种类
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
人脑的发育
(Gilbert, 1991)
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。
神经诱导 (Neural Induction)
多潜能干细胞
神经干细胞
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 (1924-1990)
2. Default Model (1992-1997)
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, 599-638, 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 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.
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.
“Default Model”
BMP inhibitors:
Noggin, Chordin,
Follistatin
Wilson & Edlund, 2001
“Default Model”
(Hemmati-Brivanlou & Melton, 1997)
The “default model” in Xenopus
BMP4
Ectoderm
Chordin
Noggin
Follistatin
Neural
Question: How do these BMP
inhibitors antagonize BMPs’
function?
Stern, Development, 2005
Default Model in Chick and Mouse
Early Development
Questions unsolved:
1. In Foxa2 (HNF3b) 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
Nature, 2001, 411, 325-330
Wilson et al.,
Summary of Experiments
An unifying mechanism of “neural induction”
?
FGF, WNT and BMP play
important roles in
neuralization of amniote
embryos (humans, rodents
and birds)
Question:
1.
How does FGF
induce neural?
2.
What about BMP
inhibitors?
Wilson et al., Nature Neurosci., 2001
Neural induction in chick embryos
---Embryologist’s view
Stage XIII-2
Stage XI-XII
Stage 3+-4
End of Stage 4
???
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
ICM
E4.5
Early mouse development
Postimplantation
(early) Epiblast
VE: visceral endoderm
AVE: anterior VE
DVE: distal VE
(late) Epiblast
(Primitive ectoderm)
Anterior
neuroectoderm
Pluripotent cell lineages in mouse embryo
E3.0
E3.5
E4.5
E5.5
Niwa, Development, 2007
Neuroectodermal fate of epiblast cells in the distal region of the
mouse egg cylinder: implication for body plan organization
during early embryogenesis
Development 121, 87-98 (1995)
Fate-mapping of the distal cap epiblast
by carbocyanine dye labeling
E6.5
Dye injection
The distal cap epiblast cells migrate to
the anterior neuroectoderm
Cell movements in early mouse embryos
Distal VE to AVE
Epiblast to
posterior
Extraembryonic
ectoderm to posterior
epiblast
Epiblast to
posterior
Movement of Otx2-positive cells from DVE to AVE
5.5 dpc
5.75 dpc
6.0 dpc
6.25 dpc
Otx2 VEcis-lacZ
Otx2 KI-lacZ
DVE: distal visceral endoderm; AVE: anterior visceral endoderm
Cell movements in early mouse embryos
Cell movements in early mouse embryos
Early post-implantation development in the mouse
Nature Rev Gen, 8, 368, 2007
Mouse Gastrulation and Germ Layer Formation
Cell 132, 661–680, 2008
Default Model in Chick and Mouse
Early Development
Questions unsolved:
1. In Foxa2 (HNF3b) 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.
Why do Xenopus and Chick or Mouse use
different mechanisms for neural induction???
New findings
BMP signaling inhibits premature neural
differentiation in the mouse embryo
Development 134, 3359-3369 (2007)
BMP-Smads Signaling Pathway
BMPR1a is essential for
BMP signaling in the early
mouse embryo
pSmad1/5/8: BMP pathway activated
Premature neural differentiation of the epiblast
occurs in BMPR1a-/- embryo
WT
Pluripotent
markers: Oct4,
Nanog and Fgf5
Bmpr1a-/-
WT
Bmpr1a-/-
Neural stem cell
markers: Six3,
Hesx1 and Sox1
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
BMP signaling is required in the epiblast for mesoderm
specification and to inhibit neural differentiation
E5.5
E6.5
E7.5
WT
Bmpr1a epiblastspecific KO at E6.5
WT
Bmpr1a epiblastspecific KO at E6.5
E6.5
E7.5
Inhibition of FGF signaling does not block neural
specification in BMPR1a-/- mouse embryo
Hesx1 (neural marker)
E5.5
E6.5
Control
Epiblast KO
Control
Epiblast KO
Bmpr1a-/-
FGFs are not acting as direct neural inducers in the early postimplantation mouse embryo.
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
Signaling centers and molecules implicated
in neural induction
AVE: Anterior visceral endoderm; MGO: Mid-gastrula organizer; EE: Extra-embryonic
region; PS: Primitive streak
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
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
Epiblast
Chordin
Noggin
Neural
A model for mouse neural induction
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.
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
Evolution of neural induction hypothesis
Default model
in mouse
(Mouse, 2007)
Default model
FGF, WNT and BMP
play important roles
(Chick, 2001)
(Xenopus, 1996)
BMP4
Ectoderm
Chordin
Noggin
Follistatin
Neural
Spemann Organizer
(Newt, 1924)
Function of BMP signaling in the epiblast of
early embryo
BMP
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?
Cell lineages in the early mouse embryo
Morula
mESC
Inner Cell Mass
Primitive
endoderm
Parietal
endoderm
Visceral
endoderm
Trophectoderm
Epiblast
Definitive
endoderm
liver
pancreas
lung
Mesoderm
blood
heart skeletal
muscle
Ectoderm
CNS
skin
Pluripotent cell lineages in mouse embryo
E3.0
E3.5
E4.5
E5.5
Morula
Early blastocyst
Late blastocyst
Egg cylinder
mES cells, 1981
Late epiblast
hES cells, 1998
Development, 134, 2007
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
E5.5-5.75, from late epiblast cells
in egg cylinder stage
Mouse ES cells and EpiSCs have distinct
gene expression and culture condition
GFs required to culture EpiSCs
Gene names shown in red were detected in hES cell cultures
Cell
type
mESC
mEpiSC
hESC
GFs
LIF
BMP4
FGF2
Activin
FGF2
Activin
Nature 448, 2007
Cell lineages in the early mouse embryo
Morula
mESC
Inner Cell Mass
Primitive
endoderm
Parietal
endoderm
Visceral
endoderm
Trophectoderm
Epiblast
Definitive
endoderm
liver
pancreas
lung
Mesoderm
blood
heart skeletal
muscle
EpiSC
Ectoderm
CNS
skin
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?
Anterior
neuroectoderm
E3.5
Markers
E4.5
E5.5
E7.5
Epiblast
Late epiblast
ICM
mES
EpiSC
NSC
Oct4
Nanog
Rex1
Oct4
Nanog
Rex1
Fgf4
Sox2
Oct4
Nanog
Fgf5
Sox1
Nestin
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
E3.5
Early blastocyst
E4.5
Late blastocyst
E5.5
Egg cylinder
Cell Stem Cell 3, 2008
Origin, culture conditions, and functional properties
of different pluripotent stem cell lines
FGF-Ativin-Bio-Blastocyst-Derived Stem Cells (FAB-SC)
Cell 135, 2008
FAB-SCs share features with EpiSCs and mESCs,
but are distinct from both
A.
C.
B.
Cell 135, 2008
Growth factor stimulation induces
FAB-SC pluripotency
A.
B.
C.
Cell 135, 2008
Capturing Pluripotency
Maintaining pluripotency
A Metastable Coalition
Cell 132, 2008
Can neural induction of ES cells recapitulate
in vivo development?
In vivo
ICM
Early epiblast
Late epiblast
LIF/BMP4
FGF2/Activin
ESC
EpiSC
?
In vitro
NSC
?
Stella-
Stella+
Rex+
Neural ectoderm
Oct4
Nanog
Fgf4
Sox2
Rex-
Oct4
Nanog
Nodal
Fgf5
Sox1
Nestin
Epiblast-like stage is crucial for BMP inhibition
of ES cell neural differentiation
Thank you!