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？
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, Neural Stem Cells)
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；神经干细
胞和胚胎干细胞神经分化的分子机制。
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.
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 and Mouse (2001)
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
Development of Xenopus embryo
Early gastrula: Dorsal blastopore lip
Fate map of the Xenopus gastrula
(Hemmati-Brivanlou & Melton, 1997)
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.
Neural Induction Hypothesis
Organizer
Mesoderm (Notochord)
Neural plate
Kessler & Melton, 1994
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.
Animal Cap Assay
A
Animal cap
In vitro
culture
V
Blastula
(stage 8)
Markers expression
(RT-PCR) :
Mesoderm
Neural ectoderm
Epidermis
Endoderm
In early 1980, TGF-b and FGF family members have been found to
have the mesodermal and neural induction activities.
Questions: Posterior neural ectoderm?
No mesoderm induction?
Inconsistency with Neural Induction Hypothesis
(Grunz & Tacke, 1989; Godsave & Slack, 1991)
A
Animal cap
In vitro
culture
V
Blastula
(stage 8)
Dissociated into
signal cell, 4-5hr
Reaggregation
and culture
Epidermis
Neural
Tissue
The idea of a positive signal involved in neural induction so
dominated thinking in the field that the significance of
results inconsistent with this idea were not widely
appreciated.
Truncated activin receptor induces neural tisse
Ali Hemmati-Brivanlou & Douglas Melton
Nature, 1992, 359, 609-614
D1XAR1
In vitro
culture
2-cell stage
Neural
Tissue
Blastula
Questions raised:
XAR1
1.
Activin is a mesoderm inducer, not a neural
inducer.
2.
The block in activin signal transduction
autoinduces cells of the animal cap to switch to a
neuronal fate in the absence of any detectable
mesoderm.
D1XAR1
Organizer
Mesoderm
???
Neural tissue
Experiments in Xenopus that support the default model
Stern, Development, 2005
“Default Model”
BMP inhibitors:
Noggin, Chordin,
Follistatin
Wilson & Edlund, 2001
“Default Model”
(Hemmati-Brivanlou & Melton, 1997)
The “default model” in Xenopus
Stern, Development, 2005
Question: How do these BMP inhibitors antagonize BMPs’ function?
By Eddy De Robertis Group in UCLA
Chordin protein is secreted by cultured cells and
by Xenopus organizer tissues
VMZ
Chordin is a 120 kDa secreted protein.
DMZ
How could Chordin antagonize BMPs’ signaling?
1. Interfere with BMP maturation or secretion;
2. Act via a parallel pathway to that of the
BMP-receptor interaction;
3. Bind to the BMP receptor;
4. Bind directly to mature BMPs.
Chordin protein inhibits the osteogenic
activity of mature BMP proteins
AP activity can also be induced by RA, but this activity could
not be inhibited by Chordin. (BMP specific inhibition)
Chordin inhibits BMP-4 binding to its receptor
(100X)
(100X)
Chordin binds to BMPs but not to Activin
Chd-BMP4 interaction
could not compete by
other GFs.
Chd-BMP4 interaction
could compete by BMP2,
but not by activin and
TGFb1.
Cross-linking analysis confirms the direct
bind of Chordin and BMP4
Chordin binds to BMP4 with high affinity
Chd could bind to BMP4 with the
same affinity as BMP4 binds to its
receptor.
Chd could bind to BMP4
homodimer and BMP4/BMP7
heterodimer with same affinity.
Neural induction and mesoderm dorsalization by
Chordin protein and was inhibited by BMP-4
Neural induction
(without mesoderm induction)
Mesoderm dorsalization
(ventral marginal zone)
Molecular mechanism of Chordin
antagonizes BMP4 signaling
By Richard Harland Group at UC Berkeley
Default Model in Chick and Mouse
Early Development
Questions unsolved:
1. In 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.
An early requirement for FGF
signaling in the acquisition of neural
cell fate in the chick embryo
Wilson et al., Curr. Biol.,
2000, 10, 421-429
Specification of cells of the stage XII chick embryo
Neural markers
Epidermal
In stage XII chick embryo, medial epiblast
differentiate into neural cells, while lateral
epiblast acquire the epidermal character.
FGF signaling and BMP expression
in stage XII epiblast cells
Medial
Bef.
Cul.
Cul.
40h
Lateral
40h+ 40h+
Bef.
SU
SU+Nog Cul.
Cul.
40h
BMP4
BMP7
FGF3
S17
FGFR2b
S17
Acquisition of neural character is
accompanied by repression of BMP4
and BMP7 expression.
BMP4 induces epidermal character in
prospective neural cells in stage XII
BMP4 1.3nM
+ Medial
The FGFR tyrosine kinase inhibitor SU5402
inhibits the acquisition of neural character
Medial
+ SU5402
Medial
+ SU5402 +Noggin
Medial +SU5402
+IB/Fc +IA/Fc
Noggin: BMP binding protein
IB/Fc & IA/Fc: soluble dominant negative BMP receptors.
The status of Wnt signaling regulates neural
and epidermal fates in the chick embryo
Nature, 2001, 411, 325-330
Wilson et al.,
Wnts, FGF3 and BMP4 expression in
medial and lateral epiblast of chick embryo
M: Medial epiblast; L: Lateral epiblast.
mFrz8CRD-IgG: Soluble Frizzle 8, Wnt receptor inhibitor.
Regulation of neural and epidermal fate in medial epiblast
Neural Markers
Epidermal Markers
Regulation of neural and epidermal fate in lateral epiblast
Neural Markers
Epidermal Markers
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)
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
Establishment of A-P axis in neural plate
Two-inducer model: Anterior and posterior neural inducers
Two-step model: Nieuwkoop's activation–transformation model
Early mouse development
Preimplantation
Early mouse development
Postimplantation
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
Molecular signals control axis formation
Nodal signaling network controls A-P axis
formation in mouse embryos
Model for visceral endoderm in forebrain development
Cell lineages in the early mouse embryo
Cell lineages in the early mouse embryo
Morula
Inner cell mass
Primitive
endoderm
Parietal
endoderm
Visceral
endoderm
Trophectoderm
Epiblast
Definitive
endoderm
Mesoderm
Ectoderm
Liver
Pancreas
Blood
Muscle
CNS
Skin
Neural stem cell lineages during early
development
FGF
???
(Wnt)
?
ICM
BMP
?
Epiblast
?
Neuroectoderm
Neural induction
Thank you!
Cell movements in early mouse embryos
Early mouse development
Early mouse development