NCAM>0, CG>0 NCAM<0, CG<0

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Laurent Kodjabachian
LGPD/IBDM
Marseille
E-mail: [email protected]
EARLY REGIONALISATION
IN VERTEBRATES
HOW TO GO FROM ONE CELL TO A FULLY
PATTERNED EMBRYO ?
fertilisation
breaking
symmetry
egg
organiser
formation
MBT
blastula
mesendoderm
and neural
inductions
gastrula
A/P
D/V
L/R
patterning
neurula
Are we all the same ? : The phylotypic stage
Romanes, 1901
Various modes of gastrulation in vertebrates
Xenopus
chick
mouse
zebrafish
Niehrs, 2004
HOW TO BREAK SYMMETRY ?
Translocation of the maternal dorsalising activity
Weaver and Kimelman, 2004
The β-catenin destruction complex
Weaver and Kimelman, 2004
Model for the transport of the dorsalising activity
Weaver and Kimelman, 2004
Embryonic inductions
Mesoderm induction
Nieuwkoop, 1969
Dorsal-ventral gradient of mesoderm inducers
Fate maps and organising centers in Xenopus
Combinatorial activation of Nodal signalling initiates
mesendoderm induction
De Robertis et al., 2000
HOW TO POSITION AND RESTRICT
EMBRYONIC AXIS FORMATION ?
Sequential inhibitions during primitive streak formation
blastula
early gastrula
Bertocchini et al., 2004
Regulation of primitive streak formation
by Nodal antagonists in mouse
blastula
early gastrula
Perea-Gomez et al., 2002
Spemann’s organiser
Spemann’s organiser transplantation
V
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Unpigmented host embryo
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neural
tube
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notochord
somite
Pigmented donor embryo
Lemaire and Kodjabachian, 1996
What does Spemann’s organiser ?
1. It induces neural tissue
2. It dorsalises the mesoderm
3. It differentiates into axial tissues
(prechordal mesoderm and notochord)
4. It orchestrates gastrulation movements
Development without organiser
UV
cold
pressure
Nocodazole
β-catenin antisense
untreated
De Robertis et al. 2000
The amount of organiser tissue influences A/P and D/V patterning
UV
LiCl
Gerhart, 2001
Spemann’s organiser is the source of
BMP and WNT antagonists:
is dorsal the ground state ?
Epidermal ectoderm
BMPs
WNTs
mesoderm
Neural ectoderm
chordin, noggin, follistatin
cerberus, dkk1, frzb
Dorsal endoderm
HOW MANY ORGANISERS ?
A/P patterning by organizer derived tissues
Mangold, 1933
Leading models of A/P patterning
Distinct organisers model
ectoderm
Spemann’s
organiser
forebrain
hindbrain
spinal
chord
Head
organiser
Trunk
organiser
Tail
organiser
Nieuwkoop two-signal model
prechordal
plate
chordamesoderm
activator
transformer
activator
forebrain
ectoderm
hindbrain
spinal
chord
transformer
Combinatorial actions of BMP, Wnt and Nodal
Niehrs, 2004
Different organiser sub-domains emit different antagonists
Frzb1
Dkk1
Wnt
Chordin
Noggin
Follistatin
cerberus
Wnt
Cerberus
Dkk1
nodal BMP
Frzb1
Dkk1
Noggin
Follistatin
BMP
Chordin
Niehrs, 1999
Two-inhibitor model
Wnts
forebrain
ectoderm
Wnts,BMPs
anti-Wnt
anti-BMP
anti-BMP
anti-Wnt
anti-BMP
anti-BMP
anti-Wnt
anti-BMP
hindbrain
spinal
chord
anti-BMP
Niehrs, 1999
The time/space translator system in the trunk
Wacker et al., 2004
NEURAL INDUCTION
The Neural Determination Pathway
neural induction
uncommitted
ectoderm
epidermal
induction
neural
precursors
consolidation
stable neural
progenitors
maturation
epidermal
precursors
epidermis
mature
neural cells
NCAM>0
Neural induction may be planar or vertical, and is
complete by the end of gastrulation
animal
A
Org
V
vegetal
Blastula
D
P
Early gastrula
Late gastrula
neuroectoderm (Sox2)
epidermis (cytokeratin 81)
mesoderm (Xbra)
Neural induction in Xenopus by inhibition of BMP
intact
epidermis
dissociated
neural
tissue
epidermis
+BMP4
noggin
BMP4
neural
tissue
Different modes of BMP inhibition involved in
neural induction
β-catenin
chordin
noggin
1
CHD
NOG...
bmp4
2
BMP4, 2...
SMAD1
neural tissue
forebrain character
ERK
3
epidermis
Neural induction in chick
epidermis
neural tissue / forebrain character
blastula stage
+ BMP inhibition
epidermis
+ BMP
epidermis
+ FGFR inhibition
epidermis
After Wilson and Edlund, 2001
The BMP pathway and ectodermal specification
Munoz-Sanjuan and Brivanlou, 2002
IS THE DEFAULT MODEL CORRECT ?
BMP inhibition is necessary for neural induction
CABR (constitutively
active BMPR)
Injection in
prospective
neural plate :
K81
Sox2
BMP inhibition in vivo is not sufficient for neural induction
nlacZ
Injection in
prospective
epidermis
nlacZ + Smad 6
K81 lacZ
nlacZ + Smad6
or
nlacZ + dnBMPR (similar results)
Sox2 lacZ
0/57
ventral view, early neurula
0/50
Ventral ectoderm cells respond to BMP inhibition
nlacZ + Smad6
uninj.
Sox2
nlacZ + Smad 6
0/21
Sox2 lacZ
15/24
Combined BMP inhibition and FGF signalling induces
neural tissue in vivo
0.16 pg
eFGF
Xbra
0/9>0
K81
Xbra
0/20>0
K81
6/10 reduced
Sox2
1/12>0
Sox2
21/21>0
Smad6 +
1pg v-ras
Smad6 +
0.16 pg
eFGF
Sox2
12/12>0
Addressing the role of FGFR signalling in Xenopus neural induction
FRS2
PKC
SU5402
RAS
MA PK
dnFGFR=truncated receptor
Phenotypic classes of SU5402 treated embryos
classI
untreated
SU 5µM
classII
classIII
SU 10µM
SU 20µM
classIV
ey
SU 40µM
cg
classV
SU 80µM
SU5402 suppresses neural induction
Sox2
(early neural )
control
SU5402
classV
NCAM
(late neural)
slug
(neural crests)
Dominant-negative FGFR4 suppresses neural induction
∆XFGFR-4a
∆XFGFR-4a
∆XFGFR-4a
+ wild type
XFGFR4
+ tR4 (consitutively
active FGFR4)
NCAM
11/14<0, 3 weak, 0>0
5/26<0, 13 weak, 8>0
4/12<0, 5 weak, 3>0
10/11>0
6/10>0
Sox2
0/12>0
Prospective neural tissue is converted into epidermis
in FGFR deficient embryos
ctrl
nlacZ
SU, classV
front view
Close up after
ISH
nlacZ
K81
Neural induction requires FGFR signalling
up until early gastrula stages
cont.
cV
cIV
cIII
Mesoderm does not form in classIV/V embryos
control
class V
somitic mesoderm
Xbra
control
Xnr2
somitic muscle
class IV
Xnot2
Neural induction requires FGF signaling in the ectoderm
n=25
n=23
n=20
n=19
n=21
st. 11
75
50
+ SU
10
25
0
Cont. DMZ
Cont. cap
SU cap
NCAM>0
NCAM<0
Mesoderm-neurectoderm conversion upon
down-regulation of FGFR activity
XBra
untreated
cIII
untreated
cIII
Sox2
Fate changes upon
down-regulation of FGFR activity
lacZ
+/- SU5402
untreated
mesoderm
cIII lateral view
neural ectoderm
FGF signalling intensity
CG
Xgal staining st30
cV lateral view
epidermis
BMP signalling may be upregulated in absence of FGF
signalling
Control
Class V
Control
Class V
Chordin
Noggin
Cerberus
Bmp4
-> FGF signalling may act in a BMP-dependent manner
FGFR signalling is required in ectoderm for neural induction
by Noggin RNA
FGFR signalling is required for neural induction by Smad6 RNA
Smad6
Smad6
+/- SU
Smad6 + SU
NCAM
FGFR signalling is required to confer the ectoderm its competence
to be neuralised by Noggin protein
100
n=20
n=20
n=38
n=36
75
NCAM>0, CG>0
50
NCAM<0, CG<0
25
SU
NOGGIN
-
+
-
+
+
+
Neural induction by Noggin protein requires intact FGFR signalling
24h
+NOGGIN (1µg/ml)
+SU5402 (80 µM)
st9.5
100
n=40
αNCAM
n=38
75
NCAM>0, CG>0
50
NCAM<0, CG<0
25
0
NOG
NOG
SU5402
BMP inhibition in vivo cannot compensate for absence of FGF signalling
NOGGIN protein
2-cell
Control
NCAM
SU
blastula
SU
SU + NOGGIN
-> FGF signalling also acts via a BMP/SMAD1-independent pathway
A new model for neural induction in Xenopus…
β-catenin
chd
nog
CHD
NOG
bmp
BMP
epidermis
neural
w
o
l
fgf
FGF
hig
h
Veg-T
xnrs
XNRs
mesoderm
Regulation between gastrulation and neurulation
Sheng et al., 2003
Conclusions
Neural induction in vivo does not occur by default
Pre-gastrula FGFR signalling is required for neural induction
and involves both BMP/SMAD1-dependent and -independent
mechanisms
Neural induction involves multiple decisions between
epidermal, neural and mesodermal identities
A now re-unified view of neural induction in chordates
implicates FGF as the initiator of this process
Opened questions
Nature of the BMP negative ventral ectoderm cells
Origin and identity of the endogenous FGF ligand(s) involved
in neural induction
Identify direct targets of FGF that are required for neural
induction and independent of BMP
Evolutionary conservation of the role of the FGF/ERK
pathway in neural specification outside chordates
BMP signalling and ectodermal cell fates
Munoz-Sanjuan and Brivanlou, 2002
Double gradient model of embryonic axis formation
Niehrs, 2004
Nieuwkoop’s Activation-Transformation Model
Stern, 2001
Modified Nieuwkoop’s model
Stern, 2001
Neural induction assays in the chick
Streit and Stern, 1999
The « Default Model » of neural induction in xenopus
Munoz-Sanjuan and Brivanlou, 2002
Mouse A/P axis establishment and patterning at gastrulation
Rossant and Tam, 2004
Inductive centers in mouse and chick
Stern, 2001
Cell movements separate prospective forebrain from posterior tissues
Wilson and Houart, 2004
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