NCAM>0, CG>0 NCAM<0, CG<0
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Laurent Kodjabachian LGPD/IBDM Marseille E-mail: kodja@ibdm.univ-mrs.fr 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 QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. QuickTime™ and a Photo - JPEG decompressor V D are needed to D see this picture. QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Unpigmented host embryo QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. QuickTime™ and a neural tube and a PhotoQuickTime™ - JPEG decompressor Photo JPEG decompressor are needed to see this picture. endoderm are needed to see this picture. 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
