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Sonic Hedgehog Regulates Integrin Activity, Cadherin Contacts, and Cell Polarity to Orchestrate Neural Tube Morphogenesis

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Sonic Hedgehog Regulates Integrin Activity, Cadherin Contacts, and Cell Polarity to Orchestrate Neural Tube Morphogenesis 12506 • The Journal of Neuroscience, October 7, 2009 • 29(40):12506–12520 Development/Plasticity/Repair Sonic Hedgehog Regulates Integrin Activity, Cadherin Contacts, and Cell Polarity to Orchestrate Neural Tube Morphogenesis Claire Fournier-Thibault,1,2 Ce´drine Blavet,1,2 Artem Jarov,1,2 Fernanda Bajanca,3,4 So´lveig Thorsteinsdo´ttir,3,4 and Jean-Loup Duband1,2 1Universite´ Pierre et Marie Curie–Paris 6, Laboratoire de Biologie du De´veloppement, and 2Centre National de la Recherche Scientifique, Laboratoire de Biologie du De´veloppement, 75005 Paris, France, 3Departamento de Biologia Animal, Centro de Biologia Ambiental, Faculdade de Cieˆncias, Universidade de Lisboa, 1749-016 Lisboa, Portugal, and 4Instituto Gulbenkian de Cieˆncia, 2781-901 Oeiras, Portugal In vertebrates, the embryonic nervous system is shaped and patterned by a series of temporally and spatially regulated cell divisions, cell specifications, and cell adhesions and movements. Morphogens of the Hedgehog, Wnt, and bone morphogenetic protein families have been shown to play a crucial role in the control of cell division and specification in the trunk neural tube, but their possible implication in theregulationofadhesiveeventshasbeenpoorlydocumented.Inthepresentstudy,wedemonstratethatSonichedgehogregulatesneural epithelialcelladhesionandpolaritythroughregulationofintegrinactivity,cadherincell–cellcontact,andcellpolaritygenesinimmature neural epithelial cells before the specification of neuronal cells. We propose that Sonic hedgehog orchestrates neural tube morphogenesis by coordinating adhesive and motility events with cell proliferation and differentiation. Introduction Novitch, 2008). Once committed, neural progenitors become lo- Development of the embryonic nervous system requires changes cated basally while NC cells, unlike other neural cells, disperse in cell fate, cell cycle, and cell movement, which are governed by through the adjacent tissues. pathways that signal to the nucleus, inducing gene transcription, Considerable progress has been made in deciphering the sig- as well as signaling that impacts the cytoskeleton. Understanding nals that regulate neural tube morphogenesis. Signaling mole- how these pathways are integrated is essential to a comprehensive cules of the Hedgehog (HH), Wnt, and bone morphogenetic knowledge of the formation of the nervous system. protein (BMP) families have been implicated in the generation of In vertebrates, neurulation proceeds as a series of steps by neural progenitors. Dorsally, BMPs specify sensory cells and NC which the neural plate is shaped into a tube that extends along the cells (Liem et al., 1997). Ventrally, Sonic hedgehog (SHH) drives anteroposterior axis (Colas and Schoenwolf, 2001). Cell division the position and identity of ventral neurons (Briscoe and Novitch, transforms the neural plate from a simple cuboidal into a 2008). In addition to BMPs, Wnt proteins also regulate dorsal pseudostratified epithelium in which neural epithelial (NE) cells progenitor proliferation and identity (Muroyama et al., 2002). contact both the basal and the luminal sides. Mitosis occurs api- Recently, evidence have been obtained showing that SHH also cally and nuclei move toward the basal side where NE cells either influences NE cell proliferation and survival (Cayuso et al., 2006), reenter the cell cycle or undergo differentiation (Wilcock et al., suggesting that gradients emanating from opposite poles in the 2007), allowing the positioning of progenitors that give rise to the neural tube, BMPs/Wnts dorsally and SHH ventrally, simulta- spinal cord. Patterning of the neural tube occurs along its dorso- neously control neural growth and patterning. ventral axis with motor neurons differentiating ventrally and sen- Coordinated morphogenesis of the neural tube would imply sory neurons and neural crest (NC) cells dorsally (Briscoe and that morphogen activities extend to the control of NE cell adhe- sion, polarity, and movement, but data are still sketchy. An inte- Received April 28, 2009; revised July 17, 2009; accepted Aug. 1, 2009. grated control has only been provided for NC cells in which the This work was supported by Centre National de la Recherche Scientifique, Universite´ Pierre et Marie Curie–Paris activities of Wnt1, BMP4, and its antagonist Noggin have been 6, and Association pour la Recherche contre le Cancer Grants 5590 and 3524. F.B. and S.T. were supported by found to orchestrate NC cell adhesion, proliferation, and speci- Fundac¸a˜o para a Cieˆncia e a Tecnologia/FEDER via POCTI/BCI/47681/2002 and the FP6/European Union Network of fication during delamination (Kalcheim and Burstyn-Cohen, Excellence“CellsintoOrgans.”Wethankourcolleaguesforgenerouslyprovidingconstructs,probes,andantibodies. CorrespondenceshouldbeaddressedtoeitherClaireFournier-ThibaultorJean-LoupDuband,Universite´ Pierreet 2005; Duband, 2006). Conversely, in vitro, SHH has been shown Marie Curie–Paris 6, Centre National de la Recherche Scientifique, Laboratoire de Biologie du De´veloppement, to alter NE and NC cell adhesion properties, causing repression of 75005 Paris, France. E-mail: [email protected] or [email protected]. NC cell delamination (Testaz et al., 2001), but the importance of A. Jarov’s present address: Roche, Shire Park, Welwyn Garden City AL7 1TW, UK. this effect in neural tube morphogenesis has not been investi- F. Bajanca’s present address: Randall Division for Cell and Molecular Biophysics, Third Floor North, New Hunt’s House, Guy’s Campus, King’s College London, London SE1 1UL, UK. gated. In this study, we assess the role of morphogens in the DOI:10.1523/JNEUROSCI.2003-09.2009 regulation of adhesive and migratory properties of NE cells by Copyright © 2009 Society for Neuroscience 0270-6474/09/2912506-15$15.00/0 misexpressing Shh, its antagonist Hedgehog-interacting protein Fournier-Thibault et al. • SHH Regulates Neural Cell Adhesion J. Neurosci., October 7, 2009 • 29(40):12506–12520 • 12507 Figure1. PropagationofShhsignalcorrelateswithchangesinNEcelladhesionproperties.A–C,Whole-mountinsituhybridizationon20somitestageembryosforPtc1(A),Gli1(B),andGli3(C). D–L, Transverse sections at the indicated levels of the neural axis showing expression of Ptc1 (D, G, J), Gli1 (E, H, K), and Gli3 (F, I, L), and revealing the progressive diffusion of Shh signal toward the dorsal part of the neural tube. M–O, Phase contrast views after 24 h of culture of neural explants at levels corresponding to the caudal unsegmented mesoderm (M), rostral unsegmented mesoderm (N), and last-formed somites (O), showing gradual restriction of NE cell capacity to spread into a flat monolayer. At the somite level, only NC cells disperse onto the FN substratum. Scale bars: A–C, 200 ␮m; D–J, 150 ␮m; M–O, 100 ␮m. (Hip1), and Bmp4 in the avian embryonic neural tube. We pro- Kida (Tohoku University, Sendai, Japan), RhoB from I. de Curtis (Depart- vide evidence that Shh orchestrates NE cell adhesion and polarity ment of Biological and Technological Research, Milan, Italy), and Hip1 from through regulation of integrin activity, cadherin contacts, and A. McMahon (Harvard University, Cambridge, MA). The HNK1 mAb has cell polarity genes, leading to a coordinated regulation of adhe- been described previously (Tucker et al., 1984). The antibodies to the ␤1 sive capacities of NE cells during development. integrin subunit and to laminin were from K. Yamada (National Institutes of Health–National Institute of Drug Research, Bethesda, MD) and Sigma- Materials and Methods Aldrich, respectively. The GoH3 rat mAb to the ␣6 integrin subunit and the Probes and antibodies. The following probes were used: Ptc1, Gli1, and Gli3 JsE3 control rat mAb were from A. Sonnenberg (Netherlands Cancer Insti- from C. Tabin (Harvard Medical School, Boston, MA), Pax3 from P. Gruss tute, Amsterdam, The Netherlands). The phosphohistone H3 (PH3) anti- (Max Planck Institute, Göttingen, Germany), Cad6B from M. Takeichi body was from Millipore, BEN and PAX3 mAbs were from the (RIKEN Center for Developmental Biology, Kobe, Japan), Daam1 from Y. Developmental Studies Hybridoma Bank (University of Iowa), the TASC 12508 • J. Neurosci., October 7, 2009 • 29(40):12506–12520 Fournier-Thibault et al. • SHH Regulates Neural Cell Adhesion Figure2. ShhmisexpressionprovokesmorphologicaldefectsintheneuraltubeasaconsequenceofNEcellextrusionintothelumen.A–D,MorphologyofShh-(A,B)(nϭ18)andAP-(C,D)(nϭ5)electroporatedneural tubesat48hpe.E–O,MorphologyofneuraltubeselectroporatedwithShhat6hpe(E,F,L,M)(nϭ25)andat15hpe(I–K,N,O)(nϭ23),showingmassivecellextrusionintothelumenoftheneuraltube(E,F,I,J,arrows). Control,AP-electroporatedneuraltubes(G,H)(nϭ6)shownoextrudedcellsat6hpe.TUNELat6hpe(L)(nϭ6)and15hpe(N)(nϭ6)revealapoptoticcellsonlyincellsextrudedinthelumenafter15hpe(L,N,arrows). PH3immunoreactivityshowsproliferationinneuraltubeselectroporatedat6hpe(M)(nϭ6)andat15hpe(O)(nϭ5).HNK1stainingat15hpe(K)(nϭ23)andat48hpe(B)(nϭ18)(D)(nϭ5)illustratesthedefectin NCcellmigrationafterShhmisexpression,leadingtoreductionofthesizeofdorsalrootganglion(B,K,shortarrows).Scalebars,50␮m.P,QuantitativeanalysisofTUNEL-positivecellscomparedwiththetotalcellnumberinto the lumen of the electroporated neural tube. Apoptosis only occurred from 15 hpe indicating anoikis as a result of cell extrusion. Q, Quantitative analysis of PH3-positive cells in electroporated (Shh) and nonelectroporated (control)sidesoftheneuraltube.ThenumberofPH3cellswasonlysignificantlyincreasedinthetransfectedsidefrom24hpeon.ErrorsbarsindicateSD.*pϽ0.05. Fournier-Thibault
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