Gene and protein interactions in limb development : the case of Msx and Gli3 Mathura Shanmugasundaram To cite this version: Mathura Shanmugasundaram. Gene and protein interactions in limb development : the case of Msx and Gli3. Development Biology. Université Pierre et Marie Curie - Paris VI, 2015. English. NNT : 2015PA066279. tel-01368052 HAL Id: tel-01368052 https://tel.archives-ouvertes.fr/tel-01368052 Submitted on 19 Sep 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Thèse de doctorat de l’Université Paris VI - Pierre et Marie Curie Pour obtenir le titre de Docteur de l’Université Paris VI Ecole Doctorale: Complexité du Vivant Spécialité: Biologie du développement Soutenue le 17 September 2015 par Mathura SHANMUGASUNDARAM Gene And Protein Interactions In Limb Development: The Case Of Msx And Gli3 Devant le jury composé de: Dr. Sylvie Schneider-Maunoury Président du Jury Dr. Bénédicte B Durand Rapporteur Dr. Marian Ros Rapporteur Dr. Malcolm Logan Examinateur Dr. Benoît Robert Directeur de thèse Gene and protein interactions in limb development: The case of Msx and Gli3 “What can be more curious than that the hand of the man formed for grasping, that of a mole, for digging, the leg of a horse, the paddle of a porpoise and the wing of a bat, should all be constructed on the same pattern and should include similar bones and in the same relative positions?” Charles Darwin, Origin of Species. Mathura Shanmugasundaram 2 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 Acknowledgements Mathura Shanmugasundaram 3 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 Acknowledgements I would like to sincerely acknowledge and convey my heartfelt gratitude and thanks to Benoît Robert, my mentor and advisor who not only gave me the wonderful opportunity to work in the Genetic and Molecular Morphogenesis Unit at Institut Pasteur, Paris but also supervised and guided me throughout the last four years. For all this and more, I dedicate this work to my guru, Benoit, without whom I would not have achieved this. Thank you for all your support and for believing in me. I would like to extend my gratitude and thanks to Yvan Lallemand, for your unwavering support and your kind gestures of advice and scientific conversations. To Pascal, for being an amazing technician. To Juliane, for your support and advice throughout the last year. To Didier, for your daily dose of entertainment and support as well as for generously sharing your wonderful office with me. I must also acknowledge many others who have made my stay in Institut Pasteur and Paris a wonderful experience. Thank you Clémire Cimper for being there with your motherly affection. Thank you Gaya and Julie for your beautiful friendship and for supporting me throughout my journey and for helping me adapt to France and it’s culture. A special thanks to Swetha for your moral support and to all my “Friday-lunch” mates. A big thanks to the department of developmental and stem cell biology for being an awesome and vibrant community. Thanks folks! I would also like to acknowledge the International Paris- Pasteur University (PPU) Program for their support and guidance throughout the years. And of course, last but not least, thank you to my dear family for being there throughout everything. For this and more, this is for all of you. I am certainly going to miss you all. Cette thèse conclut ma grande aventure Française. Je souhaite exprimer mon infinie gratitude à Benoit Robert sans qui ce travail n’aurait pas pu aboutir. Je veux remercier Yvan Lallemand pour son encadrement scientifique. Didier Montarras, je vous remercie pour vos conseils toujours précieux, chaque journée a été agrèable à vos côtés. Merci aussi, à Pascal et Catherine pour leur aide technique. Merci Clémire, Julie et Gaya pour être toujours là pour moi. Un grand merci à Maxime, pour tout. Je souhaite exprimer ma reconnaissance à tous ceux qui de près ou de loin m’ont aidé durant mes quatre années Françaises. Vous me manquerez, tous! Cette thèse est pour vous tous. Mathura Shanmugasundaram 4 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 Msx interacts with Gli3 in modulating limb morphogenesis Abstract The vertebrate limb is a paradigmatic model for morphogenesis. The anteroposterior (AP) growth and patterning of the limb bud rely on an intricate regulatory genetic network involving many genetic players such as Hox genes, Sonic hedgehog (Shh), Gli3. Shh is produced in a posterior region of the limb mesoderm and acts as a morphogen along the AP axis. It regulates both digit number and identity of different AP positions. As the main function of Shh is to prevent proteolysis of Gli3FL into Gli3R, Gli3R thereby concentrates anteriorly which notably is also the place where apoptosis takes place. In the mouse, paradoxically, despite a quasi-symmetrical expression profile, Msx1/2 null mutants show systematic anterior defects with an overgrowth in the anterior limb domain, resulting in anterior polydactyly. Both Gli3 and Msx mutant limb defects are concentrated anteriorly in a similar fashion suggesting an interaction between these genes and a possible role of this interaction in AP patterning in the limb bud as well as dysregulation of apoptosis. Indeed, we demonstrate interactions between Msx and Gli3 genes and even proteins. Mutations of Msx and Gli3 both result in anterior polydactylous phenotypes and similar loss of anterior apoptotic domains. This morphological analysis is associated with a search for common Gli3 and Msx transcriptional targets and partners in an attempt to link gene activity with changes in cell physiology that underlie morphogenesis. We have performed a differential transcriptome (RNA-Seq) on whole limb buds of Msx1-/-Msx2-/-, which has led us to narrow down on a number of potential common targets of Gli3 and Msx. Our results demonstrate that Msx and Gli3 work together in regulating the anterior posterior axis of limb development, independent of Shh. Mathura Shanmugasundaram 5 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 Abbreviation List A Alanine ADH Alcohol dehydrogenase AER Apical Ectodermal Ridge AP Anterior-Posterior BMP Bone morphogenetic protein Bmpr Bone morphogenetic protein receptor CI Clavicle Ci Cubitus Interruptus CRD Cysteine-rich domain dhh Desert hedgehog DNA Deoxyribo nucleic acid DV Dorsal- Ventral Eh1 Engrailed homology domain 1 EN-1 Engrailed 1 FGF Fibroblast Growth Factor FL Forelimb fpp Foyer préaxial primaire G Glycine Gli3A Gli3 Activated form Gli3FL GLi3 Full form Gli3R Gli3 repressor (truncated form) GPCR G-protein-coupled-like receptors Grg Groucho Hh Hedgehog Hhat Hedgehog acyl-transferase HL Hindlimb HTH Helix-Turn-Helix Hu Humerus ICD Interdigital Cell Death Ihh Indian Hedgehog IM Intermediate mesoderm kd Kilo-daltons (unit) LPM Lateral plate mesoderm msh Muscle segment homeobox P Proline PD Proximal-distal Pdn Polydactyly Nagoya PKA Protein kinase A Mathura Shanmugasundaram 6 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 Ptch Patched PZ Progress Zone RA Retionic acid Ra Radius Raldh Retinaldehyde dehydrogenases RDH Retinol dehydrogenase Sc Scapula Shh Sonic hedgehog Skn Skinny hedgehog Smo Smoothened Tbx T-box transcription factors Ul Ulna ZPA Zone of polarising activity Mathura Shanmugasundaram 7 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 CONTENTS PAGE Acknowledgements 3 Abstract 5 Abbreviations 6 INTRODUCTION Part A Limb structure and development 10 1 The limb as a model system 11 1.1 Limb initiation 11 1.2 The difficult case of Retionic Acid (RA) 13 1.3 The three axes of limb development 14 1.4 Early limb development 17 1.5 PD patterning: Old and new models 19 1.6 Dorsoventral (DV) axis 23 1.6.1 DV patterning 23 1.6.2 DV determination and AER development 25 1.7 Anterior-Posterior (AP) patterning 26 1.8 The Hedgehog (Hh) pathway 29 1.8.1 Shh auto modification and signalling 29 1.8.2 Hh receptors 32 1.9 Transcriptional targets of Shh signalling: Gli transcription factors 33 1.9.1 Gli expression during limb development 35 1.9.2 Gli processing 35 1.9.3 Shh in AP polarity and digit patterning 37 Part B Msx genes 2 A brief introduction to Msx genes 43 2.1 The discovery of Msx genes 44 2.1.1 Msx genes are homeobox genes 45 2.1.2 The Msx gene family 46 2.1.3 Msx in evolution 47 2.2 Msx genes at a molecular level 48 2.2.1 Chromosomal localization 48 2.2.2 Msx conserved structure 48 2.3 Msx gene expressions in vertebrates 51 2.4 Msx mutations 2.4.1 Msx1 mutation 53 2.4.1.1 Msx1 mutations in mice 53 2.4.1.2 Msx1 mutation in humans 55 Mathura Shanmugasundaram 8 Doctoral Thesis, Institut Pasteur 2015 Gene and protein interactions in limb development: The case of Msx and Gli3 2.4.2 Msx2 mutation 56 2.4.2.1 Msx2 mutations in mice 56 2.4.2.2 Msx2 mutations in humans 57 2.5 Msx functions 59 2.5.1 Transcriptional activity of Msx 60 2.6 Msx and apoptosis 62 2.6.1 Apoptosis in the limb: Interdigital cell death (ICD) 62 2.6.2 Apoptosis and the anterior digit: The thumb 64 2.6.3 Msx in anterior apoptosis 65 2.7 Msx and Epithelial- Mesenchymal interactions (EMI) 66 2.8 Msx in proliferation, differentiation and regeneration 67 2.9 Msx at the crossroads of limb development 71 3.