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Thesis Ga 231107 Universitat Pompeu Fabra Facultat de Ciències experimentals i de la salut The early development of the inner ear: The Role of Notch and FGF pathway on early otic neural versus non-neural patterning Gina Abelló Sumpsi PhD Thesis supervised by Dr. Alsina and Dr. Giraldez Developmental Biology Unit Barcelona, December 2007 1 EARLY OTIC REGIONALIZATION The research carried out in this thesis has been supported by Grants BMC2002-00355 and BFU2005-03045, from Ministerio de Educación y Ciencia, and XT G03/203 from Instituto de Salud Carlos III. 2 Dipòsit legal: B.41380-2008 ISBN: 978-84-692-3464-8 EARLY OTIC REGIONALIZATION 4 TABLE OF CONTENTS ACKNOWLEDGEMENTS 01 SUMMARY 03 1. INTRODUCTION 07 1.1. The Inner Ear Of Vertebrates 07 1.1.1. Anatomy and histology of the inner ear 07 1.1.2. The inner ear in different species and throughout evolution 09 1.2. Cranial Sensory Organs Derive From The Cranial Placodes 12 1.2.1. The cranial placodes 12 1.2.2. Placode development 14 1.3. Inner Ear Development 16 1.3.1. From the otic placode to the adult organ 16 1.3.2. Otic progenitors 18 1.3.3. Inner ear induction 20 1.3.4. Otic neural development 27 1.3.4.1. An overview of neural induction 27 1.3.4.2. An overview of neurogenic placodes development 32 1.3.4.3. An overview neurogenesis: the determination of the neural fate 33 1.3.4.4. Otic neurogenesis 37 1.3.5. Otic patterning 40 1.3.5.1. General mechanisms for generating diversity 40 1.3.5.2. Inner ear patterning and regionalization 41 1.3.5.3. Otic neural versus non-neural patterning 47 1.3.5.4. Notch pathway in inner ear sensory patterning 53 2. AIMS AND SCOPE OF THIS THESIS 59 3. RESULTS 65 3.1. Regionalization During Early Steps Of The Inner Ear Development 65 3.1.1. The otic cup is regionalized into a proneural and a non-neural domain 65 3.1.2. Otic regionalization occurs at the onset of otic placode formation 68 3.2. Fate Mapping The Early Inner Ear 70 3.2.1. Common origin for geniculate placode and otic neurogenic. 70 3.2.2. Fate mapping the proneural and non-neural domains 72 3.2.3. Proneural and non-neural cells undergo limited cell mixing 74 3.2.4. Preliminary studies in otic neuroblast delamination 76 3.3. Role Of Notch Pathway On Early Otic Patterning 78 3.3.1. Expression profile of Notch family members in the early steps of inner ear development 78 3.3.2. Functional analysis of the Notch pathway: Loss of function. 83 3.3.2.1. Gamma-secretase inhibitor treatment (DAPT) 83 3.3.2.2. Electroporation of a dominant negative form of mastermind-like1 88 3.3.2.3. Mouse RBP-Jkappa mutant embryos 90 3.3.3. Functional analysis of the Notch pathway: Gain of function 92 3.3.3.1. Electroporation of the Notch1 intracellular domain 92 3.4. Role Of Sox3 On Otic Proneural Domain Establishment. 95 3.4.1. Sox3 is required for the establishment of the neural territory. 95 5 EARLY OTIC REGIONALIZATION 3.5. Role Of FGF Signals On Otic Proneural Domain Establishment 97 3.5.1. FGFs are responsible for early otic neurogenesis. 97 3.5.2. FGF8 is expressed in the correct place and at the correct time. 100 3.5.3. FGF8/FGF10 positive loop involved in giving neural competence to the otic region and driving neuroblast to differentiation. 104 3.5.4. FGF8 is sufficient for establishing the proneural otic domain. 104 3.5.5. FGFs are necessary for otic AP regionalization 106 4. DISCUSSION 111 5. MATERIAL AND METHODS 131 5.1. Embryos And Staging 131 5.2. In Vitro Embryonic Assays 132 5.2.1. Organotypic explants without collagen 132 5.2.2. Whole-embryo explants on BD matrigel matrix 133 5.3. Preparation Of Cryostat Sections 134 5.4. Assays Of Cell Proliferation By Brdu 134 5.5. Detection Of Gene Expression 135 5.5.1. Antisense rna probe synthesis 135 5.5.2. Whole-mount in situ hybridization (ISH) in chick and mouse 138 5.5.3. Whole-mount in situ hybridization (ISH) in zebrafish 140 5.5.4. Double/single fluorescent in situ hybridization (fISH) on cryostat sections 142 5.6. Immunochemistry 145 5.7. Fate Map Studies Of The Otic Cup 146 5.7.1. Double labeling with the vital dyes DiI and DiO 146 5.7.2. DiI labeling and in situ hybridization or immunochemistry 147 5.8. Overexpression Experiments In Ovo By Electroporation 147 5.9. Injection Of Morpholinos In Dr. Scotting Laboratory 148 5.10. Time-Lapse Movies In Dr. Dominguez Henrique Laboratory 148 5.11. Photography And Imaging 149 5.12. Measurement Of Relative Size Of Otic Vesicles 149 6. REFERENCES 153 7. APPENDIX 179 7.1 Alsina et al., 2004 7.2 Abelló et al., 2007 8. ANNEX 183 8.1. Abbreviations 183 8.2. Signaling Pathways 184 8.2.1. BMP signaling 184 8.2.2. Wnt signaling 186 6 7 ACKNOWLEDGEMENTS First of all I would like to thank Fernando Giraldez and Berta Alsina for transmitting their fascination for the developmental biology field, for giving me the opportunity to work in the lab as an undergraduate student, and for trust in me as a PhD student. Thanks for supervising my PhD thesis in a patient and encouraging manner. Thanks also for giving me the opportunity to work for short periods in other laboratories, which has been an experience that has greatly enriched me technically and intellectually. I would also like to thank to Fernando Giraldez, Berta Alsina and Cristina Pujades for stressing the importance of communicating science, teaching us how to do it, and promoting us to learn it in different workshops, conferences, seminaries and meetings here and abroad. I would like to thank Thomas Schimmang, Domingos Henrique and Miguel Torres for receiving me in their labs, for directing me as if I were a member more of their labs and discuss about my work. Thank you to all past and present lab members of the Developmental Biology Unit of the UPF: Ferran, Encarna, Montse, Citlali, Pedro, Anna, Andrés, Guillem, Laia, Sandra, Marta, Safia, Marija, Dora i Miguel. Thanks for all the help and companionship. Thanks to the people that I met in the Zentrum für Moleular Neurobiologie in Hamburg: Laura, Víctor, Yolanda, Nadja and Oleg. Thanks for teaching me at the beginning of my bench work, for good advices about the dark side of the science, for showing me the best of Hamburg, for the midday sandwiches in the garden. Thanks for being so kind. Thanks to the people that I met in the Institute for Molecular Medicine in Lisbon: Carolina, Susana Rocha, Rita, Geni, Tiago, Ana Faro, Gonçalo, Alina, Cristina, Susana Lopes and Claudia. Thanks for introducing me the time-lapse world, for helping me in molecular biology and embryo culturing. Thanks for all the support and for being so friendly. A més, m’agradaria agrair: Al Ferran per estar sempre preparat per escoltar els meus dilemes a la hora de plantejar experiments. A la companyona Coll, al Ferran i a la Marija pels moments de desconnexió i de bogeria. Al Ferran i a la Citlali per posar banda sonora al laboratori. A la Safia i al Xavi per ser tan pacients i positius, per ensenyar-me a baixar de revolucions. A l’Andrés, 1 EARLY OTIC REGIONALIZATION la Joana i la Marija per qüestionar-ho quasi tot. A l’Encarna, per portar-nos a toc de “pito” amb les qüestions d’ordre de laboratori, per ser un “maniatis” i per la feina ben feta. A la Arrate i al Xavi per guiar-me en la microscòpia de fluorescència. A la Enri, a la Montse i al Constantí pel “catxondeo”. A la Montse i a la Maria per fer-me desconnectar a l’hora de dinar, pel recolzament incondicional durant les diferents fases del doctorat, pel volei i pel futbol. Al Jordi, a l’Alberto i a la Vane per organitzar el Volei. Als ex-veïns de passadís (immuno, bio cel, micro, fisio i neuroframaco) per l’ajuda i companyonia. Al nou edifici per les seves terrasses. També m’agradaria agrair a la família, als amics i al Jani per tots els moments de felicitat que m’heu donat. Als pares i germans, per recolzar-me, per entendre’m i respectar-me, per fer que la vida sigui fàcil. Als amics que tenen poc, o res a veure amb la ciència: les orlandines, la Raquel, la Marta, la Inés, els de hoquei i els de cerdanya per poder comptar amb vosaltres, per existir. I, finalment al Jani, gràcies. 2 SUMMARY Cranial sensory neurons are responsible for sensing environment inputs of the head and present a dual origin; they derive either from the neural crest cells or from the neurogenic placodes. Otic neuronal precursors are specified in the otic placode but interestingly do so only in the anterior domain of the otic placode, the proneural domain. In the present study, we have explored why only this territory has the competence to undergo neurogenesis, this means the early events of otic proneural regionalization and neural commitment. The proneural and non-neural domains presented complementary gene expression patterns of transcription factors and members of the Notch pathway. Moreover, this work showed that the otic territory begins to be regionalized in the AP axis at the time of otic placode formation, much before it was reported.
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