Development of Auditory and Vestibular Systems

By Raymond Romand

Development of Auditory and Vestibular Systems fourth edition presents a global and synthetic view of the main aspects of the development of the stato-acoustic system. Unique to this volume is the joint discussion of two sensory systems that, although close at the embryological stage, present divergences during development and later reveal conspicuous functional differences at the adult stage. This work covers the development of auditory receptors up to the central auditory system from several animal models, including humans. Coverage of the vestibular system, spanning amphibians to effects of altered gravity during development in different species, offers examples of the diversity and complexity of life at all levels, from genes through anatomical form and function to, ultimately, behavior.

The new edition of Development of Auditory and Vestibular Systems will continue to be an indispensable resource for beginning scientists in this area and experienced researchers alike.

• Full-color figures illustrate the development of the stato-acoustic system pathway
• Covers a broad range of species, from drosophila to humans, demonstrating the diversity of morphological development despite similarities in molecular processes involved at the cellular level
• Discusses a variety of approaches, from genetic-molecular biology to psychophysics, enabling the investigation of ontogenesis and functional development

• Language: English
• Publisher: Academic Press
• Release date: May 23, 2014
• ISBN: 9780124081086

Book preview

Development of Auditory and Vestibular Systems

Fourth Edition

Editors

Raymond Romand

Institut de Génétique et de Biologie moléculaire et cellulaire (IGBMC), Illkirch, France

Isabel Varela-Nieto

Instituto de Investigaciones Biomedicas, Madrid, Spain

Table of Contents

Cover image

Title page

Copyright

Contributors

Chapter 1. Early Development of the Vertebrate Inner Ear

Summary

1. The Adult Inner Ear

2. Development of the Inner Ear

Chapter 2. Development of the Auditory Organ (Johnston’s Organ) in Drosophila

Summary

1. Johnston’s Organ as a Specialized Chordotonal Organ

2. Structure and Function of Johnston’s Organ

3. Development of Johnston’s Organ

4. Genetic Control of Early Patterning

5. Regulation of Atonal and Sense Organ Precursor Specification in JO Development

6. Generating Scolopidia: Division of the Sense Organ Precursors

7. Chordotonal Neuron Differentiation in JO Development

8. Structure of JO Neurons

9. Gene Activation During JO Neuron Differentiation

10. Transcriptional Regulation of Ciliogenesis and Assembly of the Mechanosensory Apparatus

11. Developmental Emergence of Mechanosensory Specialization

12. How much of the Developmental Regulatory Network is Conserved in Vertebrate Hair Cells?

13. Support Cell Differentiation

14. JO Sensory Subspecializations Map to Distinct Subgroups of JO Neurons

15. The Development of JO Neuronal Subgroups

16. Sound Detection and Chordotonal Diversity in the Drosophila Larva

17. Conclusions

Chapter 3. Zebrafish Inner Ear Development and Function

Summary

1. Introduction

2. Development of the Zebrafish Inner Ear

3. Zebrafish Hearing and Vestibular Function

4. Disease Models

5. New Technologies

6. Conclusions and Future Directions

Chapter 4. Human Gene Discovery for Understanding Development of the Inner Ear and Hearing Loss

1. Introduction – The Genetics of Hereditary Hearing Loss

2. Identification of Deafness Genes

3. Mechanisms of Hearing – Lessons from Mice

4. Development of the Inner Ear

5. The Hair Bundle and Stereocilia

6. Junctions Between Cells: Gap and Tight Junctions

7. Planar Cell Polarity of the Hair Bundle

8. Gene Expression and Regulation

9. Summary

Chapter 5. Planar Cell Polarity in the Cochlea

1. Introduction

2. Planar Cell Polarity in the Inner Ear

3. Planar Cell Polarity Regulation in the Cochlea

4. Conclusions and Perspectives

Chapter 6. Functional Development of Hair Cells in the Mammalian Inner Ear

1. Introduction

2. Mammalian Auditory System

3. Mammalian Vestibular System

4. Conclusions and Remarks

Chapter 7. Neuronal Circuitries During Inner Ear Development

1. Introduction

2. Development of the Afferent Innervation Pattern

3. Development of the Efferent Innervation Pattern

4. Conclusions

Chapter 8. Recapitulating Inner Ear Development with Pluripotent Stem Cells: Biology and Translation

1. Introduction

2. Why Stem Cells?

3. Progress Towards Reconstitution of Inner Ear Development in vitro

4. Prospects for Clinical Translation

5. Conclusion

Chapter 9. Development of Mammalian Primary Sound Localization Circuits

1. Introduction

2. Overview of Brainstem Primary Sound Localization Circuits

3. Early Development of the Auditory Brainstem

4. Activity in the Developing Auditory System

5. Development of Soma-Dendritic Morphology and Innervation

6. Maturation of Intrinsic Cell Excitability and Voltage-Dependent Channels

7. Maturation of Synaptic Transmission and the Role of Neuronal Activity

8. Refinement of Circuits and the Role of Neuronal Activity

9. Concluding Remarks and Future Directions

Chapter 10. Development of Fundamental Aspects of Human Auditory Perception

1. Introduction

2. The Methodological Challenge

3. Auditory Sensitivity in Quiet

4. Frequency and Pitch Discrimination

5. Intensity Discrimination

6. Duration Discrimination

7. Spectral Shape and Timbre Discrimination

8. Loudness Perception

9. Frequency Selectivity and Simultaneous Masking

10. Temporal Processing

11. Informational and Distracting Masking

12. Auditory Scene Analysis

13. Sound Localization

14. Summary and Conclusions

Chapter 11. Developmental Plasticity of the Central Auditory System: Evidence from Deaf Children Fitted with Cochlear Implants

1. Introduction

2. Developmental Plasticity in a System Deprived of Appropriate Stimulation

3. Harnessing Developmental Plasticity for Improved Clinical Outcomes

4. Summary

Chapter 12. Development of the Mammalian ‘Vestibular’ System: Evolution of Form to Detect Angular and Gravity Acceleration

1. Introduction

2. Evolving a Labyrinth: Transforming a Gravistatic Otocyst into a Multi-Sensory Organ

3. Morphological Development of the Mouse Ear: Transforming an Otocyst into a Labyrinth

4. Formation of Hair Cells and Sensory Epithelia

5. Neuronal Development of the Mouse Ear: Making and Routing Unique Subtypes from Endorgans to the Central Nervous System

6. Conclusion and Outlook

Chapter 13. Development of the Statoacoustic System of Amphibians

1. Introduction

2. Inner Ear Induction

3. Morphogenesis of the Inner Ear

4. Development of Sensory Epithelia and their Innervation

5. Development of the Middle Ear

6. Functional Development

7. Summary and Conclusions

Chapter 14. Development of the Central Vestibular System

1. Introduction

2. Early Developmental Studies

3. Chick Tangential Nucleus as a Model

4. Emergence of Movements in Embryos

5. Recent Studies on the Development of Neuron Structure

6. Recent Studies on the Organization of the Vestibular Nuclei

7. Recent Studies on the Emergence of Neuron Electrophysiological Properties

8. Conclusions

Chapter 15. Functional Development of the Vestibular System: Sensorimotor Pathways for Stabilization of Gaze and Posture

1. General Introduction to the Inner Ear Functions: Audition and Balance in Different Species

2. Ontogeny of the Vestibular System

3. Developmental Questions

4. Conclusion: Understanding the Inner Ear Physiology Through Interspecies Comparison Using an Ecophysiological perspective

Chapter 16. Development of Vestibular Systems in Altered Gravity

1. Introduction

2. Studies in Aquatic Vertebrates

3. Studies in Higher Vertebrates

4. Invertebrates

5. General Considerations

6. Conclusions

Index

Copyright

Academic Press is an imprint of Elsevier

The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK

225 Wyman Street, Waltham, MA 02451, USA

Copyright © 2014 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangement with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

British Library Cataloguing in Publication Data

A catalogue record for this book is available from the British Library

Library of Congress Cataloguing in Publication Data

A catalogue record for this book is available from the Library of Congress

ISBN: 978-0-12-408088-1

For information on all Academic Press publications visit our website at store.elsevier.com

Printed and bound in the United States

14 15 16 17 10 9 8 7 6 5 4 3 2 1

Contributors

Erika E.  Alexander ,      Brown University, Department of Cognitive, Linguistic, and Psychological Sciences, Providence, RI, USA

Stefanie C.  Altieri ,      Richard King Mellon Foundation Institute for Pediatric Research, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Karen B.  Avraham ,      Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

Tanaya  Bardhan ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Sarah  Baxendale ,      MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield, UK

Mathieu  Beraneck ,      Centre National de la Recherche Scientifique and Université Paris Descartes, Sorbonne Paris Cité, Paris, France

Yoni  Bhonker ,      Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

Garrett  Cardon ,      University of Colorado at Boulder, Speech, Language, and Hearing Science Department, Institute of Cognitive Science, Center for Neuroscience, Boulder, CO, USA

Ping  Chen ,      Department of Cell Biology, Emory University, Atlanta, GA, USA

Amanda  Clause ,      Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Department of Otology and Laryngology, Harvard Medical School, Boston, MA, USA

Julio  Contreras

Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain

Centro de Investigacion Biomedica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain

Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

Laura F.  Corns ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Jean  Defourny ,      GIGA-Neurosciences, Developmental Neurobiology Unit, University of Liège, Liège, Belgium

Laurence  Delacroix ,      GIGA-Neurosciences, Developmental Neurobiology Unit, University of Liège, Liège, Belgium

Jeremy S.  Duncan ,      University of Iowa College of Liberal Arts and Sciences, Department of Biology, Iowa City, IA, USA

Bernd  Fritzsch ,      University of Iowa College of Liberal Arts and Sciences, Department of Biology, Iowa City, IA, USA

Cynthia M.  Grimsley-Myers ,      Department of Cell Biology, Emory University, Atlanta, GA, USA

Eri  Hashino ,      Medical Neuroscience Graduate Program; Stark Neurosciences Research Institute; Department of Otolaryngology – Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

Matthew C.  Holley ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Eberhard R.  Horn ,      Zoological Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

Oliver  Houston ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Andrew P.  Jarman ,      Center for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, UK

Stuart L.  Johnson ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Karl  Kandler ,      Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Karl R.  Koehler ,      Medical Neuroscience Graduate Program; Stark Neurosciences Research Institute; Department of Otolaryngology – Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

Benjamin J.  Kopecky ,      University of Iowa College of Liberal Arts and Sciences, Department of Biology, Iowa City, IA, USA

François M.  Lambert ,      Institute of Basic Medical Sciences, Department of Physiology, University of Oslo, Oslo, Norway

Enrique A.  Lopez-Poveda ,      Instituto de Neurociencias de Castilla y León, Instituto de Investigación Biomédica de Salamanca, Departamento de Cirugía, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain

Marta  Magariños

Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain

Centro de Investigacion Biomedica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain

Departamento de Biologia, Universidad Autonoma de Madrid, Madrid, Spain

Brigitte  Malgrange ,      GIGA-Neurosciences, Developmental Neurobiology Unit, University of Liège, Liège, Belgium

Alexander K.  Malone

Department of Otolaryngology – Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

Department of Otolaryngology – Head and Neck Surgery, University of South Florida, Tampa, FL, USA

Walter  Marcotti ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Stephen M.  Maricich ,      Richard King Mellon Foundation Institute for Pediatric Research, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Sergio  Masetto ,      Department of Physiological and Pharmacological Science, University of Pavia, Pavia, Italy

Jennifer  Olt ,      Department of Biomedical Science, University of Sheffield, Sheffield, UK

Kenna D.  Peusner ,      Department of Anatomy and Regenerative Biology, George Washington University School of Medicine, Washington, DC, USA

Padmashree C.G.  Rida

Department of Cell Biology, Emory University, Atlanta, GA, USA

Department of Biology, Georgia State University, Atlanta, GA, USA

Soroush G.  Sadeghi ,      Center for Hearing and Deafness, Department of Communicative Disorders and Sciences, State University of New York at Buffalo, Buffalo, NY, USA

Anu  Sharma ,      University of Colorado at Boulder, Speech, Language, and Hearing Science Department, Institute of Cognitive Science, Center for Neuroscience, Boulder, CO, USA

Andrea Megela  Simmons ,      Brown University, Department of Cognitive, Linguistic, and Psychological Sciences, Providence, RI, USA

Joshua  Sturm ,      Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Kathy  Ushakov ,      Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

Isabel  Varela-Nieto

Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain

Centro de Investigacion Biomedica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain

IdiPAZ, Madrid, Spain

Tanya T.  Whitfield ,      MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield, UK

Chapter 1

Early Development of the Vertebrate Inner Ear

Marta  Magariños ¹ , ² , ³ ,  Julio  Contreras ¹ , ² , ⁴ ,   and  Isabel  Varela-Nieto ¹ , ² , ⁵       ¹ Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain      ² Centro de Investigacion Biomedica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain      ³ Departamento de Biologia, Universidad Autonoma de Madrid, Madrid, Spain      ⁴ Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain      ⁵ IdiPAZ, Madrid, Spain

Abstract

The auditory and vestibular receptors of vertebrates are located in the inner ear and connected to the brain by the VIIIth cranial nerve. The inner ear is a complex and integrated system, damage to which causes hearing and/or balance impairment. Understanding the genetic, cellular, and molecular bases of inner ear development will enhance our understanding of adult inner ear physiology and pathology. Cells of the sensory receptors have a common embryonic origin in the ectodermal otic placode. The three main otic lineages of sensory hair cells, non-sensory support cells, and spiral and vestibular neurons have common otic progenitors. Apoptosis, proliferation, autophagy, and cell differentiation processes interact during early otic development to generate the structures and functionally distinct cell types of the adult inner ear. Groundbreaking work has begun to delineate the signaling networks that regulate early inner ear development, and this will be discussed in detail in this chapter.

Keywords

acoustic-vestibular ganglion ; AKT ; apoptosis ; autophagy ; IGF signaling ; otic progenitors ; otic vesicle

Outline

Summary 2

1. The Adult Inner Ear 2

1.1 Anatomy of the Adult Inner Ear 2

1.2 Comparative Anatomy of the Adult Inner Ear 3

2. Development of the Inner Ear 5

2.1 Placode Induction and Otocyst Early Patterning 7

2.2 Neurosensory Precursors 10

2.3 MicroRNAs in Inner Ear Development 11

2.4 Apoptosis, Survival, and Proliferation in Inner Ear Development 12

2.5 Autophagy in Inner Ear Development 16

2.6 Cell-type Specification of Otic Neurosensory Components 17

2.6.1 Specification of Hair and Supporting Cells 17

2.6.2 Development of Acoustic and Vestibular Neurons 19

Acknowledgments 22

References 22

Summary

The auditory and vestibular receptors of vertebrates are located in the inner ear and connected to the brain by the VIIIth cranial nerve. The inner ear is a complex and integrated system, damage to which causes hearing and/or balance impairment. Understanding the genetic, cellular, and molecular bases of inner ear development will enhance our understanding of adult inner ear physiology and pathology. Cells of the sensory receptors have a common embryonic origin in the ectodermal otic placode. The three main otic lineages of sensory hair cells, non-sensory support cells, and spiral and vestibular neurons have common otic progenitors. Apoptosis, proliferation, autophagy, and cell differentiation processes interact during early otic development to generate the structures and functionally distinct cell types of the adult inner ear. Groundbreaking work has begun to delineate the signaling networks that regulate early inner ear development, and this will be discussed in detail in this chapter.

1. The Adult Inner Ear

1.1. Anatomy of the Adult Inner Ear

The mammalian inner ear is formed by fluid-filled canals and cavities, named the membranous labyrinth, that are encased within the bony labyrinth and located inside the temporal bone (Fig. 1.1 A). The auditory (hearing) and vestibular (balance) organs are located in the inner ear, and they are connected to the brain by the fibers of the VIIIth cranial nerve. The cochlear, or hearing part, is divided into the three parallel helical scalas, tympanic, vestibular, and media, filled with lymph (Fig. 1.1A–C). The organ of Corti is located inside the scala media. This is the sensory receptor, where the hair cells transform the mechanical input elicited by sounds into an electrochemical signal (Hudspeth, 2008). The organ of Corti is formed by two main types of functional cells: sensory hair cells and non-sensory support cells (Fig. 1.1D–F). The hair cells are the sensory receptor cells and possess a set of sterocilia in their apical surface that allow mechanotransduction. There are two types of hair cells that exhibit specific functions: the inner hair cells (IHC) and the outer hair cells (OHC), which are arranged in one and three rows, respectively. IHC and OHC rows are separated by support pillar cells that form the tunnel of Corti. Deiters’, Hensen’s, and Claudius’s cells are other specialized non-sensory support cells that participate in ionic and metabolic cochlear homeostasis (Forge and Wright, 2002; Lefebvre and Van De Water, 2000).

The bipolar auditory neurons of the spiral ganglion are connected to the hair cells and convey the encoded sound information to the central nervous system (Nayagam et al., 2011; Raphael and Altschuler, 2003) (Fig. 1.1G–I). The dendritic ends of type I neurons connect to the IHCs, whereas those of the type II innervate the OHCs. The axons of the spiral neurons leave the spiral ganglion and pass through the base of the modiolus to form the cochlear division of the cochleo-vestibular nerve toward the cochlear nuclei in the brainstem. Sound information progresses in a complex, multisynaptic, parallel, and ascendant pathway from the cochlea through the brainstem nuclei to the auditory cortex (Webster et al., 1992). The tonotopic organization present in the cochlea is maintained along the pathway up to the auditory cortex. Neurons from the superior olivary complex at the brainstem also contact hair cells in a centrifugal control mechanism of the auditory pathway.

The spiral ligament and the stria vascularis form the lateral wall, and both are central to hearing physiopathology (Fig. 1.1J–L). The stria vascularis is a three-layered vascular epithelium that regulates intracochlear ion transport and maintains the endocochlear potential. The intermediate cells of the stria vascularis are melanocyte-like cells (Murillo-Cuesta et al., 2010; Patuzzi, 2011; Takeuchi et al., 2000).

The vestibular part of the inner ear contains the balance receptors, which are formed by specialized balance mechanoreceptor hair cells, similar to those of the organ of Corti, and organized into several sensory organs. The three cristae are located at the base of the semicircular canals and detect angular acceleration, whereas the two maculae detect linear acceleration and gravity (Goldberg, 1991; Highstein and Fay, 2004) (Fig. 1.1).

Hearing loss and balance impairment are consequences of adult inner ear injury. Understanding the genetic, cellular, and molecular bases of inner ear development is the first step in unraveling adult inner ear physiology and pathology.

1.2. Comparative Anatomy of the Adult Inner Ear

The auditory sensory organs have been highly modified along the phylogenetic tree (Fritzsch et al., 2013) (Fig. 1.2 ) but in contrast the vestibular sensory organs are highly comparable among species. The cochlea is absent in fish and amphibians, and its functions have been replaced by alternative auditory organs, the saccule and lagena, respectively. The saccule has a vestibular function in birds and mammals, whereas the functions of the lagena in birds are only now beginning to be understood (Mahmoud et al., 2013), and it is not present at all in placental and marsupial mammals. Even in invertebrates, hearing is mediated by a mechanosensory organ, the Johnston’s organ, which presents some developmental genetic similarities to that of vertebrates (Senthilan et al., 2012), although the sensory neurons themselves have mechanosensitive cilliary specializations, as is discussed in detail in Chapter 2.

FIGURE 1.1   Anatomy of the adult mouse inner ear.  (A) Lateral view of paint-filled inner ear. Abbreviations are: Co, cochlea; V, vestibule; Asc, Lsc, and Psc, anterior, lateral, and posterior semicircular canals; Do, dorsal; Cd, caudal. (B) Lateral view of a whole-mounted cochlea showing the pigmented stria vascularis (arrows) in the lateral wall and the round (RW) and oval (OW) windows. (C) Midmodiolar section of the cochlea and the surrounding osseous otic capsule showing the three fluid-filled scales: the scala vestibuli (SV), the scala media (SM) with the auditory receptor (black box), and the scala tympani (ST). (D) Detail of a cochlear turn highlighting the spiral ganglion (SG, left black box) and the auditory receptor (organ of Corti, right black box). (E) Phalloidin histochemistry (Phal) of the organ of Corti, labeling F-actin in the stereocilia and cuticular plate of hair cells (IHC, OHC), the reticular lamina, and pillar cells. (F) Detail of organ of Corti, showing the myosin (MyoVIIa) expression at the hair cells and the SOX2 expression at the supporting cells. (G) Semi-thin section showing a detail of the spiral ganglion (SG). (H) Electron micrograph of a spiral ganglion neuron type I (SG-TI). (I) Detail of the external compact (CM) and internal loose (LM) myelin sheaths in a SG type I neuron. (J) Detail of the marginal (MC) and basal (BC) cells in the stria vascularis. The spiral ligament (SpL) is close to the otic capsule. (K–L) Immunostaining showing the expression of Kir4.1, a K+ channel related to the production of the endocochlear potential (K), and Na+K+ATPase (L) expression in the stria vascularis (StV). (M–N) Sensory epithelium of the vestibular inner ear, a detail of the utricular macula and the cristae gross anatomy. (O–P) Detail of the macula (O) and cristae ampullaris (P) showing the myosin VIIa expression (green, labeling sensory hair cells) and neurofilament expression (red, labeling macula and cristae nerve fibers). Arrows show the afferent calyx of type I hair cells. Scale bars: A-C, K, 0.5 mm; D, 100 μm; E,F,J,L,M, 50 μm; G, 30 μm; H, 5 μm; I, 0.1 μm; J,K,L,M,N,O,P, 50 μm.

FIGURE 1.2   Comparative anatomy of the adult inner ear of different vertebrates.  Schematic view of the inner ear of Danio rerio, Rana perezi, Gallus gallus, and Mus musculus. The scheme shows the cochlear and vestibular parts, as well as the sensory areas: crista (anterior, AC, posterior, PC, and lateral, LC), macula (utricle, U, and saccule, S), basilar papilla (BP), lagena (L), and organ of Corti (Co).

2. Development of the Inner Ear

The early development of the inner ear is very similar among all vertebrates. The sensory and supporting cells of the inner ear and the spiral ganglion neurons develop from the ectodermal embryonic otic placode (Fig. 1.3 ). The cells for the spiral ligament, otic capsule, and modiolus originate from the surrounding mesenchymal cells, whereas the melanocyte-like cells of the stria vascularis and ganglionar Schwann cells have a neural crest origin (Chang et al., 2002; D’Amico-Martel and Noden, 1983; Fritzsch et al., 2011).

FIGURE 1.3   Development of the mouse inner ear.  The inner ear develops from the otic placode (A, E7.5). The otic placode invaginates to form the otic cup (B, E8–9) that later pinches off to form the otic vesicle or otocyst (C–D). This is a transitory embryonic structure that undergoes complex morphological changes and cell differentiation processes to generate almost all the cell types that conform the adult inner ear, including the neurons for the spiral and vestibular ganglions. Neural precursors delaminate and migrate from the ventral epithelium to form the acoustic-vestibular ganglion (AVG) (B–D). The cochlear duct is generated from the ventromedial region of the otic vesicle as an evagination and together with the spiral ganglion (SG) is the hearing part of the primitive AVG (yellow part, E–H). This duct elongates and grows to form a coiled tube that originates the scala media (SM). The mesenchymal cells surrounding the labyrinth differentiate to form the scala vestibularis (SV) and scala tympanic (ST) (E–H). The cochlear duct proceeds in development with the specification of a prosensory patch, which will later become the primitive organ of Corti (OC). Cells reorganize to form the greater and lesser epithelial ridges (GER and LER, I–J) that will develop to become the inner sulcus (IS), the spiral limbus, the inner and outer hair cells (IHC, OHC), and the support cells (K–L). Non-sensory support cells include inner phalangeal cells (IPC), pillar cells (PC), Deiter’s cells (DC), and Hensen’s cells (HC), which form the different substructures located on the basilar membrane (BM) within the scala media. TM: tectorial membrane, LW: lateral wall, StV: stria vascularis, SPL: spiral ligament.

The otic placode is induced from the head surface ectoderm adjacent to the hindbrain between the rhombomeres 5 and 6. The otic placode invaginates to form the otic cup, which later closes to form the otic vesicle or otocyst. The otic vesicle is a transitory autonomous structure that contains all the information required to undergo the differentiation program and thus to generate most cell types of the adult inner ear (Bissonnette and Fekete, 1996; Sanchez-Calderon et al., 2007). Vestibular structures develop from the dorsal aspect of the otic vesicle, whereas the ventral aspect will form the auditory part of the inner ear. Otic neuroblasts delaminate from the otic cup and otic vesicle to form the acoustic-vestibular ganglion (AVG), which contains the neural precursors of the spiral auditory and vestibular ganglions. Otic neurons extend their processes and connect the sensory epithelium to the brainstem nuclei (Hemond and Morest, 1991; Rubel and Fritzsch, 2002). The otic vesicle undergoes a series of morphogenetic movements and developmental processes that modify the simple epithelial sac and finally produce the complex three-dimensional membranous labyrinth (Kelly and Chen, 2009). In parallel, surrounding mesenchymal cells form a cartilaginous capsule that will become the bony labyrinth (Chang et al., 2002). Spiral ganglion axon fasciculation is also regulated by otic mesenchymal cells (Coate and Kelley, 2013).

Growth and trophic factors, transcription factors, and microRNAs orchestrate the processes that shape the inner ear. Here we will discuss the main functions of some of these molecules.

2.1. Placode Induction and Otocyst Early Patterning

Placode induction and otocyst early patterning are modulated by secreted signals and transcription factors. Of the former, FGFs are particularly important for the inner ear. The FGF family consists of at least 20 genes encoding secreted ligands that regulate cell survival, differentiation, proliferation, and migration during development by binding to seven membrane receptors encoded by four genes (Tulin and Stathopoulos, 2010). FGF signaling plays a key role during the early induction of the otocyst and participates in the formation of the otic vesicle (Ohyama et al., 2007; Schimmang, 2007), in prosensory specification (reviewed by Sanchez-Calderon et al., 2007), in otic neurogenesis and neuritogenesis (Nicholl et al., 2005; Wei et al., 2007), in the formation of auditory hair cells (Pirvola et al., 2002), and in pillar cell differentiation (Doetzlhofer et al., 2009; Sanchez-Calderon et al., 2010). During otic placode induction (Fig. 1.4 A) several FGF ligands (FGF3/8/10 in mice) act as secreted signals from the mesoderm and neural plate together with intrinsic signals coming from the ectoderm itself to proceed with otic placode determination and invagination.

FGF ligands show some functional redundancy, but there are ligands, e.g., FGF3 and FGF10, which play a more specific role (reviewed by Alvarez et al., 2003; Wright and Mansour, 2003). Fgf3/Fgf10 double mouse mutants do not develop an otic vesicle, and early otic markers as Pax family genes are mis-expressed (Léger and Brand, 2002). FGF signaling needs to be down-regulated, and thus expression of the FGF signaling inhibitors Sprouty (Spry) and Dusp6 are increased after otic placode induction of chicken and mouse embryos, respectively (Chambers and Mason, 2000; Urness et al., 2008). The restriction of otic cell fate (Freter et al., 2008) and, later on, the maintenance of supporting cell identity (Jacques et al., 2012) also depend on the attenuation of FGF signaling. Notch and Wnt signaling pathways also cooperate to define otocyst patterning and the size of the otic placode (Jayasena et al., 2008; Ohyama et al., 2006).

FIGURE 1.4   Schematic view of the molecular mechanisms regulating early inner ear development.  (A) Induction and formation of the mouse otic placode. Otic placode induction requires both intrinsic and extrinsic signals from the surrounding mesenchyme and from the neural tube (low FGF activity and high Wnt and Notch signaling). The expression of several members of the FGF family (FGF3/10) is reduced by the action of Dusp6. Pax2 expression defines the pre-otic area, and within it Notch signaling is high, whereas Wnt signaling is increased in the otic region. (B) Dorso-ventral patterning of the otocyst. Wnt signaling from the hindbrain plays an important role to establish the dorsal fate; the expression of Hmx2/Hmx3 and of Dlx5/6 (that depends on Gbx2 and BMP4 function) is also determining the dorsal territory of the otocyst, which will give rise to the vestibule and to the endolymphatic duct. A precise amount of Gli3 repressor protein is required to correctly shape the dorsal part. The ventral region of the otocyst will generate the cochlear fate that is specified by a gradient of secreted Shh from the notochord, the expression of the transcription factors Pax2, Otx2, and the action of the gene network Six1-Eya1-Dach. GATA3 loss generates a shortened cochlear duct. (C) Specification of the neurosensory components. The scheme summarizes some of the agents that are expressed in the neurosensory region to produce hair cells, supporting cells, and otic neurons. SOX2 and Neurog1 are the best-known transcription factors involved in the generation of the three types of cells. The Notch pathway is defining the early neurosensory region and will also play a role later in the differentiation of hair cells. Tbx1 is required to delimit the neurogenic domain in the otocyst and to correct cochlear morphogenesis (Xu et al., 2007). The miR-200 miRNA family has been suggested to play a role in the generation of the pool of neurosensory cells. The IGF1R might be also implicated in cell fate determination at these stages. HB: hindbrain; NT: notochorda; OV: otic vesicle; AVG: acoustic-vestibular ganglion.

The paired homeobox containing family ( Pax) genes (Pax1–9) are also early players in vertebrate otic development (Groves and Bronner-Fraser, 2000; Heller and Brändli, 1999; Torres et al., 1996). Pax8 mouse mutants present a phenotype with no inner ear, but double Pax2/Pax8 mutants show developmental arrest at the otic vesicle stage (Bouchard et al., 2010). Pax2 is activated by Sonic hedgehog (Shh) , a secreted signal derived from the floor plate and the notochord that induces ventral identity in the otocyst (Fig. 1.4B) (Riccomagno et al., 2002, 2005), as well as the proliferation of the neural progenitors for the AVG (Brown and Epstein, 2011). Pax genes together with Six1, Eye absent (Eya) , and Dachshund (Dach) are part of the gene network that provides ventral identity to the otic placode (Ozaki et al., 2004; Zheng et al., 2003). Synergistic regulation of this network has also been reported in eye and muscle development (Heanue et al., 1999). The specification of otic ventral identity is also mediated by the activation of GATA3 and Otx2 and repression of the Wnt signaling pathway (Groves and Fekete, 2012; Lawoko-Kerali et al., 2002; Morsli et al., 1999). Thus, null mutants for the zinc finger protein GATA3 show abnormal inner ear morphology (Karis et al., 2001). Interestingly, as the lethality of GATA3 mutants is also associated with a noradrenaline deficiency, vestibular morphogenesis could be rescued by treatment with a precursor of catecholamines (Lim et al., 2000). In contrast, GATA3 is absolutely essential for the development of cochlear neurosensory cells (Duncan et al., 2011).

Otic placode dorsal identity is promoted by Gbx2 and Dlx5/6 , which inhibit ventral identity and are required for vestibular development (Lin et al., 2005; Robledo and Lufkin, 2006). Both are regulated by Wnt signaling coming from the roof plate (Fig. 1.4B). The action of the gastrulation brain homeobox family member Gbx2 (Steventon et al., 2012) is antagonized by Otx2, which plays this role in the specification of sensory placodes. The loss of the homeobox genes Otx1 and Otx2 is partly responsible for the cochlear developmental defects observed in Shh null mutant mice. The expression of Hmx2/Hmx3 also helps to specify the otic dorsal identity (Wang et al., 2001; 2004); double mouse mutants for either Dlx5/6 or Hmx2/Hmx3 lack a vestibular region (Robledo and Lufkin, 2006; Wang et al., 2004). In addition, the zinc finger transcription factor Gli3 (Bok et al., 2007) and signaling by the bone morphogenetic protein BMP4 (Chang et al., 2008) promote vestibular specification by antagonizing Shh signaling and blocking its ventralizing role.

2.2. Neurosensory Precursors

Neurosensory precursors are generated from multipotent otic progenitors. Sequential cell fate specification and differentiation of the pool of otic vesicle neuroepithelial cells will generate sensory hair cells and non-sensory support cells (Driver et al., 2013), plus both the auditory and vestibular neurons. The way that these three lineages are established, and whether they have common cellular progenitors or not, has been a matter of open discussion nicely covered in Fritzsch and Beisel (2004), Kelley (2006), Matei et al. (2005), and Satoh and Fekete (2005). Here, we will discuss the main factors and signaling pathways that regulate otic cell fate specification (Fig. 1.4C).

BMP4 is differentially expressed during early development, and it has been proposed that it plays a key role in the specification of prosensory otic regions in several species of vertebrates (Cole et al., 2000; Li et al., 2005; Oh et al., 1996). Notch signaling also participates in the earliest induction stages and specification of the otic prosensory domain. Indeed, ectopic induction of Notch signaling in the chicken basilar papilla or in mouse otic domains generates ectopic sensory areas (Daudet and Lewis, 2005; Hartman et al., 2010). In fact, not only Notch1 but other components of the pathway, such as Lunatic fringe (Lfng), Delta-like1 (Dll1), and Hes5, are differentially expressed in the early, developing inner ear (Adam et al., 1998; Cole et al., 2000; Daudet and Lewis, 2005; Groves and Bronner-Fraser, 2000; Jeon et al., 2011). Although early activation of Notch signaling in the chicken otocyst has been reported to be required to specify a prosensory profile (Neves et al., 2013), this is still under examination in the mouse inner ear (Basch et al., 2011; Yamamoto et al., 2011).

Within the otic epithelium, the HMG-box transcription factor Sox2 is expressed by proliferating cells of the neurosensory domain. Once hair cells have differentiated, Sox2 expression becomes restricted to the supporting cells (Kiernan et al., 2005; Neves et al., 2007). This trait is common to several genes; for example, Jag1, Lfng, and p27kip, which are widely expressed in the early prosensory domain, but later in development their expression is restricted to non-sensory support cells. Light coat circling (Lcc) and Yellow submarine (Ysb) are two Sox2 mutants that exhibit hearing and balance defects and lack hair and support cells as well as neurons (Kiernan et al., 2005; Puligilla et al., 2010), strongly suggesting the existence of a common progenitor in the neurosensory domain for the three otic cell lineages. Human SOX2 mutations cause deafness (Hagstrom et al., 2005). In Jag1 mice, Sox2 expression is lost, thus SOX2 actions might be regulated by the Notch signaling pathway (Dabdoub et al., 2008).

The proneural gene Neurogenin1 (Neurog1) initiates the program of neural determination that will be discussed later in this chapter; however it is interesting to note here that Neurog1 inactivation causes loss of both sensory neurons and hair cells (Ma et al., 2000), as reported for Sox2 mutants (Kiernan et al., 2005). In addition, Neurog1 progenitors have been reported to generate both neurons and hair cells (Raft et al., 2007). Therefore these data further suggest that there is a common neurosensory domain. In contrast, cell lineage studies in the chicken otocyst suggest that most otic neurosensory cells have either a proneural or a prosensory origin (Kelley, 2006; Satoh and Fekete, 2005). Further work is required to define this matter of special relevance for the design of future regeneration therapies (Koehler et al., 2013).

2.3. MicroRNAs in Inner Ear Development

MicroRNAs (miRNA) are small non-coding RNA molecules, which bind to the 3’UTR of target mRNA and regulate gene expression by suppressing their translation (Kloosterman and Plasterk, 2006). They are usually located in introns in the same orientation as their host messenger RNA, generally using their promoter region, although they can also be transcribed by other promoters. It is very interesting to note that just one miRNA can regulate the expression of a multitude of target genes (Guo et al., 2010). miRNA of all phyla have been connected with developmental processes and disease (Bartel, 2009; Lewis and Steel, 2010). miRNA were first found to be involved in inner ear development and function when the expression of the miR-183 family (composed by miR-182, miR-96, and miR-183) was reported in the inner ear of zebrafish embryos (Wienholds et al., 2005). Soon after, it was found that these miRNA are expressed in hair cells of chicken and mouse, as well as in the mouse sensory otic neurons (Darnell et al., 2006; Li and Fekete, 2010; Weston et al., 2006). Moreover, loss of function mutations in miR-96 causes hearing loss in mice and men (Lewis et al., 2009; Mencía et al., 2009). miRNA defects have also been found in other ear diseases such as vestibular schwannomas (Cioffi et al., 2010), cholesteatomas (Friedland et al., 2009), and otitis media (Song et al., 2011). The key functions of miRNA in inner ear development and hearing pathophysiology have been recently reviewed in Rudnicki and Avraham (2012).

The study of the specific expression of miRNA in the inner ear has been approached by using different platforms of microarrays and complementary techniques (Barad et al., 2004; Liu et al., 2004; Søkilde et al., 2011). miRNA expression has been shown to be highly variable over time and space (Elkan-Miller et al., 2011; Friedman et al., 2009; Rudnicki and Avraham, 2012; Sacheli et al., 2009) and, interestingly, to correlate with specific developmental events (Sacheli et al., 2009). These data suggest that miRNAs have a central regulatory role in inner ear development that remains largely unexplored.

Further evidence of the importance of miRNA in inner ear development has been shown by interrupting the maturation process of miRNA through the cell-type-specific conditional deletion of Dicer1. The enzyme Dicer1 participates in the processing of the pre-miRNA that is required to acquire a mature and functional state (Bernstein et al., 2001). The use of specific otic Cre-lines such as Pax2-Cre (Soukup, 2009), Pou4f3-Cre (Friedman et al., 2009), and Foxg1-Cre (Kersigo et al., 2011) to abolish in specific inner ear domains the function of Dicer1 has shown different degrees of inner ear malformations. Phenotypes varied from evident morphological defects, such as the absence of the coiled structure of the cochlea, to more subtle defects, such as alteration in the shape of hair cell stereocilia.

The miR-183 family begins to be expressed at the otocyst stage and has the highest expression in differentiating hair cells, suggesting that this family is involved in hair cell differentiation and maturation (Sacheli et al., 2009). The three miRNAs that comprise this family are expected to be co-expressed in the same cells and transcribed in the same orientation (Weston et al., 2006; Wienholds et al., 2005). Other miRNAs associated with early inner ear development include miR-124, which is expressed in the spiral-vestibular ganglia (Weston et al., 2006), and the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429), which is expressed in the otic epithelia of zebrafish, chicken, and mouse (Darnell et al., 2006; Weston et al., 2006; Wienholds et al., 2005). The miR-200 family has been suggested to play a key role in establishing the prosensory epithelial domains (Soukup, 2009).

2.4. Apoptosis, Survival, and Proliferation in Inner Ear Development

Specific apoptosis, survival, and proliferation events shape the emerging otic vesicle. Programmed cell death contributes to the early development of the inner ear by shaping the otocyst (Lang et al., 2000; León et al., 2004). Apoptosis helps morphogenesis, contributes to get final proper cell numbers of otic cell populations, and removes defective cells. Blocking apoptosis with the pan-caspase inhibitor Boc-D-FMK caused an increased in the otocyst size, a thickening of the otic epithelium, and a reduced size of the AVG. These data suggest that apoptosis is required for otic morphogenesis and neurogenesis (Fig. 1.5 B) (Aburto et al., 2012a). Further, recent data suggest that cell death by apoptosis might allow the detachment and posterior migration of epithelial neuroblasts to populate the AVG (Aburto et al., 2012a).

Several growth and trophic factors have been reported to regulate otic cell survival and neurogenesis. IGF-I is single-chain 70 amino acid peptide that is implicated in inner ear embryonic development and adult cochlear homeostasis (Murillo-Cuesta et al., 2011; Varela-Nieto et al., 2013). IGF-I is secreted by the liver to exert endocrine functions, but it is also secreted locally to maintain local cellular homeostasis. Igf1 expression has been reported in the chicken and mouse developing inner ear (Camarero et al., 2002; Sanchez-Calderon et al., 2010). Its high affinity tyrosine kinase receptor, Igf1r, is expressed in the sensory patches of the HH19 chicken otocyst (Aburto et al., 2012a) and in the inner ear of E13.5-E15.5 mouse embryos (Okano et al., 2011; Sanchez-Calderon et al., 2010). Upstream Igf1 and Igf1r, genes involved in the specification of the neurosensory domain as Neurog1, promote the expression of proneural genes NeuroD and NeuroM, which in turn may up-regulate Igf1 expression to support otic neural progenitor survival and proliferation. IGF-I is transported and presented to its receptor by a family of IGF binding proteins. Igfbp2–5 show distinct expression patterns during cochlear development, suggesting that a local modulation of IGF-I signaling further occurs in the cochlea (Okano and Kelley, 2013).

To proceed with development, otic cells require the actions of several factors including IGF-I to survive (Fig. 1.5A). When local IGF-I actions are blocked in the cultured chicken otic vesicle, a reduction in neurogenesis is evidenced that was concomitant to an increase in TUNEL staining (Camarero et al., 2003). These data showed for the first time that IGF-I is a neuroprotector for the population of otic neural precursors. Even in the presence of added IGF-I, otic vesicles show apoptotic cell death in certain regions, indicating that IGF-I survival actions on otic progenitors are spatiotemporally regulated by downstream signaling. In contrast, early post-mitotic neurons seem to be independent of IGF-I as they become dependent on neurotrophins and to express the neurotrophins receptor TrkC (Aburto et al., 2012a). IGF-I regulates neuroblast survival through PI3K-AKT signaling pathway, and its actions can be blocked by LY294002, a chemical inhibitor of the pathway (Kong and Yamori, 2008) (LY; Fig. 1.5A). AVG size was severely reduced, with the neuroblast population the most affected cell type. Therefore, in the chicken embryo, IGF-I/AKT signaling is fundamental for the establishment of the absolute number of neurons as well as for defining the timing of neuron generation during otic development. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and, hence, the AKT signaling pathway. PTEN is a signaling node that also interacts with Wnt, Notch, and BMP pathways. An inner ear-specific Pten conditional knockout mouse has further confirmed the influence of PTEN/AKT/GSK3b signaling on the development of the spiral ganglion (SG) by showing cochlear defects; the data by these authors suggest that PTEN is required for the maintenance of neuroblast population number, neural precursors, and differentiation in the inner ear (Kim et al., 2013).

FIGURE 1.5   Cell survival, death, and proliferation during early inner ear development.  Representative experiments showing in cultured HH18 chicken otic vesicles the downstream signaling pathways involved in the modulation of otic cell survival, apoptosis, and proliferation, adapted from Magariños et al., 2012.   (A) Survival of specific cell populations in the otic vesicle is promoted by growth factors. Otic vesicles were isolated from embryos and cultured for 20 h in 0S medium without additions (a), 10 nM IGF-I (b), or in the presence of 25 μM PI3K/AKT inhibitor LY294002 (LY) (c). TUNEL levels decreased markedly in the presence of IGF-I and increased dramatically with LY (quantification in d). The data are shown as the mean+SEM, statistical significance between the different conditions was estimated by ANOVA: ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.005, versus IGF-I. (e) Levels of pAKT increase in otic vesicle lysates in the presence of IGF-I. Otic vesicles were incubated for 30 min with or without IGF-I, LY, 50 μM AKTi, or combinations of IGF-I and the inhibitors. Proteins levels were measured by Western blotting.   (B) Apoptosis is required for morphogenesis and AVG neurogenesis. (a-c) Blocking apoptosis in organotypic cultures of otic vesicles with a pan-caspase inhibitor Boc-D-FMK (BOC), 50 μM; b) caused a reduction in the AVG size and a thickening of the otic epithelium when compared with control conditions (a). (c) Cryostat section of a HH18 chicken embryo visualized by TUNEL (green throughout the figure) showing that programmed cell death occurs in vivo in the developing otic vesicle. (d-f, d’) Cultured otic vesicles from HH18 chicken embryos without additions show increased apoptosis with nuclear TUNEL staining (green) surrounded by cytoplasmic activated-caspase-3 (red) immunoreactivity.   (C) Cell proliferation in the developing otic vesicle. (a-c) Proliferation was measured by BrdU incorporation (1h; red). Cultured otic vesicles in 0S medium (a), with IGF-I (10 nM; b), or in the presence of the RAF inhibitor Sorafenib (Sor, 5 μM; c) showed the promotion of otic proliferation by IGF-I (b) and the impairment of the S-phase by Sor (c). As a result, the size of Sor-treated otic vesicles was reduced. (d) M-phase was also affected in Sor-treated otic vesicles as indicated by the quantification of phospho-Histone-3 (PH3) in cultured otic vesicles upon IGF-I exposure or Sor inhibition. (e) Levels of pERK were reduced in chicken otic vesicles cultured in presence of Sor. Representative blots are shown. Scale bar: 300 μm. Abbreviations: BOC: Boc-D-FMK (pan-caspase inhibitor); LY: LY294002 (PI3K/AKT inhibitor); Sor: Sorafenib (RAF-MEK-ERK inhibitor). Statistical significance was estimated with the Student’s t-test: ∗∗∗P<0.005 versus 0S; ###P<0.005 versus Sor.   Adapted from Magariños et al., 2010 and Aburto et al., 2012a.

To achieve inner ear organogenesis, it is necessary to strictly control the number of rounds of proliferation and cell cycle exit. Therefore, there are several signaling pathways implicated in the regulation of the cell cycle of otic progenitors, which include IGF-I, Notch, and Wnt (Beukelaers et al., 2012). We will focus here in IGF-I signaling (Fig. 1.5C).

IGF-I promotes proliferation of neural progenitors (Aberg et al., 2003; Mairet-Coello et al., 2009) and of cultured neural stem cells (Arsenijevic et al., 2001). These actions of IGF-I are spatiotemporally mediated by distinct signaling pathways (Magariños et al., 2010; Murillo-Cuesta et al., 2011; Varela-Nieto et al., 2013; Ye and D’Ercole, 2006). IGF-I promotes G1/S cell cycle progression by decreasing G1 phase length and increasing cell cycle entry (Hodge et al., 2004; Mairet-Coello et al., 2009). In chicken cultures of explanted otic vesicles, IGF-I promotes proliferation that can be followed by complementary cellular and molecular methodologies (Frago et al., 1998; Magariños et al., 2010; Sanz et al., 1999). The RAF-MEK-ERK pathway is known for its actions on cell proliferation due to its crucial role in the G1/S transition (Chambard et al., 2007; Schreck and Rapp, 2006), consequently silencing the RAF pathway with Sorafenib in cultured otic vesicles (Schreck and Rapp, 2006) and resulting in a dramatic decrease in otic cell proliferation (Fig. 1.5C) (Magariños et al., 2010). Exogenous IGF-I and the concomitant activation of the PI3K-AKT pathway does not rescue these proliferation defects, which show that RAF-MEK-ERK activity is essential.

The study of Igf1 and Igfr1 deficient mice has also shed light on the developmental actions of IGF-I. The Igf1 null mouse showed reduced levels of the activated forms of ERK1/2 in the cochlea in prenatal stages, as well as increased FoxM1 and decreased p27Kip1 transcript expression (Sanchez-Calderon et al., 2010). The Igfr1 deficient mouse exhibited a reduced rate of proliferation in precursors cells (Okano et al., 2011).

To sum up, IGF-I coordinates cell proliferation and survival in the otic vesicle through different pathways that cross talk to balance cell survival, proliferation, and differentiation (Magariños et al., 2010).

2.5. Autophagy in Inner Ear Development

Autophagy is a lysosomal degradation pathway of the cellular cytosolic constituents that is essential for regulating sources of energy in response to a variety of stimuli and for the elimination of protein aggregates and damaged organelles (Glick et al., 2010; Qu et al., 2007; Ravikumar et al., 2010; Rubinsztein et al., 2012). Autophagy in vertebrates has key functions during development (Levine and Klionsky, 2004; Mizushima and Levine, 2010). Accordingly, autophagy has been shown to be a key player for the development of vestibular function of mice (Mariño et al., 2010). Furthermore, autophagy seems to be central in the adult response to cochlear damage (Taylor et al., 2008) and in the premature age-related hearing loss of senescence-accelerated mouse prone 8 (SAMP8) mice (Menardo et al., 2012). Recently, the importance of autophagy during early inner ear development has been demonstrated experimentally [Fig. 1.6 ; (Aburto et al., 2012b)]. Autophagy inhibition by either genetic or pharmacological factors abolishes the elimination of dying cells, which dramatically impairs neurogenesis. These data suggest that activation of autophagy is required to generate the energy necessary for the migration of the neuronal precursors. An appealing hypothesis is that autophagy activation could be a general mechanism to provide energy in other singular developmental periods such as the differentiation of hair cells.

2.6. Cell-type Specification of Otic Neurosensory Components

2.6.1. Specification of Hair and Supporting Cells

After the specification of the neurosensory domain, hair and supporting cells differentiate (Chonko et al., 2013; Driver and Kelley, 2009). The first cells to be specified are the hair cells that in turn, by lateral inhibition, block the hair cell fate in the adjacent cells (Fig. 1.7 ). The basic helix–loop–helix (bHLH) transcription factor Atoh1 is one key player in hair cell determination. Atoh1 expression patterns have been reported in the inner ear of several species during development (Bermingham et al., 1999; Chen et al., 2002; Lanford et al., 2000). Loss and gain of function studies have shown that Atoh1 induces hair cell formation in sensory and non-sensory cells (Bermingham et al., 1999; Jones et al., 2006; Kawamoto et al., 2003; Woods et al., 2004; Zheng and Gao, 2000). Moreover, Atoh1 expressing cells can develop into hair cells or supporting cells (Driver et al., 2013; Matei et al., 2005; Yang et al., 2010). Recently it has been suggested that specific dosages of Atoh1 define hair cell types (Jahan et al., 2013).

Atoh1 expression is prevented by the Id proteins (inhibitors of differentiation) before hair cell formation starts (Jones et al., 2006). Ids do not have a DNA binding domain but contain an HLH domain that competes with Atoh1 for its dimerization partners E47 and E2A. Thus, they can impede the formation of a functional Atoh1 heterodimer (Norton, 2000). As development proceeds, Id expression is reduced in hair cells, thus allowing Atoh1 activity and consequently the differentiation of hair cells (Jones et al., 2006). When hair cells have differentiated, the neighboring cells develop as supporting cells by lateral inhibition. The Notch pathway is also implicated in individual cell differentiation, in addition to its previous roles in otic placode determination and neurosensory specification. The Atoh1 positive cells start to express the Notch ligands Jagged2 (Jag2) and Dll1 (Hartman et al., 2007; Lanford et al., 1999; Morrison et al., 1999). This leads to Notch1 activation in the future supporting cells and to expression of Notch targets such as Hes1, Hes5, Hey1, and Hey2 (Doetzlhofer et al., 2009; Hayashi et al., 2008; Lanford et al., 2000; Li et al., 2008; Murata et al., 2006; Zheng et al., 2000; Zine et al., 2001). The relevance of the Notch pathway has been recently enhanced by showing that the inhibition of Notch signaling with a gamma secretase inhibitor in the adult cochlea is able to restore hair cells in noise-exposed mice. Notch inhibition resulted in an increased level of Atoh1 that ended in hair cell generation by transdifferentiation of supporting cells (Mizutari et al., 2013). Atoh1-induced new hair cells supported a partial rescue of hearing, opening new perspectives for hearing loss therapies.

FIGURE 1.6   Autophagy as a new process in early otic development.  (A) Autophagy machinery is expressed in the developing inner ear. (a) Transmission electron microscopy from the neurogenic region of the otic vesicle at stage HH18 showing an omegasome (a’ arrow) and an autophagic vesicle containing cytoplasmic fragments (a’’ arrow). Bars 0.2 μm. (b) The autophagic genes Beclin-1 and Atg5 are expressed at early stages in the chicken inner ear. Gene expression was calculated as 2-ΔΔCt and normalized to the levels at HH17. (c) Immunofluorescence of LC3B (green) in autophagy-inhibited cultured otic vesicles. Otic vesicles were incubated in the 0S condition or with the inhibitor 3-methyladenine (3-MA; 10 mM). 3-MA-exposed otic vesicles show highly reduced levels of LC3B inmunostaining. (d) Cultured otic vesicle lysates exposed to 3-MA or Cloroquine (CQ; 10mM) were analyzed by Western blotting to determine the levels of LC3-I and LC3-II; β-Tubulin (β-Tub) was used as a loading control. A representative blot is shown. There was LC3I/LC3-II conversion in 0S otic vesicle cultures, which was increased by addition of CQ and blocked by treatment with 3-MA, indicating that there is autophaghic flux under these conditions.   (B) Autophagy provides energy for the clearance of the apoptotic cells and for neurogenesis. (a–c) Otic vesicles were cultured without (a) or with 3-MA (10 mM; b), which caused a reduction in the expression of the neural markers Islet-1 (red) and TuJ-1 (green). This phenotype is partially restored by supplying the culture media with methyl pyruvate, an exogenous energy source (MP; 10 mM; c). (d–h) Otic vesicles incubated without additions, 0S, treated with 3-MA, or in the presence of 3-MA in combination with MP, and then incubated with 1 mM lysotracker (LTR, red) for the last 15 min of culture. Apoptotic cell death was visualized by TUNEL (green). When autophagy is inhibited (e), TUNEL labeling increases and LTR is reduced, suggesting that the required apoptotic cell elimination is impaired. The addition of MP partially restores the accumulation of apoptotic staining (f; quantified in g and h). The results are shown as the mean ± S.E.M. relative to the 0S condition. Statistical significance was estimated with the Student’s t-test: ∗P<0.05, ∗∗P<0.01 and ∗∗∗P<0.005 versus 0S and ##P<0.01, ###P<0.005 versus 3-MA. Orientation: A, anterior; D, dorsal; Scale bar: 300 μm applies to a-c; 150 μm applies to d-f. Abbreviations: AVG: Acoustic-vestibular ganglion; LTR: lysotracker; MP: methyl pyruvate; 3-MA: 3-methyladenine.   Adapted from Aburto et al., 2012b.

2.6.2. Development of Acoustic and Vestibular Neurons

Epithelial neuroblasts are the common progenitors of otic auditory and vestibular neurons that populate the AVG. Later in development, auditory and vestibular neurons segregate; the specific mechanisms regulating early auditory and vestibular neuron specification and differentiation are largely unknown. In the adult, auditory and vestibular neurons will have defined traits and will connect their specific peripheral sensory targets with different brain centers.

The AVG is formed by neuroblasts from the otic neuroepithelia that delaminate and migrate to the adjacent mesenchymal space and transit through diverse stages to finally become post-mitotic neurons (Fig. 1.8 A). Thus, otic neurons in specific developmental stages can be identified by the distinct expression of a combination of molecular markers. Following the initial Neurog1 expression, the proneural genes NeuroD and NeuroM define the next stage in neural differentiation: the epithelial neuroblast population (Chae et al., 2004; Sanchez-Calderon et al., 2007). The migration of the neuroblasts to the mesenchyme has been studied in chicken and mouse embryos (Alvarez et al., 1989; Davies, 2011; Vemaraju et al., 2012).

The ganglionic neuroblast population can be recognized by their position, shape, and by the appearance of a distinct set of transcription factors, neurofilaments, and neurotrophin receptors. The LIM-HD transcription factor Islet-1 is expressed in epithelial and ganglionic neuroblasts and, later in development, in hair and supporting cells (Aburto et al., 2012a; Camarero et al., 2003; Li et al., 2004). Ganglionic neuroblasts can still proliferate and increase their population; accordingly they express cell cycle activator factors as FoxM1. Neuroblasts exit from the cell cycle and become post-mitotic neurons that will generate and project their neurites to peripheral and central targets (Appler and Goodrich, 2011; Fantetti and Fekete, 2011, 2012). This process is regulated by a different subset of trophic factors and signaling pathways (Fig. 1.8B). Thus, whereas IGF-I promotes survival by proliferation of neuroblasts (Aburto et al., 2012a; Magariños et al., 2010), post-mitotic otic neurons express neurotrophin receptors TrkB and TrkC (Avila et al., 1993; Pirvola and Ylikoski, 2003; Ramekers et al., 2012; Represa et al., 1993).

FIGURE 1.7   Notch function during hair cell differentiation.  Schematic view showing the regulation of the differentiation of hair cells and supporting cells promoted by Notch signaling and the process of lateral inhibition. Hair cells express the transcription factor Atoh1 and the ligands Dl1 and Jag2. These ligands activate Notch in the adjacent cells that will now assume the supporting cell identity. The Notch receptor cleaves upon ligand binding and the intracellular domain travels to the nucleus, where it will transcribe Hes/Hey genes that abrogate the hair cell fate. Supporting cells preserve Jag1 expression and Notch activation. Sox2 expression is also sustained only in the supporting cells. It has recently been hypothesized that the miR-183 is also playing a role in this differentiation process. Abbreviations: HC: hair cell; SC: supporting cell.

FIGURE 1.8   Early otic neurogenesis.  (A) In vivo early otic neurogenesis. (a-b) Cryostat section of an HH19 chicken embryo showing the immunostaining for the transcription factor Islet-1 (green) and the neuronal marker G4 (magenta) (a). Panel b corresponds to the boxed area in (a). (c) Schematic illustration of several stages of development of otic neurons and the concomitant temporal expression of extrinsic and intrinsic factors. Post-mitotic neurons depend on neurotrophins for survival, but the factors expanding neuroblast populations are still to be fully defined (adapted from Sanchez-Calderon et al., 2007). (B) Signaling during otic neuritogenesis. The AVG can be explanted at HH19, cultured in the presence of inhibitors, and then immunostained for neural markers such as Islet-1 (green), G4 (red or green, see legends in the figure), or TuJ-1 (magenta). Inhibition of both IGF-I signaling pathways, RAF-MERK-ERK and PI3K/AKT, impaired otic neuritogenesis. Sor-treated AVG have shorter processes without affecting the size of the AVG soma (left panel). LY-treated AVG showed that both the neuronal soma area and the length of the neurites of the LY culture are smaller (middle panel). Inhibition of autophagy with 3-MA also alters AVG neuritogenesis (right panel). Scale bar: 300 μm. Abbreviations: AVG: Acoustic-vestibular ganglion; Del: delamination; IN: immature neuron; LY: LY294002 (PI3K/AKT inhibitor); MP: multipotent progenitor; NBe: epithelial neuroblast; NBg: ganglionar neuroblast; ON: otic neuron; Sor: Sorafenib (RAF-MEK-ERK inhibitor); 3-MA: 3-methyladenine. Orientation: A, anterior; D, dorsal.   Adapted from Magariños et al., 2010 and Aburto et al., 2012a.

Recently, the in vitro generation of otic neurons from embryonic stem cells that were able to populate a damaged spiral ganglion and to restore hearing has been reported (Chen et al., 2012). These promising results along with others (Kwan et al., 2009) are paving the way to design feasible hearing loss therapies based on regenerative medicine, and they reinforce the need to expand our knowledge on how auditory neurons originate, differentiate, survive, and relate to their targets.

Acknowledgments

We thank the critical comments and generous sharing of results of our colleagues at the Neurobiology of Hearing group. This research was supported by grants from the Spanish Government SAF2011–24391, Fundación de Investigación Médica Mutua Madrileña, and AFHELO (FP7, European Union) to I V-N.

References

Aberg M.A.I., Aberg N.D., Palmer T.D., Alborn A.-M., Carlsson-Skwirut C., Bang P., Rosengren L.E., Olsson T., Gage F.H., Eriksson P.S. IGF-I has a direct proliferative effect in adult hippocampal progenitor cells.  Mol. Cell. Neurosci . 2003;24:23–40.

Aburto M.R., Magariños M., Leon Y., Varela-Nieto I., Sanchez-Calderon H. AKT signaling mediates IGF-I survival actions on otic neural progenitors.  PloS One . 2012;7:e30790.

Aburto M.R., Sánchez-Calderón H., Hurlé J.M., Varela-Nieto I., Magariños M. Early otic development depends on autophagy for apoptotic cell clearance and neural differentiation.  Cell Death Dis . 2012;3:e394.

Adam J., Myat A., Le Roux I., Eddison M., Henrique D., Ish-Horowicz D., Lewis J. Cell fate choices and the expression of Notch, Delta and Serrate homologues in the chick inner ear: parallels with Drosophila sense-organ development.  Development . 1998;125:4645–4654.

Alvarez I.S., Martín-Partido G., Rodríguez-Gallardo L., González-Ramos C., Navascués J. Cell proliferation during early development of the chick embryo otic anlage: quantitative comparison of migratory and nonmigratory regions of the otic epithelium.  J. Comp. Neurol . 1989;290:278–288.

Alvarez Y., Alonso M.T., Vendrell V., Zelarayan L.C., Chamero P., Theil T., Bösl M.R., Kato S., Maconochie M., Riethmacher D.