The cellular role of Atoh1 in development and regeneration in the mammalian cochlea Open Access

Kelly, Michael C (2011)

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Intricate neuroanatomical structures within our inner ears impart us with the ability to
detect sound with incredible detail. This ability is largely due to the function of the
auditory sensory end organ, known as the organ of Corti, which is made up of an
exquisitely arranged mosaic pattern of specialized mechanosensitive hair cells that
convert sound into a neuronal signal and non-sensory supporting cells. Over the course of
development, multiple pathways converge to generate and pattern the cells of organ of
Corti. Auditory hair cell damage can result from a variety of genetic and environmental
factors, and their loss results in sensorineural hearing impairment. Since the mature
mammalian ear does not spontaneously regenerate hair cells, these associated hearing
deficits are permanent.

The transcription factor Atoh1, which has an essential role during development
directing the differentiation of sensory hair cells within the inner ear, under certain
conditions can promote the generation of new hair cells when delivered to cochlear
epithelial cells. Atoh1 has become an important target for promoting
hair cells regeneration. However, the potential and limitations of Atoh1-mediated hair
cell generation have not yet been systematically tested. In order to determine which cells
within the cochlear epithelium are competent for Atoh1-mediated hair cell generation at
various developmental timepoints, I generated inducible constructs and transgenic mouse
models to direct the temporal and spatial activity of Atoh1.

Using these models, I show that Atoh1 can generate new hair cells within the cochlear
epithelium, but that the competency for hair cell generation changes significantly
depending on cell type and developmental stage. Atoh1-generated hair cells
arise within patterned ectopic sensory regions that are reminiscent of endogenous sensory
regions, and that this patterning is mediated through Notch signaling. Extended Atoh1
induction results in an expansion of regional competency for hair cell differentiation in
non-sensory regions and a conversion of supporting cells. Intriguingly, Atoh1 induction
can also cause the normally post-mitotic cochlear epithelium to re-enter cell cycle. These
studies outline parameters for Atoh1 hair cell generation and suggest that
Atoh1 can help pattern the cochlear epithelium through cell fate decisions and
proliferation control.

Table of Contents

Chapter I: General Introduction 1

1.1 Basic Mammalian Inner Ear Anatomy and Auditory Function 2

A. Anatomy of the Inner Ear 2

B. Sensory Region Architecture: Hair Cells and Supporting Cells 7

C. Pathway of Hearing: From Sound to Hair Cell Response 11

1.2 Development of the Auditory Inner Ear 16

A. Inner Ear Induction & Cochlear Duct Outgrowth 17

B. Auditory Sensory Domain Specification & Cell Cycle Exit 19

C. Cell Fate Decisions, Arrangement & Patterning in the Cochlea 24
D. Postnatal Cochlear Maturation and Hearing Onset 29

1.3 Sensorineural Hearing Loss & Regeneration 31

A. Hair Cell Death 32

B. Hair Cell Regeneration in Non-Mammalian Species 35

C. Strategies to Promote Hearing Recovery in Mammals 38

1.4 Atoh1: An Incomplete Story in Development and Regeneration 42

Chapter II: Potency of Atoh1 Delivery to Cochlear Explants 47

2.1 Introduction 47

2.2 Methods 49

2.3 Results 54

A. Tamoxifen inducible Atoh1-ER generates ectopic hair cells 54

B. Lentiviral Transfection of Cochlear Explants with Atoh1 58

2.4 Discussion 61

Chapter III: Potency of Atoh1 in the Entire Cochlea In Vivo 64

3. 1 Introduction 64

3. 2 Methods 66

3. 3 Results 71

A. Generation of an Inducible Transgenic Atoh1 Mouse 71

B. In Vivo Generation of Functional Ectopic Hair Cells 75

C. Age-Related Changes in Competency 81

D. Cellular Patterning in Ectopic Sensory Regions 85

E. Effects of Extended Atoh1 Induction in the Mammalian Cochlea 88

3.4 Discussion 97

Chapter IV: Targeting Atoh1 to Sensory Region Cells 101

4. 1 Introduction 101

4.2 Methods 103
4.3 Results 106
A. GFAPCre Directs Dox-Inducible TgAtoh1 to Supporting Cells 106

B. BAC Transgenic Drives Atoh1 Throughout Prosensory Domain 109

C. Atoh1-ER BAC Transgenic for Targeting Supporting Cells In Vivo 113

4.4 Discussion 116

Chapter VI: General Discussion of Atoh1's Cochlear Effects 119

References 146

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