Olivia Bermingham-McDonogh

Hair cell regeneration: winging our way towards a sound future.

The discovery of hair cell regeneration in the inner ear of birds provides new optimism that there may be a treatment for hearing and balance disorders. In this review we describe the process of hair cell regeneration in birds; including restoration of function, recovery of perception and what is currently known about molecular events, such as growth factors and signalling systems. We examine some of the key recent findings in both birds and mammals.

Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear

During inner ear morphogenesis, the process of prosensory specification defines the specific regions of the otic epithelium that will give rise to the six separate inner ear organs essential for hearing and balance. The mechanism of prosensory specification is not fully understood, but there is evidence that the Notch intercellular signaling pathway plays a critical role. The Notch ligand Jagged1 (Jag1) is expressed in the prosensory domains, and mutation of Jag1 impairs sensory formation. Furthermore, pharmacological inhibition of Notch in vitro during prosensory specification disrupts the prosensory process. Additionally, activation of Notch by cDNA electroporation in chick otocysts results in formation of ectopic sensory patches. Here we test whether Notch activity is sufficient for prosensory specification in the mouse, using a Cre-/loxP approach to conditionally activate the Notch pathway in nonsensory regions of the inner ear epithelia during different stages of otic vesicle morphogenesis. We find that broad ectopic activation of Notch at very early developmental stages causes induction of prosensory markers throughout the entire otic epithelium. At later stages of development, activation of Notch in nonsensory regions leads to induction of sensory patches that later differentiate to form complete ectopic sensory structures. Activation of Notch in isolated nonsensory cells results in lateral induction of Jag1 expression in neighboring cells and spreading of prosensory specification to the adjacent cells through an intercellular mechanism. These results support a model where activation of Notch and propagation through lateral induction promote prosensory character in specific regions of the developing otocyst.

Fgf20 Is Required for Sensory Epithelial Specification in the Developing Cochlea

Tissue-specific deletion of Fgfr1 results in severe defects in the development of both hair cells and support cells (Pirvola et al., 2002). Despite the importance of Fgfr1 in this early phase of cochlear development, the timing for the requirement for FGF signaling at this stage is not known. Therefore, we investigated the requirement for FGF signaling at early stages of cochlear development using an FGF receptor inhibitor. We find that inhibition of FGF signaling from embryonic day 14 (E14) to E16 has a dramatic effect on the development of the sensory epithelium, causing a severe reduction in hair cells and support cells, similar to that reported for the Fgfr1 deletion. The effects of inhibition of FGF signaling on sensory specification are not explained by increases in cell death or changes in proliferation but lead to a rapid reduction in Pea3 and Erm and a loss of Math1 expression. We also show that a specific FGF, FGF20, is the likely ligand for FGFR1 at this sensory specification phase of cochlear development; Fgf20 is expressed at the right time and place to mediate sensory cell specification, and blocking FGF20 with a specific antibody inhibits hair cell and support cell development in a manner similar to the FGF receptor inhibitor. Our results thus define the period of FGF-dependent sensory cell specification and the ligand that mediates this step in cochlear development.

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea.

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.

Regulated Reprogramming in the Regeneration of Sensory Receptor Cells

Vision, olfaction, hearing, and balance are mediated by receptors that reside in specialized sensory epithelial organs. Age-related degeneration of the photoreceptors in the retina and the hair cells in the cochlea, caused by macular degeneration and sensorineural hearing loss, respectively, affect a growing number of individuals. Although sensory receptor cells in the mammalian retina and inner ear show only limited or no regeneration, in many nonmammalian vertebrates, these sensory epithelia show remarkable regenerative potential. We summarize the current state of knowledge of regeneration in the specialized sense organs in both nonmammalian vertebrates and mammals and discuss possible areas where new advances in regenerative medicine might provide approaches to successfully stimulate sensory receptor cell regeneration. The field of regenerative medicine is still in its infancy, but new approaches using stem cells and reprogramming suggest ways in which the potential for regeneration may be restored in individuals suffering from sensory loss.

Neuregulin Expression in PNS Neurons: Isoforms and Regulation by Target Interactions

Neuregulins have several important functions in the development of the peripheral nervous system, acting on both developing Schwann cells and muscle fibers. To determine whether these factors are also important for peripheral nerve regeneration, we have analyzed neuregulin expression in motor and sensory neurons by Northern blots and in situ hybridization. The results of this analysis show that the predominant neuregulin isoform expressed in these neurons is a novel transmembrane splice variant. After axotomy, there is a rapid decline in neuregulin expression in both motor and sensory neurons, but following reinnervation of target tissues, neuregulin expression returns to near normal levels. These results indicate that the normal expression of neuregulins in these neurons is maintained by the interactions with target tissues.

Acheate-scute like 1 (Ascl1) is required for normal delta-like (Dll) gene expression and notch signaling during retinal development

Delta gene expression in Drosophila is regulated by proneural basic helix–loop–helix (bHLH) transcription factors, such as acheate‐scute. In vertebrates, multiple Delta‐like and proneural bHLH genes are expressed during neurogenesis, especially in the retina. We recently uncovered a relationship between Acheate‐scute like 1 (Ascl1), Delta‐like genes, and Notch in chick retinal progenitors. Here, we report that mammalian retinal progenitors are also the primary source of Delta‐like genes, likely signaling through Notch among themselves, while differentiating neurons expressed Jagged2. Ascl1 is coexpressed in Delta‐like and Notch active progenitors, and required for normal Delta‐like gene expression and Notch signaling. We also reveal a role for Ascl1 in the regulation of Hes6, a proneurogenic factor that inhibits Notch signaling to promote neural rather than glial differentiation. Thus, these results suggest a molecular mechanism whereby attenuated Notch levels coupled with reduced proneurogenic activity in progenitors leads to increased gliogenesis and decreased neurogenesis in the Ascl1‐deficient retina. Developmental Dynamics 238:2163–2178, 2009.

Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A. Detailed evaluation of inner ear phenotype in the Clrn1 knockout mouse (Clrn1(-/-)) coupled with expression pattern of Clrn1 in the inner ear are presented here. Clrn1 was expressed as early as embryonic day 16.5 in the auditory and vestibular hair cells and associated ganglionic neurons, with its expression being higher in outer hair cells (OHCs) than inner hair cells. Clrn1(-/-) mice showed early onset hearing loss that rapidly progressed to severe levels. Two to three weeks after birth (P14-P21), Clrn1(-/-) mice showed elevated auditory-evoked brainstem response (ABR) thresholds and prolonged peak and interpeak latencies. By P21, approximately 70% of Clrn1(-/-) mice had no detectable ABR and by P30 these mice were deaf. Distortion product otoacoustic emissions were not recordable from Clrn1(-/-) mice. Vestibular function in Clrn1(-/-) mice mirrored the cochlear phenotype, although it deteriorated more gradually than cochlear function. Disorganization of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cell dysfunction and prolonged peak latencies in vestibular and cochlear evoked potentials in Clrn1(-/-) mice strongly indicate that Clrn1 is necessary for hair cell function and associated neural activation.

CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.

Ontogenetic expression of trk neurotrophin receptors in the chick auditory system

Neurotrophins and their cognate receptors are critical to normal nervous system development. Trk receptors are high‐affinity receptors for nerve‐growth factor (trkA), brain‐derived neurotrophic factor and neurotrophin‐4/5 (trkB), and neurotrophin‐3 (trkC). We examine the expression of these three neurotrophin tyrosine kinase receptors in the chick auditory system throughout most of development. Trks were localized in the auditory brainstem, the cochlear ganglion, and the basilar papilla of chicks from embryonic (E) day 5 to E21, by using antibodies and standard immunocytochemical methods. TrkB mRNA was localized in brainstem nuclei by in situ hybridization.TrkB and trkC are highly expressed in the embryonic auditory brainstem, and their patterns of expression are both spatially and temporally dynamic. During early brainstem development, trkB and trkC are localized in the neuronal cell bodies and in the surrounding neuropil of nucleus magnocellularis (NM) and nucleus laminaris (NL). During later development, trkC is expressed in the cell bodies of NM and NL, whereas trkB is expressed in the nerve calyces surrounding NM neurons and in the ventral, but not the dorsal, dendrites of NL. In the periphery, trkB and trkC are located in the cochlear ganglion neurons and in peripheral fibers innervating the basilar papilla and synapsing at the base of hair cells.The protracted expression of trks seen in our materials is consistent with the hypothesis that the neurotrophins/tyrosine kinase receptors play one or several roles in the development of auditory circuitry. In particular, the polarized expression of trkB in NL is coincident with refinement of NM terminal arborizations on NL. J. Comp. Neurol. 413:271–288, 1999.