Учебники / Genetic Hearing Loss Willems 2004

regulator Unc-86 (63,64). Brn-3a and Brn-3b have been shown to play a role in spiral ganglion neuron development (65,66). In developing and adult mice, Brn-3c/Brn-3.1/POU4F3 displays strong expression in the sensory HCs of both the cochlear and vestibular systems. Targeted null mutations of this gene lead to loss of all the cochlear and vestibular HCs, resulting in complete deafness and profound deficit in the vestibular system in the Brn- 3c-/- mice (65,67–70). Therefore, Brn-3c seems to play a pivotal role in the development of inner ear sensory HCs. The examination of the development of the cochlea in Brn-3c-/- mice during embryogenesis indicates that prior to degeneration, HCs become committed and pass through the early stages of development (71). These data indicate that Brn-3c is required for maturation, survival, and proper position of HCs, whereas it plays little role in commitment and initial di erentiation of HCs.

2.bHLH Transcription Factors

Regulatory cascades of positive and negative basic helix-loop-helix (bHLH) transcription factors play essential roles in mammalian neurogenesis (72– 74). The bHLH genes, such as Math1, Mash-1, and NeuroD, are thought to positively regulate neuronal development, i.e., proneural genes, at the level of commitment and postmitotic di erentiation (75,76). Other bHLH genes, like Hes1 and Hes5, negatively regulate the transcription of the positivebHLH genes (77).

Math1, a mouse homolog of the Drosophila proneural gene atonal, is essential for HC development. Embryonic Math1-null mice fail to generate cochlear and vestibular HCs (78). In addition, temporal expression patterns of Math1 correlate well with the time period for HC di erentiation. Math1 is expressed in di erentiating HCs at early embryonic stages, but is downregulated in SCs and absent in cells outside the sensory epithelium at late embryonic stages (78). In addition, overexpression of Math1 in postnatal rat organs of Corti induces a production of supernumerary HCs in the greater epithelial ridge (52). These findings demonstrate that Math1 is necessary and su cient for the development of cells as HCs, and strongly suggest that Math1 acts as a ‘‘prosensory’’ gene within the organ of Corti and that the enhancement of Math1 expression level in the inner ear might be used to stimulate HC regeneration.

The negative bHLH transcription factors Hes1 and Hes5 are homologs to the products of Drosophila hairy and Enhancer-of-split [E(spl)] and have been demonstrated to a ect cell fate determination by inhibiting the action of bHLH-positive regulators, such as Math1, during neuronal development (74,77). Cochleae from Hes1and Hes5-null mice show a significant increase, respectively, in the number of inner and outer HCs (79,80). Cotransfection

experiments in cultured postnatal rat organ of Corti explants show that overexpression of Hes1 prevents HC di erentiation induced by Math1 (79). Therefore, Hes1 can negatively regulate HC di erentiation by antagonizing Math1, as has been shown during neurogenesis. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear HCs.

3.Steroid/Thyroid Receptor Family

Steroid/thyroid receptor superfamily genes act as nuclear transcription factors and are characterized by a stereotype molecular structure that includes a zinc-finger DNA-binding domain and a conserved ligand-binding/ dimerization region (81). This growing family currently includes more than 50 di erent genes, including retinoic acid receptors (RAR) and retinoid X receptors (RXR) and thyroid hormone receptors (TR).

Retinoid Signaling and HC Di erentiation. Retinoids are a family of molecules derived from vitamin A and include the biologically active metabolite retinoic acid (RA). The cellular e ects of RA are mediated through the action of two classes of receptors, the RARs, which are activated by both all-trans-RA (tRA) and 9-cis-RA, and the RXRs, which are activated only by 9-cis-RA (82,83). Three major subtypes, a, h, and, g, of these receptors have been identified with multiple isoforms due to alternative splicing and di erential promoter usage (84). The RARs mediate gene expression by forming heterodimers with the RXRs, while the RXRs can mediate gene expression as homodimers or by forming heterodimers with a variety of orphan receptors (85). Both excess and deficiency of retinoids lead to teratogenic e ects including at the inner ear level (86,87). Some of the RAR and RXR receptors are expressed within the developing cochlear duct (88–90). As development continues, the expression of each receptor becomes more intense in cells that will develop as HCs (91). RAR-null mutant studies have shown that these nuclear receptors are critical for normal inner ear development (92). These results clearly suggest that signaling by RAR plays a direct role in the development of the cochlea. Indeed, several lines of evidence have implicated the intracellular receptors for RA in controlling critical steps in cochlear di erentiation. RA has been shown by an in vitro reporter assay to be produced endogenously in the inner ear (93). In vitro assays using cultured chick otocysts indicate that RA can inhibit mitogen-induced cellular proliferation and alter normal otocyst morphogenesis as well as induce premature histological di erentiation of the cultured cochlear epithelium (94,95). In organotypic cultures of the developing mouse cochlea, exposure to RA results in the formation of supernumerary HCs and SCs (93). In

addition, blocking the activation of retinoid receptors in cultures of the embryonic cochlea, with receptor-specific antagonists or inhibitors of retinoic acid synthesis, results in a significant decrease in the number of cells that develop as HCs and a disruption in the development of the organ of Corti (91).

The potential role of RA in HC regeneration has also been studied. As previously described, HC regeneration occurs following ototoxin treatment in the vestibular epithelia of adult mammals but not in the cochleas (96,97). Interestingly, RA and cellular RA-binding protein (CRABP), which controls the level of free intracellular RA and is involved in the transfert of RA from the cytoplasm to the nucleus, are expressed in the adult vestibular epithelia but not in the adult organ of Corti (98,99). In addition, it has been shown that an upregulation in the expression of the RARh gene appears after noise-induced damage to the chick basilar papilla (100). We have also reported that RA can induce cellular proliferation and HC regeneration in the postnatal rat organ of Corti in culture (20,101).

Thyroid Hormone and Inner Ear Development. Thyroid hormone (triiodothyronine, T3) and its receptors are essential for the development of hearing. Congenital thyroid disorders impair hearing, and profound deafness is common when there is a prevalence of iodine deficiency (102,103). Also, hypothyroidism in rodents causes a pronounced disruption of the organ of Corti especially at the level of the HCs (104,105). T3 receptors (TRs) are ligand-dependent transcription factors encoded by two genes,a and h c-erbA, that give rise to three functional TRs, a1, h1, and h2, and to nonthyroid-hormone-binding isotype a2 (106,107). Both TRa1 and TRh are expressed during embryonic and postnatal development of the cochlea indicating that cochlea is the direct site of T3 action (108,109). TRhnull mice are severely deaf as assessed by defective auditory-evoked brainstem responses (ABR) whereas TRa1-null mice have normal ABR (110). These results clearly suggest that T3 plays a role in the development of the cochlea.

4.Other Transcription Factors Implicated in HC Differentiation

GATA3. The GATA family of zinc finger transcription factors includes six related proteins and are involved in the development of many di erent systems including the hematopoietic and the respiratory systems. Among these factors, GATA3 has been shown to be expressed in both the brain and the inner ear (111–114). GATA3 is expressed in all cells of the developing sensory epithelium before HC di erentiation, i.e., at E14 in mice. Expression decreases selectively in the HCs as they di erentiate progressively from base to apex in the developing organ of Corti. GATA3 sub-

sequently decreases in the SCs and cannot be detected in the adult sensory epithelium. GATA3 has also been shown to be involved in the human hypoparathyroidism, sensorineural deafness, and renal abnormalities (HRD) syndrome (115). Taken together, these results suggest that GATA3 is important for cochlear development and its expression pattern may reflect an inhibitory e ect on cell di erentiation.

Sox10. More than 20 members of the Sox gene family of transcription factors, characterized by a high-mobility group (HMG) domain as DNAbinding motif, play important roles in diverse developmental processes such as chondrogenic di erentiation or hematopoiesis (116). Among these factors, the Sox10 gene is selectively expressed in neural crest cells during early stages of development, in glia cells of the peripheral and central nervous systems, and in the otic vesicle (117,118). Sox10 mRNA is expressed in the entire epithelia of the developing cochlea at E13.5. At P0 and later, Sox10 expression is restricted to the SCs of the organ of Corti (119). Sox10 is also defective in Waardenburg-Shah syndrome characterized by an aganglionic colon as well as sensorineural deafness and pigmentation abnormalities (120). These data suggest that Sox10 is also important for development of the cochlea.

C.Myosin Superfamily and HC Differentiation

Myosins form a superfamily of proteins that includes the conventional class II myosins and 14 classes of unconventional myosins that are distinguished based on the homology of the motor domains (121). Multiple myosin isozymes are present in the cochlea specifically in the HCs, and they appear to have important nonredundant functions, as evidenced by the di erent deaf mutants described in both humans and mice. Mutations in the Myo7a gene have been found in shaker1 mouse mutants, Usher type 1B in humans, and both dominant (DNFA11) and recessive (DFNB2) forms of nonsyndromic deafness in humans (122,123). Myo15 has been reported to underlie both the shaker2 mouse mutant and another form of human recessively inherited deafness (DNFB3) (124,125). Murine myosin VI (Myo6) mutations are responsible for the inner ear abnormalities in the deaf mouse mutant known as Snell’s waltzer mouse (126). The di erent myosin gene mutations that result in deafness in both mice and humans underline the crucial role that myosins exert in the actin-rich environment of the cilia of the HCs in the inner ear. The specific functions of the di erent myosins within the HCs, however, have not yet been determined. Ultrastructural studies of the HCs of all mutant mice, i.e., shaker 1, shaker 2, and Snell’s waltzer, have revealed that the stereocilia bundles are disorganized (127– 129). This observation suggests that Myo6, Myo7a, and Myo15 are neces-

sary for normal development of the stereocilia; however, the precise molecular events perturbed by defective specific myosins remain undefined.

D.Cyclin-Dependent Kinase Inhibitors and Cell Differentiation

Inhibitors of cell cycle progression include the cyclin-dependent kinase (CDK) inhibitors, which function in both cell-cycle arrest and di erentiation in many cell types. They exert these functions by binding to and inactivating CDK complexes. Two classes of CDK inhibitors have been identified: the INK4 family, which are specific inhibitors of cyclin D–CDK (i.e., CDK4 and CDK6), and the Cip/Kip family including p21, p27, and p57, which inhibit all types of cyclin-CDK complexes (130,131). Cip/Kip proteins have been

implicated as critical terminal e ectors of the signal transduction pathway that controls cell di erentiation. p27KIP1 has been shown to be an essential

mediator of oligodendrocyte terminal di erentiation (132). p27KIP1 expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the HCs and SCs (133). In wild-type animals, p27KIP1 expression is downregulated during subsequent HC di erentiation, but persists at high levels in di erentiated SCs of the mature organ of Corti. In mice with a targeted deletion of the p27KIP1 gene, ongoing cell proliferation occurs in the postnatal organ of Corti at time points well after the normal cessation of mitosis, leading to the appearance of supernumerary HCs and SCs (133,134). In the absence of p27KIP1, mitotically active cells are still observed in the organ of Corti of P6 animals, suggesting that the persistence of p27KIP1 expression in mature SCs may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. In addition, it has recently been shown that p27KIP1 antisense oligonucleotides trigger supporting cell proliferation in the mature organ of Corti after aminoglycoside insult (135). Therefore, p27KIP1 may become a major target for HC regeneration.

E.Growth Factors and Their Receptors: Implication in HC Regeneration

Several investigators have attempted to stimulate HC regeneration in the chick and mammalian inner ear epithelia by addition of peptidic growth factors or through the manipulation of various second-messenger pathways.

1.Fibroblast Growth Factors

Several fibroblast growth factors (FGFs) and their corresponding receptors have been detected in inner ear sensory epithelium (136–140). FGF-2 has

been shown to stimulate mitosis in rat utricular macula in culture (141). Among FGF receptors, recent studies point to a specific receptor tyrosine kinase, FGFR3, as playing a key role in the development and maintenance of the auditory epithelium. FGFR3 is specifically expressed in SCs, and not in HCs, in the basilar papilla and in the organ of Corti of mammals (136). Following HC trauma to the chick or mammalian cochlea, FGFR3 is rapidly modulated in the SCs, suggesting that this receptor plays a role in the response to HC loss (136,142). In addition, deletion of this gene through homologous recombination in mice results in abnormal cochlear development; a particular type of support cell, the pillar cell, fails to develop in these knockout mice (143). These results suggest a possible role for FGFs in HC and SC regeneration.

2.Other Growth Factors

Insulin growth factor-I (IGF-I) and insulin have been shown to induce cell proliferation in the mature avian vestibular sensory epithelium (144). In mammals, studies using organotypic culture indicate that IGF-I (141), transforming growth factor a (TGFa), and epidermal growth factor (EGF) supplemented with insulin (145,146) are mitogenic for SCs in the postnatal and the mature mammalian vestibular sensory epithelium. Perilymphatic infusion of TGFa with insulin induced cell proliferation in the utricular sensory epithelium of adult rats (147). These e ects on cell proliferation may be the first step towards HC regeneration. Very recently, it has been shown that growth factor treatment can enhance vestibular HC regeneration in vivo. Infusion of a combination of TGFa, IGF-I, and RA into the perilymphatic space of adult guinea pig vestibule after gentamicin administration results in a significant enhancement of the regeneration of the vestibular HCs (17). Factors able to stimulate cell proliferation in the mammalian cochlea have not been yet identified. In addition, TGFa has been implicated as a factor driving HC regeneration in neonatal organ of Corti cultures (21,148).

3.Second-Messenger Pathways

Binding of growth factors to receptor tyrosine kinases leads to the activation of intracellular cascades composed of enzymes and signaling intermediates that could also be potential targets for therapeutic induction of HC regeneration. High concentrations of forskolin stimulate SC proliferation in chicken cochleae and in mammalian vestibular sensory epithelium in vitro, and inhibitors of protein kinase A reduce that e ect (149,150). Phosphatidylinositol 3-kinase (P13-K), the target of rapamycin (TOR), the mitogenactivated protein kinase pathway (MAPK), and some protein kinase C

(PKCs) participate in the induction of S-phase entry in cultured vestibular epithelia from chickens and rats (151). Future studies will be needed to identify the specific isoforms of the kinases that function in the proliferation cascades possibly making it possible to define specific drug targets suitable for the modulation of progenitor cell proliferation and the capacity for regenerative replacement of sensory HCs in mammalian inner ears.

IV. CONCLUSIONS

This brief overview illustrates that significant progress has been achieved to molecularly dissect vertebrate inner ear development and regeneration. However, the patterning process of the ear is not su ciently known at the moment to provide clues for human HC regeneration. Avian and mammalian auditory epithelia share many anatomical and functional features: they have similar sorts of specialized cell types and mechanisms of sensory signal transduction. Despite these similarities, these two classes of vertebrates clearly possess di erences with respect to the ability to form new cells after birth. Studies on nonmammalian vertebrates have shown that HCs can be formed postembryologically, enabling the return of normal structure and function in mature auditory and vestibular organs after damage. In mammals, all cells in the organ of Corti become terminally mitotic by E16 and experimentally induced HC loss does not cause renewed mitotic activity. The failure of renewed proliferation in the mammalian auditory epithelium may be caused by persistent inhibition of mitotic activity among progenitor cells or by the absence of postmitotic stimuli in response to HC loss. However, the recent demonstration that cultured immature nestin-positive cells present in the newborn rat organ of Corti can proliferate in vitro and subsequently di erentiate into HCs and SCs, together with the detection of nestin cells in vivo at the spiral limbus in the P15 mature organ of Corti (53), opens new prospects with regard to regeneration of the injured organ of Corti.

Our ability to regenerate or repair damaged HCs therapeutically will certainly be dependent on a better understanding of the many signaling pathways involved in fate specification. Many genes implied in HC development or regeneration have been recently identified, including the Notch

signaling pathway, various transcription factors, especially Math1, Brn-3c, RA, and thyroid hormone, cell cycle protein such as p27KIP1, and growth

factors and their receptors. Activating these systems in the mammalian inner ear will provide real potential for stimulating HC replacement. However, many questions remain unanswered including the ways in which the factors themselves are regulated and interact, the identify of their target genes, and the manner in which the transcription of these genes is influenced. The