Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential

The development of the vertebrate inner ear is an emergent process. Its progression from a relatively simple disk of thickened epithelium within head ectoderm into a complex organ capable of sensing sound and balance is controlled by sequential molecular and cellular interactions. Fibroblast growth factor (FGF) and Wnt signals emanating from mesoderm and neural ectoderm have been shown to direct inner ear fate. However, the role of these multiple signals during inner ear induction is unclear. We demonstrate that the action of the FGFs and Wnts is sequential, and that their roles support a model of hierarchical fate decisions that progressively restrict the developmental potential of the ectoderm until otic commitment. We show that signalling by Fgf3 and Fgf19 is required to initiate a proliferative progenitor region that is a precursor to both the inner ear and the neurogenic epibranchial placodes. Significantly, we find that only after FGF action is attenuated can the subsequent action of Wnt signalling allow otic differentiation to proceed. In addition, gain and loss of function of Wnt-signalling components show a role for this signalling in repressing epibranchial fate. This interplay of signalling factors ensures the correct and ordered differentiation of both inner ear and epibranchial systems.     

Nerve growth factor and serum differentially regulate development of the embryonic otic vesicle and cochleovestibular ganglion in vitro

The preceding paper (P. Bernd and J. Represa, 1989, Dev. Biol. 134) describes the characterization and localization of nerve growth factor (NGF) receptors in inner ear primordia, the otic vesicle (OV) and cochleovestibular ganglion (CVG), obtained from 72-hr (stage 19-20) quail embryos. The studies described in this paper investigated whether NGF serves as a mitogen, a survival factor, and/or a differentiation factor in this system. Explants of isolated OV and CVG were maintained for 24 hr in serum-free medium alone (M-199), M-199 containing serum, M-199 containing NGF, or M-199 containing both serum and NGF. [3H]Thymidine was also present for the entire culture period. Both OV and CVG incorporated greater amounts of [3H]thymidine in the presence of serum or NGF, and their combined effect was additive. NGF's effects were dose dependent, saturable, and specific (blocked by anti-NGF). NGF caused little or no morphological differentiation of OV and no increase in protein levels, in contrast to OV grown in the presence of serum. CVG had both cochlear and vestibular portions present in all cases, but the apparent size and protein content of CVG was increased in the presence of either serum or NGF. Effects of serum and NGF were completely, but reversibly, blocked by amiloride, suggesting that the Na+-H+ exchange system had been activated. In order to determine whether increases in [3H]thymidine incorporation were due to increased cell survival or perhaps to an increase in proliferation, explants were initially grown for a 24-hr period in serum-free medium, followed by reactivation for an additional 24 hr in medium containing serum and/or NGF. It is likely that cells requiring either serum or NGF for survival would die during a 24-hr period in their absence. Our results revealed that the level of [3H]thymidine incorporation in OV was the same after reactivation. In the case of CVG, only NGF treatment yielded similar results; [3H]thymidine incorporation was lower in CVG reactivated with serum. It appears, therefore, that serum has probable proliferative effects upon OV and CVG, as well as survival effects for CVG. NGF, however, does not appear to affect survival in either OV or CVG, so that increases in [3H]thymidine incorporation in response to NGF are most likely due to proliferative effects upon OV or CVG, at least at this embryonic stage.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

BMP pathways are involved in otic capsule formation and epithelial-mesenchymal signaling in the developing chicken inner ear

The vertebrate inner ear consists of a complex labyrinth of epithelial cells that is surrounded by a bony capsule. The molecular mechanisms coordinating the development of the membranous and bony labyrinths are largely unknown. Previously, using avian retrovirus encoding Noggin (RCAS-Noggin) or beads soaked with Noggin protein, we have shown that bone morphogenetic proteins (BMPs) are important for the development of the otic epithelium in the chicken inner ear. Here, using two additional recombinant avian retroviruses, dominant negative and constitutively active forms of BMP receptors IB (BMPRIB), we show that BMPs, possibly acting through BMPRIB, are important for otic capsule formation. We also show that Bmp2 is strongly expressed in the prospective semicircular canals starting from the canal outpouch stage, suggesting that BMP2 plays an important role in canal formation. In addition, by correlating expression patterns of Bmps, their receptors, and localization of phosphorylated R-Smad (phospho R-Smad) immunoreactivity, an indicator of BMP activation, we show that BMPs emanating from the otic epithelium influence chondrogenesis of the otic capsule including the cartilage surrounding the semicircular canals.     

In vitro development of the embryonic mouse inner ear following exposure to trypsin

Trypsin has been shown to disrupt normal in vitro morphogenesis of embryonic organ rudiments. Otic tissues derived from 11-, 12-, and 13-day-old mouse embryos were exposed to either Ca++- and Mg++-free PBS or 0.25% trypsin dissolved in Ca++- and Mg++-free PBS prior to explanation into organ culture. Trypsin treatment of otic explants disrupted the expression of the normal pattern of inner-ear development in vitro. There was a direct correlation between the embryonic age at time of exposure to trypsin and the severity of dysmorphogenesis of the inner ear. The younger explants showed abnormalities of both vestibular and auditory structures, whereas with increasing embryonic age, abnormalities were confined more to the auditory portion of the inner ear. The results suggest that integrity of the otocyst basal lamina and epitheliomesenchymal tissue interactions are important factors in early otic development. It is postulated that the major effect of trypsin on inner-ear morphogenesis is through disruption of these factors, which may act to regulate the progressive expression of early otic development.     

MicroRNAs and epigenetic regulation in the mammalian inner ear: implications for deafness

Sensorineural hearing loss is the most common sensory disorder in humans and derives, in most cases, from inner-ear defects or degeneration of the cochlear sensory neuroepithelial hair cells. Genetic factors make a significant contribution to hearing impairment. While mutations in 51 genes have been associated with hereditary sensorineural nonsyndromic hearing loss (NSHL) in humans, the responsible mutations in many other chromosomal loci linked with NSHL have not been identified yet. Recently, mutations in a noncoding microRNA (miRNA) gene, MIR96, which is expressed specifically in the inner-ear hair cells, were linked with progressive hearing loss in humans and mice. Furthermore, additional miRNAs were found to have essential roles in the development and survival of inner-ear hair cells. Epigenetic mechanisms, in particular, DNA methylation and histone modifications, have also been implicated in human deafness, suggesting that several layers of noncoding genes that have never been studied systematically in the inner-ear sensory epithelia are required for normal hearing. This review aims to summarize the current knowledge about the roles of miRNAs and epigenetic regulatory mechanisms in the development, survival, and function of the inner ear, specifically in the sensory epithelia, tectorial membrane, and innervation, and their contribution to hearing.     

The developing lamprey ear closely resembles the zebrafish otic vesicle: otx1 expression can account for all major patterning differences

The inner ear of adult agnathan vertebrates is relatively symmetric about the anteroposterior axis, with only two semicircular canals and a single sensory macula. This contrasts with the highly asymmetric gnathostome arrangement of three canals and several separate maculae. Symmetric ears can be obtained experimentally in gnathostomes in several ways, including by manipulation of zebrafish Hedgehog signalling, and it has been suggested that these phenotypes might represent an atavistic condition. We have found, however, that the symmetry of the adult lamprey inner ear is not reflected in its early development; the lamprey otic vesicle is highly asymmetric about the anteroposterior axis, both morphologically and molecularly, and bears a striking resemblance to the zebrafish otic vesicle. The single sensory macula originates as two foci of hair cells, and later shows regions of homology to the zebrafish utricular and saccular maculae. It is likely, therefore, that the last common ancestor of lampreys and gnathostomes already had well-defined otic anteroposterior asymmetries. Both lamprey and zebrafish otic vesicles express a target of Hedgehog signalling, patched, indicating that both are responsive to Hedgehog signalling. One significant distinction between agnathans and gnathostomes, however, is the acquisition of otic Otx1 expression in the gnathostome lineage. We show that Otx1 knockdown in zebrafish, as in Otx1(-/-) mice, gives rise to lamprey-like inner ears. The role of Otx1 in the gnathostome ear is therefore highly conserved; otic Otx1 expression is likely to account not only for the gain of a third semicircular canal and crista in gnathostomes, but also for the separation of the zones of the single macula into distinct regions.     

Line up and listen: Planar cell polarity regulation in the mammalian inner ear

The inner ear sensory organs possess extraordinary structural features necessary to conduct mechanosensory transduction for hearing and balance. Their structural beauty has fascinated scientists since the dawn of modern science and ensured a rigorous pursuit of the understanding of mechanotransduction. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity and resolution, and represent perhaps the most distinctive form of a type of cellular polarity, known as planar cell polarity (PCP). Until recently, however, it was not known how the precise PCP of the inner ear sensory organs was achieved during development. Here, we review the PCP of the inner ear and recent advances in the quest for an understanding of its formation.     

Dysmorphogenesis of the inner ear: disruption of extracellular matrix (ECM) formation by an L-proline analog in otic explants

L-azetidine-2-carboxylic acid (LACA), a l-proline analog, disrupts collagen secretion by cells and prevents normal morphogenesis of in vitro developing organ rudiments. Otic explants derived from 10.5-through 14-day-old mouse embryos were continuously exposed to LACA in the nutrient medium at concentrations of 75, 150, and 300 micrograms/ml. LACA disrupted normal in vitro otic morphogenesis in inner ears explanted from embryos of 10.5 through 13 days' gestation. Development of 14-day-old otic explants were not affected by LACA at the concentrations tested. There was a direct correlation between the embryonic age of the explant when exposed to LACA, and the severity of otic dysmorphogenesis. The younger explants (10.5-to 12-day-old) developed abnormalities of both vestibular and auditory structures, but with increasing embryonic age of the explants (12-to 13.5-day-old) abnormalities were confined more to the auditory portion of the inner ear. Disruption of collagen secretion of connective tissue cells of the otic explants are a major teratogenic action of LACA on inner ear development. Disrupted collagen secretion alters otic extracellular matrix production, which in turn affects the tissue interactions that regulate the progressive expression of otic morphogenesis and differentiation.     

Axon guidance in the inner ear

Statoacoustic ganglion (SAG) neurons send their peripheral processes to navigate into the inner ear sensory organs where they will ultimately become post-synaptic to mature hair cells. During early ear development, neuroblasts delaminate from a restricted region of the ventral otocyst and migrate to form the SAG. The pathfinding mechanisms employed by the processes of SAG neurons as they search for their targets in the periphery are the topic of this review. Multiple lines of evidence exist to support the hypothesis that a combination of cues are working to guide otic axons to their target sensory organs. Some pioneer neurites may retrace their neuronal migratory pathway back to the periphery, yet additional guidance mechanisms likely complement this process. The presence of chemoattractants in the ear is supported by in vitro data showing that the otic epithelium exerts both trophic and tropic effects on the statoacoustic ganglion. The innervation of ectopic hair cells, generated after gene misexpression experiments, is further evidence for chemoattractant involvement in the pathfinding of SAG axons. While the source(s) of chemoattractants in the ear remains unknown, candidate molecules, including neurotrophins, appear to attract otic axons during specific time points in their development. Data also suggest that classical axon repellents such as Semaphorins, Eph/ephrins and Slit/Robos may be involved in the pathfinding of otic axons. Morphogens have recently been implicated in guiding axonal trajectories in many other systems and therefore a role for these molecules in otic axon guidance must also be explored.     

Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle

Currently, few factors have been identified that provide the inductive signals necessary to transform the simple otic placode into the complex asymmetric structure of the adult vertebrate inner ear. We provide evidence that Hedgehog signalling from ventral midline structures acts directly on the zebrafish otic vesicle to induce posterior otic identity. We demonstrate that two strong Hedgehog pathway mutants, chameleon (con(tf18b)) and slow muscle omitted (smu(b641)) exhibit a striking partial mirror image duplication of anterior otic structures, concomitant with a loss of posterior otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of the Hedgehog pathway, by injection of sonic hedgehog or dominant-negative protein kinase A RNA, has the reverse effect: ears lose anterior otic structures and show a mirror image duplication of posterior regions. By using double mutants and antisense morpholino analysis, we also show that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in anteroposterior patterning of the zebrafish otic vesicle.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.