Canonical Wnt signaling regulates the proliferative expansion and differentiation of fibrocytes in the murine inner ear

Otic fibrocytes tether the cochlear duct to the surrounding otic capsule but are also critically involved in maintenance of ion homeostasis in the cochlea, thus, perception of sound. The molecular pathways that regulate the development of this heterogenous group of cells from mesenchymal precursors are poorly understood. Here, we identified epithelial Wnt7a and Wnt7b as possible ligands of Fzd-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated periotic mesenchyme (POM). Mice with a conditional deletion of Ctnnb1 in the POM exhibited a complete failure of fibrocyte differentiation, a severe reduction of mesenchymal cells surrounding the cochlear duct, loss of pericochlear spaces, a thickening and partial loss of the bony capsule and a secondary disturbance of cochlear duct coiling shortly before birth. Analysis at earlier stages revealed that radial patterning of the POM in two domains with highly condensed cartilaginous precursors and more loosely arranged inner mesenchymal cells occurred normally but that proliferation in the inner domain was reduced and cytodifferentiation failed. Cells with mis/overexpression of a stabilized form of Ctnnb1 in the entire POM mesenchyme sorted to the inner mesenchymal compartment and exhibited increased proliferation. Our analysis suggests that Wnt signals from the cochlear duct epithelium are crucial to induce differentiation and expansion of fibrocyte precursor cells. Our findings emphasize the importance of epithelial-mesenchymal signaling in inner ear development.     

Comprehensive Expression of Wnt Signaling Pathway Genes during Development and Maturation of the Mouse Cochlea

Background

In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points.

Results

We identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity.

Conclusion

A large number of Wnt signaling molecules were dynamically expressed during cochlear development and in the early postnatal period, suggesting complex regulation of Wnt transduction. The data revealed several potential key regulators for further study.     

Fgf3 is required for dorsal patterning and morphogenesis of the inner ear epithelium

The inner ear, which contains sensory organs specialized for hearing and balance, develops from an ectodermal placode that invaginates lateral to hindbrain rhombomeres (r) 5-6 to form the otic vesicle. Under the influence of signals from intra- and extraotic sources, the vesicle is molecularly patterned and undergoes morphogenesis and cell-type differentiation to acquire its distinct functional compartments. We show in mouse that Fgf3, which is expressed in the hindbrain from otic induction through endolymphatic duct outgrowth, and in the prospective neurosensory domain of the otic epithelium as morphogenesis initiates, is required for both auditory and vestibular function. We provide new morphologic data on otic dysmorphogenesis in Fgf3 mutants, which show a range of malformations similar to those of Mafb (Kreisler), Hoxa1 and Gbx2 mutants, the most common phenotype being failure of endolymphatic duct and common crus formation, accompanied by epithelial dilatation and reduced cochlear coiling. The malformations have close parallels with those seen in hearing-impaired patients. The morphologic data, together with an analysis of changes in the molecular patterning of Fgf3 mutant otic vesicles, and comparisons with other mutations affecting otic morphogenesis, allow placement of Fgf3 between hindbrain-expressed Hoxa1 and Mafb, and otic vesicle-expressed Gbx2, in the genetic cascade initiated by WNT signaling that leads to dorsal otic patterning and endolymphatic duct formation. Finally, we show that Fgf3 prevents ventral expansion of r5-6 neurectodermal Wnt3a, serving to focus inductive WNT signals on the dorsal otic vesicle and highlighting a new example of cross-talk between the two signaling systems.     

Redundant functions of Rac GTPases in inner ear morphogenesis

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.     

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius'' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

Molecular mechanisms underlying inner ear patterning defects in kreisler mutants

Prior studies have shown that kreisler mutants display early inner ear defects that are related to abnormal hindbrain development and signaling. These defects in kreisler mice have been linked to mutation of the kr/mafB gene. To investigate potential relevance of kr/mafB and abnormal hindbrain development in inner ear patterning, we analyzed the ear morphogenesis in kreisler mice using a paint-fill technique. We also examined the expression patterns of a battery of genes important for normal inner ear patterning and development. Our results indicate that the loss of dorsal otic structures such as the endolymphatic duct and sac is attributable to the downregulation of Gbx2, Dlx5 and Wnt2b in the dorsal region of the otocyst. In contrast, the expanded expression domain of Otx2 in the ventral otic region likely contributes to the cochlear phenotype seen in kreisler mutants. Sensory organ development is also markedly disrupted in kreisler mutants. This pattern of defects and gene expression changes is remarkably similar to that observed in Gbx2 mutants. Taken together, the data show an important role for hindbrain cues, and indirectly, kr/mafB, in guiding inner ear morphogenesis. The data also identify Gbx2, Dlx5, Wnt2b and Otx2 as key otic genes ultimately affected by perturbation of the kr/mafB-hindbrain pathway.     

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. We show here that, during the precartilage and cartilage stages, Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, we analyzed the inner ears of mice homozygous for Fgf9 null alleles. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. Our results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis.     

Expression of cytokeratin polypeptides during development of the rat inner ear

Summary The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells.     

Expression of cytokeratin polypeptides during development of the rat inner ear.

The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells.     

Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear

The vertebrate inner ear, a complex sensory organ with vestibular and auditory functions, is derived from a single ectoderm structure called the otic placode. Currently, the molecular mechanisms governing the differentiation and specification of the otic epithelium are poorly understood. We present here a detailed expression study of LMO1-4 in the developing mouse inner ear using a combination of in situ hybridization and immunohistochemistry. LMO1 is specifically expressed in the vestibular and cochlear hair cells as well as the vestibular ganglia of the developing inner ear. LMO2 expression is detected in the periotic mesenchyme of the developing mouse cochlea from E12.5 to E14.5. The expression of LMO3 expression is first observed in the cochlea at E13.5 and becomes confined to the lesser epithelial ridge (LER) from E14.5 to E17.5. LMO3 is also expressed in some of the vestibular ganglion cells. LMO4 is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista, and the spiral ganglia throughout embryogenesis. Thus, the regionalized expression patterns of LMO1-4 are closely associated with the morphogenesis of the inner ear.     

Wnt signaling during cochlear development

Wnt signaling is a hallmark of all embryonic development with multiple roles at multiple developmental time points. Wnt signaling is also important in the development of several organs, one of which is the inner ear, where it participates in otic specification, the formation of vestibular structures, and the development of the cochlea. In particular, we focus on Wnt signaling in the auditory organ, the cochlea. Attempting to dissect the multiple Wnt signaling pathways in the mammalian cochlea is a challenging task due to limited expression data, particularly at proliferating stages. To offer predictions about Wnt activity, we compare cochlear development with that of other biological systems such as Xenopus retina, brain, cancer cells and osteoblasts. Wnts are likely to regulate development through crosstalk with other signaling pathways, particularly Notch and FGF, leading to changes in the expression of Sox2 and proneural (pro-hair cell) genes. In this review we have consolidated the known signaling pathways in the cochlea with known developmental roles of Wnts from other systems to generate a potential timeline of cochlear development.     

Pax2 expression patterns in the developing chick inner ear

The fate specification of the developing vertebrate inner ear could be determined by complex regulatory genetic pathways involving the Pax2/5/8 genes. Pax2 expression has been reported in the otic placode and vesicle of all vertebrates that have been studied. Loss-of-function experiments suggest that the Pax2 gene plays a key role in the development of the cochlear duct and acoustic ganglion. Despite all these data, the role of Pax2 gene in the specification of the otic epithelium is still only poorly defined. In the present work, we report a detailed study of the spatial and temporal Pax2 expression patterns during the development of the chick inner ear. In the period analysed, Pax2 is expressed only in some presumptive sensory patches, but not all, even though all sensory patches show the scattered Pax2 expression pattern later on. We also show that Pax2 is also expressed in several non-sensory structures.     

The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm

In the current analysis, we have investigated both the cytoskeletal and signaling roles of beta-catenin during the early phases of lens development using conditional loss- and gain-of-function strategies. Conditional loss of beta-catenin in the presumptive lens does not perturb the normal sequential appearance of lens fate markers but results in a dramatic failure of the coordinated epithelial cell behavior that constitutes lens morphogenesis. Similarly, loss-of-function for Lrp6, the Wnt pathway coreceptor expressed in the eye primordium, does not prevent expression of lens induction markers. Surprisingly, conditional deletion of beta-catenin in periocular ectoderm results in the formation of Prox-1 and beta-crystallin-positive ectopic lentoid bodies. Combined with the observation that the Wnt pathway reporter TOPGAL is expressed in nasal periocular ectoderm, these data suggest that, in this location, the canonical Wnt signaling pathway normally suppresses lens fate in favor of other structures. Consistent with this proposal, a dominant-active form of beta-catenin causes a loss of lens fate and a complete absence of lens development when expressed in the presumptive lens ectoderm.     

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.     

A transgenic Lef1/beta-catenin-dependent reporter is expressed in spatially restricted domains throughout zebrafish development.

The Wnt/beta-catenin signaling pathway plays multiple roles during embryonic development, only a few of which have been extensively characterized. Although domains of Wnt expression have been identified throughout embryogenesis, anatomical and molecular characterization of responding cells has been mostly unexplored. We have generated a transgenic zebrafish line that expresses a destabilized green fluorescent protein (GFP) variant under the control of a beta-catenin responsive promoter. Early zygotic expression of this transgene (TOPdGFP) mirrors known domains of Wnt signaling in the embryo. Loss of Lef1 activity results in decreased reporter expression and posterior defects, while loss of Tcf3 (Headless, Hdl) activity does not alter reporter expression, even though it results in loss of forebrain structures. In addition, ectopic Wnt1 expression can activate the reporter. In older embryos, we identify a number of transgene-expressing cell populations as novel sites of beta-catenin signaling. We conclude that our TOP-dGFP reporter line faithfully illustrates domains of beta-catenin activity and enables the identification of responsive cell populations.     

Dual embryonic origin of the mammalian otic vesicle forming the inner ear

The inner ear and cochleovestibular ganglion (CVG) derive from a specialized region of head ectoderm termed the otic placode. During embryogenesis, the otic placode invaginates into the head to form the otic vesicle (OV), the primordium of the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV is required for inner ear morphogenesis and neurogenesis. In this study, we show that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute directly to the OV from the neural tube. Using Wnt1-Cre, Pax3(Cre/+) and Hoxb1(Cre/+) mice to label and fate map cranial NEC lineages, we have demonstrated that cells from the neural tube incorporate into the otic epithelium after otic placode induction has occurred. Pax3(Cre/+) labeled a more extensive population of NEC derivatives in the OV than did Wnt1-Cre. NEC derivatives constitute a significant population of the OV and, moreover, are regionalized specifically to proneurosensory domains. Descendents of Pax3(Cre/+) and Wnt1-Cre labeled cells are localized within sensory epithelia of the saccule, utricle and cochlea throughout development and into adulthood, where they differentiate into hair cells and supporting cells. Some NEC derivatives give rise to neuroblasts in the OV and CVG, in addition to their known contribution to glial cells. This study defines a dual cellular origin of the inner ear from sensory placode ectoderm and NECs, and changes the current paradigm of inner ear neurosensory development.