Retrograde ERK activation waves drive base-to-apex multicellular flow in murine cochlear duct morphogenesis.

A notable example of spiral architecture in organs is the mammalian cochlear duct, where the morphology is critical for hearing function. Genetic studies have revealed necessary signaling molecules, but it remains unclear how cellular dynamics generate elongating, bending, and coiling of the cochlear duct. Here, we show that extracellular signal-regulated kinase (ERK) activation waves control collective cell migration during the murine cochlear duct development using deep tissue live-cell imaging, Förster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. Long-term FRET imaging reveals that helical ERK activation propagates from the apex duct tip concomitant with the reverse multicellular flow on the lateral side of the developing cochlear duct, resulting in advection-based duct elongation. Moreover, model simulations, together with experiments, explain that the oscillatory wave trains of ERK activity and the cell flow are generated by mechanochemical feedback. Our findings propose a regulatory mechanism to coordinate the multicellular behaviors underlying the duct elongation during 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.

Hair cell regeneration or the expression of related factors that regulate the fate specification of supporting cells in the cochlear ducts of embryonic and posthatch chickens.

Hair cells in posthatch chickens regenerate spontaneously through mitosis or the transdifferentiation of supporting cells in response to antibiotic injury. However, how embryonic chicken cochleae respond to antibiotic treatment remains unknown. This study is the first to indicate that unlike hair cells in posthatch chickens, the auditory epithelium was free from antibiotic injury (25-250 mg gentamicin/kg) in embryonic chickens, although FITC-conjugated gentamicin actually reached embryonic hair cells. Next, we examined and counted the cells and performed labeling for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) (triple or double labeling) in the injured cochlea ducts after gentamicin treatment at 2 h (h), 15 h, 24 h, 2 days (d), 3 d and 7 d after BrdU treatment in posthatch chickens. Our results indicated that following gentamicin administration, proliferating cells (BrdU+) were labeled for Atoh1/Math1 in the damaged areas 3d after gentamicin administration, whereas hair cells (PV+) renewed through mitosis (BrdU+) or direct transdifferentiation (BrdU-) were evident only after 5 d of gentamicin administration. In addition, Sox2 expression was up-regulated in triggered supporting cells at an early stage of regeneration, but stopped at the advent of mature hair cells. Our study also indicated that p27(kip1) was expressed in both hair cells and supporting cells but was down-regulated in a subgroup of the supporting cells that gave rise to hair cells. These data and the obtained dynamic changes of the cells labeled for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) are useful for understanding supporting cell behaviors and their fate specification during hair cell regeneration.

Neurosensory development and cell fate determination in the human cochlea.

BACKGROUND: Hearing depends on correct functioning of the cochlear hair cells, and their innervation by spiral ganglion neurons. Most of the insight into the embryological and molecular development of this sensory system has been derived from animal studies. In contrast, little is known about the molecular expression patterns and dynamics of signaling molecules during normal fetal development of the human cochlea. In this study, we investigated the onset of hair cell differentiation and innervation in the human fetal cochlea at various stages of development. RESULTS: At 10 weeks of gestation, we observed a prosensory domain expressing SOX2 and SOX9/SOX10 within the cochlear duct epithelium. In this domain, hair cell differentiation was consistently present from 12 weeks, coinciding with downregulation of SOX9/SOX10, to be followed several weeks later by downregulation of SOX2. Outgrowing neurites from spiral ganglion neurons were found penetrating into the cochlear duct epithelium prior to hair cell differentiation, and directly targeted the hair cells as they developed. Ubiquitous Peripherin expression by spiral ganglion neurons gradually diminished and became restricted to the type II spiral ganglion neurons by 18 weeks. At 20 weeks, when the onset of human hearing is thought to take place, the expression profiles in hair cells and spiral ganglion neurons matched the expression patterns of the adult mammalian cochleae. CONCLUSIONS: Our study provides new insights into the fetal development of the human cochlea, contributing to our understanding of deafness and to the development of new therapeutic strategies to restore hearing.

Expression and function of scleraxis in the developing auditory system.

A study of genes expressed in the developing inner ear identified the bHLH transcription factor Scleraxis (Scx) in the developing cochlea. Previous work has demonstrated an essential role for Scx in the differentiation and development of tendons, ligaments and cells of chondrogenic lineage. Expression in the cochlea has been shown previously, however the functional role for Scx in the cochlea is unknown. Using a Scx-GFP reporter mouse line we examined the spatial and temporal patterns of Scx expression in the developing cochlea between embryonic day 13.5 and postnatal day 25. Embryonically, Scx is expressed broadly throughout the cochlear duct and surrounding mesenchyme and at postnatal ages becomes restricted to the inner hair cells and the interdental cells of the spiral limbus. Deletion of Scx results in hearing impairment indicated by elevated auditory brainstem response (ABR) thresholds and diminished distortion product otoacoustic emission (DPOAE) amplitudes, across a range of frequencies. No changes in either gross cochlear morphology or expression of the Scx target genes Col2A, Bmp4 or Sox9 were observed in Scx(-/-) mutants, suggesting that the auditory defects observed in these animals may be a result of unidentified Scx-dependent processes within the cochlea.

GATA3 controls the specification of prosensory domain and neuronal survival in the mouse cochlea.

HDR syndrome (also known as Barakat syndrome) is a developmental disorder characterized by hypoparathyroidism, sensorineural deafness and renal disease. Although genetic mapping and subsequent functional studies indicate that GATA3 haplo-insufficiency causes human HDR syndrome, the role of Gata3 in sensorineural deafness and auditory system development is largely unknown. In this study, we show that Gata3 is continuously expressed in the developing mouse inner ear. Conditional knockout of Gata3 in the developing inner ear disrupts the morphogenesis of mouse inner ear, resulting in a disorganized and shortened cochlear duct with significant fewer hair cells and supporting cells. Loss of Gata3 function leads to the failure in the specification of prosensory domain and subsequently, to increased cell death in the cochlear duct. Moreover, though the initial generation of cochleovestibular ganglion (CVG) cells is not affected in Gata3-null mice, spiral ganglion neurons (SGNs) are nearly depleted due to apoptosis. Our results demonstrate the essential role of Gata3 in specifying the prosensory domain in the cochlea and in regulating the survival of SGNs, thus identifying a molecular mechanism underlying human HDR syndrome.

Selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve (1). Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components (2,3). Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Somatostatin receptor types 1 and 2 in the developing mammalian cochlea.

The neuropeptide somatostatin (SST) exerts several important physiological actions in the adult central nervous system through interactions with membrane-bound receptors. Transient expression of SST and its receptors has been described in several brain areas during early ontogeny. It is therefore believed that SST may play a role in neural maturation. The present study provides the first evidence for the developmental expression of SST receptors in the mammalian cochlea, emphasizing their possible roles in cochlear maturation. In the developing mouse cochlea, cells immunoreactive to somatostatin receptor 1 (SSTR1) and somatostatin receptor 2 (SSTR2) were located in the embryonic cochlear duct on Kolliker's organ as early as embryonic day (E) 14 (E14). At E17, the expression of both receptors was high and already located at the hair cells and supporting cells along the length of the cochlear duct, which have become arranged into the characteristic pattern for the organ of Corti (OC) at this stage. At birth, SSTR1- and SSTR2-containing cells were only localized in the OC. In general, immunoreactivity for both receptors increased in the mouse cochlea from postnatal day (P) 0 (P0) to P10; the majority of immunostained cells were inner hair cells, outer hair cells, and supporting cells. Finally, a peak in the mRNA and protein expression of both receptors is present near the time when they respond to physiological hearing (i.e., hearing of airborne sound) at P14. At P21, SSTR1 and SSTR2 levels decrease dramatically. A similar developmental pattern was observed for SSTR1 and SSTR2 mRNA, suggesting that the expression of the SSTR1 and SSTR2 genes is controlled at the transcriptional level throughout development. In addition, we observed reduced levels of phospho-Akt and total Akt in SSTR1 knockout and SSTR1/SSTR2 double-knockout mice compared with wild-type mice. We know from previous studies that Akt is involved in hair cell survival. Taken together, the dynamic nature of SSTR1 and SSTR2 expression at a time of major developmental changes in the cochlea suggests that SSTR1 and SSTR2 (and possibly other members of this family) are involved in the maturation of the mammalian cochlea.

Expression patterns of FGF receptors in the developing mammalian cochlea.

Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1-4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from embryonic day 13.5 through postnatal ages. We find that Fgfr1, 2, and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27kip1) and the early hair cells (math1).

Distinct contributions from the hindbrain and mesenchyme to inner ear morphogenesis.

A mature inner ear is a complex structure consisting of vestibular and auditory components. Microsurgical ablations, rotations, and translocations were performed in ovo to identify the tissues that control inner ear morphogenesis. We show that mesenchyme/ectoderm adjacent to the developing ear specifically governs the shape of vestibular components - the semicircular canals and ampullae - by conferring anteroposterior axial information to these structures. In contrast, removal of individual hindbrain rhombomeres adjacent to the developing ear preferentially affects the growth and morphogenesis of the auditory subdivision, the cochlear duct, or basilar papilla. Removal of rhombomere 5 affects cochlear duct growth, while rhombomere 6 removal affects cochlear growth and morphogenesis. Rotating rhombomeres 5 and 6 along the anteroposterior axis also impacts cochlear duct morphogenesis but has little effect on the vestibular components. Our studies indicate that discrete tissues, acting at a distance, control the morphogenesis of distinct elements of the inner ear. These results provide a basis for identifying factors that are essential to vestibular and auditory development in vertebrates.

Lmx1a maintains proper neurogenic, sensory, and non-sensory domains in the mammalian inner ear.

Lmx1a is a LIM homeodomain-containing transcription factor, which is required for the formation of multiple organs. Lmx1a is broadly expressed in early stages of the developing inner ear, but its expression is soon restricted to the non-sensory regions of the developing ear. In an Lmx1a functional null mutant, dreher (dr(J)/dr(J)), the inner ears lack a non-sensory structure, the endolymphatic duct, and the membranous labyrinth is poorly developed. These phenotypes are consistent with Lmx1a's role as a selector gene. More importantly, while all three primary fates of the inner ear - neural, sensory, and non-sensory - are specified in dr(J)/dr(J), normal boundaries among these tissues are often violated. For example, the neurogenic domain of the ear epithelium, from which cells delaminate to form the cochleovestibular ganglion, is expanded. Within the neurogenic domain, the demarcation between the vestibular and auditory neurogenic domains is most likely disrupted as well, based on the increased numbers of vestibular neuroblasts and ectopic expression of Fgf3, which normally is associated specifically with the vestibular neurogenic region. Furthermore, aberrant and ectopic sensory organs are observed; most striking among these is vestibular-like hair cells located in the cochlear duct.

Myosin II regulates extension, growth and patterning in the mammalian cochlear duct.

The sensory epithelium of the mammalian cochlea comprises mechanosensory hair cells that are arranged into four ordered rows extending along the length of the cochlear spiral. The factors that regulate the alignment of these rows are unknown. Results presented here demonstrate that cellular patterning within the cochlea, including the formation of ordered rows of hair cells, arises through morphological remodeling that is consistent with the mediolateral component of convergent extension. Non-muscle myosin II is shown to be expressed in a pattern that is consistent with an active role in cellular remodeling within the cochlea, and genetic or pharmacological inhibition of myosin II results in defects in cellular patterning that are consistent with a disruption in convergence and extension. These results identify the first molecule, myosin II, which directly regulates cellular patterning and alignment within the cochlear sensory epithelium. Our results also provide insights into the cellular mechanisms that are required for the formation of highly ordered cellular patterns.

Fgf16(IRESCre) mice: a tool to inactivate genes expressed in inner ear cristae and spiral prominence epithelium.

Fibroblast growth factors play important roles in inner ear development. Previous studies showed that mouse Fgf16 is expressed asymmetrically during the otic cup and vesicle stages of development, suggesting roles in regulating or responding to anteroposterior axial cues. Here, we studied otic Fgf16 expression throughout embryonic development and found transcripts in the developing cristae and in a few cells in the lateral wall of the cochlear duct. To determine the otic function of Fgf16 and to follow the fate of Fgf16-expressing cells, we generated an Fgf16(IRESCre) allele. We show that Fgf16 does not have a unique role in inner ear development and that the Fgf16 lineage is found throughout the three cristae, in portions of the semicircular canal ducts, and in the cochlear spiral prominence epithelial cells. This strain will be useful for gene ablations in these tissues.

Comprehensive Wnt-related gene expression during cochlear duct development in chicken.

The avian cochlear duct houses both a vestibular and auditory sensory organ (the lagena macula and basilar papilla, respectively), which each have a distinct structure and function. Comparative mRNA in situ hybridization mapping conducted over the time course of chicken cochlear duct development reveals that Wnt-related gene expression is concomitant with various developmental processes such as regionalization, convergent extension of the cochlear duct, cell fate specification, synaptogenesis, and the establishment of planar cell polarity. Wnts mostly originate from nonsensory tissue domains, whereas the sensory primordia preferentially transcribe Frizzled receptors, suggesting that paracrine Wnt signaling predominates in the cochlear duct. Superimposed over this is the strong expression of two secreted Frizzled-related Wnt inhibitors that tend to show complementary expression patterns. Frzb (SFRP3) is confined to the nonsensory cochlear duct and the lagena macula, whereas SFRP2 is maintained in the basilar papilla along with Fzd10 and Wnt7b. Flanking the basilar papilla are Wnt7a, Wnt9a, Wnt11, and SFRP2 on the neural side and Wnt5a, Wnt5b, and Wnt7a on the abneural side. The lateral nonsensory cochlear duct continuously expresses Frzb and temporarily expresses Wnt6 and SFRP1. Characteristic for the entire lagena is the expression of Frzb; in the lagena macula are Fzd1, Fzd7, and Wnt7b, and in the nonsensory tissues are Wnt4 and Wnt5a. Auditory hair cells preferentially express Fzd2 and Fzd9, whereas the main receptors expressed in vestibular hair cells are Fzd1 and Fzd7, in addition to Fzd2 and Fzd9.

Cellular commitment and differentiation in the organ of Corti.

The organ of Corti, the sensory epithelium of the mammalian cochlea, develops from a subset of cells located along the dorsal side (referred to as the floor) of the cochlear duct. Over the course of embryonic development, cells within the developing organ of Corti become committed to develop as each of the unique cell types within the organ, including inner and outer hair cells, and at least four different types of supporting cells. Moreover, these different cell types are subsequently arranged into a highly rigorous cellular mosaic that includes the formation of ordered rows of both hair cells and supporting cells. The events that regulate both the location of the organ of Corti within the cochlear duct, the specification of each cell type and cellular patterning remain poorly understood. However, recent results have significantly improved our understanding of the molecular, genetic and cellular factors that mediate some of the decisions required for the development of this structure. In this review I will present an overview of cochlear development and then discuss some of the most recent and enlightening results regarding the molecular mechanism underlying the formation of this remarkable structure.

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.

Dan is required for normal morphogenesis and patterning in the developing chick inner ear.

During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1.

Comparative analysis of Gata3 and Gata2 expression during chicken inner ear development.

The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory-neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken.

Partially overlapping expression of Gata2 and Gata3 during inner ear development.

Gata2 and Gata3 belong to the Gata family of transcription factors in vertebrates that bind to a consensus "GATA" DNA sequence. The Gata3 gene is one of the earliest markers for the developing mouse inner ear. Ear morphogenesis is blocked in Gata3-deficient embryos, whereas nothing was known of the role of Gata2 in mouse inner ear. Here, we have compared the expression patterns of Gata2 and Gata3 during normal inner ear development and investigated their relationship in mice where either Gata3 or Gata2 has been inactivated. The expression of the two Gata genes is highly overlapping at embryonic day (E)10.5 but becomes increasingly distinct later. Whereas Gata2 is predominantly expressed in the dorsal vestibular system, Gata3 was detected mainly in the ventral cochlear duct and ganglion. No phenotypic abnormalities were observed in the inner ear of Gata2-/- embryos before lethality at E10.5 and Gata3 expression was unchanged. In contrast, a delay and strong reduction of Gata2 expression was detected in Gata3-/- otic epithelium.

TRAF6-dependent NF-kB transcriptional activity during mouse development.

Nuclear factor-kappa B (NF-kB) transcriptional activity is induced by numerous stimuli. To identify tissues exhibiting NF-kB transcriptional activity during development, we analyzed transgenic reporter mice that express beta-galactosidase from an NF-kB-responsive element. We report that NF-kB activation is widespread and present in numerous epithelial structures and within vasculature. Several regions of the developing central nervous system, including the roof plate and floor plate of the midbrain, show prominent NF-kB activation. To assess the role of the TRAF6 adaptor protein in developmental NF-kB activity, we analyzed NF-kB activation in reporter mice rendered null for TRAF6. Deletion of TRAF6 resulted in the loss of NF-kB activity in epithelia, in vasculature, and in roof and floor plate but had no effect on NF-kB activity developing telencephalon, choroid plexus, cochlear canal, and thymus. These data indicate that NF-kB transcriptional activity is present in a broad range of structures during development and that TRAF6 plays a critical role mediating developmental NF-kB activation in many but not all tissues.