Transcriptional response to Wnt activation regulates the regenerative capacity of the mammalian cochlea.

Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.

Molecular cloning and functional characterisation of chicken Atonal homologue 1: a comparison with human Atoh1.

BACKGROUND INFORMATION: The vertebrate basic helix-loop-helix transcription factor Atoh1 is essential for maturation and survival of mechanosensory hair cells of the inner ear, neurogenesis, differentiation of the intestine, homeostasis of the colon and is implicated in cancer progression. Given that mutations in Atoh1 are detected in malignant tumours, study of functionally different Atoh1 alleles and homologues might yield useful avenues for investigation. The predicted sequence of chicken Atoh1 (cAtoh1) has large regions of dissimilarity to that of mammalian Atoh1 homologues. We hypothesise that cAtoh1 might have intrinsic functional differences to mammalian Atoh1. RESULTS: In this study, we cloned and sequenced the full open reading frame of cAtoh1. In overexpression experiments, we show that this sequence is sufficient to generate a cAtoh1 protein capable of inducing hair cell markers when expressed in nonsensory regions of the developing inner ear, and that morpholino-mediated knock-down using a section of the sequence 5' to the start codon inhibits differentiation of hair cells in the chicken basilar papilla. Furthermore, we compare the behaviour of cAtoh1 and human Atoh1 (hAtoh1) in embryonic mouse cochlear explants, showing that cAtoh1 is a potent inducer of hair cell differentiation and that it can overcome Sox2-mediated repression of hair cell differentiation more effectively than hAtoh1. CONCLUSIONS: cAtoh1 is both necessary and sufficient for avian mechanosensory hair cell differentiation. The non-conserved regions of the cAtoh1 coding region have functional consequences on its behaviour.

ß-Catenin is required for hair-cell differentiation in the cochlea.

The development of hair cells in the auditory system can be separated into steps; first, the establishment of progenitors for the sensory epithelium, and second, the differentiation of hair cells. Although the differentiation of hair cells is known to require the expression of basic helix-loop-helix transcription factor, Atoh1, the control of cell proliferation in the region of the developing cochlea that will ultimately become the sensory epithelium and the cues that initiate Atoh1 expression remain obscure. We assessed the role of Wnt/ß-catenin in both steps in gain- and loss-of-function models in mice. The canonical Wnt pathway mediator, ß-catenin, controls the expression of Atoh1. Knock-out of ß-catenin inhibited hair-cell, as well as pillar-cell, differentiation from sensory progenitors but was not required to maintain a hair-cell fate once specified. Constitutive activation of ß-catenin expanded sensory progenitors by inducing additional cell division and resulted in the differentiation of extra hair cells. Our data demonstrate that ß-catenin plays a role in cell division and differentiation in the cochlear sensory epithelium.

Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development.

The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.

Frizzled-7 is required for Xenopus heart development.

Wnt signalling regulates cardiogenesis during specification of heart tissue and the morphogenetic movements necessary to form the linear heart. Wnt11-mediated non-canonical signalling promotes early cardiac development whilst Wnt11-R, which is expressed later, also signals through the non-canonical pathway to promote heart development. It is unclear which Frizzled proteins mediate these interactions. Frizzled-7 (fzd7) is expressed during gastrulation in the mesodermal cells fated to become heart, and then in the primary heart field. This expression is complementary to the expression of wnt11 and wnt11-R We further show co-localisation of fzd7 with other early- and late-heart-specific markers using double in situ hybridisation. We have used loss of function analysis to determine the role of fzd7 during heart development. Morpholino antisense oligonucleotide-mediated knockdown of Fzd7 results in effects on heart development, similar to that caused by Wnt11 loss of function. Surprisingly, overexpression of dominant-negative Fzd7 cysteine rich domain (Fzd7 CRD) results in a cardia bifida phenotype, similar to the loss of wnt11-R phenotype. Overexpression of Fzd7 and activation of non-canonical wnt signalling can rescue the effect of Fzd7 CRD. We propose that Fzd7 has an important role during Xenopus heart 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.

Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis.

Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1-Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes fit a model of cell intercalation along differential Dchs1-Fat4 activity that drives a simultaneous narrowing, thickening and elongation of the sternum. Our results identify the regulation of cellular polarity within the early pre-chondrogenic mesenchyme, when skeletal shape is established, and provide the first demonstration that Fat4 and Dchs1 establish polarized cell behaviour intrinsically within the mesenchyme. Our data also reveal the first indication that cell intercalation processes occur during ventral body wall elongation and closure.

Kremen1 regulates mechanosensory hair cell development in the mammalian cochlea and the zebrafish lateral line.

Here we present spatio-temporal localization of Kremen1, a transmembrane receptor, in the mammalian cochlea, and investigate its role in the formation of sensory organs in mammal and fish model organisms. We show that Kremen1 is expressed in prosensory cells during cochlear development and in supporting cells of the adult mouse cochlea. Based on this expression pattern, we investigated whether Kremen1 functions to modulate cell fate decisions in the prosensory domain of the developing cochlea. We used gain and loss-of-function experiments to show that Kremen1 is sufficient to bias cells towards supporting cell fate, and is implicated in suppression of hair cell formation. In addition to our findings in the mouse cochlea, we examined the effects of over expression and loss of Kremen1 in the zebrafish lateral line. In agreement with our mouse data, we show that over expression of Kremen1 has a negative effect on the number of mechanosensory cells that form in the zebrafish neuromasts, and that fish lacking Kremen1 protein develop more hair cells per neuromast compared to wild type fish. Collectively, these data support an inhibitory role for Kremen1 in hair cell fate specification.

Long-term time lapse imaging of mouse cochlear explants.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Atoh1, an essential transcription factor in neurogenesis and intestinal and inner ear development: function, regulation, and context dependency.

Atoh1 (also known as Math1, Hath1, and Cath1 in mouse, human, and chicken, respectively) is a proneural basic helix-loop-helix (bHLH) transcription factor that is required in a variety of developmental contexts. Atoh1 is involved in differentiation of neurons, secretory cells in the gut, and mechanoreceptors including auditory hair cells. Together with the two closely related bHLH genes, Neurog1 and NeuroD1, Atoh1 regulates neurosensory development in the ear as well as neurogenesis in the cerebellum. Atoh1 activity in the cochlea is both necessary and sufficient to drive auditory hair cell differentiation, in keeping with its known role as a regulator of various genes that are markers of terminal differentiation. Atoh1 is known in other fields as an oncogene and a tumor suppressor involved in regulation of cell cycle control and apoptosis. Aberrant Atoh1 activity in adult tissue is implicated in cancer progression, specifically in medullablastoma and adenomatous polyposis carcinoma. We demonstrate through protein sequence comparison that Atoh1 contains conserved phosphorylation sites outside the bHLH domain, which may allow regulation through post-translational modification. With such diverse roles, tight regulation of Atoh1 at both the transcriptional and protein level is essential.