MOSAIC ATOH1 DELETION IN THE CHICK AUDITORY EPITHELIUM REVEALS A HOMEOSTATIC MECHANISM TO RESTORE HAIR CELL NUMBER.

The mechanosensory hair cell of the vertebrate inner ear responds to the mechanical deflections that result from hearing or change in the acceleration due to gravity, to allow us to perceive and interpret sounds, maintain balance and spatial orientation. In mammals, ototoxic compounds, disease, and acoustic trauma can result in damage and extrusion of hair cells, without replacement, resulting in hearing loss. In contrast, non-mammalian vertebrates can regenerate sensory hair cells. Upon damage, hair cells are extruded and an associated cell type, the supporting cell is transformed into a hair cell. The mechanisms that can trigger regeneration are not known. Using mosaic deletion of the hair cell master gene, Atoh1, in the embryonic avian inner ear, we find that despite hair cells depletion at E9, by E12, hair cell number is restored in sensory epithelium. Our study suggests a homeostatic mechanism can restores hair cell number in the basilar papilla, that is activated when juxtracrine signalling is disrupted. Restoration of hair cell numbers during development may mirror regenerative processes, and our work provides insights into the mechanisms that trigger regeneration.

FGFR1-mediated protocadherin-15 loading mediates cargo specificity during intraflagellar transport in inner ear hair-cell kinocilia.

The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.

Changing shape and shaping change: Inducing the inner ear.

The inner ear arises from non-neural ectoderm as a result of instructions sent by surrounding tissues. These interactions progressively restrict the potential of the ectoderm, resulting in the formation of the otic placode, a disk of thickened ectoderm that will give rise to all of the inner ear derivatives and its neurons. While otic placode is a surface structure, the inner ear is internalised, embedded within the cranial mesenchyme. Here, the cellular and molecular interactions that restrict the lineage of non-neural ectoderm in its transition to otic placode are reviewed, and how these interactions impinge on the coordination of otic placodal cell shape that drive the dramatic morphogenesis of the placode, as it becomes the otocyst.

Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development.

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation.

FGFR1-Frs2/3 signalling maintains sensory progenitors during inner ear hair cell formation.

Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27(Kip1), Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.

Deficient FGF signaling causes optic nerve dysgenesis and ocular coloboma.

FGF signaling plays a pivotal role in eye development. Previous studies using in vitro chick models and systemic zebrafish mutants have suggested that FGF signaling is required for the patterning and specification of the optic vesicle, but due to a lack of genetic models, its role in mammalian retinal development remains elusive. In this study, we show that specific deletion of Fgfr1 and Fgfr2 in the optic vesicle disrupts ERK signaling, which results in optic disc and nerve dysgenesis and, ultimately, ocular coloboma. Defective FGF signaling does not abrogate Shh or BMP signaling, nor does it affect axial patterning of the optic vesicle. Instead, FGF signaling regulates Mitf and Pax2 in coordinating the closure of the optic fissure and optic disc specification, which is necessary for the outgrowth of the optic nerve. Genetic evidence further supports that the formation of an Frs2α-Shp2 complex and its recruitment to FGF receptors are crucial for downstream ERK signaling in this process, whereas constitutively active Ras signaling can rescue ocular coloboma in the FGF signaling mutants. Our results thus reveal a previously unappreciated role of FGF-Frs2α-Shp2-Ras-ERK signaling axis in preventing ocular coloboma. These findings suggest that components of FGF signaling pathway may be novel targets in the diagnosis of and the therapeutic interventions for congenital ocular anomalies.

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.

Expression of the heparan sulfate 6-O-endosulfatases, Sulf1 and Sulf2, in the avian and mammalian inner ear suggests a role for sulfation during inner ear development.

BACKGROUND: Inner ear morphogenesis is tightly regulated by the temporally and spatially coordinated action of signaling ligands and their receptors. Ligand-receptor interactions are influenced by heparan sulfate proteoglycans (HSPGs), cell surface molecules that consist of glycosaminoglycan chains bound to a protein core. Diversity in the sulfation pattern within glycosaminoglycan chains creates binding sites for numerous cell signaling factors, whose activities and distribution are modified by their association with HSPGs. RESULTS: Here we describe the expression patterns of two extracellular 6-O-endosulfatases, Sulf1 and Sulf2, whose activity modifies the 6-O-sulfation pattern of HSPGs. We use in situ hybridization to determine the temporal and spatial distribution of transcripts during the development of the chick and mouse inner ear. We also use immunocytochemistry to determine the cellular localization of Sulf1 and Sulf2 within the sensory epithelia. Furthermore, we analyze the organ of Corti in Sulf1/Sulf2 double knockout mice and describe an increase in the number of mechanosensory hair cells. CONCLUSIONS: Our results suggest that the tuning of intracellular signaling, mediated by Sulf activity, plays an important role in the development of the inner ear.

Zebra finch as a developmental model.

The domesticated zebra finch (Taeniopygia guttata) is a well-established animal model for studying vocal learning. It is also a tractable model for developmental analyses. The finch genome has been sequenced and methods for its transgenesis have been reported. Hatching and sexual maturation in this species takes only two weeks and three months, respectively. Finch colonies can be established relatively easily and its eggs are laid at a stage earlier than in other common avian experimental models, facilitating the analysis of very early avian development. Representing the Neoaves to which 95% of all bird species belong, the finch can potentially complement two existing, Galloanserae developmental models, the chick, and quail. Here, we provide a step-by-step guide for how to set up a finch colony in a conventional laboratory environment. Technical tips are offered to optimize hens' productivity and ensure a constant supply of fertilized finch eggs. Methods of handling finch eggs and embryos for subsequent embryological, cellular, or molecular analyses are also discussed. We conclude by emphasizing scientific values and cost effectiveness of maintaining a finch colony for avian developmental studies. genesis 53:669-677, 2015. © 2015 Wiley Periodicals, Inc.

Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination.

After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination.

Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis.

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.

From placode to labyrinth: culture of the chicken inner ear.

The inner ear transduces the mechanical stimuli that are associated with sound and balance perception. Missteps during its formation often result in deafness, and thus understanding otic development has a profound clinical relevance. The intricate complexity of the inner ear is derived from a simple epithelial sheet during embryogenesis. Study of this process in vitro has provided insight into the mechanisms of otic induction, patterning and differentiation. This article details methods for the culture of otic placode, otocyst, and basilar papilla, providing a toolkit for the investigation of multiple facets of otic organogenesis, for regeneration studies and for setting up small molecule screens to identify possible therapeutic targets.

From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes.

The inner ear and the epibranchial ganglia constitute much of the sensory system in the caudal vertebrate head. The inner ear consists of mechanosensory hair cells, their neurons, and structures necessary for sound and balance sensation. The epibranchial ganglia are knots of neurons that innervate and relay sensory signals from several visceral organs and the taste buds. Their development was once thought to be independent, in line with their independent functions. However, recent studies indicate that both systems arise from a morphologically distinct common precursor domain: the posterior placodal area. This review summarises recent studies into the induction, morphogenesis and innervation of these systems and discusses lineage restriction and cell specification in the context of their common origin.

Pax2 modulates proliferation during specification of the otic and epibranchial placodes.

BACKGROUND: The inner ear and epibranchial ganglia of vertebrates arise from a shared progenitor domain that is induced by FGF signalling, the posterior placodal area (PPA), before being segregated by Wnt signalling. One of the first genes activated in the PPA is the transcription factor Pax2. Loss-of- and gain-of function studies have defined a role for Pax2 in placodal morphogenesis and later inner ear development, but have not addressed the role Pax2 plays during the formation and maintenance of the PPA. RESULTS: To understand the role of Pax2 during the development of the PPA, we used over-expression and repression of Pax2. Both gave rise to a smaller otocyst and repressed the formation of epibranchial placodes. In addition, cell cycle analysis revealed that Pax2 suppression reduced proliferation of the PPA. CONCLUSIONS: Our results suggest that Pax2 functions in the maintenance but not the induction of the PPA. One role of Pax2 is to maintain proper cell cycle proliferation in the PPA.

Induction of the chick columella and its integration with the inner ear.

BACKGROUND: The auditory complex of the chick, like that of humans, is made of intimate and highly ordered connections between the inner ear, the middle ear, and the outer ear. Unlike mammals, the middle ear of chick has only one ossicle, known as the columella. The independent lineages of the two suggest that some mechanism must exist that ensures the connectivity between the inner ear and the columella; however, the basis of integration is not known. RESULTS: Using quail-chick chimeras, we demonstrate that columella development depends on signaling interactions. Specifically, both pharyngeal endoderm and cranial paraxial mesoderm can alter the morphology of the columella. Only a discrete region of pharyngeal endoderm exerts this patterning activity, and this region is specified by the overlying paraxial mesoderm. CONCLUSIONS: Paraxial mesoderm is also used in the induction of the inner ear, thus we propose that this overlapping source of signalling cues in both middle and inner ear development may underlie the integration of these structures.

Embryonic development of the emu, Dromaius novaehollandiae.

The chick, Gallus gallus, is the traditional model in avian developmental studies. Data on other bird species are scarce. Here, we present a comparative study of the embryonic development of the chick and the emu Dromaius novaehollandiae, a member of Paleognathae, which also includes the ostrich, rhea, tinamou, kiwi, and cassowary. Emu embryos ranging from Hamburger and Hamilton (HH) equivalent stages 1 to 43 were collected and their gross morphology analyzed. Its early development was studied in detail with time-lapse imaging and molecular techniques. Emu embryos in general take 2-3 times longer incubation time to reach equivalent chicken stages, requiring 1 day for HH2, 2.5 days for HH4, 7 days for limb bud initiation, 23 days for feather germ appearance, and approximately 50-56 days for hatching. Chordin gene expression is similar in emu and chick embryos, and emu Brachyury is not expressed until HH3. Circulation is established at approximately the 27- to 30-somite stage. Forelimb buds are formed and patterned initially, but their growth is severely retarded. The size difference between an emu and a chick embryo only becomes apparent after limb bud formation. Overall, emu and chick embryogenesis proceeds through similar stages, but developmental heterochrony between these two species is widely observed.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

FGF signaling regulates cytoskeletal remodeling during epithelial morphogenesis.

Changes in the cytoskeletal architecture underpin the dynamic changes in tissue shape that occur during development. It is clear that such changes must be coordinated so that individual cell behaviors are synchronized; however, the mechanisms by which morphogenesis is instructed and coordinated are unknown. After its induction in non-neural ectoderm, the inner ear undergoes morphogenesis, being transformed from a flat ectodermal disk on the surface of the embryo to a hollowed sphere embedded in the head. We provide evidence that this shape change relies on extrinsic signals subsequent to genetic specification. By using specific inhibitors, we find that local fibroblast growth factor (FGF) signaling triggers a phosphorylation cascade that activates basal myosin II through the activation of phospholipase Cgamma. Myosin II exhibits a noncanonical activity that results in the local depletion of actin filaments. Significantly, the resulting apical actin enrichment drives morphogenesis of the inner ear. Thus, FGF signaling directly exerts profound cytoskeletal effects on otic cells, coordinating the morphogenesis of the inner ear. The iteration of this morphogenetic signaling system suggests that it is a more generally applicable mechanism in other epithelial tissues undergoing shape change.

Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway.

The eye field is initially a large single domain at the anterior end of the neural plate and is the first indication of optic potential in the vertebrate embryo. During the course of development, this domain is subject to interactions that shape and refine the organogenic field. The action of the prechordal mesoderm in bisecting this single region into two bilateral domains has been well described, however the role of signalling interactions in the further restriction and refinement of this domain has not been previously characterised. Here we describe a role for the rostral cephalic paraxial mesoderm in limiting the extent of the eye field. The anterior transposition of this mesoderm or its ablation disrupted normal development of the eye. Importantly, perturbation of optic vesicle development occurred in the absence of any detectable changes in the pattern of neighbouring regions of the neural tube. Furthermore, negative regulation of eye development is a property unique to the rostral paraxial mesoderm. The rostral paraxial mesoderm expresses members of the bone morphogenetic protein (BMP) family of signalling molecules and manipulation of endogenous BMP signalling resulted in abnormalities of the early optic primordia.

Hair cell differentiation becomes tissue specific by E9.5 in mouse inner ear.

Tissue culture is a standard method to study tissue interactions during embryogenesis. The serum that is usually used in culture can, however, confound results as it includes unidentified factors. In this study, we used a serum-free otocyst culture to investigate the tissue interactions that determine hair cell fate in mice otocysts. Otocysts cultured with surrounding tissues have the ability to produce mature hair cells in serum-free otocyst culture. Although isolated otocysts from E9.5 mice produced hair cells, those from E9.0 mice could not. This indicates that the mouse otocyst gains the ability to generate hair cells between E9.0 and E9.5 and that this ability depends on signals from the surrounding mesenchyme and/or the hindbrain.