Developmental maturation of presynaptic ribbon numbers in chicken basilar-papilla hair cells and its perturbation by long-term overexpression of Wnt9a.

The avian basilar papilla is a valuable model system for exploring the developmental determination and differentiation of sensory hair cells and their innervation. In the mature basilar papilla, hair cells form a well-known continuum between two extreme types-tall and short hair cells-that differ strikingly in their innervation. Previous work identified Wnt9a as a crucial factor in this differentiation. Here, we quantified the number and volume of immunolabelled presynaptic ribbons in tall and short hair cells of chickens, from developmental stages shortly after ribbons first appear to the mature posthatching condition. Two longitudinal locations were sampled, responding to best frequencies of approximately 1 kHz and approximately 5.5 kHz when mature. We found significant reductions of ribbon number during normal development in the tall-hair-cell domains, but stable, low numbers in the short-hair-cell domains. Exposing developing hair cells to continuous, excessive Wnt9a levels (through virus-mediated overexpression) led to transiently abnormal high numbers of ribbons and a delayed reduction of ribbon numbers at all sampled locations. Thus, (normally) short-hair-cell domains also showed tall-hair-cell like behaviour, confirming previous findings (Munnamalai et al., 2017). However, at 3 weeks posthatching, ribbon numbers had decreased to the location-specific typical values of control hair cells at all sampled locations. Furthermore, as shown previously, mature hair cells at the basal, high-frequency location harboured larger ribbons than more apically located hair cells. This was true for both normal and Wnt9a-overexposed basilar papillae.

Wnt9a Can Influence Cell Fates and Neural Connectivity across the Radial Axis of the Developing Cochlea.

Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses.SIGNIFICANCE STATEMENT Wnts are a class of secreted factors that are best known for stimulating cell division in development and cancer. However, in certain contexts during development, Wnt-expressing cells can direct neighboring cells to take on specific fates. This study suggests that the Wnt9a ligand may play such a role in the developing hearing organ of the bird cochlea. This was shown through patterning defects that occur in response to the overexpression of Wnt9a. This manipulation increased one type of sensory hair cell (tall HCs) at the expense of another (short HCs) that is usually located furthest from the Wnt9a source. The extraneous tall HCs that replaced short HCs showed some physiological properties and neuronal connections consistent with a fate switch.

Current concepts of hair cell differentiation and planar cell polarity in inner ear sensory organs.

Phylogenetically and ontogenetically, vertebrate development led to the generation of several inner ear sensory organs. During embryogenesis, cell fate specification determines whether each progenitor cell differentiates into a sensory hair cell or a supporting cell within the common sensory primordium. Finally, all sensory epithelia of the inner ear consist of a hair cell/supporting cell mosaic, albeit with anatomical differences depending on the sensory organ type. Hair cells develop a polarized bundle of stereovilli that is of functional importance for mechanotransduction. After initiating stereovillar development, hair cells align their bundles in a coordinated fashion, generating a characteristic hair cell orientation pattern, a process referred to as planar cell polarity (PCP). The pathway that controls PCP in the inner ear needs both to establish the development of a polarized morphology of the stereovillar bundle of the hair cell and to organize a systematic hair cell alignment. Because the hair cell orientation patterns of the various inner ear organs and vertebrate species differ fundamentally, it becomes apparent that in vertebrates, different aspects of PCP need to be independently controlled. In spite of important progress recently gained in the field of PCP research, we still need to identify the mechanisms (1) that initiate molecular asymmetries in cells, (2) that guide the transmission of polarity information from cell to cell, and (3) that consistently translate such polarity information into morphological asymmetries of hair cells.

Evolution and development of hair cell polarity and efferent function in the inner ear.

The function of the inner ear critically depends on mechanoelectrically transducing hair cells and their afferent and efferent innervation. The first part of this review presents data on the evolution and development of polarized vertebrate hair cells that generate a sensitive axis for mechanical stimulation, an essential part of the function of hair cells. Beyond the cellular level, a coordinated alignment of polarized hair cells across a sensory epithelium, a phenomenon called planar cell polarity (PCP), is essential for the organ's function. The coordinated alignment of hair cells leads to hair cell orientation patterns that are characteristic of the different sensory epithelia of the vertebrate inner ear. Here, we review the developmental mechanisms that potentially generate molecular and morphological asymmetries necessary for the control of PCP. In the second part, this review concentrates on the evolution, development and function of the enigmatic efferent neurons terminating on hair cells. We present evidence suggestive of efferents being derived from motoneurons and synapsing predominantly onto a unique but ancient cholinergic receptor. A review of functional data shows that the plesiomorphic role of the efferent system likely was to globally shut down and protect the peripheral sensors, be they vestibular, lateral line or auditory hair cells, from desensitization and damage during situations of self-induced sensory overload. The addition of a dedicated auditory papilla in land vertebrates appears to have favored the separation of vestibular and auditory efferents and specializations for more sophisticated and more diverse functions.

Developmental origin and fate of middle ear structures.

Results from developmental and phylogenetic studies have converged to facilitate insight into two important steps in vertebrate evolution: (1) the ontogenetic origin of articulating elements of the buccal skeleton, i.e., jaws, and (2) the later origins of middle ear impedance-matching systems that convey air-borne sound to the inner ear fluids. Middle ear ossicles and other skeletal elements of the viscerocranium (i.e., gill suspensory arches and jaw bones) share a common origin both phylogenetically and ontogenetically. The intention of this brief overview of middle-ear development is to emphasize the intimate connection between evolution and embryogenesis. Examples of developmental situations are discussed in which cells of different provenance, such as neural crest, mesoderm or endoderm, gather together and reciprocal interactions finally determine cell fate. Effects of targeted mutagenesis on middle ear development are described to illustrate how the alteration of molecularly-controlled morphogenetic programs led to phylogenetic modifications of skeletal development. Ontogenetic plasticity has enabled the diversification of jaw elements as well as middle ear structures during evolution. This article is part of a special issue entitled "MEMRO 2012".

Non-cell-autonomous planar cell polarity propagation in the auditory sensory epithelium of vertebrates.

Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates.

Mapping of Wnt, frizzled, and Wnt inhibitor gene expression domains in the avian otic primordium.

Wnt signaling activates at least three different pathways involved in development and disease. Interactions of secreted ligands and inhibitors with cell-surface receptors result in the activation or regulation of particular downstream intracellular cascades. During the developmental stages of otic vesicle closure and beginning morphogenesis, the forming inner ear transcribes a plethora of Wnt-related genes. We report expression of 23 genes out of 25 tested in situ hybridization probes on tissue serial sections. Sensory primordia and Frizzled gene expression share domains, with Fzd1 being a continuous marker. Prospective nonsensory domains express Wnts, whose transcripts mainly flank prosensory regions. Finally, Wnt inhibitor domains are superimposed over both prosensory and nonsensory otic regions. Three Wnt antagonists, Dkk1, SFRP2, and Frzb are prominent. Their gene expression patterns partly overlap and change over time, which adds to the diversity of molecular microenvironments. Strikingly, prosensory domains express Wnts transiently. This includes: 1) the prosensory otic region of high proliferation, neuroblast delamination, and programmed cell death at stage 20/21 (Wnt3, -5b, -7b, -8b, -9a, and -11); and 2) sensory primordia at stage 25 (Wnt7a and Wnt9a). In summary, robust Wnt-related gene expression shows both spatial and temporal tuning during inner ear development as the otic vesicle initiates morphogenesis and prosensory cell fate determination.

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.