Exploring the Expression and Function of cTyro3, a Candidate Zika Virus Receptor, in the Embryonic Chicken Brain and Inner Ear.

The transmembrane protein Axl was proposed as an entry receptor for Zika virus (ZIKV) infection in vitro, but conflicting results from in vivo studies have made it difficult to establish Axl as a physiologically relevant ZIKV receptor. Both the functional redundancy of receptors and the experimental model used can lead to variable results. Therefore, it can be informative to explore alternative animal models to analyze ZIKV receptor candidates as an aid in discovering antivirals. This study used chicken embryos to examine the role of chicken Tyro3 (cTyro3), the equivalent of human Axl. Results show that endogenous cTyro3 mRNA expression overlaps with previously described hot spots of ZIKV infectivity in the brain and inner ear. We asked if ectopic expression or knockdown of cTyro3 influenced ZIKV infection in embryos. Tol2 vectors or replication-competent avian retroviruses were used in ovo to introduce full-length or truncated (presumed dominant-negative) cTyro3, respectively, into the neural tube on embryonic day two (E2). ZIKV was delivered to the brain 24 h later. cTyro3 manipulations did not alter ZIKV infection or cell death in the E5/E6 brain. Moreover, delivery of truncated cTyro3 variants to the E3 otocyst had no effect on inner ear formation on E6 or E10.

Embryonic and Neonatal Mouse Cochleae Are Susceptible to Zika Virus Infection.

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.

Zika virus can directly infect and damage the auditory and vestibular components of the embryonic chicken inner ear.

BACKGROUND: Sensorineural hearing loss is an understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. Also lacking is information about the susceptibility and the pathogenesis of the developing inner ear following Zika virus (ZIKV) exposure. To address this, ZIKV was delivered directly into the otic cup/otocyst of chicken embryos and infection of inner ear tissues was evaluated using immunohistochemistry. RESULTS: After injections on embryonic days 2 to 5, ZIKV infection was observed in 90% of the samples harvested 2 to 8 days later; however, the degree of infection was highly variable across individuals. ZIKV was detected in all regions of the inner ear, associated ganglia, and in the surrounding periotic mesenchyme. Detection of virus peaked earlier in the ganglion and vestibular compartments, and later in the cochlea. ZIKV infection increased cell death robustly in the auditory ganglion, and modestly in the auditory sensory organ. Macrophage accumulation was found to overlap with dense viral infection in some tissues. Additionally, dysmorphogenesis of the semicircular canals and ganglion was observed for a subset of injection conditions. CONCLUSIONS: This article presents evidence of direct ZIKV infection of developing inner ear epithelium and shows previously unknown inner ear dysmorphogenesis phenotypes.

The acquisition of positional information across the radial axis of the cochlea.

The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss.

Expression of class III Semaphorins and their receptors in the developing chicken (Gallus gallus) inner ear.

Class III Semaphorin (Sema) secreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used in situ hybridization or immunohistochemistry to map the expression of Sema3D, Sema3F, Nrp1, Nrp2, and PlxnA1 in the chicken (Gallus gallus) inner ear from embryonic day (E)5-E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. Sema3D expression flanking the sensory tissue in vestibular organs suggests that it may repel Nrp2- and PlxnA1-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8-E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, Sema3D in the future tegmentum vasculosum opposes Nrp1 and PlxnA1 in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. Sema3D immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.

Zika Virus Can Strongly Infect and Disrupt Secondary Organizers in the Ventricular Zone of the Embryonic Chicken Brain.

Zika virus (ZIKV) is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly. Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in nonuniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8 expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced, and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell-autonomous changes in brain patterning.

Viral Ubiquitin Ligase Stimulates Selective Host MicroRNA Expression by Targeting ZEB Transcriptional Repressors.

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.

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.

Distinct functions for netrin 1 in chicken and murine semicircular canal morphogenesis.

The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of the inner ear. In the center of each pouch, a fusion plate forms where cells lose their epithelial morphology and the basement membrane breaks down. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse.

Notch-Wnt-Bmp crosstalk regulates radial patterning in the mouse cochlea in a spatiotemporal manner.

The sensory cells of the mammalian organ of Corti assume a precise mosaic arrangement during embryonic development. Manipulation of Wnt signaling can modulate the proliferation of cochlear progenitors, but whether Wnts are responsible for patterning compartments, or specific hair cells within them, is unclear. To address how the precise timing of Wnt signaling impacts patterning across the radial axis, mouse cochlear cultures were initiated at embryonic day 12.5 and subjected to pharmacological treatments at different stages. Early changes in major patterning genes were assessed to understand the mechanisms underlying alterations of compartments. Results show that Wnt activation can promote medial cell fates by regulating medially expressed Notch genes in a spatiotemporal manner. Wnts can also suppress lateral cell fates by antagonizing Bmp4 expression. Perturbation of the Notch and Bmp pathways revealed which secondary effects were linked to these pathways. Importantly, these effects on cochlear development are dependent on the timing of drug delivery. In conclusion, Wnt signaling in the cochlea influences patterning through complex crosstalk with the Notch and Bmp pathways at several stages of embryonic development.

Organotypic Culture of the Mouse Cochlea from Embryonic Day 12 to the Neonate.

The development of the mammalian cochlea is a complex process involving several intersecting signaling pathways to ultimately generate its highly organized cellular architecture. In humans, and in the mouse, there is one row of inner hair cells aligned next to three rows of outer hair cells. The support cells intercalate between the hair cells to create a cellular mosaic across the organ of Corti (OC). Organotypic culture of the cochlea is a valuable technique for investigating the early stages of OC development. Cultures can be established at proliferative stages and maintained in vitro until cellular differentiation commences. It is straightforward to monitor differentiation in response to perturbations of key signaling pathways using pharmacological agents. While postnatal cochlea organ cultures have already been adapted for in vitro studies, it is more challenging to establish cultures at early embryonic stages due to the small size of the organ primordium. The protocol described in this chapter is permissive for all stages of development from E12 cochleas to day 5 neonatal murine cochleas, allowing culture survival up to 2 weeks in vitro.

Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation.

The avian embryo has a well-documented history as a model system for the study of neurogenesis, morphogenesis, and cell fate specification. This includes studies of the chicken inner ear that employ in ovo electroporation, in conjunction with the Tol2 system, to yield robust long-term transgene expression. Capitalizing on the success of this delivery method, we describe a modified version of the Tol2 expression vector that readily accepts the insertion of a microRNA-encoding artificial intron. This offers a strategy to investigate the possible roles of different candidate microRNAs in ear development by overexpression. Here, we describe the general design of this modified vector and the electroporation procedure. This approach is expected to facilitate phenotypic screening of candidate miRNAs to explore their bioactivity in vivo.

Expression and Misexpression of the miR-183 Family in the Developing Hearing Organ of the Chicken.

The miR-183 family consists of 3 related microRNAs (miR-183, miR-96, miR-182) that are required to complete maturation of primary sensory cells in the mammalian inner ear. Because the level of these microRNAs is not uniform across hair cell subtypes in the murine cochlea, the question arises as to whether hair cell phenotypes are influenced by microRNA expression levels. To address this, we used the chicken embryo to study expression and misexpression of this gene family. By in situ hybridization, expression of all 3 microRNAs is robust in immature hair cells of both auditory and vestibular organs and is present in the statoacoustic ganglion. The auditory organ, called the basilar papilla, shows a weak radial gradient (highest on the neural side) in prosensory cells near the base on embryonic day 7. About nine days later, the basilar papilla also displays a longitudinal gradient (highest in apical hair cells) for the 3 microRNAs. Tol2-mediated gene delivery was used to ask whether cell phenotypes are malleable when the prosensory epithelium was forced to overexpress the miR-183 family. The expression plasmid included EGFP as a reporter located upstream of an intron carrying the microRNA genes. The vectors were electroporated into the otic cup/vesicle, resulting in strong co-expression of EGFP and the miR-183 family that persisted for at least 2 weeks. This manipulation did not generate ectopic hair cells in non-sensory territories of the cochlear duct, although within the basilar papilla, hair cells were over-represented relative to supporting cells. There was no evidence for a change in hair cell phenotypes, such as short-to-tall, or basal-to-apical hair cell features. Therefore, while increasing expression of the miR-183 family was sufficient to influence cell lineage decisions, it did not redirect the differentiation of hair cells towards alternative radial or longitudinal phenotypes.

A subset of chicken statoacoustic ganglion neurites are repelled by Slit1 and Slit2.

Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2-3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 h in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 h confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst.

Bicistronic gene transfer tools for delivery of miRNAs and protein coding sequences.

MicroRNAs (miRNAs) are a category of small RNAs that modulate levels of proteins via post-transcriptional inhibition. Currently, a standard strategy to overexpress miRNAs is as mature miRNA duplexes, although this method is cumbersome if multiple miRNAs need to be delivered. Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway. We have designed a vector to exploit this naturally-occurring intronic pathway to deliver the three members of the sensory-specific miR-183 family from an artificial intron. In one version of the vector, the downstream exon encodes the reporter (GFP) while another version encodes a fusion protein created between the transcription factor Atoh1 and the hemaglutinin epitope, to distinguish it from endogenous Atoh1. In vitro analysis shows that the miRNAs contained within the artificial intron are processed and bind to their targets with specificity. The genes downstream are successfully translated into protein and identifiable through immunofluorescence. More importantly, Atoh1 is proven functional through in vitro assays. These results suggest that this cassette allows expression of miRNAs and proteins simultaneously, which provides the opportunity for joint delivery of specific translational repressors (miRNA) and possibly transcriptional activators (transcription factors). This ability is attractive for future gene therapy use.

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 genetics of hair cell development and regeneration.

Sensory hair cells are exquisitely sensitive vertebrate mechanoreceptors that mediate the senses of hearing and balance. Understanding the factors that regulate the development of these cells is important, not only to increase our understanding of ear development and its functional physiology but also to shed light on how these cells may be replaced therapeutically. In this review, we describe the signals and molecular mechanisms that initiate hair cell development in vertebrates, with particular emphasis on the transcription factor Atoh1, which is both necessary and sufficient for hair cell development. We then discuss recent findings on how microRNAs may modulate the formation and maturation of hair cells. Last, we review recent work on how hair cells are regenerated in many vertebrate groups and the factors that conspire to prevent this regeneration in mammals.

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.