Kirk W. Beisel

Smaller inner ear sensory epithelia in Neurog1 null mice are related to earlier hair cell cycle exit

We investigated whether co‐expression of Neurog1 and Atoh1 in common neurosensory precursors could explain the loss of hair cells in Neurog1 null mice. Analysis of terminal mitosis, using BrdU, supports previous findings regarding timing of exit from cell cycle. Specifically, we show that cell cycle exit occurs in spiral sensory neurons in a base‐to‐apex progression followed by cell cycle exit of hair cells in the organ of Corti in an apex‐to‐base progression, with some overlap of cell cycle exit in the apex for both hair cells and spiral sensory neurons. Hair cells in Neurog1 null mice show cell cycle exit in an apex‐to‐base progression about 1–2 days earlier. Atoh1 is expressed in an apex‐to‐base progression rather then a base‐to‐apex progression as in wildtype littermates. We tested the possible expression of Atoh1 in neurosensory precursors using two Atoh1‐Cre lines. We show Atoh1‐Cre mediated β‐galactosidase expression in delaminating sensory neuron precursors as well as undifferentiated epithelial cells at E11 and E12.5. PCR analysis shows expression of Atoh1 in the otocyst as early as E10.5, prior to any histology‐based detection techniques. Combined, these data suggest that low levels of Atoh1 exist much earlier in precursors of hair cells and sensory neurons, possibly including neurosensory precursors. Analysis of Atoh1‐Cre expression in E18.5 embryos and P31 mice reveal β‐galactosidase stain in all hair cells but also in vestibular and cochlear sensory neurons and some supporting cells. A similar expression of Atoh1‐LacZ exists in postnatal and adult vestibular and cochlear sensory neurons, and Atoh1 expression in vestibular sensory neurons is confirmed with RT‐PCR. We propose that the absence of NEUROG1 protein leads to loss of sensory neuron formation through a phenotypic switch of cycling neurosensory precursors from sensory neuron to hair cell fate. Neurog1 null mice show a truncation of clonal expansion of hair cell precursors through temporally altered terminal mitosis, thereby resulting in smaller sensory epithelia. Developmental Dynamics 234:633–650, 2005.

Expression and function of FGF10 in mammalian inner ear development

We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10s main receptors. Developmental Dynamics 227:203–215, 2003.

MicroRNA gene expression in the mouse inner ear.

MicroRNAs (miRNAs) are small non-coding RNAs that function through the RNA interference (RNAi) pathway and post-transcriptionally regulate gene expression in eukaryotic organisms. While miRNAs are known to affect cellular proliferation, differentiation, and morphological development, neither their expression nor roles in mammalian inner ear development have been characterized. We have investigated the extent of miRNA expression at various time points throughout maturation of the postnatal mouse inner ear by microarray analysis. Approximately one third of known miRNAs are detected in the inner ear, and their expression persists to adulthood. Expression of such miRNAs is validated by quantitative PCR and northern blot analysis. Further analysis by in situ hybridization demonstrates that certain miRNAs exhibit cell-specific expression patterns in the mouse inner ear. Notably, we demonstrate that miRNAs previously associated with mechanosensory cells in zebrafish are also expressed in hair cells of the auditory and vestibular endorgans. Our results demonstrate that miRNA expression is abundant in the mammalian inner ear and that certain miRNAs are evolutionarily associated with mechanosensory cell development and/or function. The data suggest that miRNAs contribute substantially to genetic programs intrinsic to development and function of the mammalian inner ear and that specific miRNAs might influence formation of sensory epithelia from the primitive otic neuroepithelium.

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Atoh1 Null Mice Show Directed Afferent Fiber Growth to Undifferentiated Ear Sensory Epithelia Followed by Incomplete Fiber Retention

Inner ear hair cells have been suggested as attractors for growing afferent fibers, possibly through the release of the neurotrophin brain‐derived neurotrophic factor (BDNF). Atoh1 null mice never fully differentiate hair cells and supporting cells and, therefore, may show aberrations in the growth and/or retention of their innervation. We investigated the distribution of cells positive for Atoh1‐ or Bdnf‐mediated β‐galactosidase expression in Atoh1 null and Atoh1 heterozygotic mice and correlated the distribution of these cells with their innervation. Embryonic day (E) 18.5 Atoh1 null and heterozygotic littermates show Atoh1‐ and BDNF‐β‐galactosidase–positive cells in comparable distributions in the canal cristae and the cochlea apex. Atoh1‐β‐galactosidase–positive but only occasional Bdnf‐β‐galactosidase–positive cells are found in the utricle, saccule, and cochlea base of Atoh1 null mutant mice. Absence of Bdnf‐β‐galactosidase expression in the utricle and saccule of Atoh1 null mice is first noted at E12.5, a time when Atoh1‐β‐galactosidase expression is also first detected in these epithelia. These data suggest that expression of Bdnf is dependent on ATOH1 protein in some but does not require ATOH1 protein in other inner ear cells. Overall, the undifferentiated Atoh1‐ and Bdnf‐β‐galactosidase–positive cells show a distribution reminiscent of that in the six sensory epithelia in control mice, suggesting that ear patterning processes can form discrete patches of Atoh1 and Bdnf expression in the absence of ATOH1 protein. The almost normal growth of afferent and efferent fibers in younger embryos suggests that neither fully differentiated hair cells nor BDNF are necessary for the initial targeted growth of fibers. E18.5 Atoh1 null mice have many afferent fibers to the apex of the cochlea, the anterior and the posterior crista, all areas with numerous Bdnf‐β‐galactosidase–positive cells. Few fibers remain to the saccule, utricle, and the base of the cochlea, all areas with few or no Bdnf‐β‐galactosidase–positive cells. Thus, retention of fibers is possible with BDNF, even in the absence of differentiated hair cells. Developmental Dynamics 233:570–583, 2005.

Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1adr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a “cochlear-gravistatic” endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.

MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival

MicroRNAs (miRNAs) post‐transcriptionally repress complementary target gene expression and can contribute to cell differentiation. The coordinate expression of miRNA‐183 family members (miR‐183, miR‐96, and miR‐182) has been demonstrated in sensory cells of the mouse inner ear and other vertebrate sensory organs. To further examine hair cell miRNA expression in the mouse inner ear, we have analyzed miR‐183 family expression in wild type animals and various mutants with defects in neurosensory development. miR‐183 family member expression follows neurosensory cell specification, exhibits longitudinal (basal‐apical) gradients in maturating cochlear hair cells, and is maintained in sensory neurons and most hair cells into adulthood. Depletion of hair cell miRNAs resulting from Dicer1 conditional knockout (CKO) in Atoh1‐Cre transgenic mice leads to more disparate basal‐apical gene expression profiles and eventual hair cell loss. Results suggest that hair cell miRNAs subdue cochlear gradient gene expression and are required for hair cell maintenance and survival. Developmental Dynamics 240:808–819, 2011.

Differential expression of KCNQ4 in inner hair cells and sensory neurons is the basis of progressive high-frequency hearing loss.

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9-11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to high-frequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis

In mouse ear development, two bHLH genes, Atoh1 and Neurog1, are essential for hair cell and sensory neuron differentiation. Evolution converted the original simple atonal-dependent neurosensory cell formation program of diploblasts into the derived developmental program of vertebrates that generates two neurosensory cell types, the sensory neuron and the sensory hair cell. This transformation was achieved through gene multiplication in ancestral triploblasts resulting in the expansion of the atonalbHLH gene family. Novel genes of the Neurogenin and NeuroD families are upregulated prior to the expression of Atoh1. Recent data suggest that NeuroD and Neurogenin were lost or their function in neuronal specification reduced in flies, thus changing our perception of the evolution of these genes. This sequence of expression changes was accompanied by modification of the E-box binding sites of these genes to regulate different downstream genes and to form inhibitory loops among each other, thus fine-tuning expression transitions.

Characterization of transcriptomes of cochlear inner and outer hair cells.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two types of sensory receptor cells that are critical for hearing in the mammalian cochlea. IHCs and OHCs have different morphology and function. The genetic mechanisms that define their morphological and functional specializations are essentially unknown. The transcriptome reflects the genes that are being actively expressed in a cell and holds the key to understanding the molecular mechanisms of the biological properties of the cell. Using DNA microarray, we examined the transcriptome of 2000 individually collected IHCs and OHCs from adult mouse cochleae. We show that 16,647 and 17,711 transcripts are expressed in IHCs and OHCs, respectively. Of those genes, ∼73% are known genes, 22% are uncharacterized sequences, and 5.0% are noncoding RNAs in both populations. A total of 16,117 transcripts are expressed in both populations. Uniquely and differentially expressed genes account for <15% of all genes in either cell type. The top 10 differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, and Map4k4 in IHCs and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, and Ankrd22 in OHCs. We analyzed commonly and differentially expressed genes with the focus on genes related to hair cell specializations in the apical, basolateral, and synaptic membranes. Eighty-three percent of the known deafness-related genes are expressed in hair cells. We also analyzed genes involved in cell-cycle regulation. Our dataset holds an extraordinary trove of information about the molecular mechanisms underlying hair cell morphology, function, pathology, and cell-cycle control.