Biology subjects

Kelley, M. W.

Publications and source records attributed to Kelley, M. W..

7 recordsLinked to original sources

bioRxiv2026

Single-Cell Profiling of the Developing Organ of Corti Identifies Etv4/5/1 as Key Regulators of Pillar Cell Identity

The mammalian auditory sensory epithelium, the organ of Corti, contains a number of unique cell types, including the inner and outer pillar cells that form the walls of the tunnel of Corti. The limited number of pillar cells and their close physical proximity to other cells within the organ of Corti has limited efforts to transcriptionally characterize their development. To identify potential regulators of pillar cell formation, we isolated cochlear duct epithelial cells at time points between embryonic day 11 and 16 for single cell RNA-sequencing. The resulting data were used to build a developmental trajectory from undifferentiated precursor cells to each of the major cell types in the organ of Corti including inner pillar cells. Bioinformatic analyses including SCENIC, TradeSeq and CellOracle identified the Etv4/5/1 transcription factors as likely regulators of inner pillar cell development. To specifically examine the role of Etv4/5/1 in pillar cell development, conditional triple mutants were generated. Results indicate defects in the formation of inner pillar cells as well as changes in other cochlear cells likely as a result of secondary interactions. Finally, we demonstrate that expression of Etv4/5/1 in pillar cells is dependent on Fgfr3 and identify downstream targets of Fgfr3/Etv signaling in inner pillar cells. These results provide significant insights regarding the specification and early development of inner pillar cells, which will have implications for understanding congenital deficits and potential applications in the development of regenerative strategies.

developmental biology↗

bioRxiv2025

CASZ1 regulates the rate at which outer hair cells mature and is required for hearing

The transcriptional activator ATOH1 is a master regulator of the development of mechanosensory hair-cells (HCs) in the ear. We report that the ATOH1 target gene Casz1 encodes a transcription factor that regulates the rate of outer HC (OHC) maturation by gene repression. Genetic deletion of Casz1 during (but not after) development of the mouse cochlea caused: hearing loss; abnormal organization of mechanosensory stereocilia bundles in OHCs; abnormally low F-actin density in OHC cuticular plates; progressive loss of OHCs; and mild morphological alterations in inner HCs. RNA sequencing revealed that Casz1 deletion delayed downregulation of genes expressed in immature OHCs, including the actin regulator-encoding gene Coro2a, and accelerated upregulation of genes expressed in mature OHCs. Coro2a knockdown restored the density of cuticular plate F-actin in Casz1 mutant OHCs. Our data indicate that CASZ1 regulates transcriptional and morphological maturation of OHCs, and that CASZ1 in maturing HCs is necessary for hearing.

developmental biology↗

bioRxiv2024

Jag1 represses Notch activation in lateral supporting cells and inhibits an outer hair cell fate in the medial compartment of the developing cochlea

Notch signaling regulates both inner and middle ear morphogenesis and establishes a strict pattern of sensory cells in the organ of Corti in the mammalian cochlea. Patients with Alagille syndrome have impaired Notch signaling ([~]94% with JAG1 mutations) resulting in sensorineural and conductive hearing loss. Here, we investigate the function of Jag1-mediated Notch activation in cochlear patterning and signaling using the Jag1 "Nodder" (Jag1Ndr/Ndr) mouse model of Alagille syndrome. Jag1Ndr/Ndr mice exhibited severe vestibular and auditory deficits and a dose-dependent increase in ectopic inner hair cells and a reduction in outer hair cells. Single cell RNA sequencing of the organ of Corti demonstrated a global dysregulation of genes associated with inner ear development and deafness. Analysis of individual cell types indicated a novel role for Jag1 in repressing Notch activation in lateral supporting cells and revealed a function for Jag1 in gene regulation and development of outer hair cells. Additionally, "outer hair cell-like" SLC26A5 (Prestin) positive cells were present in the medial compartment and pillar cell region of Jag1Ndr/Ndr mice and exhibited location-dependent expression of the inner hair cell fate-regulator Tbx2, revisiting the potency of Tbx2 in driving inner hair cell commitment in "outer hair cell-like" cells in the Jag1-mutant IHC compartment. Our data reveals a novel role for Jag1 in repressing Notch activation in lateral supporting cells and highlights involvement for Notch signaling in inner versus outer hair cell specification and positioning.

developmental biology↗

bioRxiv2022

ZBTB20 is Essential for Cochlear Maturation and Hearing in Mice

The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral non-sensory region. Here we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear non-sensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane, a flattened spiral prominence, and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the non-sensory epithelium covering the outer border Claudius cells, outer sulcus root cells and spiral prominence epithelial cells. Transcriptome analysis shows ZBTB20 regulates genes coding for tectorial membrane proteins in the greater epithelial ridge, and those preferentially expressed in root cells and spiral prominence epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral non-sensory domain.

developmental biology↗

bioRxiv2021

A nesprin-4/kinesin-1 cargo model for nucleus positioning in cochlear outer hair cells

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

cell biology↗

bioRxiv2020

gEAR: gene Expression Analysis Resource portal for community-driven, multi-omic data exploration

The gEAR portal (gene Expression Analysis Resource, umgear.org) is an open access community-driven tool for multi-omic and multi-species data visualization, analysis and sharing. The gEAR supports visualization of multiple RNA-seq data types (bulk, sorted, single cell/nucleus) and epigenomics data, from multiple species, time points and tissues in a single-page, user-friendly browsable format. An integrated scRNA-seq workbench provides access to raw data of scRNA-seq datasets for de novo analysis, as well as marker-gene and cluster comparisons of pre-assigned clusters. Users can upload, view, analyze and privately share their own data in the context of previously published datasets. Short, permanent URLs can be generated for dissemination of individual or collections of datasets in published manuscripts. While the gEAR is currently curated for auditory research with over 90 high-value datasets organized in thematic profiles, the gEAR also supports the BRAIN initiative (via nemoanalytics.org) and is easily adaptable for other research domains.

bioinformatics↗

bioRxiv2019

Characterizing Adult cochlear supporting cell transcriptional diversity using single-cell RNA-Seq: Validation in the adult mouse and translational implications for the adult human cochlea

Hearing loss is a problem that impacts a significant proportion of the adult population. Cochlear hair cell loss due to loud noise, chemotherapy and aging is the major underlying cause. A significant proportion of these individuals are dissatisfied with available treatment options which include hearing aids and cochlear implants. An alternative approach to restore hearing would be to regenerate hair cells. Such therapy would require recapitulation of the complex architecture of the organ of Corti, necessitating regeneration of both mature hair cells and supporting cells. Transcriptional profiles of the mature cell types in the cochlea are necessary to can provide a metric for eventual regeneration therapies. To assist in this effort, we sought to provide the first single-cell characterization of the adult cochlear supporting cell transcriptome. We performed single-cell RNA-Seq on FACS-purified adult cochlear supporting cells from the LfngEGFP adult mouse, in which supporting cells express GFP. We demonstrate that adult cochlear supporting cells are transcriptionally distinct from their perinatal counterparts. We establish cell type-specific adult cochlear supporting cell transcriptome profiles, and we validate these expression profiles through a combination of both fluorescent immunohistochemistry and in situ hybridization co-localization and qPCR of adult cochlear supporting cells. Furthermore, we demonstrate the relevance of these profiles to the adult human cochlea through immunofluorescent human temporal bone histopathology. Finally, we demonstrate cell cycle regulator expression in adult supporting cells and perform pathway analyses to identify potential mechanisms for facilitating mitotic regeneration (cell proliferation, differentiation, and eventually regeneration) in the adult mammalian cochlea. Our findings demonstrate the importance of characterizing mature as opposed to perinatal supporting cells.

genomics↗