An Efficient Method to Detect Messenger RNA (mRNA) in the Inner Ear by RNAscope In Situ Hybridization

Ghosh, Sumana; Casey, Graham; Stansak, Kendra; Thapa, Punam; Walters, Bradley J.

doi:10.1007/978-1-0716-2022-9_6

Cited by 3 publications

(3 citation statements)

References 48 publications

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“…It is worth noting that despite evidence from scRNA-Seq showing high expression of innexins in muscle cells, there is no corresponding evidence in our whole-mount in situ hybridization results. This is not entirely unexpected given the limited resolution of standard whole-mount in situ hybridization (e.g., Ghosh et al 2022 ) and that no previous studies have reported expression in M. leidyi muscle cells based solely on in situ hybridization.…”

Section: Resultsmentioning

confidence: 81%

Independent Innexin Radiation Shaped Signaling in Ctenophores

Ortiz

Bobkov

DeBiasse

et al. 2023

Molecular Biology and Evolution

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Innexins facilitate cell-cell communication by forming gap junctions or non-junctional hemichannels, which play important roles in metabolic, chemical, ionic, and electrical coupling. The lack of knowledge regarding the evolution and role of these channels in ctenophores (comb jellies), the likely sister group to the rest of animals, represents a substantial gap in our understanding of the evolution of intercellular communication in animals. Here we identify and phylogenetically characterize the complete set of innexins of four ctenophores: Mnemiopsis leidyi, Hormiphora californensis, Pleurobrachia bachei, and Beroe ovata. Our phylogenetic analyses suggest that ctenophore innexins diversified independently from those of other animals and were established early in the emergence of ctenophores. We identified a four-innexin genomic cluster, which was present in the last common ancestor of these four species and has been largely maintained in these lineages. Evidence from correlated spatial and temporal gene expression of the M. leidyi innexin cluster suggest that this cluster has been maintained due to constraints related to gene regulation. We describe basic electrophysiological properties of putative ctenophore hemichannels from muscle cells using intracellular recording techniques, showing substantial overlap with the properties of bilaterian innexin channels. Together, our results suggest that the last common ancestor of animals had gap junctional channels also capable of forming functional innexin hemichannels, and that innexin genes have independently evolved in major lineages throughout Metazoa.

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Section: Resultsmentioning

confidence: 81%

Independent Innexin Radiation Shaped Signaling in Ctenophores

Ortiz

Bobkov

DeBiasse

et al. 2023

Molecular Biology and Evolution

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“…RNAscope in-situ hybridization labeling and analysis. The RNA labeling procedure was carried out as previously described (Ghosh et al, 2022). Mouse temporal bones were fixed in 4% PFA at room temperature for 3 hours.…”

Section: Methodsmentioning

confidence: 99%

PKHD1L1 is required for stereocilia bundle maintenance, durable hearing function and resilience to noise exposure

Strelkova,

Osgood,

Tian

et al. 2024

Preprint

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Sensory hair cells of the cochlea are essential for hearing, relying on the mechanosensitive stereocilia bundle at their apical pole for their function. Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1) is a stereocilia protein required for normal hearing in mice, and for the formation of the transient stereocilia surface coat, expressed during early postnatal development. While the function of the stereocilia coat remains unclear, growing evidence supports PKHD1L1 as a human deafness gene. In this study we carry out in depth characterization of PKHD1L1 expression in mice during development and adulthood, analyze hair-cell bundle morphology and hearing function in aging PKHD1L1-defficient mouse lines, and assess their susceptibility to noise damage. Our findings reveal that PKHD1L1-deficient mice display no disruption to bundle cohesion or tectorial membrane attachment-crown formation during development. However, starting from 6 weeks of age, PKHD1L1-defficient mice display missing stereocilia and disruptions to bundle coherence. Both conditional and constitutive PKHD1L1 knock-out mice develop high-frequency hearing loss progressing to lower frequencies with age. Furthermore, PKHD1L1-deficient mice are susceptible to permanent hearing loss following low-level acoustic overexposure, which typically induces only temporary shifts in hearing thresholds. These results suggest a role for PKHD1L1 in establishing robust sensory hair bundles during development, necessary for maintaining bundle cohesion and function in response to acoustic trauma and aging.

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“…RNA in situ hybridization was conducted using the BaseScope assay (Advanced Cell Diagnostics) according to the manufacturer's instructions with a modified protocol previously described (Ghosh et al, 2022). Mouse cochleae were fixed in 4% PFA for 3 hours and microdissected in cold 20% RNAlater (Thermo Fisher Scientific) in PBS.…”

Section: Rna In Situ Hybridizationmentioning

confidence: 99%

A Mutation inTmem135Causes Progressive Sensorineural Hearing Loss

Kim,

Simms,

Behnammanesh

et al. 2024

Preprint

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Transmembrane protein 135 (TMEM135) is a 52 kDa protein with five predicted transmembrane domains that is highly conserved across species. Previous studies have shown that TMEM135 is involved in mitochondrial dynamics, thermogenesis, and lipid metabolism in multiple tissues; however, its role in the inner ear or the auditory system is unknown. We investigated the function of TMEM135 in hearing using wild-type (WT) and Tmem135FUN025/FUN025 (FUN025) mutant mice on a CBA/CaJ background, a normal-hearing mouse strain. Although FUN025 mice displayed normal auditory brainstem response (ABR) at 1 month, we observed significantly elevated ABR thresholds at 8, 16, and 64 kHz by 3 months, which progressed to profound hearing loss by 12 months. Consistent with our auditory testing, 13-month-old FUN025 mice exhibited a severe loss of outer hair cells and spiral ganglion neurons in the cochlea. Our results using BaseScope in situ hybridization indicate that TMEM135 is expressed in the inner hair cells, outer hair cells, and supporting cells. Together, these results demonstrate that the FUN025 mutation in Tmem135 causes progressive sensorineural hearing loss, and suggest that TMEM135 is crucial for maintaining key cochlear cell types and normal sensory function in the aging cochlea.

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An Efficient Method to Detect Messenger RNA (mRNA) in the Inner Ear by RNAscope In Situ Hybridization

Cited by 3 publications

References 48 publications

Independent Innexin Radiation Shaped Signaling in Ctenophores

Independent Innexin Radiation Shaped Signaling in Ctenophores

PKHD1L1 is required for stereocilia bundle maintenance, durable hearing function and resilience to noise exposure

A Mutation inTmem135Causes Progressive Sensorineural Hearing Loss

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