The limitations of these enrichment approaches are that they usually require large numbers of input cells, well-characterized transgenic mouse drivers, and prolonged cell preparation ( Paul and Huang, 2018). Recently, transgenic RiboTag mice were successfully used to purify RNAs from specific cochlear cell types ( Milon et al., 2021). Fluorescence-activated cell sorting (FACS) or manual cell sorting is often performed to enrich and isolate individual cells based on fluorescence labeling or morphological characteristics ( Rueda et al., 2019 West et al., 2022). However, such studies are mainly based on dissociated pooled cochlear cells, such as IHCs, OHCs, supporting cells, spiral ganglion neurons (SGNs), and striatal cells ( Scheffer et al., 2015 Tang et al., 2020). Next-generation RNA-sequencing (RNA-seq) has been powerful to evaluate the expression dynamics of critical genes in various vulnerable-low-abundance cochlear cell types ( Yan et al., 2013 Slatko et al., 2018). These constraints pose a significant challenge in dissecting cochlear cells during the maturation processes at the molecular level. Moreover, these sensory epithelial cells are extremely vulnerable during isolation, especially considering they are embedded in a calcified temporal bone at juvenile and mature ages. In contrast to ∼100 million photoreceptors (i.e., rods and cones) in the retina, the mouse cochlea contains only ∼700 inner hair cells (IHCs) and ∼2,000 outer hair cells (OHCs) ( Ehret and Frankenreiter, 1977), the two types of sensory hair cells (HCs). Additionally, many non-syndromic hearing losses are progressive during cochlear maturation ( Zhang et al., 2020), thus highlighting the significance of understanding the molecular dynamics of cochlear maturation at the single-cell level. ![]() However, cochlear development does not stop at postnatal day 7 (P7) but continues in mice after calcification of the temporal bone when hearing sensitivity improves gradually from P14 to P21 ( Lim and Anniko, 1985). Previous studies have shed light on the development of cochlear epithelia at embryonic and early postnatal ages in mice ( Kolla et al., 2020). The perception of sound is mediated by the sensory epithelium located in the cochlea. The vertebrate inner ear comprises auditory and vestibular sensory end organs responsible for hearing and balance, respectively. Our transcriptomes of juvenile and mature mouse cochlear cells provide the sequel to those previously published at late embryonic and early postnatal ages and will be valuable resources to investigate cochlear maturation at the single-cell resolution. We further identified and confirmed a long non-coding RNA gene Miat to be expressed during maturation in cochlear hair cells and spiral ganglia neurons, and Pcp4 to be expressed during maturation in cochlear hair cells. Importantly, pseudotime trajectory analysis revealed that inner hair cell maturation peaks at P14 while outer hair cells continue development until P28. We also mapped known deafness genes to corresponding cochlear cell types. Our hair cell scRNA-seq datasets are consistent with published transcripts from bulk RNA-seq. We attained the transcriptomes of multiple cell types, including hair cells, supporting cells, spiral ganglia, stria fibrocytes, and immune cells. Here we performed the 10x Genomics single-cell RNA sequencing (scRNA-seq) of mouse cochleae at postnatal days 14 (P14) and 28. ![]() Juvenile and mature mouse cochleae contain various low-abundant, vulnerable sensory epithelial cells embedded in the calcified temporal bone, making it challenging to profile the dynamic transcriptome changes of these cells during maturation at the single-cell level. 2Lynch Comprehensive Cancer Research Center, Creighton University School of Medicine, Omaha, NE, United States.1Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, United States.Zhenhang Xu 1 Shu Tu 1† Caroline Pass 1† Yan Zhang 1 Huizhan Liu 1 Jack Diers 1 Yusi Fu 2 David Z.
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