Abstract
Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells.
Highlights
The sensory and neuronal cells of the vertebrate inner ear arise from the otic vesicle (OV), a spheroid epithelial structure formed during midgestation embryogenesis through invagination of the otic placode
Whole E10.5 embryos with the greater otic regions removed were used to represent a wide range of non-otic lineages from all three germ layers, and periotic tissues represented non-otic tissues proximally associated with the OV, such as dorsal neural tissues and periotic mesenchyme
Multivariate analyses including principal component analysis (PCA) and Spearman’s correlation demonstrated that samples from the three otic populations shared transcriptome-wide correlations in expression profiles that distinguish this lineage from non-otic populations
Summary
The sensory and neuronal cells of the vertebrate inner ear arise from the otic vesicle (OV), a spheroid epithelial structure formed during midgestation embryogenesis through invagination of the otic placode. By E10.5 otic vesicle invagination has encapsulated the otic placode epithelia into a vesicle where subsequent processes of morphogenesis and differentiation give rise to cell fates of the inner ear, including neural, sensory (hair cells and supporting cells) and non-sensory fates (Figures 1B,C). A progressive lineage bifurcation model of otic differentiation is consistent with experimental observations and illustrates how pan-otic progenitor cells of the otic vesicle could give rise to non-sensory, neural, supporting cell, and hair cell populations (Figure 1C). Classification of genes that enable rigorous identification of otic sensory lineage cells would greatly benefit in vitro studies of inner ear development and regeneration, which currently rely on combinatorial expression of transient non-specific markers (Oshima et al, 2010; Koehler et al, 2013)
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