Abstract
Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type–specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants.
Highlights
Genome-wide expression profiling is a valuable tool for gaining systems-level understanding of biological processes during development, response to stress, and pathological conditions
Our protocol is based on flow cytometry to sort and capture cells labeled with commercially available antibodies to endogenously expressed cluster of differentiation (CD) antigens
We identified Zeb1 and miR200b as regulators of epithelial and mesenchymal identity in the mouse inner ear, and we further identified the signaling pathway disrupted by the Zeb1 mutation in the Twirler mouse mutant
Summary
Genome-wide expression profiling is a valuable tool for gaining systems-level understanding of biological processes during development, response to stress, and pathological conditions. Accurate interpretation of expression profiles from complex tissues such as neuroepithelia is often complicated and hindered by cellular heterogeneity. Such cellular complexity has made it difficult to identify relevant transcriptional networks from the auditory and vestibular systems of mammalian inner ears, which are composed of hair cells, multiple types of supporting cells, neurons, mesenchymal cells and vascular endothelium. The complexity of the auditory and vestibular systems is reflected in over 250 genes which, when mutated, underlie inner ear malformations or dysfunction in mice (http://hearingimpairment.jax.org/master_table.html). There are over 118 syndromes that include hearing loss as part of their phenotype [1], and over 100 genes – roughly half of which have been cloned which underlie hereditary non-syndromic hearing loss in human (http://hereditaryhearingloss.org/) and [2]
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