Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.

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Published in Nature Communications, v. 6, article 8549, p. 1-14.

© 2015 Macmillan Publishers Limited. All rights reserved.

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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This work was supported by the Triologic Society Career Development grant (R.H.), NIDCD/NIH R01DC013817 and R01DC03544 (R.H.), DOD CDMRP MR130240 (R.H.), the Swiss National Science Foundation grant 310030B-144085, 3100-06489 (W.R.), Fondation pour la Recherche Me´dicale FRM DEQ20131029168 (B.D.), ANR Ciliopath-X (B.D.), Nebraska Tobacco Settlement Biomedical Research Development Funds (S.M.J.) and NIDCD/NIH, R01DC014658 (G.I.F.), K01DK092402 (N.A.Z.) and R01DK102001 (N.A.Z.).

ncomms9549-s1.pdf (2527 kB)
Supplementary Figures 1-13 and Supplementary References.

ncomms9549-s2.xlsx (4799 kB)
Supplementary Data 1: Gene expression profiles in the inner ear measured using microarrays. Expression profiles were recorded from six cellular populations (three cell-types: HCs, ENHCs and NECs, isolated from two inner ear organs: the cochlea and the vestibular system) using independent biological triplicates. This dataset contains quantile-normalized expression levels for microarray probes that were flagged as expressed in at least two samples. Assignment of probes to the gene clusters shown in Figure 1 is indicated in column V.

ncomms9549-s3.xlsx (1406 kB)
Supplementary Data 2: Gene expression profiles in the inner ear measured using RNA-seq. Gene expression was recorded in hair-cells (HC) and non-HCs populations. This dataset contains expression levels for genes covered by at least 20 reads. Expression levels were normalized by quantile normalization and a floor value of 20 was set. Assignment of genes to the clusters shown in supplementary Figure 4 is indicated in column K.

ncomms9549-s4.xlsx (12 kB)
Supplementary Data 3: Assignment of the mouse orthologs of known human deafness-causing genes to gene expression clusters.

ncomms9549-s5.xlsx (80 kB)
Supplementary Data 4: Marker genes of inner-ear HCs.

ncomms9549-s6.xlsx (67 kB)
Supplementary Data 5: Putative HC-enriched RFX target genes.

ncomms9549-s7.xlsx (1273 kB)
Supplementary Data 6: Gene expression profiles in Zebrafish hair cells. Expression levels measured using RNA-seq in transgenic zebrafish which express GFP predominantly in hair cells. Data were recorded for sorted GFP-positive (+) and negative (−) cells from 5 day post fertilization (dpf) larvae using flow cytometry.

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