A Triple Line of Defense: The Role of the Group 1 RFX Transcription Factors in the Inner Ear

Abstract

The group 1 RFX transcription factors (TFs), RFX1, RFX2, and RFX3, are master regulators of ciliogenesis. Our laboratory showed that conditional deletion of both Rfx1 and Rfx3 (Rfx1/3;Gfi1-Cre) from hair cells (HCs) of mice results in profound hearing loss, an abrupt loss of all outer hair cells shortly after the onset of hearing, and a mild, late onset vestibular phenotype. However, Rfx1/3;Gfi1-Cre mutant HCs do not have kinocilia or planar cell polarity defects. Due to significant homology in functional domains, a similar role in ciliogenesis, and expression in cochlear and vestibular HCs, we hypothesized that RFX2 functions to compensate for the loss of RFX1/3 in inner ear HCs. Here, we investigated the compensatory role of RFX2 for RFX1/3 in kinocilia development and maintenance and its function in the vestibular system. Additionally, we explored the signaling cascade downstream of the group 1 RFX transcription factors in the vestibular system.

I generated a triple conditional knockout (cKO) mouse of the group 1 RFX TFs (Rfx1/2/3 cKO), crossing an Rfx2 gene trap mouse model (Rfx2Gt) with the Rfx1/3;Gfi1-Cre mouse. These mice underwent vestibular sensory evoked potential (VsEP) testing at 1 month old (mo), 3mo, and 6mo to test vestibular function. Rfx1/2/3 cKO mice had significantly elevated VsEP thresholds as early as 1mo. As observed with immunohistochemistry, kinocilia were shortened in Rfx1/2/3 cKO mice in the saccule and utricle at postnatal day(P) 10 and 6mo, suggesting a role of the group 1 RFX TFs in kinocilia development or maintenance. In addition to a kinociliary phenotype, a single cell RNA-sequencing (scRNA-seq) experiment of P5 Rfx1/2/3 cKO vestibular HCs, using the 10x Genomics platform, identified significantly downregulated genes in Rfx1/2/3 cKO that have a known role in ciliogeneis, with genes that were uniquely downregulated in the striolar HCs. These data show that RFX2 has a compensatory role for RFX1/3 within the vestibular system and that the group 1 RFX TFs have an essential role in vestibular function, potentially through vestibular kinocilia development or maintenance. Finally, we reveal part of the group 1 RFX signaling pathway in vestibular HCs, identifying candidate genes for maintaining vestibular function.