Significance: Our understanding of the molecular basis of our sense of hearing and balance has improved significantly, although some of the key players in sensory hair cells have yet to be identified. Sensory hair cells depend on extracellular filaments known as tip links to transduce mechanical stimuli into electrical signals. We demonstrate that the tip link protein PCDH15 interacts with two integral member proteins, TMC1 and TMC2, which have recently been put forth as candidates for the mechanotransduction channel.

Sensory hair cells are exquisitely sensitive to mechanical stimuli and as such, are prone to damage and apoptosis during dissections or in vitro manipulations. Thiouracil (TU)-tagging is a noninvasive method to label cell type-specific transcripts in an intact organism, thereby meeting the challenge of how to analyze gene expression in hair cells without the need to sort cells. We adapted TU-tagging to zebrafish to identify novel transcripts expressed in the sensory hair cells of the developing acoustico-lateralis organs. We created a transgenic line of zebrafish expressing the T.gondii uracil phospho-ribosyltransferase (UPRT) enzyme specifically in the hair cells of the inner ear and lateral line organ. RNA was labeled by exposing 3 days post-fertilization (dpf) UPRT transgenic larvae to 2.5 mM 4-thiouracil (4TU) for 15 hours. Following total RNA isolation, poly(A) mRNA enrichment, and purification of TU-tagged RNA, deep sequencing was performed on the input and TU-tagged RNA samples. Analysis of the RNA sequencing data revealed the expression of 28 transcripts that were significantly enriched (adjusted p-value < 0.05) in the UPRT TU-tagged RNA relative to the input sample. Of the 25 TU-tagged transcripts with mammalian homologs, the expression of 18 had not been previously demonstrated in zebrafish hair cells. The hair cell-restricted expression for 17 of these transcripts was confirmed by whole mount mRNA in situ hybridization in 3 dpf larvae. The hair cell-restricted pattern of expression of these genes offers insight into the biology of this receptor cell type and may serve as useful markers to study the development and function of sensory hair cells. In addition, our study demonstrates the utility of TU-tagging to study nascent transcripts in specific cell types that are relatively rare in the context of the whole zebrafish larvae.

Background: During visual system development, multiple signalling pathways cooperate to specify axial polarity within the retina and optic tectum. This information is required for the topographic mapping of retinal ganglion cell axons on the tectum. Meis1 is a TALE-class homeodomain transcription factor known to specify anterior-posterior identity in the hindbrain, but its role in visual system patterning has not been investigated. Results: meis1 is expressed in both the presumptive retina and tectum. An analysis of retinal patterning reveals that Meis1 is required to correctly specify both dorsal-ventral and nasal-temporal identity in the zebrafish retina. Meis1-knockdown results in a loss of smad1 expression and an upregulation in follistatin expression, thereby causing lower levels of Bmp signalling and a partial ventralization of the retina. Additionally, Meis1-deficient embryos exhibit ectopic Fgf signalling in the developing retina and a corresponding loss of temporal identity. Meis1 also positively regulates ephrin gene expression in the tectum. Consistent with these patterning phenotypes, a knockdown of Meis1 ultimately results in retinotectal mapping defects. Conclusions: In this work we describe a novel role for Meis1 in regulating Bmp signalling and in specifying temporal identity in the retina. By patterning both the retina and tectum, Meis1 plays an important role in establishing the retinotectal map and organizing the visual system.

Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.

Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. Thein vivosignificance of these protein–protein interaction domains of PCDH15 in hair cells has not been determined. Here, we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) inpcdh15a-null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior and hair cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype and that the CD1- or CD3-specific regions were dispensable for hair bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain was deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for thein vivofunction of Pcdh15a in zebrafish hair cells.SIGNIFICANCE STATEMENTTip links transmit force to mechanotransduction channels at the tip of hair bundles in sensory hair cells. One component of tip links is Protocadherin 15 (PCDH15). Here, we demonstrate that, when transgenically expressed, either zebrafish Pcdh15a-cytodomain 1 (CD1) or Pcdh15a-CD3 can rescue the phenotype of apcdh15a-null mutant. Even when lacking the specific regions for CD1 or CD3, truncated Pcdh15a that contains the so-called common region (CR) at the cytoplasmic/membrane interface still has the ability to rescue similar to full-length Pcdh15a. In contrast, Pcdh15a lacking the entire cytoplasmic domain is not functional. These results demonstrate that the CR plays a key role in the mechanotransduction complex in hair cells.