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Abstract
BackgroundSensory 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.MethodsWe 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. ResultsAnalysis 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2072-5) contains supplementary material, which is available to authorized users.
Highlights
Sensory hair cells are exquisitely sensitive to mechanical stimuli and as such, are prone to damage and apoptosis during dissections or in vitro manipulations
To further our understanding of the biology of zebrafish hair cells, and to develop an alternative to invasive cell-purification techniques, in this study we describe the use of TUtagging in zebrafish to label hair cell-expressed transcripts in vivo
We found 28 significantly enriched transcripts, only seven of which were known to be expressed in zebrafish hair cells
Summary
Sensory hair cells are exquisitely sensitive to mechanical stimuli and as such, are prone to damage and apoptosis during dissections or in vitro manipulations. We adapted TU-tagging to zebrafish to identify novel transcripts expressed in the sensory hair cells of the developing acoustico-lateralis organs. Sensory hair cells are the highly specialized mechanoreceptors of the auditory, vestibular, and lateral line (acousticolateralis) organs in vertebrates. Due to their mechanical sensitivity, relative scarcity, and the complex anatomy of the acoustico-lateralis organs, hair cells have been a difficult cell type to dissociate, purify and analyze. Uncovering additional hair cell-specific transcripts would both increase the usefulness of zebrafish as a model for hearing and balance disorders, and deepen our understanding of the development and function of vertebrate hair cells
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