Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, USA. ericksti@ohsu.edu.
Timothy
T
Erickson
Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, USA. nicolson@ohsu.edu.
Teresa
T
Nicolson
D000818:Animals; D030801:Animals, Genetically Modified; D005786:Gene Expression Regulation; D006199:Hair Cells, Auditory, Inner / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D007814:Larva / Q000235:genetics* / Q000254:growth & development; D009928:Organ Specificity / Q000235:genetics; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D013889:Thiouracil / Q000008:administration & dosage / Q000031:analogs & derivatives; D015027:Zebrafish / Q000235:genetics
D012333:RNA, Messenger; C114719:4-thiouracil; D013889:Thiouracil; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase