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

Abstract

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.

Fig. 1

UPRT expression and activity in zebrafish hair cells. a Diagram of the construct used for Tol2 Gateway-mediated transgenesis. A minimal myo6b promoter was used to drive expression of the HA-UPRT-P2A-mCherry transgene in auditory, vestibular, and lateral line hair cells. b-b” Maximum projection images of immunolabeled HA-UPRT and fixed mCherry fluoresecence in inner ear hair cells of a 3 dpf Tg(myo6b:UPRT) larva. AC, anterior crista; AM, anterior macula; LC, lateral crista; PC, posterior crista. The focal plane includes a neuromast (NM-MI1). c Dot blot for TU-tagged, biotinylated total RNA demonstrating the enzymatic activity of UPRT in the hair cells of 5 dpf zebrafish larvae. Nontransgenic wild-type (WT) larvae exhibited low levels of 4TU incorporation in contrast to Tg(myo6b:UPRT) larvae when exposed to 5 mM 4TU for 3 h. RNA from Tg(myo6b:UPRT) larvae exposed to DMSO only did not exhibit any detectable biotinylation

Fig. 2

TU-tagging workflow diagram. Larvae (3 dpf) were exposed to 2.5 mM 4TU for 15 h and then homogenized to isolate total RNA. Purified Poly (a) mRNA was then fragmented and biotinylated for strepavidin-mediated pull down. RNAseq libraries were constructed and sequenced for comparison of transcript abundance between TU-tagged and input control RNA

Fig. 3

Enriched nascent transcripts in larval hair cells. SeqMonk scatter plot showing the correlation between the log₂-transformed reads-per-million mapped reads (RPM) values for each zebrafish gene from the TU-tagged and input mRNA samples. The 28 significantly enriched TU-tagged genes (DESeq adjusted p-value < 0.05; Table 1) are indicated by the red points to the upper left of the diagonal. The top ten significantly enriched genes are annotated by name, with previously known zebrafish hair cell transcripts underlined

Fig. 4

Validation of hair-cell specific gene expression by whole mount mRNA in situ hybridization. Panels a-q show the mRNA in situ hybridization patterns for 17 of the uncharacterized TU-enriched genes. Lateral views (dorsal up; anterior to the left) of the head and anterior trunk of 3 dpf larvae are depicted. In each panel, the focal plane includes sensory epithelia of the inner ear and neuromasts (NM), as indicated in panel a. The DESeq adjusted p-value (padj) and fold-enrichment of the transcript in the TU-tagged mRNA sample are indicated for each gene. Scale bar in A = 100 μm