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

Timothy Erickson, Teresa Nicolson

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 theT.gondiiuracil phospho-ribosyltransferase (UPRT) enzyme specifically in the hair cells of the inner ear and lateral line organ. RNA was labeled by exposing 3days post-fertilization (dpf) UPRT transgenic larvae to 2.5 mM 4-thiouracil (4TU) for 15hours. 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.

Original languageEnglish (US)
Article number842
JournalBMC Genomics
Volume16
Issue number1
DOIs
StatePublished - Oct 23 2015

Fingerprint

Thiouracil
Zebrafish
RNA
RNA Sequence Analysis
Larva
Lateral Line System
Gene Expression
High-Throughput Nucleotide Sequencing
Messenger RNA
Inner Ear
Fertilization
In Situ Hybridization
Cell Biology
Dissection

Keywords

  • Gene expression
  • Hair cell
  • Inner ear
  • Lateral line
  • Neuromast
  • Sensory hair cells
  • Transcriptional profiling
  • Transcriptomics
  • TU-tagging
  • UPRT
  • Zebrafish

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish. / Erickson, Timothy; Nicolson, Teresa.

In: BMC Genomics, Vol. 16, No. 1, 842, 23.10.2015.

Research output: Contribution to journalArticle

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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 theT.gondiiuracil phospho-ribosyltransferase (UPRT) enzyme specifically in the hair cells of the inner ear and lateral line organ. RNA was labeled by exposing 3days post-fertilization (dpf) UPRT transgenic larvae to 2.5 mM 4-thiouracil (4TU) for 15hours. 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.",
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