Transcriptomic Profiling of Zebrafish Hair Cells Using RiboTag.

Maggie S Matern,Yang Song,Alisha Beirl,Yoko Ogawa,Katie S Kindt,Nikhil Paladugu,Ronna Hertzano

doi:10.3389/fcell.2018.00047

Abstract

The zebrafish inner ear organs and lateral line neuromasts are comprised of a variety of cell types, including mechanosensitive hair cells. Zebrafish hair cells are evolutionarily homologous to mammalian hair cells, and have been particularly useful for studying normal hair cell development and function. However, the relative scarcity of hair cells within these complex organs, as well as the difficulty of fine dissection at early developmental time points, makes hair cell-specific gene expression profiling technically challenging. Cell sorting methods, as well as single-cell RNA-Seq, have proved to be very informative in studying hair cell-specific gene expression. However, these methods require that tissues are dissociated, the processing for which can lead to changes in gene expression prior to RNA extraction. To bypass this problem, we have developed a transgenic zebrafish model to evaluate the translatome of the inner ear and lateral line hair cells in their native tissue environment; the Tg(myo6b:RiboTag) zebrafish. This model expresses both GFP and a hemagglutinin (HA) tagged rpl10a gene under control of the myo6b promoter (myo6b:GFP-2A-rpl10a-3xHA), resulting in HA-tagged ribosomes expressed specifically in hair cells. Consequently, intact zebrafish larvae can be used to enrich for actively translated hair cell mRNA via an immunoprecipitation protocol using an antibody for the HA-tag (similar to the RiboTag mice). We demonstrate that this model can be used to reliably enrich for actively translated zebrafish hair cell mRNA. Additionally, we perform a global hair cell translatome analysis using RNA-Seq and show enrichment of known hair cell expressed transcripts and depletion of non-hair cell expressed transcripts in the immunoprecipitated material compared with mRNA extracted from whole fish (input). Our results show that our model can identify novel hair cell expressed genes in intact zebrafish, without inducing changes to gene expression that result from tissue dissociation and delays during cell sorting. Overall, we believe that this model will be highly useful for studying changes in zebrafish hair cell-specific gene expression in response to developmental progression, mutations, as well as hair cell damage by noise or ototoxic drug exposure.

Highlights

Hearing loss is a genetically heterogeneous disorder, with mutations in over 150 genes estimated to underlie genetic nonsyndromic hearing deficits (Van Camp and Smith, 2017)
To more effectively isolate RNA from zebrafish hair cells (HCs), we have developed a transgenic zebrafish RiboTag model to evaluate the translatome of zebrafish inner ear and lateral line HCs; the Tg(myo6b:GFP-2A-rpl10a-3xHA) zebrafish [from here on referred to as Tg(myo6b:RiboTag)]
To create a RiboTag zebrafish model that would allow for enrichment of the inner ear and lateral line HC translatome, we utilized the rpl10a gene, which has previously been used to effectively immunoprecipitate ribosomes in zebrafish via a GFP tag (Tryon et al, 2013)

Summary

Introduction

Hearing loss is a genetically heterogeneous disorder, with mutations in over 150 genes estimated to underlie genetic nonsyndromic hearing deficits (Van Camp and Smith, 2017). Across vertebrate species, the auditory, vestibular, and lateral line sensory organs are comprised of a variety of cell types, of which HCs make up only a small percentage (Hertzano and Elkon, 2012; Jiang et al, 2014; Matern et al, 2017) Due to their relative scarcity, cell typespecific approaches such as manual cell sorting, fluorescence activated cell sorting (FACS), or single cell RNA-Seq (scRNASeq) are necessary to analyze gene expression in HCs. due to their relative scarcity, cell typespecific approaches such as manual cell sorting, fluorescence activated cell sorting (FACS), or single cell RNA-Seq (scRNASeq) are necessary to analyze gene expression in HCs These methods have been used in both mice and zebrafish to analyze HC gene expression changes that occur in mutant animals, during development and regeneration, or after exposure to noise or ototoxic drugs (McDermott et al, 2007; Hertzano and Elkon, 2012; Jiang et al, 2014; Steiner et al, 2014; Burns et al, 2015; Elkon et al, 2015; Scheffer et al, 2015). Tissue dissociation can induce significant cellular stress due to loss of lateral inhibition and cell-cell contact, and combined with the prolonged time associated with tissue processing, may lead to confounding changes in gene expression (Sanz et al, 2009; Gay et al, 2013, 2014)

Methods

Results

Conclusion