Allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants.

Richard J White,Elisabeth M Busch-Nentwich,Eirinn Mackay,Stephen W Wilson

doi:10.7554/elife.72825

Richard J White, Elisabeth M Busch-Nentwich + Show 2 more

Open Access

https://doi.org/10.7554/elife.72825

Copy DOI

Abstract

In model organisms, RNA-sequencing (RNA-seq) is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often over-represented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that the differential expression of genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

Highlights

Large scale genetic screens to identify gene function by randomly introducing mutations have been a staple of zebrafish genetics for several decades (Driever et al, 1996; Haffter et al, 1996; Kettleborough et al, 2013)
The F3 clutches contain the novel mutations in Mendelian ratios, and in a forward genetics approach are screened for recessive phenotypes of interest which appear in approximately 25% of embryos (Mullins et al, 1994)
We report that the highly polymorphic nature of zebrafish strains can lead to gene expression differences between mutant and sibling embryos through allele-specific expression (ASE)

Summary

Introduction

Large scale genetic screens to identify gene function by randomly introducing mutations have been a staple of zebrafish genetics for several decades (Driever et al, 1996; Haffter et al, 1996; Kettleborough et al, 2013). The advent of RNA-sequencing (RNA-seq) has enabled investigators to estimate the location of such mutations in the genome, while providing information regarding gene expression levels and affected cellular pathways in the mutants. The F3 clutches contain the novel mutations in Mendelian ratios, and in a forward genetics approach are screened for recessive phenotypes of interest which appear in approximately 25% of embryos (Mullins et al, 1994). These embryos are referred to as “mutants” whereas those without phenotypes are “siblings”

Methods

Results

Discussion

Conclusion