Abstract
The ribosome consists of protein and RNA components. Deletion of genes encoding specific ribosomal proteins has revealed that heterogeneity in the ribosome must exist in vertebrates; however, this has not been tested for ribosomal RNA (rRNA). In zebrafish (Danio rerio), the '45S-M' ribosomal RNA-encoding locus undergoes massive extrachromosomal amplification during oocyte growth and ovary differentiation and is distinct from the regular ribosomal DNA (rDNA) locus encoding somatic rRNA (45S-S). Although the 45S-M rDNA locus falls within the only described sex-linked region in multiple wild zebrafish strains, its role in sexual differentiation is unclear. We used CRISPR-Cas9 gene editing to alter 45S-M rDNA sequences in zygotes and found that although there was no effect on growth or male development, there was dramatic suppression of female differentiation. Males with edited 45S-M rDNA produced phenotypically normal sperm and were able to fertilize eggs from wild-type females, with resulting embryos once more displaying normal development. Our work supports the hypothesis that specialized 45S-M rDNA is the elusive apical sex-determining locus in zebrafish and that this region represents the most tractable genetic system to date for studying ribosomal RNA heterogeneity and function in a vertebrate.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.