Variant ribosomal DNA is essential for female differentiation in zebrafish

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 (<i>Danio rerio</i>), 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'.