Knockout of dead end 1 by CRISPR/Cas9 leads to loss of germ cells and male-biased sex development in freshwater angelfish ( Pterophyllum scalare)

Abstract

In this study, we established target mutagenesis of the dnd1 gene using CRISPR/Cas9 genome editing technology in freshwater angelfish ( Pterophyllum scalare). Our loss-of-function analysis revealed that dnd1 homozygotes mutants exhibited infertility, characterized by empty gonad somatic structure without germ cells. Notably, we observed a significant male-biased sex development in these infertile individuals, with most displaying male secondary sexual characteristics and normal courtship behaviors. However, some dnd1 mutants exhibited testis- like gonads but lacked clear male secondary characteristics, representing sex-unknown phenotypes. This discrepancy suggests that while germ cells are essential for promoting or stabilizing female sexual fate, they may also influence the full expression of male secondary traits, highlighting the complexity of sex determination mechanisms in teleosts. Furthermore, we identified a significant upregulation of amh (a Sertoli cell marker gene) in germ cell-deficient gonads of dnd1 mutants, indicating that amh may play a central role in driving male sexual fate in the absence of germ cells. These findings underscore the crucial role of dnd1 in fertility and female sexual development in freshwater angelfish, advancing our understanding of reproduction biology and providing a valuable platform for precision breeding in this popular ornamental fish.