Abstract
The zebrafish, Danio rerio, is a powerhouse of fecundity, females can produce hundreds of rapidly developing, transparent, semipermeable eggs each week, for months on end. The zebrafish is considered a flagship model for vertebrate development, ideally suited for high-throughput, large-scale screening assays that range from classic gene expression and function studies to drug toxicity screens. Despite its extensive use in modern research, the fundamental biological process of sexual determination in this species is still considered an elusive, complex riddle. Contrary to individuals found in the wild, most modern laboratory strains have lost their sex chromosomes, resulting in sexually isogenic males and females. A large volume of research has revealed that environmental factors, unknown genetic traits, and DNA methylation levels can determine an individual’s sexual fate.
This thesis aims to enhance research capabilities regarding sex determination and differentiation in domesticated zebrafish and to expand our understanding of this fundamental biological process. By developing and applying novel methodologies, significant advancements in investigating sex determination at both genetic and epigenetic levels are provided.
One of the major innovations presented in this thesis is the combination of a 3D-printed well plate with a vas::EGFP reporter line, enabling high-throughput sex skewing experiments. Employing this method led to the development of a second approach for sexing juvenile zebrafish, based on the detection of a 45S rDNA variant known for female-specific amplification during sex differentiation. Both approaches offer more efficient and scalable solutions compared to traditional methods relying on external phenotypes or histology.
The thesis further explores the use of DNA methylation-modifying drugs, such as decitabine, as tools in the exploration of the epigenetic factors affecting zebrafish sex determination. To address the limitations encountered when using non-specific methylation modifiers, a CRISPR-based epigenetic editing method was adapted for use in zebrafish. The successful demonstration of this method opens new avenues for future research investigating the causal relationships between local 5mC levels and sex determination.
In the final research chapter, 45S-M rDNA, which shares a locus with the only sex-linked region found in wild zebrafish, is investigated as a potential apex sex-determining gene. The extensive CRISPR knockout experiments conducted during this study revealed this 45S rDNA variant to be essential for feminisation and at the same time inconsequential to male development, thus adding a significant piece to the puzzle of sex determination in zebrafish.