Mechanisms of Sex Determination in Zebrafish

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The Problem: Differences in (or Disorders of) Sex Development (DSD) are complex medical issues that involve multiple interacting cell types and organ systems. DSDs impact disease susceptibility, psychological development, and sense of person. Knowledge of mechanisms by which genetic factors interact with each other and with the environment to direct sex determination (SD), however, is incomplete. Zebrafish is an important medical model with a SD system that is sensitive to unidentified genetic variation and poorly understood environmental influences. Our research showed that zebrafish lab strains have weak, polymorphic, or non-genetic SD mechanisms, but natural zebrafish from India have a single, strong Sex- Associated Region on chromosome-4 (sar4) with chromosomally ZZ males and ZW females. ZZ fish are always males and most ZW fish are females, but a minority are fertile `neomales', likely due to genetic modifiers or environmental factors. Rare WW fish can be either males or females. We and others have made mutations in many candidate SD genes, but none represent the sar4 SD factor. We also showed that meiotic oocytes are necessary in lab strains to maintain female development, presumably by sending a signal to the gonadal soma. Our overall goals are to identify the molecular genetic basis of the sar4 sex determinant, and to discover its molecular, cellular, and organ level mechanisms to direct male or female development. Under one hypothesis, sar4-W has a dominant, variably penetrant, female-determining gene. An alternative hypothesis invokes sar4-Z dosage, with two doses resulting in a male and one dose or no doses usually giving females. In either case, we want to know how it specifies sex. In preliminary results, we: 1) collected four independently derived strains with the natural sar4 SD mechanism; 2) made genome assemblies for several natural strains; 3) developed PCR primers to assay sar4-Z and sar4-W; 4) made Bacterial Artificial Chromosomes (BACs) for ZZ and for ZW fish and isolated many sar4 BACs; and 5) performed single-cell RNA-seq on ZZ and ZW genotypes, which identified ZZ- and ZW-specific cell clusters. A comparison of sar4-Z to sar4-W identified a large recombination-suppressing inversion, many indels, and other polymorphisms. The overall vision of the research program is to manipulate genomes and environments to uncover the mysteries of zebrafish SD. Work includes: 1) identifying SD factor(s) in sar4 by performing necessity experiments that examine the effects of CRISPR/Cas9-induced long deletions and targeted knockouts on SD and sufficiency experiments using BAC transgenesis to identify clones that alter SD; and 2) to learn the pathways of male and female SD in zebrafish by single-cell gene expression studies in a variety of developmental stages, mutant genotypes, and environmental manipulations in ZZ and ZW fish. The significance will be a better understanding of gene interactions and environmental influences on zebrafish sex determination that will inform human DSDs.