Genomic analysis detects polygenic sex determination and signs of early gonad differentiation in zebrafish

Zebrafish is a popular research organism in many areas of developmental biology. However, knowledge on its sexual development is lacking. Before this project there were conflicting reports on zebrafish sex determination (SD) mechanism. There were reports that suggested zebrafish SD depended on environmental factors, while others claimed the presence of sex chromosomes. In addition to the confusion on SD mechanism, there was also a lack of information on when the bipotential gonad starts the testis or ovary differentiation process. This Ph.D. project aimed to address these two areas of zebrafish sexual development by using a combination of classical breeding and molecular methods. The first part of this project sought to unravel the SD mechanism of zebrafish. Using classical breeding experiments, it was observed that progeny sex ratio across 62 zebrafish families were wide ranging (4.8% to 97.3% male) and included several sex-biased families. On the other hand, broods that were produced by repeated crossing of the same breeding pair had very similar sex ratios indicating strong influence from parental genotypes. In the selective breeding experiment, a sex ratio shift was observed and an all-male family was produced in four generations. These responses to selective breeding indicate that zebrafish SD mechanism is genetically based. To find out whether there is any substantial structural difference between male and female genomes, an indication for the presence of sex chromosomes, two molecular methods (namely FluoMEP and array comparative genomic hybridization) were developed and customized, respectively, for use in zebrafish. Extensive comparisons between genomes of the two sexes did not identify any universal structural difference. The lack of substantial sex-linked difference between male and female genomes and the presence of sex-biased families are both incompatible with the chromosomal sex determination system. Together with the observations of wide ranging family sex ratios, strong parental genotype influence and responses to selective breeding show that zebrafish primarily uses a polygenic sex determination system. After establishing the SD mechanism of zebrafish, the second part of this project was to attempt to specify the SD timing by finding the earliest sign of gonadal transcriptome bifurcation. Using Tg(vasa:vasa-EGFP) transgenic line, the gonadal transcriptome of zebrafish that showed strong (mostly future female) or weak (future male) vasa-EGFP fluorescence at 28, 35, 42 and 105 dpf were analyzed by a custom-made expression microarray. A total of 16,524 transcripts were found to be differentially expressed between the strong or weak fluorescence groups at one (or more) time point(s). Eleven of the up-regulated transcripts in the strong fluorescence groups from 28 to 105 dpf and nine candidate sex genes were used to make a qPCR array for analyzing zebrafish larvae (7, 14 and 21 dpf). Based on the expression profiles of the twenty selected genes, zebrafish larvae could be clustered into at least two groups from 7 dpf onward. To summarize, the data obtained through the use of classical breeding experiments and customized genomic screening tools clearly indicate that the primary SD mechanism of zebrafish is a polygenic sex determination (PSD) system. From gonadal transcriptome profiling using a selected set of genes, divergent expression patterns were observed in zebrafish larvae as young as 7 dpf. This shows that gonadal differentiation begins prior to juvenile ovary phase. Therefore, we hypothesize that zebrafish sex is determined between the period of fertilization to 7 dpf, before any sexually dimorphic histological phenotypes can be observed.DOCTOR OF PHILOSOPHY (SBS