The Role of DNA Methylation and ZBTB Transcription Factors in Sex Differentiation, Maintenance and Heat‐Induced Masculinization in Zebrafish

Sex differentiation, the process by which an organism develops as a male or female, can be influenced by the environment in many species. High temperature, or pollutant exposure, can lead to skewed sex-ratios and genotype-phenotype mismatches that can seriously threaten populations and biodiversity. Understanding the mechanisms involved in sex differentiation is thus of crucial importance. In the present study, we analyzed the DNA methylation and transcriptional changes that occur during sex differentiation in zebrafish. Our results support the fact that DNA methylation is not only involved in sex differentiation but also in sex maintenance. Initiation of sex differentiation was associated with a DNA demethylation event. Subsequently, a second DNA methylation remodeling event, which coincided with the loss of bi-potentiality, was observed in males. Among the genes under study, the methylation level of the aromatase gene was found to be the most robust sex marker. However, genes encoding for transcription factors of the ZBTB family offered a more precocious marker since sex-specific changes in their transcription levels preceded changes in DNA methylation. In addition, their transcription levels were found be sensitive to a known masculinizing factor, i.e. temperature. Our results thus support an unexpected role of ZBTB proteins in sex differentiation.