PROJECT SUMMARY Development of separate sexes is a fundamental process throughout vertebrates. In many species, male and female differentiation is triggered early during embryogenesis by a master sex determination (MSD) gene that resides on the Y chromosome. Although sex differentiation is a remarkably conserved process among vertebrates, the MSD gene that initiates the process is incredibly variable. It remains unclear how a diverse assortment of MSD genes integrates with the canonical vertebrate sex differentiation network. A clear understanding of the genetic regulation of sex determination and differentiation is essential because disruptions in this process can lead to several disorders of sexual development. Our long-term goals are to use the diverse types of sex chromosomes among closely related species of stickleback fish to determine how anti-Müllerian hormone (Amh) has been repeatedly co-opted into a role as an MSD gene. Work in my lab has established stickleback fish as a premier vertebrate model system to understand how sex chromosomes and sex determination evolves. We have identified the independent acquisition of a Y-linked Amh (Amhy) in two species of stickleback fish that evolved through separate duplications and translocations. Our central hypothesis is that sex-linked copies of Amh (Amhy) can elicit a similar transcriptional response in the sex differentiation network across species. This occurs once duplicates have independently gained regulatory elements necessary for expression during early primordial gonad development. In this proposal we will use an innovative combination of functional genetics and comparative genomics to explore our hypothesis through three aims: (1) Determine if Amhy is necessary and sufficient for male development in threespine and brook stickleback fish, (2) Identify how Amhy is regulated during the time of sex determination, and (3) Characterize how the downstream sex differentiation regulatory network responds to the acquisition of Amhy as an MSD gene. This project will have a significant impact on the field as it will reveal the genetic mechanisms responsible for the observed plasticity in sex determination across vertebrates. This work will also lead to broader insights into the common function of Amh across vertebrates as a whole and will provide key genomic resources that will further enable stickleback fish as a leading vertebrate model system to understand how sex chromosomes evolve.