Genetic Basis of Gonad Determination in Anolis Lizards

Vertebrate animals determine whether the gonads develop into male testes or female ovaries through a variety of ways, yet scientists still do not fully understand how these processes evolved or how they operate across different species. Reptiles, in particular, display an extraordinary range of gonad-determining systems, making them an important group for uncovering fundamental principles of biology. This project focuses on Anolis lizards, which have an ancient genetic system of gonad determination that is distinct from the genetic systems used by mammals and birds. By investigating how testis versus ovary formation is determined in these lizards, the research will provide new insights into the development of reproductive systems across vertebrates. This work will advance fundamental knowledge in genetics and developmental biology, while also supporting the training of undergraduate and graduate students, fostering hands-on research experiences, and expanding participation in science through collaborative training opportunities. These efforts contribute to the national interest by promoting scientific progress, strengthening the research workforce in gene editing biotechnology, and enhancing understanding of biological systems relevant to health and biodiversity. This project advances NSF’s priorities in Biotechnology. This project will investigate the molecular mechanisms of testis determination in the brown anole lizard, Anolis sagrei, with a focus on the Y-linked gene rpl6y and its X-linked counterpart rpl6. Preliminary data suggest that rpl6y is required for testis development and that downregulation of rpl6, potentially mediated by a gonad-specific antisense transcript (astra), is necessary for this process. To test these hypotheses, Aim 1 will use HCR RNA FISH to identify the cell types in which the rpl6y gene is expressed, and CRISPR/Cas9-mediated gene disruption will be performed to determine the functional requirement for rpl6y in testis determination. Aim 2 will manipulate astra expression in vivo and in cell culture to assess its role in regulating rpl6 and its contribution to gonadal differentiation. Aim 3 will evaluate whether RPL6Y and RPL6 proteins differentially influence protein translation using ribosome profiling and quantitative proteomics. Together, these studies will establish whether a translation-based mechanism underlies gonad determination in anoles, potentially revealing a previously unknown mode of vertebrate gonad determination and expanding understanding of how testis versus ovary development is controlled at the molecular level. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2539278 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Douglas Menke | Institution: University of Georgia Research Foundation Inc, ATHENS, GA | Award Amount: $980,133 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2539278 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2539278.html