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Abstract
The primary goal of this research is to increase the repertoire of molecular tools and resources for functional and genetic experiments in Anolis sagrei, a reptilian model system for developmental and evolutionary studies. I applied these new tools to investigate mechanisms of Hedgehog (Hh) pathway induction in reptiles and the role of a limb specific shh enhancer in reptilian limb development. To understand the mechanism of Hh induction in reptiles, I evaluated the role of primary cilia in Hh signaling using a novel cell culture system that I generated for this purpose: an immortalized, clonal lizard embryonic fibroblast cell line (ASEC-1). Using CRISPR/Cas9, I successfully created targeted mutations in the ift88 gene, which is known to be required for ciliogenesis in other species. The ift88 mutant cell lines that I generated lack cilia, have lost their sensitivity towards Hh signaling, and show increased basal expression of gli1. My data suggests that in reptiles gli1 might be the major activator of the Hh pathway and, potentially, the predominant role of primary cilia reptiles may be to silence the pathway.Beyond my cell culture studies, I tested the functional importance of ZRS, a deeply conserved limb enhancer for the shh gene. Recent studies in snakes suggest that the ZRS underwent gradual sequence degradation that contributed to the loss of shh expression in the developing limbs and the evolution of limb reduction in this group of squamate reptiles. However, functional tests of snake ZRS elements have only been performed in mice. Ideally, the phenotypic consequences of sequence changes in the snake ZRS should be assessed in limbed reptiles rather than mammals. Using the CRISPR/Cas9 and CRISPR/Cas12a system, I have successfully deleted portions of the brown anole ZRS and ~33% of these mutants show a range of limb-related phenotypes.
Along with these projects, I worked on an education research project related to group work from the instructor’s point of view in an Undergraduate Biology laboratory class. My work explores differences in students’ behavior in self-selected vs randomized groups and suggests a strategy to identify poor group members for instructor intervention.