Collaborative Research: Enhancers and the Convergent Evolution of Limb Deduction in Squamates

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Although the development of arms and legs is a key evolutionary innovation that allowed animals to move onto land, many species of snakes and lizards subsequently lost these limbs. Indeed, limb loss has evolved at least 24 times among lizards. How limb loss has occurred repeatedly in lizard lineages is a question that can be addressed with the replicated natural experiments that will be the focus of this study. This project will reveal whether the same or different genetic changes are associated with limb loss among these different lizard groups. This research will combine technologies from different fields of biology in novel ways to explore this extraordinary example of repeated evolution and gain new insight into genome function. This project will greatly improve knowledge of the genes and genetic control elements involved in limb development. Additionally, in the course of this project, thousands of genetic regions that are shared by humans and other animals will be characterized. Therefore, project data will deliver insights that can be applied to better understand other functional elements within the genomes of humans and different animals. The methods developed will pave the way for other researchers who wish to leverage the power of natural evolutionary experiments to understand genome function. Finally, materials for educators will be developed for use in teaching students how information is encoded in genomes and how natural evolutionary experiments can be used to reveal how genetic sequences yield unique biological diversity.This project will use chromatin-immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify regulatory elements (enhancers) in squamate genomes that control the embryonic development of the limbs. This information will be used to design squamate-specific DNA sequence capture baits targeting these regulatory regions. Additional capture baits will be designed to target the coding regions of genes active in the developing limbs. Together, these baits will be used to survey both noncoding regulatory elements and coding exons of key developmental genes from every lineage of limb-reduced squamates. DNA sequence changes in limb enhancers and limb development genes will be compared in limb-reduced species and their fully limbed relatives, and functional changes in regulatory activity will be assessed in vivo with transgenic mouse reporter assays. This will provide insight into the relative frequency of regulatory changes compared to gene coding sequence changes contributing to the evolution of limb loss. Finally, the functional evolution of an ancient amniote limb-genital enhancer for the gene Tbx4 will be dissected molecularly by taking advantage of sequence variation across the multiple lineages of limb-reduced species. This will grant insight into the fate of enhancers that have lost their regulatory roles and determine whether these elements are recruited to new functions.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.