Elucidation of the Pax/Six gene regulatory network in Ephydatia muelleri

This thesis has been accepted as part of the honors requirements in the Program in Biochemistry and Molecular Biology. _____________________________________ ____________________ (advisor signature) (date) _____________________________________ ____________________ (reader signature) (date) 2 Table of Contents • Acknowledgments • Abstract • Introduction • Materials and Methods • Results • Discussion • Figures and Tables Acknowledgements I would like to thank my two mentors April Hill and Lisa Gentile for all their work on this project, with special thanks to my advisor April Hill who has helped me discover my passion for biology research. I would also like to acknowledge the members of the Hill and Gentile labs who have contributed to this project. Abstract The evolution of gene regulatory networks accounts for much of the diversity we observe in the animal kingdom. In particular, the Pax/Six/Eya/Dac gene network has been known to play critical roles in development of all bilaterians and cnidarians. However, this network has not been characterized in Porifera. These studies focus on tracing the evolutionary history of the PSED network back to the most basal organism in the metazoan phylogeny through demonstrating the presence of a direct gene regulatory network between PaxB and Six1/2 orthologs in sponges. To this end, putatitve Six1/2 cis-regulatory elements were identified while the PaxBprd transcription factor was isolated and purified. Binding assays demonstrated that the PaxBprd protein bound directly to upstream cis-regulatory regions of the Six1/2 gene, as well as a conserved Six1/2 intron site in two different species of sponges. Additionally, we focused on broadening the Pax gene regulatory network in sponges by looking at additional downstream gene targets of 3 Pax. We identified a possible indirect regulatory network between Pax and four novel target genes through the use of RNAi and qRT-PCR techniques. This study helps to elucidate the origins of the PSED network as well as provide the beginnings of a whole gene regulatory network in sponges.