Choanoflagellate transcriptional regulation and the origin of animal cell types

Animal bodies develop through complex developmental pathways in which cells are programmed for particular fates and functions. Transcriptional regulation has been shown to be central to this process, but we know little about how transcriptional regulatory programs evolved along the animal stem lineage. Can we trace animal developmental programs to their unicellular, pre-animal roots? Which mechanistic aspects of transcriptional regulation are unique to animals and which are more deeply conserved? My doctoral research explored these questions through bioinformatic and genetic approaches in choanoflagellates, the closest living relatives of animals. Through a better understanding of transcription factors and cell type specification in these organisms, I strove to help us triangulate the transcriptional regulatory capacity of the common ancestor of animals and choanoflagellates, which lived hundreds of millions of years in the past.Chapter 1 reviews how transcriptional regulation has evolved along the animal stem lineage. It has been frequently proposed that animal origins required the evolution of increasingly “complex” transcriptional regulation. I break this idea of complexity into specific mechanisms and trace what is known about the evolution of these mechanisms in animals and their closest living relatives.In Chapter 2, I present an example of how functional interrogation of choanoflagellate transcriptional networks can help us better understand the ancient roles of specific transcription factors as well as the regulatory architecture of cell differentiation. I explored the function of the RFX family of transcription factors in choanoflagellates, identifying a particular sub-family (cRFXa) that has a functionally conserved role in regulating dozens of genes required for ciliogenesis. By generating genome-edited mutant strains, I show that cRFXa is essential for proper ciliogenesis in the model choanoflagellate Salpingoeca rosetta, and that this defect is coupled with the loss of full expression of dozens of highly conserved ciliary genes. Coupled with existing data from animals, this work shows that the RFX/ciliogenesis regulatory module dates before the divergence of animals and choanoflagellates. It also helps us to understand the regulatory changes that might have been required for the differentiation of ciliated and non-ciliated cells early in animal evolution.Finally, in the Appendix I present work from early in my dissertation on a novel virus I helped to discover in Entomophthora muscae, a behavior-manipulating fungal pathogen of dipteran flies, including Drosophila melanogaster. We identified this virus through sequence analysis, including small RNA sequencing signatures generated by host Dicer processing, as well as through electron microscopy to directly visualize viral capsids in both cell-free extract and within fungal cells themselves