The roles of RNA Polymerase I and III subunits Polr1a, Polr1c, and Polr1d in craniofacial development BY © 2016 Kristin Emily Noack Watt Submitted to the graduate degree program in Anatomy and Cell Biology and to the Graduate Faculty of The University of Kansas Medical Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Paul Trainor, Co-Chairperson Brenda Rongish, Co-Chairperson Brian Andrews Jennifer Gerton Tatjana Piotrowski Russell Swerdlow Date Defended: January 26, 2016 The Dissertation Committee for Kristin Watt certifies that this is the approved version of the following dissertation: The roles of RNA Polymerase I and III subunits Polr1a, Polr1c, and Polr1d in craniofacial development Paul Trainor, Co-Chairperson Brenda Rongish, Co-Chairperson Date approved: February 2, 2016 ii Abstract Craniofacial anomalies account for approximately one-third of all birth defects. Two examples of syndromes associated with craniofacial malformations are Treacher Collins syndrome and Acrofacial Dysostosis, Cincinnati type which have phenotypic overlap including deformities of the eyes, ears, and facial bones. Mutations in TCOF1, POLR1C or POLR1D may cause Treacher Collins syndrome while mutations in POLR1A may cause Acrofacial Dysostosis, Cincinnati type. TCOF1 encodes the nucleolar phosphoprotein Treacle, which functions in rRNA transcription and modification. Previous studies demonstrated that Tcof1 mutations in mice result in reduced ribosome biogenesis and increased neuroepithelial apoptosis. This diminishes the neural crest cell (NCC) progenitor population which contribute to the development of the cranial skeleton. In contrast, apart from being subunits of RNA Polymerases (RNAP) I and/or III, nothing is known about the function of POLR1A, POLR1C, and POLR1D during embryonic and craniofacial development. I therefore examined zebrafish with mutations in polr1a, polr1c, and polr1d to understand the function of RNAPI during embryogenesis. Remarkably, these genes are dynamically expressed during embryonic development and display enriched expression in craniofacial regions, such as the pharyngeal arches, demonstrating that they may have tissue specific functions. Homozygous mutant polr1a, polr1c, and polr1d zebrafish display reduced cranial cartilage formation due to elevated tp53-dependent cell death of NCC progenitors and a diminished population of cranial NCC. In addition, mutant embryos have altered ribosome biogenesis, as demonstrated by reduced rRNA transcription and polysome profiling, which impact the survival and proliferation of cranial iii NCC. Taken together, this suggests diminished ribosome biogenesis, nucleolar stress, and Tp53-dependent neuroepithelial apoptosis underlie the loss of NCC needed to generate the normal cranioskeletal elements, consistent with the Tcof1 studies. In support of this mechanism, genetic inhibition of tp53 is one approach for improving the skeletal deficiencies in polr1c and polr1d mutants. These zebrafish models represent the first study of RNAPI during craniofacial development and demonstrate that tissue specific phenotypes can arise from disruptions of global processes like ribosome biogenesis. The polr1a, polr1c, and polr1d zebrafish will be useful for further examining RNAPI function as well as finding potential therapies for the prevention of craniofacial anomalies. iv Acknowledgements In the course of my PhD research, I have been helped by so many wonderful people. I would like to thank all those who kindly provided reagents. The Nancy Hopkins lab generated the polr1a, polr1c, and polr1d zebrafish and Dr. Adam Amsterdam kindly provided primers for genotyping. Dr. Tom Schilling provided sox10:gfp transgenic zebrafish to aid in my studies of neural crest cells, and Dr. Tatjana Piotrowski provided the tp53 mutant zebrafish for my rescue experiments. The core facilities at the Stowers Institute were also immensely helpful. I thank the SIMR Aquatics team for zebrafish care and maintenance – especially Jamie, Jessica, Lily, Xia, and Darin – for completion of numerous fin clip requests so I could genotype my fish and maintain the colony. The microscopy team provided excellent training and assistance with confocal microscopy and I especially thank Cindy Maddera for her assistance as well as Richard Alexander for his assistance with image quantification and the IMARIS software. I thank the Molecular Biology core for assistance with qPCR experiments and RNA-Seq, especially Willie, Brian, and Allie. Finally, I thank Chris Seidel for his analysis of the RNA-Seq data. I would next like to thank our collaborators at University of Southern California, Cynthia Neben and Dr. Amy Merrill-Brugger for their assistance with polysome profiling. I thank Cynthia for running numerous polysome samples, optimizing the protocol, and then kindly showing me the process and Amy for helping to make the collaboration possible and for discussions about ribosome biogenesis. I thank my committee members for their guidance, support, and suggestions. My project benefited greatly from the input of each of my committee members – to Dr. Piotrowski for her zebrafish expertise, to Dr. Gerton for her expertise in the ribosome field, v to Dr. Andrews for his medical knowledge, to Dr. Rongish for her knowledge in embryology, and to Dr. Swerdlow for his insightful perspectives on a project outside his area. Dr. Swerdlow’s comments on my project aided greatly in the successful resubmission of my grant. I especially thank Dr. Rongish for her guidance and comments throughout my time at KUMC – first as graduate student advisor when I started in the department and her continued guidance as a member of my committee. I owe extra thanks to Dr. Piotrowski and Dr. Gerton and the members of their labs for sharing reagents and protocols. I was lucky to have a lab full of zebrafish researchers across the hall to answer my questions. I especially thank Julia in the Piotrowski lab for giving me numerous protocols and suggestions, and I also thank Mark and Marina for answering my questions about zebrafish development. I thank Baoshan from the Gerton lab for kindly sharing protocols for biochemical techniques and information on leucine treatments. I next thank the members of the Trainor lab for making the lab a truly wonderful environment. I had the pleasure of working with Dr. Lisa Sandell during my rotation in the lab and found her excitement for science truly inspiring. I thank past post-docs of the lab: Kim for all of her help in answering my questions about jaws, Hiroshi for teaching me Western blotting, and Kaz for immunostaining protocols. Annita was helpful in getting me started in the lab and worked on some of the zebrafish project with me. Later on, as our projects were so similar, she was a great person to talk to about ribosomes and neural crest cells and proof-read all of my abstracts. I am grateful to my fellow graduate students Aude, Naomi, and Shachi for being great lab-mates and making the lab such a fun place to be; and for the technicians in the lab, especially Jen who showed me where everything was when I started in the lab and Shawn who helped me a great deal with protein work. I also thank the students who contributed to the zebrafish research: Stéphanie, Angie, Joaquin, vi and Elena. These students each helped me become a better teacher and asked wonderful questions. I am thankful for all of the support from the current members of the lab as I prepared for my defense – William, Shachi, Karla, Stephen, Melissa, and George. And finally I thank Carolyn for being a wonderful administrator and for the countless number of practice talks and committee meeting that she scheduled for me. I especially thank Paul for giving me the opportunity to work in his lab and also letting me take on a zebrafish project. I don’t think a lot of people would be willing to let a graduate student start a project on a different model organism than the rest of the lab, and I think it says a lot about Paul’s willingness to support his students. His training in presenting and writing helped me improve immensely in how I approach these things. And of course, Paul was always willing to challenge me in different ways – whether it was about how to think about an experiment, or train a student, or write – which helped me grow as a scientist. I would never have written a successful grant application without his assistance and I learned a lot through writing a book chapter. I am incredibly grateful that he gave me the opportunity to attend and present at numerous conference during my time in graduate school and for all of the connections I made at these conferences. I also have a lot of people to thank for preparing me for graduate school. I gained the majority of my lab experience working for Dr. Beitz, who genuinely cares about the members of his lab and still asks about how I am doing. I also thank Jeff and Maura for introducing me to developmental biology research in zebrafish. The year I spent in the lab made me so excited about research and I have Jeff to thank for encouraging me to attend graduate school. I also have to thank Julie Kuhlman, who started her lab at Iowa State when I was working with Jeff. When I said I was looking at graduate schools in the Kansas City area, she told me, “I have this friend, Paul Trainor…” and I am so glad she told me to look him up; I think it worked out very well for me. vii Finally, I have to thank my family. My parents, Max and Jeanne, have always stressed the importance of education and worked so hard to provide learning opportunities as I was growing up. They are two of the hardest working people I know and taught me the value of doing the job right and doing it well. Their example, love, and support over the years have helped me become the person I am to which I fell incredibly blessed.