Genetic Determinants of the Development and Evolution of Drosophila Pigmentation By Abigail M. Lamb A disseratation submitted in partial fulfillment Of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and Developmental Biology) In the University of Michigan 2021 Doctoral Committee: Professor Patricia Wittkopp, Chair Associate Professor Scott Barolo Associate Professor Laura Buttitta Professor Kenneth Cadigan Abigail M. Lamb [email protected] ORCID iD: 0000-0001-5184-4180 Dedication To everyone who started graduate school but, for any reason, didn’t continue to the point of dissertation defense. I was lucky to have the support I had, otherwise I would not have made it to this point. I have seen too many wonderful, talented people pushed out of academic science, and they deserved so much better than what they experienced. ii Acknowledgments I would first like to thank Trisha Wittkopp, not only for her scientific and professional mentorship, but especially for her support, kindness, and understanding during (possibly too many) years of struggle and success – both at the bench and in life. Going to college in the first place felt like a stretch goal given the circumstances of my early life, and graduate school even now somehow sounds impossibly unlikely, even as I complete my dissertation. I truly believe that my ability to succeed in graduate school was dependent on finding an environment and culture where I could thrive, and the Wittkopp Lab became Home to an extent that is difficult to describe. For fear of forgetting someone, I will not attempt to individually name all of the many, many labmates I spent time with both in and out of the lab. However, I will give special mention to Elizabeth Walker for being the reassuring and welcoming presence I needed when I showed up not even knowing how to PCR, Andrea Hodgins-Davis for being one of the kindest human beings I’ve ever met, Jen Lachowiec for having the best laugh and talking me into playing hockey, Mark Hill for all the chats over beverages and horror cinema, Jade Diaz for being extra and venting with me, Lisa Kim for helping me keep things weird, and Mo Siddiq even though he spends too much time giving me genuine compliments as a form of torture. Special thanks to Anati Azhar and Patty Simmer for allowing me the privilege of being your mentor through the emotional roller coaster of academic science research, and to Alicia Wang, Lisa Kim, Alisha John, and Elizabeth Walker for making everything work while simultaneously being delightful. iii Outside of my home lab: Thank you to Scott Barolo, Laura Buttitta, and Ken Cadigan for serving on my thesis committee and always having excellent feedback and advice. In MCDB, I am grateful to all the helpful and supportive staff, the Graduate Student Council, and everyone who poured themselves into building the culture they want for themselves and those who come after them. Special shout out to Shyama Nandakumar for being a fellow chaotic softie with whom I could commiserate over drinks and houseplants after we said “yes” to too many things. Thank you to my collaborators, Jennifer Kennell, Zinan Wang, and Henry Chung, and my funding sources: The National Science Foundation Graduate Research Fellowship Program and The Michigan Predoctoral Training Program in Genetics. I’ve had the privilege to form a family of widely varying degrees of genetic relatedness, especially Jennifer, Jon, Glenna, Dorris, Bob, Shannon, Laura, Maureen, Jessica, Meg, Anthony, Garrett, Abigail, Bella, and Kali. Thank you for getting me through the (many) rough patches and making it possible for me to keep going. Thank you to the science educators and mentors who helped me find my path: Cindy Bagwill, Jerry Dunaway, and Tony Frankino. Thank you to all the students I’ve had the privilege to teach: y’all were a joy. And lastly, there is no chance I could have done any of this without Michael, who occupies his own category and is the single most important person in my life. And thank you to Strobel, because I wouldn’t have gotten in the door without him. iv Table of Contents Dedication ii Acknowledgments iii List of Tables viii List of Figures ix List of Appendices xiii Abstract xiv Chapter 1: Introduction 1 Chapter 2: Tools and Strategies for Scarless Allele Replacement in Drosophila Using CRISPR/Cas9 30 Abstract 30 Introduction 31 Results 35 Discussion 41 Materials and methods 45 Acknowledgments 49 v Abbreviations 49 References 50 Chapter 3: ebony Affects Pigmentation Divergence and Cuticular Hydrocarbons in Drosophila americana and D. novamexicana 56 Abstract 56 Introduction 57 Materials and Methods 60 Results and Discussion 67 Conclusions 80 Acknowledgements 83 References 83 Chapter 4: microRNAs Are Necessary Components of the Genetic Architecture Underlying Adult Cuticle Pigmentation in Drosophila melanogaster 88 Abstract 88 Introduction 89 Results and Discussion 91 Conclusion 113 Materials and Methods 114 References 117 Chapter 5: Conclusions and Future Directions 123 References 133 vi Appendices 137 vii List of Tables Table 1-1: Cases of phenotypic evolution attributed to differences in miRNA regulation in GePheBase. ................................................................................................ 7 Table 4-1 Summary of phenotype data from all miRNA overexpression crosses. .......................................................................................................................................... 95 Table 4-2. Summary of phenotypic effects of overexpression and competitive inhibition of all microRNAs included in competitive inhibition screen. .............. 101 Supplementary Table S2-1: Oligonucleotides and primers used in reagent construction and molecular screening. .................................................................... 144 Supplementary Table S2-2: Genome editing rates in the first stage of the allele swap. .............................................................................................................................. 148 Supplementary Table S2-3: Genome editing rates in the second stage of the allele swap. .............................................................................................................................. 149 Supplementary Table S3-1: Oligonucleotides used to generate sgRNAs for CRISPR/Cas9 genome editing and nos-Cas9-nos transgene................................. 156 Supplementary Table S4-1: Phenotypes and notes on all individual flies collected in miRNA overexpression screen. ............................................................................. 166 Supplementary Table S4-2: Phenotypes and notes on all individual flies collected in miRNA competitive inhibition screen. ................................................................... 167 viii Supplementary Table S4-3: Summary data from overexpression and competitive inhibition screens. ........................................................................................................ 168 Supplementary Table S4-4: List of pigmentation genes. ........................................ 169 ix List of Figures Figure 1-1: miRNA deletions affecting phenotypes in Caenorabditis elegans and Drosophila melanogaster. ............................................................................................. 12 Figure 1-2: Schematic of biosynthetic pathway and genetic network underlying Drosophila melanogaster pigmentation, reproduced from Massey and Wittkopp 2016. ................................................................................................................................ 14 Figure 1-3: Looking under the lamppost. .................................................................... 21 Figure 2-1: Challenges for single-stage allele replacement strategies using CRISPR. ........................................................................................................................... 32 Figure 2-2: Schematic of marker-assisted, 2-stage allele swap within the D. americana tan transgene in D. melanogaster. ............................................................ 35 Figure 2-3: Representative pigmentation and fluorescence phenotypes of flies at each stage of the tan allele swap process. ................................................................. 39 Figure 2-4: Workflow for 2-stage marker assisted allele swap. ............................... 42 Figure 3-1: Drosophila novamexicana shows divergent body color within the virilis group. .................................................................................................................... 59 Figure 3-2: ebony affects body, wing, and pupal pigmentation in D. novamexicana and D. americana. ................................................................................ 69 Figure 3-3: CRISPR/Cas9-induced mutations created null alleles of the D. novamexicana and D. americana ebony genes. ........................................................ 72 x Figure 3-4: Reciprocal hemizygosity testing shows effects of ebony divergence between D. americana and D. novamexicana on body pigmentation. ................... 74 Figure 3-5: Cuticular hydrocarbons (CHCs) are affected by ebony and differ between D. americana and D. novamexicana. ........................................................... 77 Figure 3-6: ebony does not contribute to divergence of CHCs between D. americana and D. novamexicana. ............................................................................... 79 Figure 4-1: Schematic of miRNA overexpression