The Evolutionary Transition from Lungs to a Gas Bladder: Evidence from Immunohistochemistry, Rna-Seq, and Morphology
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THE EVOLUTIONARY TRANSITION FROM LUNGS TO A GAS BLADDER: EVIDENCE FROM IMMUNOHISTOCHEMISTRY, RNA-SEQ, AND MORPHOLOGY A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Emily Funk December 2020 © 2020 Emily Funk THE EVOLUTIONARY TRANSITION FROM LUNGS TO A GAS BLADDER: EVIDENCE FROM IMMUNOHISTOCHEMISTRY, RNA-SEQ, AND MORPHOLOGY Emily Funk, Ph. D. Cornell University 2020 Key to understanding the evolutionary origin and modification of phenotypic traits is revealing the underlying developmental genetic mechanisms. An important morphological trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe- finned fishes. While gas bladders and lungs are similar in many ways, the distinguishing morphological difference between these organs is the general direction of budding from the foregut during development. Lungs bud ventrally and the gas bladder buds dorsally from the foregut endoderm. To compare lung and gasbladder development, the relevant taxa include bichir and bowfin. Bichir are the only living ray-finned fish that develops ventrally budding lungs. Bowfin, an early-diverging lineage, sister to teleosts, develops a gas bladder and exhibits a number of ancestral characteristics. Additionally, we included zebrafish as a representative of teleost fishes. I investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction using immunohistochemistry and RNA sequencing to determine whether gene expression patterns show a dorsoventral inversion paralleling the morphological inversion in budding direction. I also characterize morphological budding direction in bowfin, a purported transitional form, using nano-CT scanning. Taken together, the results of our gene expression and morphological studies of gasbladder development suggest that the inversion and modification of expression patterns of an ancestral lung-gene network underlies the evolution of a dorsal gas bladder from ventral lungs. The bowfin gas bladder does indeed bud dorsally from the foregut and does not represent an intermediate, laterally-budding morphology between ventral lungs and a dorsal gas bladder. We suggest that a regulatory change producing the dorsoventral inversion of expression of Tbx5, a known lung-regulatory gene, in the foregut during gasbladder development could facilitate the inversion of expression of downstream genes, such as Tbx4, Wnt2, Fgf10, and Bmp-signaling. Furthermore, this gene network may have been modified during the evolution of the gas bladder leading to the expression of Bmp16, rather than the orthologous Bmp4, to regulate gasbladder development in ray-finned fishes. Changes in the timing and spatial expression of lung-regulatory genes appear to induce the dorsal budding of the gas bladder during development. BIOGRAPHICAL SKETCH Emily Funk received her a Bachelor of Science in Ecology and Evolutionary Biology from the University of Connecticut in 2013. While at UConn, Emily completed her Honors Thesis research with Dr. Eric Schultz on the genetic basis for osmoregulatory adaptation to freshwater in alewife (Alosa pseudoharengus). Emily’s experience during her undergraduate education sparked her interest in the genetic underpinnings of evolution especially in fishes. After graduating, Emily joined Amy McCune’s lab in the Department of Ecology and Evolutionary Biology at Cornell to study the evolution of morphological novelty. v ACKNOWLEDGMENTS Many thanks to my adviser, Amy McCune, and committee members, Natasza Kurpios and Bob Reed, for stimulating discussions, methods-development advice, and invaluable feedback throughout my graduate career. Thank you to Ezra Lencer for being a great lab mate and for sharing his bioinformatics expertise as well as providing thoughtful feedback on this thesis. I am grateful to all members of the Kurpios Lab for welcoming me into their lab group and entertaining stimulating discussions. My sincere thanks goes to my undergraduate research assistants, Catie Breen, who assisted with immunohistochemistry assays and Eda Birol, who helped with the nano-CT image analyses. Thank you to Ken Zeedyk, Dr. Joe Fetcho, Nikki McGuire, for graciously providing fish embryo samples, and thanks to Francis Feng for boat access to bowfin spawning grounds. I would also like to thank Drs. Ingo Braasch and Andrew Thomson for early access to the bowfin genome. Many thanks to the Cornell Biotechnology Resource Center Imaging Facility, and in particular, Teresa Porri, for guidance with nano-CT scanning and image analyses. Thanks to the great friends and colleagues who have supported me throughout grad school including, Andrea Attenasio, Lina Arcila, Annie Scofield, Cora Demler, Joe Welkin, Jake Berv, Michelle Wong, Bridget Darby, and many more. A special thanks to the SeaPigs, Erin Larson, Lauren Brzozowski, Lizzie Lombardi, Jenny Uehling, Kelsey Jensen, Karin Vanderburg, Emily Rodekohr, Olivia Graham, and Kara Andres, for being the most awesome running and adventure friends ever. I cannot thank my family enough for supporting me in anything and everything I choose vi to pursue in life. Thank you to my Mom and Dad, and my brothers, Tom and Jeff. This work was made possible by support from National Science Foundation Graduate Research Fellowship, Cornell Presidential Life Sciences Fellowship, Sigma Xi Cornell Chapter, the Cornell Center for Vertebrate Genomics Scholar’s Program, American Society for Ichthyology and Herpetology Edward C. Raney Fund, McCune Lab funds, and Cornell EEB Departmental Funds. vii TABLE OF CONTENTS BIOGRAPHICAL SKETCH..........................................................................................v ACKNOWLEDGMENTS.............................................................................................vi TABLE OF CONTENTS ...........................................................................................viii LIST OF FIGURES ......................................................................................................ix LIST OF TABLES.........................................................................................................xi PREFACE.....................................................................................................................xii CHAPTER 1...................................................................................................................1 CHAPTER 2.................................................................................................................48 CHAPTER 3.................................................................................................................86 APPENDIX.................................................................................................................119 viii LIST OF FIGURES Figure 1.1 : Basic morphology and phylogenetic distribution of lungs and the gas bladder…..4 Figure 1.2: Diagram of known gene interactions regulating lung development……………….8 Figure 1.3: Sox2 and Nkx2.1 expression in bichir lungs………………………………………20 Figure 1.4: Sox2 and Nkx2.1 expression in bowfin gas bladder………………………………21 Figure 1.5: Sox2 and Nkx2.1 expression in zebrafish gas bladder…………………………….22 Figure 1.6: Bmp4 expression and Smad phosphorylation in bichir lungs…………………….25 Figure 1.7: Bmp16 gene tree…………………………………………………………………..26 Figure 1.8: Bmp16 expression and Smad phosphorylation in bowfin gas bladder…………...29 Figure 1.9: Gene expression changes during the lung-to-gas bladder transition in ray-finned fishes………………………………………………………………………………………….32 Figure 2.1……………………………………………………………………………………..52 Figure 2.2: Principle Component Analysis (PCA) plots……………………………………...60 Figure 2.3: Differentially expressed genes between dorsal and ventral tissues of the foregut during gas bladder development………………………………………………………………63 Figure 2.4: Tissue-specific expression of known lung-development genes, differentially expressed at budding, during gas bladder development in bowfin……………………………68 Figure 2.5: Tissue-specific expression of known lung-development genes, differentially expressed at outgrowth, during gas bladder development in bowfin……………………...….69 Figure 2.6: Dorsoventral expression of Tbx5 during gas bladder development in bowfin……70 Figure 2.7: Expression of Tbx4, Wnt2ba, and Fgf10 during gas bladder development in bowfin………………………………………………………………………………………....72 Figure 3.1: Original and modified versions of the iconic 19th century morphological transformation series of lung and gasbladder morphology……………………………………90 Figure 3.2: Lung and gas bladder phenotypes arrayed in a modern phylogenetic framework..94 Figure 3.3: Location and morphology of the gas bladder in a juvenile bowfin……………….96 Figure 3.4: Paired photographs and nano-CT renderings of external morphology of developing bowfin larvae, stages 25 to 29……………………………………………………………….100 ix Figure 3.5: Morphology of the developing gas bladder in bowfin, stages 25, 26, and 27…..102 Figure 3.6: Morphology of the developing gas bladder in bowfin, stages 28 and 29……….104 Figure 3.7: Angular measurements of the right-twisted gas bladder bud……………………108 Appendix Appendix Figure A1.1: Bowfin Bmp16 epitope for custom Bmp16 antibody design………121 Appendix Figure A1.2: Bmp4 expression in bowfin and zebrafish………………………….122 Appendix Figure A1.3: Bmp16 Immunofluorescence negative control in mouse…………..123 Appendix Figure A2.1: Bmp4 and Bmp16 expression