Investigation of candidate sex determination and sex differentiation genes in sea lamprey, Petromyzon marinus, and Pacific lamprey, Entosphenus tridentatus By Arfa A. Khan A thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements of the degree of MASTER OF SCIENCE Department of Biological Sciences University of Manitoba Winnipeg, Manitoba Copyright © 2017 by Arfa Khan i Abstract Genes associated with sex differentiation (the process by which undifferentiated gonads develop into testes or ovaries) are relatively conserved among vertebrates, but those associated with sex determination (the genetic and/or environmental “switch” that activates differentiation) are more variable, particularly in fishes. Virtually nothing is known regarding the genetic basis of these processes in lampreys, one of the two extant groups of jawless fishes. Therefore, I tested whether 19 candidate sex determination genes identified from other vertebrates showed sex specific sequence differences in sea lamprey, Petromyzon marinus, and Pacific lamprey, Entosphenus tridentatus; 11 and seven genes amplified in sea and Pacific lampreys, respectively, but none showed sex specific differences. I also used qRT-PCR to measure expression of seven candidate sex differentiation genes (e.g., SOX9, DMRT1, WT1, DAZAP1) prior to and during testicular differentiation in sea lamprey, and found that expression patterns were consistent with their presumed role in other vertebrates. ii Acknowledgements I would like to thank my advisor, Dr. Margaret Docker, for all her support. I couldn’t have done it without her mentoring and passion for learning more about the mystery that is lampreys. You have been influential in my growth as a student during my time at the University of Manitoba and have motivated me every step of the way. I would also like to thank my committee, Drs. Alberto Civetta and Colin Garroway, for their comments and suggestions that have contributed greatly to the development of my thesis. I thank all my past and present lab mates for their help along the way. I am especially grateful to Erin Spice for teaching me lab techniques and her constant support. Special thanks to Nisha Ajmani for teaching me how to navigate through the sea lamprey genome and how to use different Ensembl features and tools. A very special thanks to our collaborators, Drs. Richard Manzon (University of Regina), Michael Wilkie and Jonathan Wilson and their students (Wilfrid Laurier University), John Hume (Michigan State University) and Dr. Jon Hess (Columbia River Inter-Tribal Fish Commission), for lamprey sampling. This research was made possible by funding from the University of Manitoba Graduate Enhancement of Tri-council Stipends (GETS) and Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. Finally and most importantly, I would like to thank my family, husband and friends for their encouragement and patience. I am very thankful for my parents’ courage leaving the comfort of their families and home, and immigrating with my brother, sister and me to Canada 17 years iii ago. We have been blessed with wonderful opportunities which we could have only dreamt of; thank you for everything Mama and Papa. iv Table of Contents Abstract ……………………………………………………………………………...……………ii Acknowledgements ……………………………………………………………………………...iii Table of Contents …………………………………………………………………………………v List of Tables…………………………………………………………………………………….vii List of Figures…………………………………………………………………………………….ix 1. Chapter 1: General introduction……………………………………………..1 1.1. Lamprey biology: life-cycle……………………………………………………………...2 1.2. Lamprey biology: conservation and control……………………………………………...6 1.2.1. Control of the invasive sea lamprey in the Great Lakes…………………………..6 1.2.2. Conservation of the Pacific lamprey………………………………………………7 1.2.3. Conservation of the anadromous sea lamprey in their native range………………9 1.3. Sex determination in vertebrates………………………………………………………..12 1.3.1. Genetic basis of sex determination of model vertebrates………………………..14 1.3.2. Sex determination in fish………………………………………………...………16 1.3.3. Sex determination in lampreys…………………………………………………...19 1.4. Genes involved in sex differentiation in………………………………………………...21 1.4.1. Sex differentiation in model vertebrates…………………………………………21 1.4.2. Sex differentiation in fish………………………………………………………...22 1.4.3. Sex differentiation in lampreys…………………………………………………..25 1.5. Testicular differentiation………………………………………………………………..28 1.5.1. Spermatogenesis…………………………………………………………………28 1.5.2. Testicular differentiation in lampreys……………………………………………29 1.6. Candidate genes for sex determination in vertebrates and conserved sex differentiation genes in lampreys.………………………………………………………………………32 1.7. Thesis objectives…………………………………………………………………...……42 1.8. Significance……………………………………………………………………………..43 1.9. Literature cited…………………………………………………………………………..46 2. Chapter 2: Candidate sex determination gene sequencing in lampreys: Pacific lamprey, Entosphenus tridentatus and sea lamprey, Petromyzon marinus……………………………………………………………………..…76 2.1. Abstract………………………………………………………………………………….76 2.2. Introduction………………………………………………………………………...……78 2.3. Methods…………………………………………………………………………………81 2.3.1. Sample collection………………………………………...………………………81 2.3.2. DNA isolation and RNase elimination…………………………………………..82 2.3.3. Identifying target genes and PCR………………………………………………..83 2.3.4. Sequencing preparation and analysis…………………………………………….85 2.3.4.1. Propanol precipitate, cycle sequencing, and ethanol precipitation……....85 2.3.4.2. Sequencing alignment and analysis……………………………………...87 2.4. Results…………………………………………………………………………………...88 2.5. Discussion……………………………………………………………………………….90 2.6. Literature cited…………………………………………………………………………103 v 2.7. Tables and Figures……………………………………………………………………..111 3. Chapter 3: Gene expression in sea lamprey, Petromyzon marinus during testis differentiation and development………………………………..……187 3.1. Abstract…………………………………………………………………………..……187 3.2. Introduction…………………………………………………………………………….188 3.2.1. Stages of testicular differentiation and development in lampreys……………...190 3.2.2. Gene expression during testicular differentiation………………...…………….194 3.3. Methods………………………………………………………………………………..197 3.3.1. Sample collection and preservation…………………………………………….197 3.3.2. Identification of conserved testis differentiation genes and reference genes…..199 3.3.3. RNA isolation and cDNA synthesis……………………………………………201 3.3.4. Quantitative reverse-transcriptase polymerase chain reaction qRT-PCR………202 3.3.5. Gene expression analysis……………………………………………………….202 3.3.6. Histological sampling and preparation…………………………………………203 3.3.7. Histological analysis……………………………………………………………205 3.4. Results………………………………………………………………………………….206 3.5. Discussion……………………………………………………………………………...209 3.6. Literature cited…………………………………………………………………………219 3.7. Tables and Figures……………………………………………………………………..229 Appendices…………………………………………………………………………….…....255 4. Chapter 4: Conclusion and future directions………………..……………267 4.1. Contributions of this study……………………………………………………………..267 4.2. Study limitations………………………….……………………………………….…...269 4.3. Future directions………………………………………..………………………….…..271 4.4. Conclusion……………………………………………………………………………..274 4.5. Literature cited…………………………………………………………………………276 vi List of Tables Table 2.1. Sampling location for Pacific lamprey and sea lamprey used for sequencing (111) Table 2.2. List of DMRT1 amino acid sequences used for constructing comparison alignment (113) Table 2.3. List of DMRTA2 amino acid sequences used for constructing comparison alignments (114) Table 2.4. List of DMRT2A amino acid sequences used for constructing comparison alignments (115) Table 2.5. List of FEM1c amino acid sequences used for constructing comparison alignments (116) Table 2.6. List of FOXL2 amino acid sequences used for constructing comparison alignments (117) Table 2.7. List of RSPO1 amino acid sequences used for constructing comparison alignments (118) Table 2.8. List of NR5A1 amino acid sequences used for constructing comparison alignments (119) Table 2.9. List of SOX2 and SOX3 amino acid sequences used for constructing comparison alignments (121) Table 2.10. List of SOX8 amino acid sequences used for constructing comparison alignments (123) Table 2.11. List of SOX9 amino acid sequences used for constructing comparison alignments (124) Table 2.12. List of HSP90b1 or TRA1 amino acid sequences used for constructing comparison alignments (125) Table 2.13. List of WNT3 amino acid sequences used for constructing comparison alignments (126) Table 2.14. List of WNT4 and WNT3 amino acid sequences used for constructing comparison alignments (127) Table 2.15. List of WNT4 and WNT5 amino acid sequences used for constructing comparison alignments (129) Table 2.16. List of WNT5 amino acid sequences used for constructing comparison alignments (131) Table 2.17. List of WNT3, 4, and 5 amino acid sequences used for constructing comparison alignments (133) vii Table 2.18. List of WT1 amino acid sequences used for constructing comparison alignments (135) Table 2.19. Primers and PCR annealing temperatures for successfully amplified sex determination genes (136) Table 2.20. Details regarding sex determining genes successfully amplified (137) Table 2.21. Primers and details for genes that did not amplify in the sea lamprey and Pacific lamprey (140) Table 2.22. Genes that were successfully amplified and that implicated roles in vertebrates (141) Table 3.1. Reference and