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Investigating Adult Stem Cell Lineage Specification in the Freshwater Flatworm Schmidtea Mediterranea

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Investigating Adult Stem Cell Lineage Specification in the Freshwater Flatworm Schmidtea Mediterranea Investigating Adult Stem Cell Lineage Specification in the Freshwater Flatworm Schmidtea mediterranea by Shu Jun Zhu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Shu Jun Zhu 2018 Investigating Adult Stem Cell Lineage Specification in the Freshwater Flatworm Schmidtea mediterranea Shu Jun Zhu Doctor of Philosophy Molecular Genetics University of Toronto 2018 Abstract Adult stem cells (ASCs) serve to facilitate tissue turnover during the lifespan of an organism. The goal of my research has been to deepen our understanding of how ASCs become specified towards differentiation to produce physiologically functional cell types. To this end, I have chosen to use the freshwater planarian Schmidtea mediterranea, which possess a large pool of ASCs that supports the constant cell turnover of all their tissues. A transcriptomic approach was used to identify a set of candidate genes to screen for potential regulators and markers of ASC progeny. Using gene expression characterization and RNAi functional analysis, I have identified a MEX3 homolog (Smed-mex3-1) and a putative MYB-type transcription factor (Smed-myb-1) as regulators of ASC differentiation. This body of work demonstrates that (A) mex3-1 is a critical factor broadly required for ASC differentiation, and (B) myb-1 is a regulator of the early temporal window of epidermal lineage progression. ii Acknowledgements This journey, which has now occupied a quarter of my life, would not have come to pass were it not for Bret’s willingness to take a chance on a new graduate to help him start his lab. Bret, thank you for believing in me, and for your mentorship and support during these many years. This experience has been nothing short of an absolute pleasure. My time at SickKids has been enriched by a wealth of individuals, and though this thesis needs no further elongation, I would like to offer special thanks to a few. To Dani and Chong, for being so welcoming and helping me get my bearings when I first arrived at SickKids as a new tech. To my committee members Derek and Mike, for their guidance throughout my graduate studies and their feedback on this thesis. To the Derry lab, for graciously accepting their repeated defeats in our yearly competitions of ultimate athleticism - curling. And to all past and present members of the Pearson lab, who have recognized it is what it is. Things would be amiss without particular acknowledgement of Alex and Ko, for nucleating the lab and starting their adventure alongside me; Rose, for her dry humour and even drier oat bars; Dave, for carrying me through Lordran; Alyssa, for being the safest place there is; and lastly to Steph and Yan, whose friendships and what that has meant for me cannot be overstated. Thank you to my friends, for the reminders that life exists outside of the lab, and giving me fortitude when it was dearly wanting. And of course, an immense thank you to my wonderful parents, who have never failed to support my endeavours, especially when I decided to leave a job to go back to school. Thank you for everything you’ve done for me. iii Table of Contents Acknowledgements……………………………………………………………………………...iii Table of Contents………………………………………………………………………………...iv List of Figures…………………………………………………………………………………..viii Chapter 1 Introduction…………………………………………………………………………....1 1. Adult stem cells…………………………………………………………………………….....1 2. Schmidtea mediterranea, an in vivo model of adult stem cell biology……………………….2 2.1. Planarians: anatomically complex and highly regenerative...…………………………....2 2.2. Progression of planarian research with molecular biology.……………………………...4 2.3. Molecular analysis of neoblasts……………………………………………………….....5 2.3.1. Molecular markers of neoblasts…………………………………………………...5 2.3.2. Establishing neoblast pluripotency………………………………………………..6 2.3.3. Neoblast lineages………………………………………………………………....7 2.3.4. Identification of neoblast subclasses……………………………………………...9 2.4. Regulation of neoblast cell fates………………………………………………………...11 2.4.1. Strategies of stem cell fate regulation……………………………………………11 2.4.2. Signaling pathways in planarians………………………………………………..12 2.4.3. Potential niche signals from differentiated tissues……………………………….13 2.4.4. Asymmetric neoblast division…………………………………………………...14 2.5. Planarian epidermis……………………………………………………………………..15 2.5.1. A model for neoblast differentiation……………………………………………..15 2.5.2. Epidermal lineage progression…………………………………………………..16 2.5.3. Regulators of epidermal lineage progression……………………………………18 iv 3. Thesis rationale: Using a transcriptomic approach to identify regulators in adult stem cell fate…………………………………………………………………………………………..19 Chapter 2 A mex3 homolog is required for differentiation during planarian stem cell lineage development …………………………………………………………………………………….21 4. RNAseq analysis of flow cytometry-isolated populations…………………………………...23 4.1. Identification and selection of candidate progeny-enriched genes……………………...23 4.2. Expression analysis by whole-mount in situ hybridization……………………………..27 5. New early progeny and late progeny markers are identified…………………………………33 5.1. Kinetics of irradiation-sensitivity of prog/AGAT-like genes……………………………33 5.2. New progenitor markers are expressed by early or late progeny……………………….34 6. RNAi screening identifies mex3-1 as a regulator of differentiation…………………………39 6.1. mex3-1(RNAi) animals display defects typical of impaired stem cell function…………39 6.2. mex3-1 is required for epidermal progenitor specification……………………………..42 7. Knockdown of mex3-1 results in expansion of the stem cell compartment…………………45 7.1. mex3-1 RNAi does not impair, but increases cell division……………………………..45 7.2. Knockdown of mex3-1 expands the stem cell compartment……………………………47 8. mex3-1 is broadly required for differentiation………………………………………………48 8.1. Epidermal regeneration and homeostatic turnover requires mex3-1……………………48 8.2. mex3-1 is required for differentiation of multiple lineages……………………………..49 8.3. mex3-1 has a broader role than p53 in neoblast differentiation…………………………52 9. RNAseq of mex3-1(RNAi) animals identifies novel progenitor transcripts………………….54 9.1. Transcriptional profiling of mex3-1(RNAi) animals…………………………………….54 9.2. Downregulated genes in mex3-1(RNAi) animals are markers of progenitors…………..56 10. Methods……………………………………………………………………………………..59 10.1. RNA sequencing........……………………………………………………….......59 v 10.2. Analysis of RNAseq data......................................................................................60 10.3. Phylogenetics and cloning.....................................................................................61 10.4. Animal husbandry and RNAi................................................................................61 10.5. Immunostaining, TUNEL, EdU, BrdU, irradiation, and in situ hybridization......62 Chapter 3 Smed-myb-1 specifies the early temporal identity during planarian epidermal differentiation...............................................................................................................................64 11. RNAi screening identifies a role for Smed-myb-1 in specification during epidermal differentiation.........................................................................................................................65 11.1. Knockdown of myb-1 ablates early epidermal progeny........................................65 11.2. myb-1 is expressed during epidermal lineage development..................................66 11.3. RNAi of myb-1 does not impair neoblast maintenance or progeny survival.........68 12. Epidermal lineage progression does not require early progenitor gene expression................71 12.1. myb-1 RNAi selectively downregulates early progeny markers...........................71 12.2. myb-1(RNAi) animals regenerate late progeny, later progeny, and epidermis......74 13. Epidermal differentiation is accelerated after myb-1 knockdown...........................................75 13.1. Stem cell descendants enter late progeny, later progeny, and epidermis earlier after myb-1 knockdown............................................................................................................75 13.2. myb-1(RNAi) animals maintain regional epidermal identity.................................78 14. Epidermal lineage progression is spatiotemporally shifted in myb-1(RNAi) animals.............79 14.1. RNAi of myb-1 temporarily increases progenitor and epidermal cells.................79 14.2. Late progeny exhibit early progeny characteristics after myb-1 RNAi.................80 15. Methods..................................................................................................................................83 15.1. Animal care, RNAi, and γ-irradiation...................................................................83 15.2. In situ hybridization, immunostaining, BrdU labeling, and TUNEL....................84 15.3. qPCR, RNAseq, and differential expression analysis...........................................84 vi Chapter 4 Discussion.....................................................................................................................86 16. X2 characterization identifies epidermal progenitor markers.................................................86 17. mex3-1 as a candidate mediator of asymmetric cell fate in planarians...................................87 17.1. Cell fate regulation by mex3 genes........................................................................87
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