Initial Molecular Characterization of Planarian Pigment Cells, a New Model for Studying Lineage Specification and

Gut Excretion

by

Xinwen He

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Molecular Genetics University of Toronto

Copyright by Xinwen He 2016

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Initial Molecular Characterization of Planarian Pigment Cells, a

New Model for Studying Lineage Specification and Gut Excretion

Xinwen He

Master of Science

Department of Molecular Genetics

University of Toronto

2016

Abstract

ASCs play crucial roles in tissue homeostasis and regeneration. Here, I established pigment cells

in Schmidtea mediterranea as a novel model to study ASC lineage specification by identification

of hmbs and alas as makers for pigment cells, and ans as a marker for their progenitors. Co-

expression analysis between hmbs and tryptophan metabolism enzyme, kmo, suggests

ommochrome as one of the responsible pigments for planarian body pigmentation. Initial

characterization of this lineage reveals an fst homolog as a negative regulator of pigment cell

regeneration and proliferation. Functional studies of light-induced depigmentation also uncover

homologs of draper and epimerase as positive regulators of gut excretion mediated pigment cell

removal. Together, these findings in pigment cell lineage and gut excretion pave ways to future

studies of ASC regulations.

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Acknowledgments

Life is about passing down messages. Primitive organisms pass down their messages in

the form of genetic materials. We human beings pass down our messages to our future

generations, not only with our genetic materials, but also in the forms of knowledge and

experiences. This thesis presented here is a recording of the knowledge and experiences I

gathered through my shallow studies in planarians, through the guidance from my supervisor,

and through working and living with my colleagues, families, and friends.

First and foremost, I would like to thank my supervisor Dr. Bret Pearson for his

continued support and guidance through my scientific endeavors. Using his own knowledge and

experiences, he showed me how to design and perform experiments, and how to think critically

and scientifically. More importantly, he is a great role model that inspires me on the meanings of

my career and personal life. It is my privilege to work with him, to do science together, and to

make discoveries that will benefit our society and the future generations. I would also like to

thank my supervisory committee, Dr. Marc Meneghini, and Dr. Nadeem Moghal, as well as my

collaborator, Dr. Jason Pellettieri. I have benefited greatly with their scientific expertise in their

fields and their different perspectives on the project. I am grateful to all past and present

members of the Pearson Lab. My work is only possible because of their optimization of

techniques, their advice on scientific problems, and the enjoyable lab environment they created.

I am sincerely thankful for my parents and grandparents. Their constant support since my

childhood has accompanied me to travel around the globe. I can never forget the encyclopedias

they bought me when I was little, which opened the doors for me to the magnificent world of

science. I am extremely lucky to have such a great family and have the resources and support for

me to freely develop my career and become the person I want to be. I would also like to thank

my girlfriend, Kelly. She is the best friend that I have ever had in my life. For over eight years,

her constant motivation, support, and love are the key to helping me staying focused on my

studies.

Last but not least, I thank all the planarians that have sacrificed for this study. Their lives

perished with a meaning. And I hope the messages of the knowledge generated can be passed on.

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Table of Contents

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ................................................................................................................................ vi

List of Appendices ........................................................................................................................ vii

Introduction .................................................................................................................................1

1.1 ASCs are sources for tissue homeostasis and regeneration .................................................1

1.2 Freshwater planarian, Schmidtea mediterranea, boasts distinct properties as a suitable model for ASC and regeneration study ................................................................................4

1.3 Advances and drawbacks of current approaches in planarian based ASC and

regeneration studies .............................................................................................................5

1.4 Ablation of planarian pigment cells by light exposure allows dissection of specific

lineage specification regulators ............................................................................................6

1.5 Previous studies on planarian pigment cells are limited and contradictory .........................6

1.6 Gut excretion of various cell types in response to RNAi and light exposure treatment ......8

1.7 Outline and rationale for the thesis research ........................................................................9

Materials and Methods ..............................................................................................................10

2.1 Animal husbandry and light-induced depigmentation .......................................................10

2.2 Intact planarian sample collection and RNA deep sequencing ..........................................10

2.3 Candidate selection and cloning for expression and functional analysis ...........................11

2.4 Riboprobe synthesis for expression analysis .....................................................................12

2.5 WISH and dFISH for expression analysis .........................................................................13

2.6 Targeted functional knockdown by RNA interference ......................................................16

Results .......................................................................................................................................17

3.1 Identified hmbs and alas as planarian pigment cell markers .............................................17

3.1.1 Selection of candidates and their expression patterns............................................17

3.1.2 Genes with sub-epidermal expressions and pigment cell-like morphologies ........19

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3.1.3 Expression of spider markers correlates to pigment status of planarian tissue ......20

3.1.4 hmbs is functionally required for pigment production and maintenance ...............22

3.1.5 hmbs is co-expressed with other spider markers and kmo .....................................23

3.1.6 Pigment cells show inward migration toward gut during depigmentation in

response to light exposure ......................................................................................26

3.1.7 Pigment cells display fast regeneration timeline during repigmentation ...............28

3.1.8 Decreasing levels of co-expression between punctate and spider markers as repigmentation progresses .....................................................................................30

3.2 Identified regulators of depigmentation and pigment cell regeneration ............................32

3.2.1 hmbs is not required for pigment cell survival .......................................................32

3.2.2 Presence of pigment is required for light-induced pigment cell loss .....................32

3.2.3 epimerase positively regulates depigmentation in response to light .....................34

3.2.4 draper positively regulates depigmentation in response to light ...........................34

3.2.5 Disruption of draper function leads to formation of wrinkles on epidermis and ectopic expressions of lineage markers ..................................................................36

3.2.6 follistatin negatively regulates pigment cell regeneration and homeostasis ..........37

Discussion .................................................................................................................................40

4.1 Identified markers, and established pigment cells as a novel model for ASC and regeneration study ..............................................................................................................40

4.2 Regulation of depigmentation and pigment cell regeneration ...........................................43

4.3 Future directions ................................................................................................................46

4.3.1 Deeper analysis of pigment cell and potential progenitors ....................................46

4.3.2 Continued screening of pigment cell line