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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

    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

  • ii

    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.

  • iii

    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.

  • iv

    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

  • v

    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 lineage markers and regulators ...................46

    4.3.3 Further analysis of depigmentation regulators .......................................................48

    4.3.4 Further analysis of pigment cell regeneration regulators .......................................48

    4.3.5 Comparative transcriptomics for identification of gut excretion regulators ..........49

    References ......................................................................................................................................50

    Appendices .....................................................................................................................................57

  • vi

    List of Figures

    Figure 1 Decision making of adult stem cells. ............................................................................ 2

    Figure 2 Planarian pigment cell as a model of lineage specification and gut excretion. ............ 7

    Figure 3 RNAseq sample collection. ........................................................................................ 11

    Figure 4 Expression patterns of genes with differential expression levels in depigmented

    animals. ...................................................................................................................... 18

    Figure 5 Expression patterns of spider and punctate genes resemble pigment cell morphology.

    .................................................................................................................................... 20

    Figure 6 Expression of spider and punctate genes directly correlates to the pigment status of

    planarian tissue. .......................................................................................................... 21

    Figure 7 Functional requirement of hmbs in pigmentation maintenance and repigmentation. 23

    Figure 8 Co-expression analysis of spider and punctate markers. ............................................ 25

    Figure 9 Pigment cells migrate towards gut during depigmentation. ....................................... 27

    Figure 10 Rapid regeneration of pigment cells during repigmentation. ..................................... 29

    Figure 11 Co-expression of spider and punctate genes decreases along repigmentation time

    course. ........................................................................................................................ 31

    Figure 12 hmbs is not required for pigment cell survival and its RNAi offers protection from

    light exposure. ............................................................................................................ 33

    Figure 13 RNAi phenotypes of epimerase, draper and fst. ........................................................ 35

    Figure 14 Ectopic expression of lineage markers in draper RNAi condition. ............................ 38

    Figure 15 Model for pigment cell lineage proliferation.............................................................. 42

    Figure 16 Model of epimerase, draper and fst functions in pigment cell lineage specification

    and excretion. ............................................................................................................. 45

  • vii

    List of Appendices

    Appendix 1 List of in situ patterns identified but not included in the main text. ..................... 57

    Appendix 2 List of genes cloned and screened in this study. ................................................... 58

    Appendix 3 List of phenotypes observed during RNAi screen. ............................................... 61

  • 1

    Introduction

    1.1 ASCs are sources for tissue homeostasis and regeneration

    Adult stem cells (ASCs) are the ultimate source for the tissue turnover of approximately 50 70

    billion cells per day in humans (Reed, 1999). Many mysteries lie behind the phenomenal power

    of ASCs, not only can they regulate the exact type, and the exact number of differentiated cells

    to replace, but also can they maintain a stable population of themselves in the long term.

    Terminal selector transcription factors play key roles in determining the types of differentiated

    cells. In some cases, a single terminal selector can regulate hundreds of downstream effector

    genes, and its ectopic expression can even reprogram cells to adopt a different fate (Eastman et

    al., 1999; Flames and Hobert, 2009; Hobert, 2011). For instance, unc-30, a C. elegans homeobox

    gene, controls the expression of crucial genes, including unc-25 and unc-47, for the identity and

    function of GABAergic neurons (Eastman et al., 1999). And the ectopic expression of unc-30 is

    capable of driving wild-type unc-25 and unc-47 promoters in other tissue types (Eastman et al.,

    1999). Despite a relatively rich knowledge of terminal selector transcription factors for the

    differentiation of neuron types, understanding of such mechanisms in many other tissue types is

    lacking. In addition to the determination of tissue types, what is even more poorly understood is

    the regulation of the exact amount of differentiated cells to replace. The feedback control

    between differentiated cells and ASCs is one interesting mechanism to be investigated, a

    mechanism that could be compromised in the case of cancer development.

    In order to maintain a stable population of ASCs, establishing an asymmetry between the

    proliferation and differentiation is crucial (Figure 1) (Sherley, 2002; Simons and Clevers, 2011).

    Such asymmetry can be achieved intrinsically, by disproportionate distribution of RNA and

    protein molecules that drive different cell fates (Bossing et al., 1996; Doe, 1996, 2008; Doe and

    Bowerman, 2001; Bayraktar, 2010). For example, Inscuteable, Prospero, and Numb are

    asymmetrically localized to the apical cortex in neuroectodermal cells in Drosophila embryos

    and physically segregated to daughter cells after neuroblast divisions (Doe, 1996). Prospero,

    with a homeodomain and nuclear localization signals, then drives the differentiation and

    suppresses the stem cell identity in the daughter cells (Doe, 1996; Bayraktar, 2010). Asymmetry

    can also be achieved extrinsically, such as receiving different signals from the stem cell and

    differentiated tissue microenvironment (Snippert et al., 2010; Sato et al., 2011). For example, the

  • 2

    Figure 1 Decision making of adult stem cells. Adult stem cells (ASCs) respond to clues such as aging and injury, then decide between symmetric and asymmetric proliferation to fulfill the different functions during homeostasis and regeneration. ASCs also receive feedbacks from daughter cells to control the rate and mode of proliferation.

    Lgr5 stem cell fate is determined by signaling in the microenvironment created by the

    differentiated Paneth cells in the mammalian intestinal crypt (Sato et al., 2011).

    Adding to the complexity of the asymmetry of ASCs is that a re-balance between proliferation

    and differentiation is necessary for the survival and well-being of an organism in response to

    injury, aging, and disease. Regeneration, an ability that we humans desired and has captured our

    imagination since Greek mythology, is a fascinating phenomenon to showcase the shift of

    balance between the different fate choices of ASCs. Regeneration is also a very poorly

    understood question, given its huge potential to shed light on tissue differentiation, remodeling,

    and the control of tissue size and proportions. One of the first scientific observations of

    regeneration is made in Hydra by Abraham Trembley in 1740. The simple organism was

    observed to completely regenerate its head after amputation (Lenhoff and Lenhoff, 1986). Soon

  • 3

    after, discoveries of regeneration in more and more complicated organisms were made, including

    planarians, amphibian tadpoles, and salamanders (Pallas, 1766; Spallanzani, 1769; Snchez

    Alvarado A, 2000). And more than 200 years later since Trembleys initial discovery, the

    regeneration ability of human liver was noticed (Widmann and Fahimi, 1975). Many different

    types of regeneration exist and can be categorized into two categories based on their requirement

    of blastema formation. The blastema is a mass of morphologically undifferentiated cells formed

    at the end of the stump during regeneration, similar in form and function to the vertebrate

    embryonic limb bud (Snchez Alvarado A, 2000; Tasaki et al., 2011). For instance, human liver

    regeneration requires limited proliferation of the surviving cells while bone regeneration requires

    proliferation and differentiation of stem cells already present in the tissue (Michalopoulos and

    DeFrances, 1997; Prockop, 1997). Both types of regeneration are non-blastema based. In

    comparison, more robust regeneration programs involve blastema formation (Snchez Alvarado

    A, 2000). Examples of this type of regeneration are used by planarians, mollusks, and also by

    vertebrates during limb and tail regeneration (Brndsted, 1969; Newth, 1958; Needham, 1952;

    Lange, 1920; Brockes, 1997; Iten and Bryant, 1976; Morgan, 1901). Previous studies have

    established the developmental programs across the animal kingdom as a conserved mechanism

    (Wolpert, 1994; De Robertis and Sasai, 1996). It has also been shown that the regeneration

    program shares a lot of similarities between the tissue differentiation and proliferation control

    during embryonic development (Needham, 1952). It is very likely that the regeneration program

    is also a conserved mechanism and has common origins to a wide variety of regeneration

    phenomena. Thus, studies of regeneration in different model animals can be cross-related.

    The understanding of ASC and regeneration biology would not only allow us to gain insights to

    the elegant developmental and differentiation programs of multicellular organisms but also to

    better appreciate many disease states and realize the therapeutic potentials in regenerative

    medicine. Despite these great implications, many areas of these subjects await further

    investigations. It is of my interests in this thesis research presented here to gain more insights on

    a few key steps toward a better understanding of the ASC biology and regeneration. Specifically,

    by identifying and investigating the regulators of ASC asymmetric proliferation and

    differentiation, regulators that determining the fate commitment during lineage specifications,

    and the mechanisms mediating the feedback control between differentiated tissue and ASCs

    during homeostasis and during regeneration (Figure 1).

  • 4

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

    ASCs are rare and hard to access in almost all model organisms, making in vivo studies of ASC

    biology extremely difficult. Common model organisms, including Drosophila melanogaster,

    Caenorhabditis elegans, Mus musculus, lack robust regeneration events to be studied. While

    animals traditionally used to study regeneration, including axolotls and salamanders, are

    extremely refractory to genetic and molecular manipulations (Snchez Alvarado A, 2000). In

    comparison, the freshwater planarian, Schmidtea mediterranea, hosts a large and accessible

    population of ASCs (neoblasts), representing 10-20% of their cells, has robust regeneration

    programs, and recent advances in molecular biology techniques in these animals really made

    them an ideal model for ASC and regeneration studies (Morgan, 1901; Krichinskaya and

    Martynova, 1975; Hayashi et al., 2006; Wagner et al., 2011; Sal and Bagu, 2002; Snchez

    Alvarado A. 2003, 2006; Cebri, 2007; Rossi et al., 2008; Sal et al., 2009; Aboobaker, 2011;

    Elliott and Snchez Alvarado, 2013; Rink, 2013). S. mediterranea is a type of free-living

    flatworm belonging to the Lophotrochozoan superphylum (Aguinaldo et al., 1997). Standing at

    this unique phylogenetic location in relation to mammals while hosting a wide variety of

    homologs of essential genes to humans, planarians are an ideal model for studies to provide us

    with valuable information in the conservation and evolution of ASCs and regeneration regulators

    (Labb et al., 2012). Showcasing their large population of ASCs, planarians display amazing

    regeneration capabilities, with reported regeneration into an entire functional animal from an

    amputated piece of as little as 1/279th of the body mass of the original animal (Morgan, 1901).

    Regeneration of an entire planarian body is extraordinary from limited ASCs in small amputation

    fragments, especially when one considers the extremely complicated anatomy of the flatworm,

    with a central brain and a PNS that contain all neuron types that can be found in an invertebrate

    system, a muscular pharynx and a triclad gut for uptake of food and nutrient intake involving

    phagocytosis, and a primitive excretory system protonephridia (Cebri, 2007; Cebri et al., 2002;

    Reddien and Snchez Alvarado, 2004; Forsthoefel et al., 2011; Scimone et al., 2011). In addition

    to the robust regeneration, planarians are also able to replace their aging tissue with a fast

    turnover rate leading to an immortal lifestyle (Eisenhoffer et al., 2008; Pellettieri and Snchez

    Alvarado, 2007). How planarians balance their regeneration/immortality without tumor

    development is an interesting question as well (Pearson and Snchez Alvarado, 2008). Finally, S.

  • 5

    mediterranea is a constitutive adult, thus allowing a number of gene functions to be investigated

    that may be required during embryonic development in other model organisms (Newmark and

    Snchez Alvarado, 2002). Combining all the above attributes, planarians are an ideal model for

    studying the molecular mechanisms underlying regeneration, and ASC proliferation and

    differentiation.

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

    Research in S. mediterranea has greatly expanded in the past decade as a result of the increasing

    availability of molecular and genetic tools. Available techniques include whole-mount in situ

    hybridization (WISH) to investigate gene expression patterns and double fluorescent WISH

    (dFISH) to perform co-localization study on gene products (Pearson et al., 2009). Planarians are

    also amenable to gene function study by RNA interference (RNAi), where double strand RNA

    (dsRNA)-expressing bacteria are fed to the animals and subsequently spreads to all tissue types

    (Snchez Alvarado and Newmark, 1999). The S. mediterranea genome has been sequenced and

    five transcriptomes of this species have been generated (Robb et al., 2008; Robb et al., 2015;

    Labb et al., 2012; Zhu et al., 2015; Solana et al., 2012). These sequencing datasets made

    possible to perform a number of comparative transcriptomic studies to dissect the involvement of

    different genes under different conditions. All differentiated planarian tissue are post-mitotic and

    the ASCs are the only dividing cells in the animal, allowing researchers to use gamma irradiation

    to specifically ablate all ASC population (Eisenhoffer et al., 2008). A number of studies have

    been published using this technique to analyze the gene expression in ASCs versus differentiated

    tissue (Eisenhoffer et al., 2008; Zhu et al., 2015; Reddien et al., 2005). Another technique is to

    induce planarian regeneration, most commonly by amputation, to observe changes in gene

    expression in wound response. A number of studies were able to identify regulators of ASC

    proliferation and migration, and of program cell death during tissue remodeling (Kao et al.,

    2013; Pellettieri et al., 2010; Gavio et al., 2013; Currie and Pearson, 2013). The amputation

    method, however, causes loss of multiple tissue types, making it difficult to dissect the

    involvement of regulators in individual lineage specification. Methods that allow identification

    for ASC regulators following a specific cell type loss, without inducing physical injuries, are

    urgently needed.

  • 6

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

    S. mediterranea displays a brownish color under normal laboratory conditions (Figure 2A)

    (Newmark and Snchez Alvarado, 2002). It was accidentally noted that planarians left under

    light exposure become depigmented over the course of several days (Figure 2A). It is also known

    that planarians display a strong negative phototaxis and prefer a dark environment. It was further

    observed that increased level of apoptosis surrounds the gut area of the animals while the gut was

    darkened at the same time, presumably enriched in excreted pigment or pigment cells (Figure

    2B, C. Apoptosis assay by terminal deoxynucleotidyl transferase dUTP nick end labeling

    (TUNEL), courtesy of Dr. Jason Pellettieri). When depigmented planarians are removed from the

    acute light exposure, however, the animals will repigment over the course of approximately two

    weeks (Figure 2A). Thus, I hypothesize that planarian pigment cells are lost in response to acute

    light exposure and that the repigmentation following acute light exposure requires rapid

    regeneration of pigment cells. Thus, the planarian light-induced depigmentation provides us with

    a possible exciting model to investigate the ASC regulation in response to a specific type of

    tissue loss, the pigment cell loss, without physical injury, allowing us to identify regulators of

    this specific lineage.

    1.5 Previous studies on planarian pigment cells are limited and contradictory

    Surprisingly little is known about planarian pigment cells, let alone regulators of their lineage

    specification. Previous studies came to different conclusions about the composition of planarian

    pigment. One study in a related planarian species, Dugesia gonocephala s. l, show that planarian

    pigment cells have process-rich morphology, similar to that of melanocyte in the mammalian

    epidermis (Palladini et al., 1979a). Enriched in these processes that extend to the sub-epidermal

    layer between muscle fibers are pigment granules (Palladini et al., 1979a). Each pigment cell is

    thus responsible for the pigmentation of a much larger surface area of the animal than that of the

    pigment cell soma alone. Interestingly, melanocyte-stimulating hormone release-inhibiting factor

    (MIF) was shown to induce a lighter pigmentation of the treated planarians (Palladini et al.,

    1979b). These results suggest melanin to be the potential pigment of planarians. On the other

    hand, one study biochemically determined the body pigment of another related planarian species,

    Dugesia ryukyuensis, to be ommochrome, with the major evidence coming from the absorption

  • 7

    Figure 2 Planarian pigment cell as a model of lineage specification and gut excretion. A) Planarians under normal laboratory conditions exhibit a brownish body pigmentation. After ~10 days of light exposure, planarians are depigmented. The body pigmentation recovers in ~2 weeks following removal of light exposure. B) Planarian gut darkens during depigmentation (lower left) and an increased level of apoptosis was detected by TUNEL (upper right). The increased level of TUNEL staining is associated with the darkened gut (upper left). C) Details of TUNEL stains overlapping with darkened gut. D) Outline of major goals for the thesis study. 1. Establishing molecular markers for planarian pigment cells and their progenitors. 2. Identifying regulators of pigment cell differentiation, regeneration and gut excretion. (Live imaging and TUNEL stain images (Panel B and C) courtesy of Dr. Jason Pellettieri.)

  • 8

    maxima of the extracted body pigment at 256nm, 367nm, and 463nm, characteristic of

    ommochromes (Hase et al., 2006). We know from studies of pigment in other organisms that

    melanin and ommochromes are derivatives of tyrosine and tryptophan metabolism, respectively

    (Ando et al., 2007; Croucher et al., 2013). It is of my interest to investigate perturbation in which

    of the two amino acid metabolism pathways would affect planarian pigmentation.

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

    Planarians are acoelomates without body cavities, neither do they have specialized circulatory or

    respiratory systems (Carranza et al., 1997). These attributes limit planarians to adopt a flattened

    body shape for diffusion of oxygen and nutrients. Their triclad gut, although having only one

    opening for both ingestion and egestion, is one of the few specialized organs that cover the entire

    length and width of the animals (Forsthoefel et al., 2011). One would speculate if planarian gut

    plays important roles that are fulfilled by the circulatory or lymphatic system in other animals.

    Indeed, planarian gut has been noted to take part in the excretion of cells under various

    conditions, a typical role played by lymphatic system in vertebrates. Under p53 RNAi condition,

    planarians display an over-proliferation of stem cells, while at the same time, an ectopic

    expression of cyclinB positive cells can be observed in the gut cavity of the animals (Pearson and

    Snchez Alvarado, 2010). It is unknown whether the cell excretion function of planarian gut is

    one of the mechanisms used to remove detrimental cells and guard against tumor development.

    In the same study, when p53 RNAi was applied to animals following amputation, head and tail

    fragments, which no longer possess a pharynx and thus no opening of the gut to the external

    environment, were noticed to develop tissue dysplasia (Pearson and Snchez Alvarado, 2010).

    No such tissue dysplasia was observed in the trunk fragment, however, presumably because the

    gut is still open due to the presence of the pharynx on these fragments and able to remove the

    detrimental cells excreted by the animal. Another event of gut excretion of cells was noticed

    under mex-3 RNAi condition, which causes loss of progeny cells and eventually the failure of

    tissue turnover (Zhu et al., 2015). Progeny cells marked by prog-2 in the parenchyma were

    ectopically found inside the gut and eventually excreted (Zhu et al., 2015). Considering these

    previous observations, the darkening and increased apoptosis activity in planarian gut during

    acute light exposure is likely to be gut excretion as well, and it is of my interest to identify

  • 9

    regulators of this process. In comparison to previous RNAi-based approaches, the light exposure

    induced depigmentation offers a novel model on gut excretion.

    1.7 Outline and rationale for the thesis research

    In collaboration with Dr. Jason Pellettieris lab (Keene State University, NH, USA), we have

    generated an RNA-deep sequencing (RNAseq) dataset of S. mediterranea, by collecting intact

    animals at several time points during the acute light exposure induced depigmentation and the

    repigmentation process 4 days after returning to the dark. Comparing the RNAseq results

    between the Day 10 (completely depigmented) time point and the control, expression levels of

    more than one hundred transcripts were either increased or decreased by more than two-folds.

    Working with these candidates of differentially-expressed transcripts, I have two goals in this

    thesis research. First, I will establish the first molecular markers for planarian pigment cells

    (Figure 2D). I hypothesize that a number of transcripts with decreased expression level in the

    depigmented animals are specifically expressed in the pigment cells and can be demonstrated to

    be pigment cell markers. My rationale is that if pigment cells are lost during light exposure, the

    specifically expressed transcripts are no longer present in the animals, thus showing a decreased

    expression level in the RNAseq data of depigmented worms. Second, I aim to identify regulators

    of planarian pigment cell lineage specification, regeneration, and gut excretion (Figure 2D). I

    hypothesize that certain genes with altered expressions, either with increased or decreased

    expression in planarians in response to light exposure are key regulators of these processes. My

    rationale is that planarians would activate specific genetic machinery to replenish pigment cells

    after sensing a reduction in the pigment cell population and that the active removal of pigment

    cells through gut excretion is mediated by genes that are specifically regulated during light

    exposure. Both of the two processes would lead to altered expressions of responsible genes that

    can be identified in the RNAseq data. To the end of these two goals, I performed a screen of 40

    up-regulated and 69 down-regulated genes in the depigmented animals. In addition, I also

    screened a few candidate genes with suspected involvement, by analyzing their expression

    patterns and their functional involvement during depigmentation, gut excretion, pigment cell

    lineage specification and regeneration.

  • 10

    Materials and Methods

    2.1 Animal husbandry and light-induced depigmentation

    A diploid, asexual CIW4 strain of Schmidtea mediterranea originally collected in Barcelona,

    Spain was reared as previously described (Benazzi et al., 1972; Newmark and Snchez Alvarado,

    2002). The animals used in this study were kept in a dark incubator at 20C under default/control

    conditions. The animals were cultured in planarian water (Milli-Q H2O supplemented with 21g/L

    Instant OceanTM, 0.9mM NaHCO3, 0.83mM MgSO4, 0.9mM CaCl2 and 0.04mM KHCO3 salts,

    as previously described; Zhu et al., 2015) Animals were fed with beef liver paste every 10 days,

    but a starvation period of one week was carried out before performing experiments on the animal

    subjects. For the experiment of collecting whole planarian samples for RNAseq during the light-

    induced depigmentation and repigmentation time course, a light exposure set up in a Styrofoam

    box using CFL bulb was used. Specifically, a 14W, 2700K CFL bulb was placed approximately

    30cm away from a tissue culturing plate containing the planarians. A piece of UV-resistant glass

    was placed between the tissue culturing plate and the light source. A fan was used in this set up

    to keep the circulation of the air and to prevent overheating of planarians. The animals were

    treated with 5 cycles of 24 hours of light exposure and 24 hours of dark exposure (dark exposure

    by placing the samples in the normal condition incubator) for a total of 10 days, at which point

    the animals were completely depigmented on their dorsal surface. The animals were then

    returned to the normal condition dark incubator for the rest of the experiment and to be observed

    for repigment. For the light-induced depigmentation performed during the RNAi functional

    screens, an alternative LED light exposure set up was used. The LED set up bares the sample

    principle of the CFL set up, and unless otherwise noted, a cycle of 15-minute light and 225-

    minute dark exposure was performed for 48 times (a total of 8 days at 6 cycles per day). This

    alternative setup and light exposure schedule were used in the RNAi screen instead for its

    shortened timeline and more consistent animal health at the end of the treatment.

    2.2 Intact planarian sample collection and RNA deep sequencing

    In collaboration with Dr. Jason Pellettieris Laboratory, 5 to 10 intact planarian animals were

    collected from 3 biological replicates under the CFL light-induced depigmentation set up for

    total RNA extraction using TRIzol Reagent (Invitrogen, catalog number 15596-026) (Figure 3).

    Two time points during the depigmentation, at Day 1 (24hr) and Day 2 (48hr), one time point

  • 11

    when the worms are completely depigmented, at Day 10, one time point when the worms are

    repigmenting, at Day 14 (since start of light exposure treatment, or Day 4 repigmentation), in

    addition to a control sample with no light exposure, for a total of five samples were collected

    (Figure 2). Poly-A-selected cDNA libraries were prepared from the purified total RNA samples

    using TruSeq kits from Illumina. We generated approximately 108 of 50-base single end reads

    for each time point sample on Illumina HiSeq2500 with v4 chemistry. Using bowtie2 alignment,

    the RNAseq reads were mapped to the 2011 Pearson Lab asexual Schmidtea mediterranea

    transcriptome and generated a coverage of approximately 8 times for every nucleotide of the

    82,188 transcripts. Pseudocounts of +1 were added to every numerator and denominator when

    calculating expression ratios from the RNAseq counts (i.e. raw counts per kilo million (RPKM)

    + 1 was used), in order to reduce the bias of lowly expressed transcripts having large fold

    changes without substantial absolute changes.

    Figure 3 RNAseq sample collection. Animals were treated with 5 cycles of 24 hours of light and 24 hours of dark exposure for a total of 10 days. After 10 days, animals were placed in the dark for recovery of pigmentation. Whole worm samples were collected at 24 hrs, 48 hrs, 10 days time points during depigmentation, at 4 days time point during repigmentation and at 10 days time point for control animals. Total RNA was extracted and then subjected to cDNA library preparation and RNA deep sequencing.

    2.3 Candidate selection and cloning for expression and functional analysis

    Candidate genes to be screened for pigment cell markers and regulators of pigment cell lineage

    specification were selected using a 2-fold expression change (RPKM + 1) cut-off, from genes

  • 12

    with either increased or decreased expression in Day 10 (depigmented) animals in comparison to

    control animals. Near full-length transcript of the established candidate genes were cloned using

    gene-specific forward and reverse primers into a double stranded RNA expression vector (pT4P)

    as previously described (Rink et al., 2009). For transcripts longer than 1.5kb, up to 1.5kb of 5

    sequences of the transcripts were cloned. Forward and reverse primers were designed to have an

    18-22 nucleotide identity to the binding region of the transcript with a melting temperature

    around 54C, then additional sequences of 5-CATTACCATCCCG-3 and

    5-CCAATTCTACCCG-3 were added to the 5 of forward and reverse primers, respectively.

    Total RNA was extracted from 7-day planarian regeneration time course and used as the

    template to generate cDNA by SuperScript III Reverse Transcriptase (Thermo Scientific, Catalog

    number: 18080-051). Polymerase chain reaction (PCR) was then performed with the forward and

    reverse primer sets using the cDNA as the template. PCR products were gel extracted and treated

    by T4 DNA polymerase (Thermo Scientific, Catalog number: EP0061) with dCTP as the only

    nucleotide available in the reaction mix. The predominant 3-5 exonuclease activity of the T4

    DNA polymerase generates 5-CATTACCATCCC-3 and 5-CCAATTCTACCC-3 overhangs

    at either end of the PCR products. Using a similar method but with the addition of dGTP instead,

    complementary overhangs were generated on the SmaI restriction enzyme digested pT4P vectors

    using T4 DNA polymerase, for directional insertion of the PCR product. The T4 DNA

    polymerase-treated PCR product and pT4P vector were then mixed and allowed spontaneous

    overhang binding, without an enzyme-mediated ligation step, and then transformed into DH5

    E. coli. Positive clones of the candidate transcripts were then verified by Sanger sequencing.

    2.4 Riboprobe synthesis for expression analysis

    Primer set AA18 (5-CATTACCATCCCGCCACCGGTTCCATGG-3) and PR244F (5-

    GGCCCCAAGGGGTTATGTGG-3) was used with the templates of cloned transcripts in pT4P

    vectors in order to generate PCR products containing one T7 site in the antisense direction to the

    3 ends of the inserted transcripts. Then using a previously published method, antisense

    riboprobes were synthesized using T7 RNA-polymerase (Thermo Scientific, Catalog number:

    EP0111) for a reaction time of 3 hours (Pearson et al., 2009). During this reaction, 400ng of the

    abovementioned PCR product was used as the DNA template. And either digoxigenin (DIG,

    Roche, Product No. 11277073910) or fluorescein (Roche, Product No. 11685619910) was used

  • 13

    for RNA riboprobe labeling. A hydrolysis reaction was performed on the synthesized riboprobes

    prior to use in whole-mount in situ hybridization (WISH) experiments.

    2.5 WISH and dFISH for expression analysis

    Whole-mount in situ hybridization (WISH) and double fluorescent in situ hybridization were

    performed by adapting previously published methods (Pearson et al., 2009). In both cases,

    planarian animals roughly 4mm in length were killed using 5% N-acetyl-cysteine (NAC; Sigma)

    in PBS, a mucolytic compound, and then fixed with 4% formaldehyde in PBSTx (formaldehyde:

    Sigma; PBSTx: 1 PBS + 0.5% Triton X-100). The fixed animals were treated with a reduction

    solution (reduction solution: 50 mM DTT, 1% NP-40, 0.5% SDS, in 1 PBS) and then subjected

    to dehydration in 50% methanol solution and 100% methanol solution sequentially (50%

    methanol solution: equal volumes of 1 PBSTx and 100% methanol). Both the reduction and

    dehydration steps function to permeabilize the fixed animal samples allowing access of

    riboprobes, antibodies, and substrates for in situ development. The dehydrated samples were then

    treated with a 6% bleach solution (6% H2O2 in Methanol) to bleach pigment present in the

    animals to help with in situ pattern imaging. After overnight treatment in 6% bleach solution, the

    animal samples were rehydrated using 50% methanol and PBSTx solutions sequentially.

    Hybridization of riboprobes was then performed after a 2-hour Prehyb incubation of the animal

    samples in riboprobe mix (Hybridization solution: 50% De-ionized Formamide (Roche); 5%

    Dextran Sulfate (Sigma); 5 SSC (from 20 stock); 1 Denhardts solution (Sigma); 100g/ml

    Heparin (Sigma, from porcine intestine, from 100 stock); 1mg/ml yeast torula RNA (Sigma);

    1% Tween-20 (Sigma); 0.6% DTT. Prehyb solution: Hybridization solution without dextran

    sulfate, Heparin or DTT. Riboprobe mix: 1:500 to 1:1000 times dilution of DIG-labeled

    riboprobe or 1:200 to 1:250 times dilution of fluorescein labeled riboprobes in Hybridization

    solution). Hybridization was performed at 56 for more than 16 hours and then the animal

    samples were washed at 56 with each of the following solutions twice, 2 SSC solution, 0.2

    SSC solution and 0.05 solution, for a duration of 30 minutes per wash (2 SSC solution: 2

    SSC + 0.1% Triton-X. 0.2 SSC solution: 0.2 SSC + 0.1% Triton-X. 0.05 SSC solution:

    0.05 SSC + 0.1% Triton-X. All using H2O as the solvent). After the SSC washes, the animal

    samples were blocked with 5% horse serum plus 5% western blocking reagent (Roche) in MABT

    (MABT: 100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20, pH to 7.5 with NaOH). After 2

    hours of blocking, the animal samples were then incubated with anti-DIG-AP, anti-DIG-POD or

  • 14

    anti-fluorescein-POD antibodies (All three of the antibodies from Roche. anti-DIG-AP, used at

    1:4,000, anti-DIG-POD, used at 1: 500, and anti-fluorescein-POD, used at 1:400 in blocking

    solution) overnight at room temperature. 6 times of washes with MABT at 20 minutes each were

    performed after the antibody incubation. The animal samples were then ready for signal

    detection by colorimetric development or fluorescent development.

    For samples with epitope detection by anti-DIG-AP undergoing colorimetric development, a pre-

    incubation of 10 minutes in AP buffer was performed (AP buffer: 100 mM Tris, pH 9.5; 100 mM

    NaCl; 50 mM MgCl2; 0.1% Tween-20 brought up to volume with 10% polyvinylalcohol solution

    (PVA; Sigma P8136)). AP buffer was replaced with Developmental buffer containing the

    NBT/BCIP substrate (Development buffer: AP buffer with 4.5L/mL NBT (Roche) and

    4.5L/mL BCIP (Roche)). The colorimetric development was carried out for a duration of 10

    minutes to 3 hours, until an optimal signal to background ratio was achieved. The

    colorimetrically developed animals were washed with PBSTx twice and post-fixed with 4%

    formaldehyde in PBSTx for 20 minutes. Another two PBSTx washes were performed after the

    fixation before the post-fixed samples were treated with 100% Ethanol. Samples were incubated

    in 100% Ethanol, which removes the unspecific background staining and turns the NBT/BCIP

    precipitate dark blue, for 5-20 minutes until optimal signal to background ratio was achieved.

    100% Ethanol was then replaced with another PBSTx wash and the samples were then placed in

    80% Glycerol solution overnight, before mounted onto slides for imaging (80% Glycerol

    solution: 80% Glycerol; 10mM Tris, pH 7.5; 1mM EDTA). The colorimetric WISH stains

    prepared according to the above procedure were imaged on a Leica M165 fluorescent dissecting

    microscope. Images were post-processed in Adobe Photoshop. All colorimetric quantification

    were performed with the freely available ImageJ software (http://rsb.info.nih.gov/ij/).

    Two types of fluorescent developments were performed for samples undergoing fluorescent

    development. FastBlue development was performed on samples with epitope detection by anti-

    DIG-AP, as previously described (Zhu et al., 2015; Lauter et al., 2011). Samples after MABT

    washes were incubated in SB8.2 buffer for 10 minutes (SB8.2 buffer: 100 mM Tris, pH 8.2; 100

    mM NaCl; 50 mM MgCl2; 0.1% Tween-20 brought up to volume with 10% polyvinylalcohol

    solution (PVA; Sigma P8136)). SB 8.2 buffer was then replaced with FastBlue development

    buffer containing the FastBlue and NAMP substrates (FastBlue development buffer: mix equal

    volume of 5L/mL Fast Blue BB (Sigma F3378) and 5L/mL of naphtol-AS-MX-phosphate

    http://rsb.info.nih.gov/ij/

  • 15

    (NAMP, Sigma N5000), for a final concentration of 2.5L/mL of each substrate). Development

    of FastBlue stain was monitored by its blue colorimetric development for a duration ranging

    from 10 minutes to 3 hours. The development was stopped as soon as a faint colorimetric pattern

    similar to that observed with NBT/BCIP development can be observed under dissecting scope.

    In the cases of epitope detection by anti-DIG-POD and anti-fluorescein-POD, the in situ stains

    were developed with FITC or Cy3 tyramides. To prepare for the tyramide development, samples

    after the MABT washes were first incubated in PBSTx for 30 minutes. Then PBSTx was

    replaced with tyramide solution for another 30-minute incubation (Tyramide solution: 1:500

    dilution of Cy3 tyramide or 1:2000 dilution of FITC tyramide in PBSTx). After the tyramide

    solution incubation, two spikes at 25 minutes apart of H2O2 at a final concentration of 1 part per

    10,000 were performed to the samples in tyramide solution. After 45 minutes of the initial spike

    of H2O2, the tyramide development was stopped by replacing the tyramide solution with PBSTx.

    Another 2 washes of PBSTx for 5 minutes each was performed before the animals were put into

    AntiFade mounting media (AntiFade mounting media: 80% Glycerol solution + 1% DABCO

    (Sigma)).

    Tyramide development was coupled with FastBlue development or another tyramide

    development to produce multicolor fluorescent in situ hybridization stains. Multiple riboprobes

    labeled with different epitopes, for instance, DIG and fluorescein, were added to the same

    riboprobe mix. The hybridization and first round tyramide development were performed

    similarly as in the case of single riboprobe in situ. In the case of a second round development

    using FastBlue, anti-DIG-AP is already added during the antibody incubation for the first round

    development, thus, the samples after first round tyramide development were incubated in SB 8.2

    buffer and proceed as normal for FastBlue development. In the case of a second round

    development using another tyramide development, the first round POD-conjugated antibody

    activity was quenched by incubating the samples in sodium azide solution for 60 minutes

    (Sodium azide solution: 100mM sodium azide in PBSTx). The sodium azide solution was then

    washed 5 times with PBSTx for 5 minutes each before the samples were incubated in the next

    round of antibody overnight in blocking solution. And the second round tyramide development

    proceeded similarly as first round development with a different tyramide. After all developments

    are done, samples were placed in AntiFade mounting media. All fluorescently developed

    samples, including FastBlue and tyramide stains, were imaged on an Olympus IX81 Quorum

  • 16

    Spinning Disk Confocal with a Hamamatsu C9100-13 EM-CCD camera and a Yokogawa CSU

    X1 scanhead. Whole-animal images were stitched together using the application Volocity, and

    all images were post-processed in Adobe Photoshop.

    2.6 Targeted functional knockdown by RNA interference

    RNA interference (RNAi) targeting cloned candidate transcripts was performed as previously

    described to knock down their functions in planarian pigmentation and pigment cell lineage

    specification (Newmark et al., 2003). RNAi in this study was performed by feeding planarians

    with RNAi food in the form of a beef liver paste containing bacteria expressing double-strand

    RNAs of the targeted transcript. In order to produce the bacteria culture to make the RNAi food,

    pT4P vectors containing target transcript sequence, as described in section 2.3, were transformed

    into HT115 E. coli strain, which is capable of RNA synthesis by T7 RNA polymerase when

    induced by isopropyl -D-1-thiogalactopyranoside (IPTG). In the pT4P vector backbone, there

    are two T7 promoters flanking and going in the direction towards the insertion site of the cloned

    transcript, which are key to produce complementary strands of the double-stranded RNA

    responsible for RNA interference. E. coli bacteria containing the cloned pT4P vectors were

    grown to an optical density at 600nm (OD600) of 0.8 and then induced with 1mM IPTG. The

    induced bacteria culture was then incubated at 37 for another 2 hours, allowing RNA

    production by T7 RNA polymerase. Bacteria were then pelleted and mixed with beef liver paste

    at a ratio of 333L of liver per 100 mL of original bacteria culture volume. After thorough

    mixing of the beef liver paste and the bacteria pellet, blue food coloring was added to the mixture

    at 1 L coloring /60 L food. The RNAi food was then aliquoted to 60L per feeding, which is

    sufficient to feed 40 worms in the size range of 2-4mm in length, and stored at -80 until use.

    The negative control RNAi was prepared in a similar way but with a pT4P vector containing

    GFP sequence at the insertion site. During the screening of all genes in this study, RNAi food

    was fed to >7 day starved planarians at a frequency of twice per week for 5 weeks, for a total of

    10 feedings. Certain phenotypes take fewer feeds and less time to display, and the time points in

    figures denote the number of feeds for the specific gene being knocked down as well as days

    after the last feeding of RNAi food. For example, 10fd14 denotes 10 RNAi feedings and 14 days

    after the last feeding, and 12fd7 corresponds to 12 RNAi feedings and 7 days after the last

    feeding.

  • 17

    Results

    3.1 Identified hmbs and alas as planarian pigment cell markers

    3.1.1 Selection of candidates and their expression patterns

    Analysis of Day 10 depigmented animal and control animal RNAseq data yields 149 genes with

    more than 2-fold decreased and 162 genes with more than 2-fold increased expression levels in

    the depigmented animals. Of these 311 candidates, 69 down-regulated and 40 up-regulated genes

    were cloned based on the descending order of their fold changes. Among the 109 cloned genes,

    60 of them give specific WISH patterns. Expression patterns can be categorized as expression in

    the gut, in the sub-epidermis with spider morphology (rich in processes extruding from cell

    soma), in the sub-epidermis with punctate morphology, in the epidermis with unknown

    morphology, in the anterior, in the pharynx, in the para-pharyngeal region, in the neural system

    and in the parenchyma with variable expressions across individual animals (Figure 4). Most sub-

    epidermal expression patterns (spider and punctate patterns) are generated by genes with down-

    regulated expression in depigmented animals. All the variable parenchyma expression patterns

    are generated by genes with up-regulated expression in depigmented animals. Other categories of

    expression patterns are generated by either down- or up-regulated genes. Up to 3 representative

    genes of each category were listed in Figure 3. The expression pattern screen of differentially

    expressed genes presented here provides guidance for later functional studies of planarian

    depigmentation and repigmentation. In order to establish molecular markers of pigment cells,

    genes with distinct sub-epidermal expressions were prioritized for RNAi screening (Figure 4,

    rows 2-4). For the identification of regulators of depigmentation and repigmentation, genes with

    distinct gut and variable parenchyma expression patterns were prioritized for screening (Figure

    4, first and last rows). Many up-regulated genes without distinct wild-type stains were also

    screen by RNAi knockdown. The rationale behind screening of these genes is that up-regulated

    genes without a distinct expression pattern in normally pigmented animals may still have a

    dramatically different expression during, and possibly be required for, depigmentation and

    pigment cell regeneration.

  • 18

    Figure 4 Expression patterns of genes with differential expression levels in depigmented animals. Up to 3 representative expression patterns by WISH of each category are listed. The number of genes that fall into each category is displayed in the brackets following category names. Categories of specific expression patterns are gut, sub-epidermal with spider morphology, sub-epidermal with punctate morphology, epidermal (other), anterior, pharynx, para-pharyngeal, neural, ubiquitous, other distinct patterns, and parenchyma variable. For genes in the parenchyma variable category,

    two representative images of expression in different individuals are shown. Scale bar: 100m.

  • 19

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

    The expression patterns of spider and punctate categories of genes resemble the anatomical

    location of pigment cells as previously proposed and are further characterized to determine

    whether they are marking pigment cells (Palladini et al., 1979a). Spider gene category includes

    an hmbs homolog (spider-1). Hydroxymethylbilane synthase (HMBS), encoded by hmbs, is an

    enzyme responsible for the third step of heme synthesis pathway catalyzing the production of

    hydroxymethylbilane from porphobilinogen (Grandchamp et al., 1987). Another spider gene is

    an alas homolog (spider-2). Aminolevulinic acid synthase (ALAS), encoded by alas, is the

    enzyme responsible for the first step of the same heme synthesis pathway as HMBS, responsible

    for the conversion of succinyl-CoA to d-Aminolevulinic acid (Bishop, 1990). HMBS functions

    in the cytoplasm while ALAS is usually found in the mitochondria (Grandchamp et al., 1987;

    Bishop, 1990). Punctate genes category includes an ans homolog (punctate-1). L-Asparaginase,

    encoded by ans, catalyzes the deamination reaction from L-asparagine to L-aspartate (Cantor et

    al., 2009). A homolog of sds (punctate-2) also belongs to the punctate category. L-serine

    dehydratase/L-threonine dehydratase, encoded by sds, is responsible for the catalytic activity of

    converting L-serine and L-threonine to pyruvate and 2-oxobutanoate, respectively (Ogawa et al.,

    1989). Both the punctate markers and spider markers display distinct expression patterns

    throughout the entire animal in the sub-epidermal layer (Figure 5A). The expression of both

    types of markers is quite uniform within the dorsal or ventral surface. However, the dorsal

    surface has a higher density of both markers over the ventral surface. spider-1, hmbs, is

    expressed at a density of 16144 cells/mm2 in the dorsal surface (n = 6). Whereas punctate-1,

    ans, is expressed at a density of 113771 cells/mm2 in the dorsal surface (n = 6). One exception

    to the generally uniform expression of spider and punctate markers is the lack of expression in

    the eye region. No hmbs expression can be detected in the patches of cells opposing the planarian

    eye cups, which include photo-sensory neurons (Figure 5B). This is a logical expression pattern

    for potential pigment cell markers, because planarian lacks pigmentation in the photo-sensory

    region, allowing unhindered light passage. It is also observed that cells expressing spider

    markers and punctate markers are not located in the outermost layer of the planarian epidermis

    (Figure 5B, C). Individual cells labeled by punctate marker ans and sds display small and round

    morphology. Individual cells labeled by hmbs and alas are observed to have cytoplasmic

    processes extending from the cell soma. All the morphological observations about cells labeled

  • 20

    by spider cell markers agree with previously published data on planarian pigment cells, thus are

    suitable candidates for pigment cell markers (Figure 5D) (Palladini et al., 1979a, 1979b).

    Figure 5 Expression patterns of spider and punctate genes resemble pigment cell morphology. A) Spider and punctate genes are expressed throughout the surface of planarian animals. The dorsal planes of animals are shown. B) Details of hmbs expression in the anterior region. Individual hmbs positive cells have extending processes from the cell soma. A gap exists between hmbs expression and the edge of the dorsal plane, suggesting expression of hmbs in the sub-epidermal layer. Patches opposing eye pigment cups lack hmbs expression. C) Details of ans expression in the posterior region. Individual ans positive cells have small and round morphology. D) Illustration of pigment cell morphology in axial cross section. E, epidermis. BM, basal membrane. MF, muscle fibre. P, parenchyma. Black dots in the pigment cell indicate pigment granules. All expression patterns stained by WISH. Scale bar: 100m.

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

    After morphologically establishing spider and punctate markers as suitable candidates for

    pigment cell markers, I next tested whether their expression patterns were affected during

    depigmentation, as predicted if they are bona fide pigment cell markers. Planarians roughly 3mm

    in length were exposed to visible light in the LED light exposure set up for 7 days. The animals

  • 21

    Figure 6 Expression of spider and punctate genes directly correlates to the pigment status of planarian tissue. A) Live image of a light-exposed planarian animal. (>8 days, completely depigmented) B) Live image of a regenerating planarian. 7 days after amputation. Newly regenerated head and tail (blastema) are mostly not pigmented. C) Normal expression of hmbs (spider-1), pepck (spider-3) and ans (punctate-1) in WT (control) diminishes in 7-day light exposed animals (mostly depigmented). D) hmbs is lowly expressed in newly regenerated blastema at 5-day post amputation. F) Details of limited expression of hmbs in 5-day blastema. All expression patterns stained by WISH. Panels D and F are courtesy of Stephanie

    Hallows. Scale bar: 100m.

    were almost completely depigmented at the end of this treatment (Figure 6A). I then performed

    WISH on these light exposed animals and determined the expression level of spider and punctate

  • 22

    markers. The expression of punctate markers is undetectable while spider marker expression is

    greatly reduced (n 3 for each WISH stains of both conditions) (Figure 6C). The positive

    correlation between the pigment status of planarians and the expression of spider and punctate

    markers supports these genes as pigment cell markers.

    Another method to generate unpigmented planarian tissue is by amputation (Figure 6B). The

    newly regenerated blastema after amputation is unpigmented and it takes about 2 weeks before

    the pigment status is restored to a similar level to that of the old body part. With assistance from

    the Pearson Laboratory technician, Stephanie Hallows, we performed WISH of spider markers in

    newly regenerated blastema at 5 days post amputation. Only a handful of cells displayed

    expression of the spider-1 marker in both the anterior and posterior blastema (n > 10, counting

    head, trunk, and tail fragments) (Figure 6D, F), further supporting the positive correlation

    between the expression of spider markers and pigment status of the planarian tissue.

    3.1.4 hmbs is functionally required for pigment production and maintenance

    For a marker with high specificity, its expression should be exclusive within the pigment cell

    population only. And logically, I hypothesized that the exclusive expression of these genes in

    pigment cells are crucial for pigment cell functions, for instance, pigment production and

    maintenance. Thus, I performed functional knockdown of several punctate and spider genes by

    RNAi and observed changes in planarian pigmentation. After 12 feeds of RNAi, functional

    knockdown of punctate genes did not show any effect on planarian pigmentation or the recovery

    of pigmentation after light exposure (n = 28). On the other hand, when spider-1, hmbs, was

    functionally knocked down by RNAi, the pigmentation of hmbs RNAi animals became much

    lighter than that of control animals (n = 35) (Figure 7A). spider-1 RNAi animals did not display

    any apparent health or behavioral defects. Although the treated animals are much lighter in

    pigmentation, residual pigmentation with similar expression morphology to that of control

    animals can still be observed.

    I next tested the functional requirement of spider-1 during the repigmentation of blastema after

    amputation. Control and spider-1 RNAi worms after 12 feeds were amputated at 7 days after the

    last feeding of RNAi food and allowed to regenerate for 30 days. Roughly 7 days is required for

    planarians to regenerate new head and tail tissue after amputation and morphallax to similar body

    proportions as before amputation (Newmark and Snchez Alvarado, 2002). In this experiment,

  • 23

    Figure 7 Functional requirement of hmbs in pigmentation maintenance and repigmentation. A) Representative live images of animals after 12 feeds of RNAi food and 7 days after the last feeding. Control animals display normal pigmentation while hmbs RNAi worms are no longer pigmented. B) Representative live images of animals after similar treatment as in A), but imaged with amputation performed at 7 days post RNAi feedings and after 30 days of regeneration. In control animals, pigmentation and boundaries of clearing around eyes are established in newly regenerated tissue. In hmbs RNAi animals, pigmentation is not established in neither the old body parts nor the newly regenerate tissue.

    an extensive 30-day regeneration period allowed the amputated animals to fully regenerate their

    pigment cells in the new tissue and produce body pigment. In the control animals, I observed that

    the newly regenerated head and tail tissue have recovered substantial pigmentation, although still

    slightly lighter than the old body parts, at the end of the regeneration period (n = 4, counting

    trunk fragments only) (Figure 7B). I also observed that the boundaries of the characteristic

    pigment-less patches in the eye region have been re-established, due to sufficient body pigment

    deposition in the surrounding tissues. In the spider-1 RNAi worms, however, the newly

    regenerated head and tail contained no detectable recovery of body pigment (n = 6, counting

    trunk fragments only) (Figure 7B). There is no discernible boundary of pigmentation between the

    eye region and the surrounding tissues to be detected, either. Furthermore, even the old body

    parts remained largely unpigmented. These results suggest that hmbs is responsible for the

    maintenance of planarian body pigmentation during homeostasis and for the pigment production

    in newly regenerated tissues, supporting hmbs as the planarian pigment cell marker functionally.

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

    After establishing hmbs as planarian pigment cell markers with morphological and functional

    evidence, I then determined its co-expression with several other spider and punctate markers

    identified in this study. By performing dFISH, I observed that less than 7% of ans positive cells

  • 24

    also express hmbs (Figure 8A). This result indicates that the two genes are mostly not co-

    expressed. On the other hand, dFISH of hmbs and alas shows that 97% of alas expressing cells

    also express hmbs (Figure 8B). Not only does the relative position of the dFISH signals of the

    two genes overlap, but also do the overlapping dFISH signals have similar morphology,

    indicating the two genes are co-expressed.

    I then performed dFISH to determine if punctate markers are co-expressed with each other. Due

    to the low yield of fluorescent signals in Tyramide Signal Amplification (TSA), however, no

    usable data was obtained. Several reasons can account for the low detectability of punctate

    markers by TSA. The punctate gene expression levels are relatively low and the accessibility to

    them by RNA probes might be hindered, depending on the subcellular localization. In addition,

    POD-conjugated antibodies are inherently ineffective at amplifying fluorescent signals from

    weak RNA probes. The enzymatic activity of POD-conjugated antibodies usually diminishes

    after 45 minutes of spiking by H2O2, making detection of punctate gene expression using POD

    extremely difficult. I then tested the method of using two rounds of alkaline phosphatase

    reactions with different fluorescent substrates, to sequentially detect the two punctate genes. This

    attempt also failed, due to the intense background generated by HNPP substrates used in this

    approach. I was able to, however, perform ans in situ alone (n = 5), sds in situ alone (n = 5), and

    in situ of ans and sds combined (n = 5) (with DIG-labeled riboprobes of both transcripts added to

    the same riboprobe mix) in three separate samples (Figure 8D). By counting cells labeled by the

    above riboprobe mixes, I discovered that ans (2472 530 cells/mm2) and sds (2764 128

    cells/mm2) positive cells are of similar density to each other (Figure 8D). Furthermore, when two

    probes are used together, there is no increase in the density of labeled cells comparing to that

    detected by using single probes alone (2484 293 cells/mm2) (Figure 8E). In addition, the

    WISH development of double probes were much quicker and stronger within a similar timeframe

    than that of single probes (Figure 8D, top left panel). These results suggest that the two punctate

    markers, ans and sds, are co-expressed in the same population of cells. Note that the density of

    punctate cells calculated here is the result of counting WISH stains on the epi-fluorescent

    microscope of both the dorsal and ventral sides of the samples, thus, the density is roughly

    doubled of that calculated from confocal images of the dorsal surface only.

    The transcript of a gene in the tryptophan metabolism pathway, kynurenine 3-monooxygenase

    (kmo), was cloned by my collaborator, Dr. Jason Pellettieri (Croucher et al., 2013; Linzen,

  • 25

    Figure 8 Co-expression analysis of spider and punctate markers. A) Most hmbs or ans positive cells do not co-express with each other. B) hmbs and alas are highly co-expressed. C) hmbs and kmo are highly co-expressed. White arrows indicate a lack of co-expression in A), and cells that are co-expressing markers in B) and C). Approximate locations on the dorsal plane of the animals imaged were shown to the right. D) ans and sds expression are similar in cellular population and morphology. WISH with combined ans and sds probes generates similar expression pattern to individual probes without apparent changes in population or morphology. E) The density of positively stained cells in each

    condition shown in D). Error bar: Standard deviation. Scale bar: 100m.

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    1974). kmo shows an expression pattern similar in morphology and density to that of other spider

    markers (Figure 8C). I then performed dFISH between kmo and hmbs and observed that hmbs is

    co-expressed in 96 4.2% of kmo positive cells (Figure 8C). The tryptophan metabolism

    pathway, in which kmo functions, is key to the production of ommochromes, one of the major

    types of pigments found in the animal kingdom. Reports of studies in mosquitos and silk worms

    show that kmo is required for proper pigmentation of the eyes and silk color, in either organism,

    respectively (Han et al., 2003; Osanai-Futahashi et al., 2012). I also tested the expression pattern

    of 6 transcripts involved in tyrosine metabolism, a pathway involved in melanin production, and

    did not find any spider cell-like expression patterns. In summary, the co-expression of kmo and

    hmbs in pigment cells strongly suggests that ommochrome is one of the responsible pigments for

    the body pigmentation of S. mediterranea. This result agrees with previous studies on purified

    planarian body pigment using spectrophotometry in the UV region (Hase et al., 2006).

    3.1.6 Pigment cells show inward migration toward gut during depigmentation in response to light exposure

    Using the established pigment cell marker hmbs, a basal timeline for planarian pigment cell loss

    during acute light exposure was established. I found that planarian pigment cells are lost

    gradually over the course of 10 days under white LED light exposure. Animals were mostly

    depigmented after 7 days of light exposure (Figure 9A). A 12-day light exposure schedule was

    used in this experiment to ensure a complete depigmentation. The population of planarian

    pigment cells, marked by hmbs, gradually reduces over the time course of the light exposure (n

    5 for each time point) (Figure 9B). However, the greatest change in pigment cell population

    was observed after Day 5 during the light exposure. The disappearance of hmbs positive cells is

    slightly slower than the disappearance of pigmentation. The most dramatic change in the body

    pigmentation status is observed before Day 5 (Figure 9A). This discrepancy between

    pigmentation and pigment cells disappearance is likely due to the hypothesized mechanism that

    pigments within individual pigment cells are bleached by light exposure before generating ROS

    and leading to cell death. At the end of the light exposure time course, hmbs expression is not

    detectable in the majority of planarian tissues (Figure 9B, right panel). Throughout the entire

    depigmentation timeline, it is also observed that the head and tail regions are the first to become

    depigmented while the para-pharyngeal region remains pigmented for longer durations (Figure

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    Figure 9 Pigment cells migrate towards gut during depigmentation. A) Representative live images of planarians during light exposure induced depigmentation time course. B) Pigment cells, marked by hmbs, decrease during the time course. Axial cross sections show no hmbs expression in the parenchyma in Day 0 (un-exposed) animals. But hmbs expression is increasingly detected in the parenchyma near the gut as the time course progresses. In the meantime, expression in the sub-epidermal layer decreases. C) Axial cross sections of planarians show the appearance of pigment/pigment cells in the gut in Day 5 light exposed animals.

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    9B). The difference of depigmentation across different regions of the body is evidenced both by

    the direct observation of body pigmentation in live worms and by labeling pigment cells with

    hmbs riboprobes.

    During the light exposure time course, I have observed darkening of the gut in the treated

    animals, especially at 5 days after the start of the light exposure. Comparing the axial cross

    sections of depigmenting planarians with those of the animals kept in dark, there is an evident

    increase of the accumulation of pigment in the gut of depigmenting planarians (n = 5, the number

    of animals used to generate cross sections) (Figure 9C). In certain cross sections of the

    depigmented animals, concentrated spots of pigmentation can be observed in addition to smears

    of pigment in the gut region, suggesting possible excretion of pigment cells instead of pigment

    alone (Figure 9C, right panel). In order to determine if pigment cells are indeed removed through

    gut excretion during light exposure, I performed experiments to detect the changes of hmbs

    positive cells in axial cross sections (n 4, the number of animals used to generate cross

    sections for each time point). In control animals without light exposure, hmbs positive cells are

    only observed in the sub-epidermal region (Figure 9B, left panel). At Day 5 of light exposure,

    however, a substantial amount of hmbs positive cells can be observed ectopically in the central

    parenchyma of the planarians, a region in proximity to the gut (Figure 9B, centre left). On day 7

    of light exposure, an overall reduction in the number of hmbs positive cells can be observed

    (Figure 9B, centre right). And among the remaining hmbs positive cells, the majority are

    positioned ectopically in the central parenchyma region close to the gut. The ectopic expression

    of hmbs expression is exacerbated in the Day 10 animals, where in many cross sections the

    central parenchyma region becomes the only location of hmbs expression (Figure 9B, right). In

    summary, the reduction of pigment cell population, marked by hmbs, and the observation of

    inward migration of pigment cells suggest that an active removal of dying pigment cells through

    gut excretion likely happens during light exposure. These findings agree with previous apoptosis

    analysis using TUNEL by my collaborator, Dr. Jason Pellettieri (Figure 2B, C).

    3.1.7 Pigment cells display fast regeneration timeline during repigmentation

    After 12 days of light exposure, the depigmented animals were placed in dark allowing for

    repigmentation. Whole worm samples were collected during repigmentation (n 5 for Day 1.5

    8.5, n = 2 for Day 12.5). And the regeneration of pigment cells was analyzed using hmbs marker.

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    The time point at the conclusion of light exposure was counted as time point zero for the

    regeneration time course. Sporadic expressions of hmbs can be observed in animal samples

    collected at as early as Day 1.5, suggesting rapid pigment cell regeneration (Figure 10B). A

    gradual increase of pigment cell population can be observed from this time point onwards. As

    early as 7 days into the regeneration, pigmentation spots can be observed on live animals with

    expected pigment cell morphology. After more than 12 days of repigmentation, pigment cells are

    regenerated throughout the entire body of the previously depigmented animals (Figure 10B,

    right). Despite this rapid regeneration of pigment cells, the pigmentation status of planarians at

    this time point is still very low and sporadic (Figure 10A). Only patches of pigmentation can be

    observed while most parts of the body remained unpigmented. This difference in the rate of

    pigment cell regeneration and repigmentation suggests the possible lengthy process of pigment

    production taken by the newly regenerated pigment cells. During the two weeks of

    repigmentation analyzed in this study, pigment cells did not display any preferences or any

    differences of the regeneration rates in the head, trunk or tail regions (Figure 10A, B).

    Figure 10 Rapid regeneration of pigment cells during repigmentation. A) Representative live images of planarians during repigmentation time course following depigmentation. B) Pigment cells, marked by hmbs, are rapidly regenerated during repigmentation. Regeneration of pigment cells precedes the appearance of pigmentation.

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

    In control animals, punctate cells labeled by ans have a density of 1137 71 cells/mm2 in the

    dorsal surface, much higher than the density of 164 44 cells/mm2 for pigment cells labeled by

    hmbs (Figure 11A, B). Only 6.99% (n = 6) of all ans positive cells in control animals also

    express hmbs (Figure 11A, B). Considering the resolution of the imaging equipment and

    technique used, false-positive likely accounts for a substantial portion of the co-expression

    observed. The convincing evidence of substantial co-expression between ans and hmbs comes

    from dFISH experiments at early time points during repigmentation. At Day 1 and Day 3

    repigmentation, 78.2% (n = 3) and 64.6% (n = 4) of ans positive cells co-express hmbs,

    respectively (Figure 11A, B). In two samples at Day 1 repigmentation, almost all hmbs positive

    cells are co-expressing ans (32 of 33 hmbs+ cells for Individual 1, and 13 of 13 for Individual 2).

    This observation strongly suggests ans expression as a key feature of newly differentiated

    pigment cells. This high level of co-expression gradually drops to a moderate level of 27.4% (n =

    2) and 29.7% (n =1) at Day 8 and Day 11 during repigmentation, respectively (Figure 11A, B).

    The repigmentation time course also shows that across all time points and all individual animals

    tested, ans always has a wider expression than hmbs (Figure 11A, B). In summary, the

    percentage of co-expression is highest at the earliest time point of repigmentation following

    punctate and spider cell depletion. And the co-expression decreases as punctate cells are more

    rapidly regenerated than pigment cells.

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    Figure 11 Co-expression of spider and punctate genes decreases along repigmentation time course. A) In control animals, most ans positive cells do not co-express hmbs. High level of co-expression exists early during repigmentation. At later time points, the percentage of co-expression drops. B) Magenta: density of ans positive cells in the dorsal plane. Green: density of hmbs positive cells in the dorsal plane. Gray: Percentage of ans positive cells that are co-expressing hmbs. Error bar: Standard deviation. Scale bar: 100m.

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    3.2 Identified regulators of depigmentation and pigment cell regeneration

    3.2.1 hmbs is not required for pigment cell survival

    Earlier, I showed that RNAi knockdown of hmbs leads to depigmentation of planarians and

    argued the finding to be a functional evidence to establish hmbs as a molecular marker for

    pigment cells. I further analyzed hmbs RNAi knockdown to study its effect on pigment cells and

    related cells (n 8 for each of the stains). It is observed that after 12 feedings of hmbs RNAi, the

    expression level of hmbs transcripts is no longer detectable by WISH (Figure 12A). On the

    contrary, the expression level and the number of cells expressing alas is similar to that in the

    control animals (Figure 12A). As mentioned earlier, alas is a redundant marker to hmbs that also

    labels pigment cells (Figure 8B). I also tested the effect of hmbs RNAi on the punctate marker

    ans, which potentially marks pigment cell progenitors, but found that the expression level and

    the population of positive cells are not affected (Figure 12A). This experiment demonstrates that

    although hmbs is required for pigment production and maintenance, it is not required for the

    survival of pigment cells or their potential progenitor cells.

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

    Several previous studies suggested that various types of animal pigment can produce reactive

    oxygen species (ROS) in response to light exposure. At high levels, ROS is detrimental to

    cellular functions and can lead to cell death (Shen et al., 2015; Pospil et al., 2014). I

    hypothesized that ROS produced by planarian pigment causes pigment cell death and removal

    during light exposure. It has been established that hmbs RNAi leads to pigment loss without loss

    of pigment cells. Thus, working with hmbs RNAi animals, I investigated the effects of light

    exposure on pigment cells without the presence of pigment. Animals were fed with control and

    hmbs RNAi food for 10 times. At the conclusion of RNAi feedings, the hmbs RNAi animals

    appeared almost unpigmented while the control animals displayed normal body pigmentation (n

    = 10 for both). The RNAi animals were then treated with light exposure for 9 days, a period that

    is sufficient to completely depigment the control animals. The hmbs RNAi animals remained

    unpigmented throughout the light exposure treatment. I then determined the population of

    pigment cells in the light exposed animals using the secondary pigment cell marker alas. As

    predicted, almost no pigment cells were detectable by WISH in the control RNAi animals

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    Figure 12 hmbs is not required for pigment cell survival and its RNAi offers protection from light exposure. A) WISH analysis of pigment cell lineage markers in control and hmbs RNAi animals. hmbs RNAi results in undetectable levels of hmbs transcripts by WISH, but does not affect the survival of pigment cells, marked by alas, or pigment cell progenitors, marked by ans. B) hmbs RNAi animals retain a population of pigment cells, marked by alas, after 9 days of depigmentation,

    while control animals lose all pigment cells. Scale bar: 100m.

    (Figure 12B). In the hmbs RNAi worms, despite appeared as unpigmented, the expression level

    of alas remained high in individual cells and the population of alas positive cells remained at a

    comparable level to untreated animals (Figure 12B). These results support the hypothesis of ROS

    generated by body pigment in response to light being the cause of planarian depigmentation and

    pigment cell loss after light exposure.

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    3.2.3 epimerase positively regulates depigmentation in response to light

    Transcript SmedASXL_011154, encoded by a gene that is homologous to epimerase, is

    exclusively expressed in the triclad-gut of planarians (Figure 4, top row, left panel). Epimerases

    are isomerase enzymes responsible for the inversion of the stereochemistry of biological

    molecules. They are reported to be involved in a wide variety of biological processes, including

    sugar metabolism and protein sialylation, which are important for signal transduction and cell

    surface adhesions (Daude et al., 1995; Keppler et al., 1999). This gene is observed to be 5 times

    down-regulated in depigmented animals according to the RNAseq data. After 10 feeds of RNAi,

    I performed light exposure treatment on the RNAi animals. At Day 5 of the light exposure

    treatment, the majority of the pigmentation of the control animals has disappeared (Figure 13A).

    Although a limited population of pigment cells generally remains at this time point (Figure 9B),

    the worms appeared mostly colorless (Figure 13A). On the other hand, the epimerase RNAi

    worms at Day 5 depigmentation retained most of their body pigmentation (Figure 13A). The

    difference of de