THE GENE EXPRESSION UNDERPINNINGS for the INDEPENDENT EVOLUTION of a DROSOPHILA PIGMENTATION TRAIT Dissertation Submitted To

THE GENE EXPRESSION UNDERPINNINGS FOR THE INDEPENDENT EVOLUTION OF

A DROSOPHILA PIGMENTATION TRAIT

Dissertation

Submitted to

The College of Arts and Sciences of the

UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for

The Degree of

Doctor of Philosophy in Biology

Sumant Grover M.Sc.

Dayton, Ohio

December 2018

THE GENE EXPRESSION UNDERPINNINGS FOR THE INDEPENDENT EVOLUTION OF

A DROSOPHILA PIGMENTATION TRAIT

Name: Grover, Sumant

APPROVED BY:

Thomas M. Williams, Ph.D. Faculty Advisor

Mark Nielsen, Ph.D. Committee Member

Amit Singh, Ph.D. Committee Member

Madhuri Kango Singh, Ph.D. Committee Member

John Yoder, Ph.D. Committee Member

Sumant Grover

2018

iii

ABSTRACT

THE GENE EXPRESSION UNDERPINNINGS FOR THE INDEPENDENT EVOLUTION OF

A DROSOPHILA PIGMENTATION TRAIT

Name: Grover, Sumant University of Dayton

Advisor: Dr. Thomas M. Williams

An innumerable number of animal species has evolved since the Cambrian period 541-485.4 million years ago. There are 35 disparate body plans of the animal phyla, for which evolutionary developmental biologists are fascinated by their wealth of morphological traits that evolved to adorn species with these varied body plans. This fascination has driven the community of scientists to seek answers to developmental questions, such as which genes’ expressions shape the formation of said traits, and evolutionary questions, such as how did these required genes become expressed in the proper manner. Lessons from genomics has revealed the general insight that animals of the same phylum possess a shockingly similar set of regulatory genes that control gene expression, and this insight is largely true for the differentiation genes whose encoded proteins contribute to building the traits. Lessons from genetics has revealed that gene expression is controlled by DNA sequences known as cis-regulatory elements (CREs) through their possession of binding sites for a combination of transcription factor proteins. For any given trait, CREs and their interacting transcription factors connect a set of genes together into a Gene Regulatory

Network (GRN) that executes orchestrated patterns of gene expressions that lead to the trait’s

iv formation. Thus, in order to understand how morphological traits evolve, it is essential to understand how GRNs and CREs evolve. A deeper understanding of the mechanisms of morphological evolution would include determining whether and under what circumstances evolution favors the modifications of certain genes over others. In order to advance the understanding of morphological evolution it is advantageous to study traits that have evolved more recently so that GRNs, genes, and CREs can be closely compared, and traits that have evolved on multiple occasions to check for any preferences in the genetic targets of evolutionary change. Male-specific patterns of melanic abdominal pigmentation among fruit flies of the

Drosophila genus are one such ideal trait, which were the focus of my thesis research.

Chapter I presents and overview of evolutionary developmental biology, the charge of this scientific community to reveal an understanding of how morphology evolves, and an introduction to the pigmentation patterns of fruit flies and their utility as a model trait to gain general insights on how GRNs and CREs have shaped the emergence of novel patterns of pigmentation. Chapter II presents research on how the male-specific melanic pigmentation evolved in the Sophophora subgenus through the use of the differentiation genes pale and Ddc.

The ancestral expression pattern for pale was found to be conserved, whereas Ddc expression underwent a substantial change that lengthened the duration and level of expression during pupal development of species from lineages with darker (melanic) abdomens. Through reporter transgene assays, we were able to identify the CRE driving this evolved pattern of Ddc expression. We showed that this CRE is pleiotropic regulated by the Grainy head transcription factor, and its ancestral function is to activate Ddc expression in response to epidermal wounding.

Chapter III presents research seeking to understand the gene expression underpinnings for the independent evolution of male-specific abdomen pigmentation possessed by both D. melanogaster and D. funebris, species that descended from a common ancestor around 60 million years ago. Through in situ hybridization assays we revealed patterns of mRNA expression for many of the key pigmentation (differentiation) genes that encode enzymes involved in a pigment

v metabolic pathway. We found that strikingly similar patterns of pigmentation gene expression

(yellow, tan, ebony, pale, and Ddc) occur for both D. funebris and D. melanogaster. At the level of regulatory genes, striking differences were found. While the transcription factor Bab1 is expressed in the female abdomen of D. melanogaster where it function to turn off the expression of pigmentation genes needed to make melanic pigments, in D. funebris Bab1 is expressed in the abdomens of both males and females. Thus, a unique transcription factor gene or genes are playing the equivalent role to Bab1 in this species pigmentation GRN. The Hox gene Abd-B is expressed in the A5 and A6 abdomen segments of D. melanogaster, where it is necessary for the development of the broad melanic pigmentation of the segments of males. In D. funebris, Abd-B expression appears to extend anterior to segment A5, including segments A4 and A3. These segments are more elaborately pigmented in D. funebris males, suggesting that the expansion of

Abd-B expression contributes to this species pattern of male-specific pigmentation. Collectively, the results of this chapter indicate that convergent evolution involved the similar deployment of pigmentation genes through the activity of a novel regulatory gene or genes, and through a different use of a common regulatory gene. This research opens avenues for future research that will make more in depth comparisons of convergent GRNs, including the depths of CREs and their interacting transcription factors.

Dedicated to my parents and elder brother

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TABLE OF CONTENTS

ABSTRACT ...... iv

DEDICATION ...... vii

LIST OF FIGURES ...... x

LIST OF TABLES ...... xiii

CHAPTER I: INTELLECTUAL FOUNDATION FOR AN INVESTIGATION INTO THE REPEATED EVOLUTION OF A FRUIT FLY PIGMENTATION TRAIT ...... 1

Morphological Diversity and the Generally Conserved Genetic Toolkit ...... 1

Genes and Gene Regulatory Networks in the Development of Morphology ...... 5

Genes and Gene Regulatory Networks in the Evolution of Morphology ...... 7

Fruit Fly Pigmentation as a Model Trait to Study Morphological Evolution ...... 8

CHAPTER II: AUGMENTATION OF A WOUND RESPONSE ELEMENT ACCOMPANIES THE ORIGIN OF A HOX-REGULATED DROSOPHILA ABDOMINAL PIGMENTATION TRAIT ...... 13

Abstract ...... 13

Introduction ...... 14

Materials and Methods ...... 16

Results ...... 21

Discussion ...... 41

Acknowledgements ...... 46

Supplementary Information ...... 48

CHAPTER III: INVESTIGATING THE SIMILARITIES AND DIFFERENCES IN THE GENE REGULATORY NETWORKS FOR CONVERGENT FRUIT FLY PIGMENTATION TRAITS ...... 56

viii

Abstract ...... 56

Introduction ...... 56

Materials and Methods ...... 60

Results ...... 63

Discussion ...... 70

BIBLIOGRAPHY ...... 78

APPENDIX A Alignment of the Ddc-MEE1 with binding site mutant versions ...... 88

APPENDIX B Sequence alignment of Ddc-MEE1 with scanning mutant versions...... 91

APPENDIX C Alignment of sequence orthologous to the D. melanogaster Ddc-MEE1 ...... 97

LIST OF FIGURES

FIGURE 1.1 Representation of genes in a Gene Regulatory Network governing the development of a hypothetical morphological trait...... 4

FIGURE 1.2 A simplistic representation of how changes in GRN at the level of a

CRE might lead to a novel pattern of gene expression and morphology ...... 6

FIGURE 1.3 Phylogenetic representation of diverse body color pattern for different

Drosophila species ...... 12

FIGURE 2.1 Pigmentation phenotypes and enzymatic pathway in the Sophophora

Subgenus ...... 23

FIGURE 2.2 The spatial and temporal expression pattern of D. melanogaster pale and Ddc ...... ….25

FIGURE 2.3 pale abdominal epidermis expression is broadly conserved in

Sophophora ...... 27

FIGURE 2.4 Ddc is broadly expressed in the abdominal epidermis of species with elaborate patterns of tergite pigmentation ...... 29

FIGURE 2.5 D. willistoni exhibits little to no Ddc expression in the abdominal epidermis of various pupal stages through eclosion ...... 30

FIGURE 2.6 An abdominal epidermis cis-regulatory element is located 5’ of the

endogenous Ddc promoter ...... 33

FIGURE 2.7 A Grh binding site is a required input in the regulatory logic driving

Ddc abdominal epidermis CRE activity...... 35

FIGURE 2.8 Male-specific pigmentation requires the activity of Grh whose

x expression has remained conserved ...... 37

FIGURE 2.9 Genetic interactions between pigmentation network transcription factors

and CREs regulating tan and Ddc expression ...... 39

FIGURE 2.10 The ancestry of the robust abdominal epidermis regulatory activity of the CRE controlling Ddc expression ...... 41

FIGURE 2.11 The evolution of a novel pigmentation trait required cis-regulatory evolution at several realizator loci ...... 46

FIGURE S2.1 Morphological markers used to differentiate the pupal stages of samples subjected to gene expression analyses ...... 48

FIGURE S2.2 Evaluation of binding site mutations on the activity of the Ddc-MEE1 ... 49

FIGURE S2.3 Mapping functional Ddc regulatory sequences through CRE scanning mutagenesis ...... 50

FIGURE S2.4 Truncated forms of the Ddc-MEE1 CRE possess most of the pattern and

activity of the large CRE version ...... 51

FIGURE S2.5 The D. melanogaster Ddc-MEE1 has a more robust response to ectopic

Abd-B ...... 52

FIGURE 3.1 Male specific pigmentation independently evolved in fruit flies ...... 59

FIGURE 3.2 The known tergite pigmentation Gene Regulatory Network for

Drosophila melanogaster...... 60

FIGURE 3.3 The expression profiles for the differentiation genes responsible for the

D. melanogaster tergite pigmentation pattern...... 65

FIGURE 3.4 The expression profiles for the differentiation genes responsible for the

D. funebris tergite pigmentation pattern ...... 66

FIGURE 3.5 Bab1 expression differs between D. melanogaster and D. funebris ...... 68

FIGURE 3.6 Comparison of Abd-B expression between D. melanogaster and D. funebris ...... 70

FIGURE 3.7 Hypotheses and their implications for possible patterns of D. funebris

Abd-B expression ...... 75

FIGURE 3.8 Model for the tergite pigmentation Gene Regulatory Network of

Drosophila funebris ...... 77

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LIST OF TABLES

TABLE S 2.1. Primers used to create in situ probes to orthologous Ddc and pale gene sequences...... 53

TABLE S 2.2 Primers used to clone D. melanogaster Ddc gene non-coding sequences for use in reporter transgene assays to test for cis-regulatory element (CRE) activity ..... 54

TABLE S.2.3 Primers used to create reporter transgenes with sequences orthologous to the D. melanogaster Ddc MEE1 cis-regulatory element ...... 55

TABLE 3.1 Primers used to PCR-amplify templates for generating antisense in situ hybridization probes ...... 63

xiii

CHAPTER I

INTELLECTUAL FOUNDATION FOR AN INVESTIGATION INTO THE REPEATED

EVOLUTION OF A FRUIT FLY PIGMENTATION TRAIT

Morphological Diversity and the Generally Conserved Genetic Toolkit

It has been estimated that there are approximately 8.7 million species on planet Earth, many which still remain to be discovered (Mora et al., 2011), all descending from a common ancestor at some point in Earth’s history. For animal species, their history dates back past the

Cambrian time period some 540 million years ago (Gould, 1989). Since the Cambrian, there have been at least 35 disparate body plans that can be identified (Carroll et al., 2004), and each body plan is considered to be a prototype for one of the 35 recognized animal phyla. Animal species sharing a similar body plan are grouped together into a phylum. Within each phylum, dozens to upwards of a million species have evolved that possess varying degrees of morphological diversity that decorate and individualize the common body plan. While the evolutionary and genetic underpinnings of animal disparity and diversity are both compelling, diversity has been the dominant theme since the Cambrian, and is the focus of this thesis.

Arguably, the most successful phylum based upon the number of species is Arthropoda.

This phylum includes over 1.1 million extant species that can be grouped into the lower taxonomic rank of subphyla, which include hexapoda, myriapoda, crustacean, and chelicerata.

Each subphyla can be successively divided into lower and lower taxonomic ranks of class, order, family, genus, and species. Arthropod species share some common features, such as segmented bodies with a head, thorax and abdomen, presence of jointed appendages, and an exoskeleton made up of chitin (Ruppert et al., 2004). In comparison, the phylum Chordata on the other hand,

1 possess over a hundred thousand species with features that are disparate from Arthropods, like the presence of a notochord, pharyngeal gill slits, and dorsal hollow nerve tube among others.

Lower and lower taxonomic ranks can be characterized by species possessing body plans that generally share fewer and fewer unique morphological traits that distinguish the overall phylotypic body plan, and that have evolved in more recent time frames. Arthropods show enormous diversity in body segment features. One such example are wings, which are found on species from the subclass Pterygota. This subclass include Beetles, which belong to the order

Coleoptera, and which have a highly modified front pair of wings (forewings) that are hardened structures called elytra that function to protect the hindwings (Wootton, 1992). Wing possessing insects from the order Diptera, like fruit flies, on the other hand have forewings that are used for flight and have highly modified hindwings, called halters, which are oscillating inertial organs(Wootton, 1992). Yet another striking modification of wings is a characteristic of the order

Lepidoptera (butterflies and moths), specifically forewings and hindwings which are covered by scales instead of hairs.

Morphological traits are made during the timeframe of development. Thus, in order to understand how morphological diversity evolves it is helpful to understand how morphology develops. Animal genomes contain several thousands of genes, for example the fruit fly species

Drosophila (D.) melanogaster has a nuclear genome with ~17,000 genes (Oliver and Leblanc,

2003) (Clark et al., 2007). In comparison, the human genome has ~22, 000 genes (Ezkurdia et al.,

2014), showing that vastly divergent animal species differ in gene content by less than an order of magnitude. This wealth of genes can be partitioned into three different categories (Carroll et al.,

2004). These include housekeeping genes, which encoded proteins that function in nearly all cell types, such as those that are a part of the DNA replication machinery. Differentiation genes, such as genes whose encoded protein functions in one or few cell types, such as the globin genes that function in red blood cells. Toolkit genes, which encoded proteins that regulate gene expression and that can be used in combinations with other toolkit genes to regulate the

2 expression of different genes and thereby participate in the development of a multitude of morphological traits. Toolkit genes prominently include those that encode transcription factor proteins that can bind to DNA sequences of the genes it regulates.

For the development of any given trait, only a subset of the species’ genes are utilized, a subset that can be considered to be a part of a Gene Regulatory Network or GRN (Figure 1.1).

Each network is hierarchical in its structure, with toolkit genes occupying the higher “regulatory” tiers of the network. This can include genes that comprise a signaling pathway (e.g. encode signaling ligands and their cognate receptors), and transcription factor genes that encode proteins that possess DNA binding domains which allow them to bind to sites within cis-regulatory elements or CREs of the genes whose expression are being activated or repressed (Figure 1.1).

The regulated genes can be other toolkit genes, but ultimately a GRN controls the expression of the differentiation genes that make the trait and are essentially the lowest GRN tier. This understanding that development involves the use of a GRN raises the question as to how these

GRNs evolve in route to a novel morphological trait. Arguably, the greatest surprise of the first

25 years of evolutionary-developmental biology research was the finding that animals within the same genus and even higher taxonomic ranks possess many of the same differentiation genes and toolkit genes (Carroll, 2008). Simply put, it is a reasonable approximation to expect that closely related species possess the same genes. Moreover, the encoded proteins for genes have generally been found to have remained conserved too, to the point where the equivalent gene from a distantly related species can function the same as when inserted into a host with a dramatically

Figure 1.1. Representation of genes in a Gene Regulatory Network governing the development of a hypothetical morphological trait. (A) Protein from a ligand gene (LG1) activates a signaling pathway resulting in the regulated expression of target genes that happen to be transcription factors (TF1 and TF2 in this scenario). TF1 and TF2 then activate and repress additional target genes, which can include other transcription factors, but ultimately the differentiation genes (DG) responsible for the formation of a trait such as mechanosensory organs. The regulation of any target gene occurs through interactions between combinations of transcription factors and binging sites sequences within a CRE. (A’) Key for the symbolism used in panel A. (B) This GRN drives spatial and temporal patterns of differentiation gene expression that ultimately results in the formation of the mechanosensory organ morphological trait.

different body plan or body plan features (Carroll, 2008). Therefore, if the gene content and the encoded proteins are not readily changing, then perhaps it is the CREs controlling gene expression (Figure 1.2). Over the past 15 years, a wealth of data from various animal species lead to first the suspicion that CRE evolution is a prominent feature for morphological evolution

(Carroll, 2008), to case studies demonstrating specific instances where indeed this is true (Martin and Orgogozo, 2013). However, it remains less well understood how entire GRNs first evolve, and whether evolution can be more predictable than expecting the involvement of gene expression changes through CRE evolution. Specifically, can we anticipate certain genes being involved in the evolution of specific traits? Such outcomes would suggest that while natural

4 selection is creative, there are some favored genetic paths for the evolution of specific traits

(Stern, 2010).

Genes and Gene Regulatory Networks in the Development of Morphology

A gene can be simply defined as a region of DNA that is inheritable and possesses information encoding a protein or a functional RNA. The process by which a gene has its final product made, translated proteins or a transcribed functional RNA, is referred to as gene expression, a process which is highly regulated both in space (cell or tissue type) and lifetime

(Davidson, 2006). For protein coding genes, their structure is generally of two basic components, coding and non-coding regions. The coding region possesses codon for the string of amino acids included in the functional protein molecules. Non-coding regions are stretches of DNA that do not code for protein. These may be non-coding RNA, introns, or sequences upstream or downstream of a gene.

Within non-coding DNA are sequences of another important functional class, collectively known as cis-regulatory elements or CREs. These sequences behave like switches through interactions with transcription factor proteins to regulate the expression of its target gene or genes (Figure 1.2). CREs that activate gene expression are often referred to as enhancers and those that repress expression are referred to as silencers (Johnson et al., 2015; Papatsenko et al.,

2009). A general property of CREs (schematized in Figure 1.1A) is the possession of DNA sequences that act as binding sites for a transcription factor protein. The specific activity of a

CRE arises from the combination of interacting transcription factors which is often referred to as a regulatory logic (Arnone and Davidson, 1997).

Figure 1.2: A simplistic representation of how changes in GRN at the level of a CRE might lead to a novel pattern of gene expression and morphology. (A) Ancestral CRE logic has too few transcription factor binding sites (colored shapes) to drive a pattern of Gene A expression in cells of the fruit fly abdomen. The gain of new binding sites by DNA sequence mutations leads to the emergence of a pattern of Gene A expression in the posterior fruit fly abdomen. (B) In the absence of Gene A expression, the ancestral state of the abdomen is non-melaninc. The gain of novel Gene A expression results in a derived melanic phenotype.

In order to make some generalizations about GRNs, I made a schematic for a hypothetical GRN whose developmental use is to generate the mechanosensory organs decorating a fruit fly body (Figure 1.1). After receiving input from signaling molecule, here LG1, several transcription factors become expressed (TF1 and TF3) which repress the differentiation genes specific for cell types (DG2 and DG6). Likewise, the expression of the transcription factors TF2,

TF4, TF5 and TF6 and their regulatory inputs leads to the expression of a set of differentiation genes including DG3 (Figure 1.1A) in specific mechanosensory organ precursor cells during development. The expression of DG3 plays a necessary role in the formation of mechanosensory

6 organs (Figure 1.1B). DG3’s expression pattern is mediated by a regulatory logic of transcription factors binding to sequences (called binding sites) within a CRE or CREs for this gene. This hypothetical mechanosesnory organ GRN is portrayed in overly simple manner compared to the complexity of well-studied GRNs and CRE regulatory logic (Arnone and Davidson, 1997; Bonn and Furlong, 2008a; Swanson et al., 2010). In general, finding CREs and resolving their regulatory logic in the context of a GRN is a quite challenging and time consuming.

Genes and Gene Regulatory Networks in the Evolution of Morphology

The formation of a trait during development is complex and requires the precise action of numerous genes in a GRN whose encoded proteins are expressed through combinations of transcription factors interacting with CREs (Levine and Davidson, 2005a). This complexity begs the question of how GRNs evolve? One conceivable type of changes is in the protein coding sequences of genes, as in the case of the origin of the vertebrate glucocorticoid receptor following a gene duplication event (Ortlund, 2008). However, a vast body of data has shown that function protein coding evolution has been rather rare, as the biochemical activity of toolkit and differentiation genes has generally remained conserved over long evolutionary timespans in large part because the encoded proteins are highly pleiotropic and changes to the proteins often encumber negative pleiotropic consequences (Carroll, 2005; Carroll, 2008).

While genes and their encoded proteins are often pleiotropic, gene expression patterns are often driven by multiple modular CREs, with each CRE driving one or a few aspects of the overall expression pattern. This makes CREs generally less pleiotropic than the protein coding sequences, and seemingly more tolerable for function modifying mutations (Carroll, 2005). Thus, it can be expected that a major way in which GRNs can evolve are through gene expression changes driven by the gain, loss, or modification of CREs. As a hypothetical example, considering Gene A, it is conceivable that an ancestral pattern of expression is absent from a body region, such as the fruit fly abdomen, due to a regulatory sequence region lacking the necessary

7 set of transcription factor binding sites (Figure 1.2A). Mutations that add additional binding sites to this region may result in a novel pattern of Gene A expression in the abdomen. If Gene A plays a key differentiation gene function, such as making black melanin pigments, the abdomen may evolve a derived melanic phenotype (Figure 1.2B). This example makes it seem as though the gain of single novel pattern of gene expression is enough for trait evolution. However, in reality it can be anticipated that such changes will be required for many genes and CREs in order for a sophisticated GRN to take form from scratch (Monteiro and Gupta, 2016). There is an alternative to the from scratch origin of GRNs, this being through the co-option of a master regulatory gene whose novel use might redeploy an existing GRN in a new context. Co-option is suspected to under the origin of beetle horns (Moczek and Rose, 2009), and the fruit posterior genital love

(Glassford et al., 2015). Ultimately, understanding how GRNs and CREs evolve during bouts of trait evolution requires empirical studies for which the genetic and molecular details of trait evolution can be teased out. Moreover, it is of great interest to understand whether the genetic targets of change are more or less random, or alternatively predisposed – favored genes for certain traits. To achieve such an understanding requires empirical studies to include traits that have evolved on multiple independent occasions to see whether the same or unique genes are staring in the evolutionary process (Kopp, 2009).

Fruit Fly Pigmentation as a Model Trait to Study Morphological Evolution

While the diversification of wings among pteygotes insects has evolved an impressive diversity and facilitated an impressive extent of ecological success, this trait is limited for studies of CRE and GRN evolution. CREs evolve at an accelerated pace (Hersh and Carroll, 2005) and the ability to compare the same DNA sequences (orthologous) for species from different orders is troubled by the overwhelming difference in sequence content (Prud’homme et al., 2007). As a consequence, the precise changes creating or modifying CREs and thereby wiring or rewiring a

GRN are essentially unresolvable. The problem is even more acute for traits that differ above the

8 level of orders. Therefore, depth in understanding of how CREs and GRNs evolve requires focusing on morphological traits that differ between species of lower taxonomic rank, such as the genus or subgenus (Stern, 2000).

One suitable trait is the body coloration patterns decorating the dorsal abdomens of fruit flies (Figure 1.3). Male and female fruit flies have six abdominal segments (A1-A6) that are not part of the genitalia. The dorsal surface of each segment is covered by a chitinous cuticle plate known as a tergite. During the ~60 million years of the Drosophila genus, the color and pattern of tergite pigmentation has widely diversified (Wittkopp et al., 2003). Drosophila species share essentially the same toolkit and differentiation genes that are involved in pigment metabolism

(Clark et al., 2007). This includes genes such as yellow and tan whose expression are needed to make black melanin pigments, and the gene ebony whose expression is needed to make more yellow colored cuticle (Wright, 1987). Other pigmentation genes include pale and Ddc, whose expression is thought to be essential for making both black and yellow colored cuticle.

One excellent evo-devo trait is the male-specific melanic (black) coloration of the posterior A5 and A6 tergites of species such as D. melanogaster and D. biarmipes (Figure 1.3) from the melanogaster species group. In contrast to males, pigmentation of the female tergites is generally limited to a posterior stripe, similar to what is present on the anterior tergites of both males and females. This trait is recognized to be the derived state, evolving from an ancestor with a monomorphic pattern of pigmentation, perhaps similar to the species D. willistoni (Rebeiz and

Williams, 2017a) (Figure 1.3). Based on this understanding of trait evolution, D. willistoni can be used as a surrogate for the monomorphic ancestor that no longer exists, allowing comparisons to be made to its tergite pigmentation GRN and the GRN of D. melanogaster. It has been shown that male-specific patterns of yellow and tan expression in the A5 and A6 segments evolved through the origin of novel CRE activities, and that the absence of ebony expression from the male A5 and A6 segments is also due to the activities of CREs (Camino et al., 2015; Rebeiz, 2010).

While the encoded pigmentation genes are needed to carry out the pigment metabolic pathway, their patterns of expression are set by the activity of the upstream regulatory genes of the GRN. For D. melanogaster, the regulatory tier of the GRN has received some attention. An

RNA-interference (RNAi) screen found 28 transcription factor genes to generate tergite pigmentation phenotypes following the reduction of their expression (Rogers et al., 2014).

Among these 28, the paralogous Bab1 and Bab2 (collectively called Bab) transcription factors are of considerable importance for the sex-specificity of this trait. Bab is expressed in a sexually dimorphic manner, with expression occurring throughout the female abdomen epidermis, but lacking in the male abdomen during pupal development when the tergite pigmentation pattern is being specified (Salomone et al., 2013a). Bab functions as dominant repressors of melanic pigmentation (Couderc et al., 2002; Kopp et al., 2000), which occurs in part by these proteins binding to the body element CRE of the yellow gene and repressing its activity in females

(Roeske et al., 2018). Bab is thought to turn off tan expression in females by exerting its influence on the t_MSE CRE by a mechanism that remains to be resolved. Broad melanic pigmentation of D. melanogaster males is spatially limited to the A5 and A6 segment tergites.

This spatial selectivity relies upon the activity of the Hox gene Abd-B that encodes a DNA- binding transcription factor. Abd-B protein is expressed in the abdominal epidermis of male and female pupae (Kopp and Duncan, 2002; Wang and Yoder, 2012), and in males where Bab is not expressed it can bind to two sites within the body element CRE to activate yellow expression

(Jeong et al., 2006). Abd-B has also been found to activate expression of tan through the t_MSE

CRE, however this positive role does not appear to involve direct interaction with binding site sequences (Camino et al., 2015).

This progress in elucidating the D. melanogaster tergite pigmentation GRN has provided many insights as to how a novel GRN evolved through gene expression and CRE evolution

(Rebeiz and Williams, 2017a). In Chapter 2 of my thesis, I directed my attention to the pigmentation genes pale and Ddc to understand their roles in this GRNs evolution. I found that

10 pale expression existed ancestrally, whereas Ddc expression was ancestrally weak and evolved to be more intense during an extended period of pupal development. With this recognition that Ddc expression evolved, I explored the CRE basis, and identified an ancestral CRE regulating the epidermal wound repair activity of Ddc (Mace et al., 2005)was augmented to drive this extended and robust activity during the development of tergite pigmentation. Thus, for D. melanogaster the tergite pigmentation GRN required evolved expression of Ddc, tan, yellow, and ebony that respond to upstream regulators such as Bab and Abd-B. In Chapter 3, I pursed the question of whether evolution was biased to modify certain genes or not. I found that the expression profiles for the pigmentation genes of D. funebris, closely matched the patterns seen in D. melanogaster.

D. funebris is from a distantly-related clade of fruit flies to the clade for D. melanogaster and D. willistoni, and which possesses a male-specific pattern of tergite pigmentation which is considered to have evolved independently (Figure 1.3) (Gompel and Carroll, 2003). This similarity in pigmentation gene expression suggests that tergite pigmentation evolution is biased to utilize these genes in a very similar manner. In contrast to the similarity in pigmentation gene expressions, Bab expression was found to be sexually monomorphic in D. funebris, and Abd-B expression appears to extend into more anterior segments. Thus, at the level of the regulatory tier, differences seem to exist suggesting GRN evolution may be more flexible at this level.

Figure 1.3: Phylogenetic representation of diverse body color pattern for different Drosophila species. The most recent common ancestor of all these fruit flies existed approximately 60 million years ago. This ancestor is likely to have possessed a sexually monomorphic pattern of abdominal tergite pigmentation. While several species (D. melanogaster, D. biarmipes, and D. funebris) have derived sexually dimorphic pigmentation patterns, others (D. willistoni) have the suspected ancestral monomorphic pattern. The dimorphic patterns of D. melanogaster and D. funebris wee inferred to have evolved independently (Gompel and Carroll, 2003).

CHAPTER II

AUGMENTATION OF A WOUND RESPONSE ELEMENT ACCOMPANIES THE ORIGIN

OF A HOX-REGULATED DROSOPHILA ABDOMINAL PIGMENTATION TRAIT

Abstract

A challenge for evolutionary research is to uncover how new morphological traits evolve the coordinated spatial and temporal expression patterns of genes that govern their formation during development. Detailed studies are often limited to characterizing how one or a few genes contributed to a trait’s emergence, and thus our knowledge of how entire GRNs evolve their coordinated expression of each gene remains unresolved. The melanic color patterns decorating the male abdominal tergites of Drosophila (D.) melanogaster evolved in part by novel expression patterns for genes acting at the terminus of a pigment metabolic pathway, driven by cis-regulatory elements (CREs) with distinct mechanisms of Hox regulation. Here, we examined the expression and evolutionary histories of two important enzymes in this pathway, encoded by the pale and

Ddc genes. We found that while both genes exhibit dynamic patterns of expression, a robust pattern of Ddc expression specifically evolved in the lineage of fruit flies with pronounced melanic abdomens. Derived Ddc expression requires the activity of a CRE previously shown to activate expression in response to epidermal wounding. We show that a binding site for the

Grainy head transcription factor that promotes the ancestral wound healing function of this CRE is also required for abdominal activity. Together with previous findings in this system, our work shows how the GRN for a novel trait emerged by assembling unique yet similarly functioning

CREs from heterogeneous starting points

Introduction

A major challenge for evolutionary developmental biology research is to explain how novel morphological traits come into existence through genetic changes and their underlying molecular mechanisms. The reigning paradigm for trait development involves the action of a gene regulatory network (GRN) in which a hierarchy of regulatory genes, encoding signaling pathway and transcription factor proteins, drive the expression of a set of realizator genes whose encoded proteins drive the physical manifestation of the trait (Davidson, 2006; Levine and Davidson,

2005b). The expression patterns of a GRN’s genes are specified by the binding of transcription factors to cis-regulatory elements (CREs) that function as enhancers or silencers of transcription

(Arnone and Davidson, 1997; Gray and Levine, 1996; Johnson et al., 2015; Stanojevic et al.,

1991). As GRNs are generally complex in realizator and regulatory gene number (Bonn and

Furlong, 2008b), the origin of a novel trait requires an explanation regarding how the full set of realizator genes arrived at their appropriate patterns of expression (García-Bellido, 1975).

A multitude of possible scenarios could explain the origination of complex GRNs, and these differ in terms of their number and distribution of necessary genetic modifications. For instance, if a top-level regulatory gene of an ancestral GRN evolves a novel domain of expression, possibly by changing a single CRE, this could co-opt an entire GRN through the pleiotropic actions of this single gene, (Monteiro and Gupta, 2016; Rebeiz and Tsiantis, 2017). In contrast, changes may be distributed throughout a GRN, involving the gain, modification, or loss of dedicated CREs for each realizator gene in the network. In order to distinguish whether the origins of novelty tend to emerge through such short or long evolutionary paths, it is necessary to characterize how GRNs and their constituent CREs evolve for a myriad of novel morphological traits in diverse lineages.

The coloration patterns adorning the cuticle of insects present ideal traits for investigation as a great diversity phenotypes exist within a tractable phylogenetic framework in which ancestral and derived character states can be reasonably inferred (Jeong et al., 2006; Wittkopp et al., 2003).

One experimentally tractable coloration trait is the male-specific melanism of the dorsal tergites that cover the 5th and 6th abdominal segments of Drosophila (D.) melanogaster (Rebeiz and

Williams, 2017a). This dimorphic trait is suspected to have evolved from a monomorphic ancestral state in the Sophophora subgenus (Figure 2.1A) (Jeong et al., 2006). In D. melanogaster, cuticle pigmentation requires the activities of several realizator genes whose protein products function in a tyrosine metabolic pathway that can produce yellow sclerotin or a black melanic cuticle (Figure 2.1B) (Wright, 1987). In males, the realizator genes yellow and tan are highly expressed in the A5 and A6 segment epidermis underlying the developing tergites, expression of which is necessary for the formation of the black tergite color (Camino et al.,

2015). In females, the realizator gene ebony is highly expressed in the A5 and A6 segment epidermis (Rebeiz et al., 2009) which leads to broadly yellow tergites. The CREs responsible for the sex- and segment-specific expression patterns of yellow, tan, and ebony have been identified

(Jeong et al., 2008b; Rebeiz et al., 2009; Wittkopp et al., 2002), as well as some of the direct interacting transcription factors (Camino et al., 2015; Jeong et al., 2006; Roeske et al., 2018).

Notably, yellow expression is directly activated by the Hox factor Abd-B binding to sites within the body element CRE, whereas the limitation of tan expression to 5th and 6th segments involves the direct binding of Abd-A and Hth to sites within the t_MSE to facilitate repression in the more anterior abdomen segments. Although tan, yellow, and ebony play essential roles in making black and yellow pigments, the pigment metabolism pathway involves several other key genes whose expression and regulation need to be understood within the context of this trait’s development and evolution.

The first steps to forming catecholamine pigments are the conversion of Tyrosine to

Dopa and then to Dopamine through the activity of Tyrosine Hydroxylase (encoded by the gene pale) (Neckameyer and White, 1993), and Dopa Decarboxylase (encoded by Ddc), respectively

(Figure 2.1B) (Wright, 1987). pale and Ddc differ from yellow, tan, and ebony in that their activities are seemingly needed to make both black and yellow pigments (Figure 2.1B). Both

15 proteins have been shown to accumulate transcripts before eclosion, but appear to be under translational control that governs the timing of tanning (Davis et al., 2007). However, the spatial distributions of pale and Ddc expression remain largely uncharacterized during pupal and adult stages when tergite pigmentation is developing, and the evolutionary origin of their CREs remain unresolved.

Here, we show that both pale and Ddc exhibit spatial and temporal patterns of expression during pupal and adult life stages in D. melanogaster. While pale expression has been broadly conserved among Sophophora species exhibiting both derived and ancestral pigmentation patterns, we show that high levels of epidermal Ddc expression evolved in the lineage of species with male-limited patterns of melanic pigmentation. This gain of abdominal Ddc expression requires the activity of a pleiotropic CRE that additionally regulates Ddc expression in the larval epidermis and in response to epidermis wounding (Mace et al., 2005; Scholnick et al., 1993). We find that the abdominal function of this CRE requires an ancestral binding site for Grainy head

(Grh) necessary for its wound-response function. However, robust pupal abdomen CRE activity required changes elsewhere to this CRE. Together with previous results, our findings illustrate how a novel trait evolved through a lengthy evolutionary path, which involved independent gains of new expression patterns for an entire pathway of realizator genes through varied mechanisms.

Materials and Methods

Fly Stocks and Genetic Crosses

Fly stocks used in this study were maintained at 22oC on a previously published sugar food medium (Salomone et al., 2013b). Species stocks used in this study were D. melanogaster

(14021-0231.04), and D. willistoni (14030-0811.24) from the National Drosophila Species Stock

Center at Cornell University, and D. melanogaster yellow and white (yw) gene mutant, D. biarmipes, and D. pseudoobscura stocks were obtained from Dr. Sean B. Carroll. Mutant allele stocks used to visualize abdomen pigmentation phenotypes were the null mutant y1 (Bloomington

Drosophila Stock Center or BDSC #169) allele for yellow, the hypomorphic t5 (BDSC #133) allele for tan, and the null e1 (BDSC #1658) allele for ebony. UAS-regulated RNA-interference

(RNAi) stocks were generated by the Transgenic RNAi Project at Harvard Medical School, and acquired from the BDSC. These stocks express double stranded RNAs that specifically target mCherry (BDSC #35785), grh (BDSC #28820), abd-A (BDSC #28739), hth (BDSC #27655),

Ddc (BDSC #51462), and pale (BDSC #65875). Experiments requiring ectopic expression of

Abd-B in the A4 abdominal segment were achieved through crosses with a stock possessing the

Abd-BMcp mutant allele (BDSC #24618). In order to use the GAL4/UAS system (Brand and

Perrimon, 1993) in the dorsal abdomen midline, a stock with the genotype pnr-GAL4/TM3, Ser1

(BDSC #3039) was used that possesses a 3rd chromosome from which GAL4 is expressed in the dorsal midline pattern of the pannier gene (Calleja et al., 2000). In order to make qualitative, and in some cases quantitative, comparisons of reporter transgene activities, transgenes were integrated into the attP2 and/or 51D attP sites via ɸC31 integrase-mediated transgenesis (Best

Gene Inc.) (Bischof et al., 2007; Groth and Calos, 2004).

Gain- and loss-of-function experiments

To assess adult pigmentation phenotypes following reduction in either pale, Ddc, and grh expression, males from the respective UAS-RNAi stocks were crossed to pnr-GAL4/TM3, Ser1 virgin female flies. Adult progeny inheriting the pnr-GAL4 chromosome and the UAS-RNAi transgene bearing chromosome express the dsRNA hairpin in the pnr gene’s dorsal-medial pattern, including the pupal dorsal abdominal epidermis. The following crosses were made in order to investigate the effects of reduced transcription factor gene expression on the ability of the t_MSE and Ddc-MEE1 CREs to drive the expression of the Enhanced Green Fluorescent Protein

(EGFP) reporter transgene in P14-P15(i) stage pupae. Virgin females from the RNAi stocks for

UAS-mCherry, UAS-abd-A, UAS-hth, and UAS-grh were collected and separately crossed to males bearing a CRE reporter transgene in the 51D attP site of the 2nd chromosome (Bischof et

17 al., 2007) and a pnr-GAL4 bearing 3rd chromosome. As the mCherry gene does not exist in the D. melanogaster genome, expression of a dsRNA targeting this gene served as a negative control.

In order to compare the responsiveness of the Ddc-MEE1 and t_MSE CREs to Abd-B in pupae, adult males possessing the Abd-BMcp allele were crossed to virgin females possessing an

EGFP reporter transgene under the regulatory control of either the t_MSE or the Ddc-MEE1 integrated into the 51D attP site (Bischof et al., 2007). As a negative control, similar crosses were made in which males from a yw stock that exhibit wild type expression of Abd-B (+) were utilized in place of the Abd-BMcp allele stock. EGFP reporter expression was assessed in P14-

P15(i) stage pupae that inherited the reporter transgene and either the wild type or Abd-B mutant genotype. in situ hybridization and immunohistochemistry

A previously describe protocol was used in the in situ hybridizations studies (Jeong et al.,

2008b). In brief, digoxigenin labeled riboprobes for Ddc and pale were prepared through in vitro transcription of PCR templates amplified from each species’ genomic DNA (Table S2.1 for probe primers). Abdomens were dissected at the pupal developmental stages P10, P12, P14-15(i) and the newly eclosed adult fly stage P15(i) (Figure S2.1). These different stages were identified by inspection for the presence and absence of various morphological markers (Ashburner et al.,

2005) (Figure S2.1). Probe hybridizations were made visible through the use of an anti- digoxigenin antibody (Roche Diagnostics), whose presence was detected by an alkaline phosphatase reaction using BCIP/NBT (Promega).

Dorsal abdomens were dissected for immunohistochemistry at the P12 and P14-P15(i) stages for D. melanogaster and D. willistoni. These stages are when we found Ddc transcription to be robust (P12) and declining P14-P15(i) in the D. melanogaster abdominal epidermis (Figure

2.2). Qualitative comparisons of Grainy head (Grh) expression patterns were made through immunohistochemical analysis using an antibody specific for Grh (Kim and Mcginnis, 2010).

Abdomens were dissected to isolate the dorsal epidermis. Samples were fixed for 35 min in PBST

(phosphate‐buffered saline with 0.3% Triton X‐100) with 4% paraformaldehyde, and then blocked in blocking buffer (PBST with 1% bovine serum albumin) for 1 hour at room temperature. The abdomens were then incubated overnight with guinea pig anti-Grh primary antibody at a dilution of 1:200 in PBST. After four washes in PBST and then an hour incubation in blocking buffer, specimens were incubated with goat anti-guinea pig Alexa Fluor 647

(Invitrogen) secondary antibody at a dilution of 1:500. After four washes in PBST, samples were incubated for 10 minutes in a 1:1 solution of glycerol mount (80% glycerol, 0.1M Tris pH 8.0) and PBST. Samples were transferred to glycerol mount, and finally situated between a cover slip and slide for imaging with a confocal microscope.

EGFP reporter transgenes and their quantitation

EGFP reporter transgenes were used to study the gene regulatory activity (Rebeiz and

Williams, 2011) of sequences controlling Ddc expression and to make comparisons with the t_MSE that controls male-specific tan expression (Camino et al., 2015). All reporter transgene were made by cloning CREs into the AscI and SbfI restriction enzyme sites for the S3aG vector

(Rogers and Williams, 2011). In this position each CRE is placed 5’ of a minimal hsp70 promoter and the coding sequence for the EGFP-NLS reporter protein (Barolo et al., 2004). The primer pairs used to amplify D. melanogaster Ddc sequences are presented in Table S2.2. The primer pairs used to amplify sequences orthologous to the D. melanogaster Ddc-MEE1 CRE are presented in Table S2.3. The details on the construction of the t_MSE EGFP reporter transgenes were discussed previously (Camino et al., 2015). Ddc-MEE1 transcription factor binding site mutant sequences (FigureS2.2 andFigureS2.3), and scanning mutant sequences (Figure S2.5) were synthesized by GenScript USA Inc. Each synthesized sequence was flanked by an AscI and

SbfI restriction enzyme site for subsequent cloning of the mutant CREs into the S3aG vector.

Reporter transgenes containing truncated version of the Ddc upstream region, and those

19 possessing Ddc-MEE1 scanning or binding site mutant sequences were each integrated in the attP2 site on the 3rd chromosome (Groth et al., 2000). The t_MSE, Ddc-MEE1, Ddc-MEE364,

Ddc-MEE130, and orthologous Ddc-MEE1 CREs were integrated into the 51D attP site on the 2nd chromosome (Bischof et al., 2007).

Quantitative comparisons of the levels of EGFP reporter gene expression driven by sequences orthologous to the D. melanogaster Ddc-MEE1 were achieved following a previously described approach (Camino et al., 2015; Rebeiz and Williams, 2011; Roeske et al., 2018; Rogers and Williams, 2011; Rogers et al., 2013). In brief, for each transgene EGFP expression was imaged from five biological replicate specimens using a confocal microscope with settings for which few pixels were saturated when EGFP expression was driven by the D. melanogaster Ddc-

MEE1. For each confocal image, a separate pixel value statistic was measured for the dorsal epidermis of the A5 segment using the Image J program (Abràmoff et al., 2004). For each reporter transgene, the regulatory activity was calculated as the mean pixel value and standard error of the mean (SEM). Activities reported in figures 5 and 9 were normalized to the activity for the wild type Ddc-MEE1 in the attP2 site, which was considered 100%. Activities reported in

Figure S2.4 were normalized to the activity for the wild type Ddc-MEE1 in the 51D site, which was considered 100% (Figure S2.4). CRE activities in the A4 abdomen segment reported in the

Abd-BMcp mutant background were normalized to the A4 activity of the species’ CRE in an Abd-B wild type background, in which CRE activity was considered 100% (Figure S2.5).

Imaging of fly abdomens and experimental replication

Images of fruit fly abdomen pigmentation patterns were taken using an Olympus SZX16

Zoom Stereoscope and Olympus DP72 digital camera. Specimens were prepared from 4 day old flies. Projection images for EGFP-NLS reporter transgene expression were generated with an

Olympus Fluoview FV 1000 confocal microscope and software. In each figure panel reporting a pigmentation pattern, gene expression pattern, or CRE activity, a representative image was

20 selected from biological replicate specimens (n≥3). In all figures where comparisons were made between images, each image was processed through the same sequence of modifications using

Photoshop CS3 (Adobe).

DNA sequence representations and alignments

Sequence visualizations for the Ddc loci of D. melanogaster (Figure 2.6) was made using the GenePalette tool (Rebeiz and Posakony, 2004; Smith et al., 2017). DNA sequence alignments of wild type, mutant, and orthologous Ddc-MEE1 sequences were made using the Chaos and

Dialign software (Brudno et al., 2004).

Results

Melanic tergite pigmentation depends on pale and Ddc activity.

The genetic and enzymatic pathway for the synthesis of the D. melanogaster cuticle pigments has been studied for several decades (Rebeiz and Williams, 2017b; Wittkopp et al.,

2003; Wright, 1987), though in recent years, much focus has been devoted to the expression and regulatory mechanisms of tan, ebony, and yellow genes which act at later steps in the pathway

(Figure 2.1B). Though the precise molecular mechanism of Yellow protein function remains unknown (Ferguson et al., 2011; Hinaux et al., 2018), the loss of yellow gene function results in a loss of black melanin pigments (Figure 2.1F and 2.1F’). ebony encodes a protein with NβAD synthetase activity which shunts Dopamine to form a yellow colored NβAD sclerotin. The loss of ebony function results in a broadly melanic phenotype (Figure 2.1H and 2.1H’). tan encodes an enzyme with NβAD hydrolase activity, which converts NβAD back to Dopamine (True et al.,

2005). Reduced tan activity results in a broad loss of melanin pigments (Figure 2.1G and 2.1G’). pale encodes an enzyme with Tyrosine hydroxylase activity, which is the initial step in the pigmentation pathway converting Tyrosine to Dopa. RNA-interference (RNAi) of pale expression in the dorsal midline of D. melanogaster results in a dramatic loss of melanic

21 pigments and to a lesser extent yellow cuticle color (Figure 2.1D and 2.1D’). Ddc encodes an enzyme with Dopa decarboxylase activity and is responsible for converting Dopa to Dopamine.

RNAi for Ddc in the dorsal midline results in a stark reduction in melanic pigments in the abdominal tergites (Figure 2.1E and 2.1E’).

Collectively, these results show that pale and Ddc are essential for the formation of the sexually dimorphic pattern of D. melanogaster abdomen pigmentation. The dramatic loss of black colored cuticle following Ddc RNAi indicates that most black color is that of Dopamine melanin rather than Dopa melanin (Figure 2.1B). ebony, tan, and yellow have been established to be expressed in complex spatial, temporal, and sex-specific manners. Thus, we sought to reveal whether the expression patterns for pale and Ddc are similarly complex.

Figure 2.1. Pigmentation phenotypes and enzymatic pathway in the Sophophora subgenus. (A) Extant species that represent the inferred diversification of abdominal pigmentation from an ancestral monomorphic state to a derived dimorphic state in the Sophophora subgenus. The tergites covering the A5 and A6 abdominal segments of D. melanogaster and D. biarmipes are sexually dimorphic for a melanic pigment color, with the male tergites being fully pigmented and pigmentation limited to posterior stripes in females. The more distantly related species such as D. pseudoobscura and D. willistoni are monomorphic, though differ greatly in the extent of their melanization. (B) The production of yellow and black cuticle depends on the activity of a metabolic pathway converting Tyrosine into NβAD sclerotin or Dopa/Dopamine melanin. (C and C’) The wild type abdomen pigmentation pathway includes a male-specific pattern of fully melanic A5 and A6 segment tergites. (D and D’) RNA-interference for pale results in a near complete absence of black cuticle and a reduction in yellow cuticle color. (E and E’) RNA- interference for Ddc results in a stark reduction in black cuticle color and a reduction in yellow color. (F and F’) The null allele phenotype for yellow is a loss of black cuticle color. (G and G’) The hypomorphic allele phenotype for tan is a reduction in black cuticle color though the stripe and midline spot region pigmentation remains. (H and H’) The null allele phenotype for ebony is a broadening of black cuticle to all abdomen segments. RNA-interference was achieved by driving a UAS-regulated dsRNA transgene for pale and Ddc by the GAL4 transcription factor that was expressed in the dorsal-medial pattern of the pnr gene.

The spatial and temporal pattern of abdominal pale expression.

During the approximately 100 hour time course of D. melanogaster pupal development at 25oC, pupae transition through roughly 15 distinct stages that can be identified by morphological markers (Figure S2.1) (Ashburner et al., 2005). By the P10 stage, pale is expressed robustly in the abdomen mechanosensory bristle cells, whereas little to no expression is seen in the surrounding epidermis (Figure 2.2A and 2.2A’). This pattern of expression is maintained through the P12 stage when some bristles have developed clear projections (Figure

2.2B and 2.2B’). By the P14-P15(i) stage, pale expression has dramatically increased in the abdominal epidermis region underlying where tergites will form, and is especially elevated in the

A5 and A6 segments. Expression of pale is comparatively absent from the epidermal cells that will be covered by flexible un-pigmented pleural membrane that will ultimately fold underneath each tergite (Figure 2.2C and 2.2C’). This epidermal pattern of expression is sustained in newly eclosed, P15(ii) stage, flies (Figure 2.2D and 2.2D’). Collectively, these results show that pale undergoes a complex temporal and spatial pattern of expression like yellow, tan, and ebony.

However, pale differs from the other pigmentation genes in that its expression is not conspicuously dimorphic.

The spatial and temporal pattern of abdominal Ddc expression.

By the P10 stage, D. melanogaster Ddc is expressed robustly in the abdomen mechanosensory bristle cells, whereas the surrounding epidermis lacks detectible expression

(Figure 2.2F and 2.2F’). By the P12 stage, Ddc expression has dramatically increased in the abdominal epidermis region underlying where tergites will form, notably in the A3-A6 segments, while expression is comparatively absent from the epidermal cells that will be covered by flexible un-pigmented pleural membrane (Figure 2.2G and 2.2G’). By the P14-P15(i) stage, Ddc expression remains in the abdominal epidermis though the level of expression has declined

(Figure 2.2H and 2.2H’). In newly eclosed flies, Ddc expression has become difficult to detect

(Figure 2.2I and 2.2I’). Collectively, these results show that Ddc undergoes a complex temporal

24 and spatial pattern of expression like yellow, tan, and ebony. However, like pale, Ddc expression is not conspicuously dimorphic. As the pattern of D. melanogaster tergite pigmentation represents a novel trait that evolved from a monomorphic ancestral state, we were curious whether the Ddc and pale expression patterns were modified during this trait’s origins.

Figure 2.2 The spatial and temporal expression pattern of D. melanogaster pale and Ddc. (A- J) Samples for D. melanogaster males and (A’-J’) females. (A and A’) pale is expressed in the mechansosensory bristle cells as early as the P10 stage of pupal development. (B and B’) Mechanosensory cell expression continues through the P12 stage. (C and C’) By the P14-P15(i) stage, pale expression has dramatically increased in the abdominal epidermis. Most notably in the A5 and A6 segments and in the epidermal cells underlying where tergites will form. (D and D’) The epidermis pattern of pale expression can still be detected in newly eclosed flies. (E and E’) A sense probe control shows the background pattern of signal observed in an eclosed fly. (F and F’) Ddc is expressed in the mechansosensory bristle cells as early as the P10 stage of pupal development. (G and G’) By the P12 stage, Ddc expression has dramatically increased in the abdominal epidermis, most notably cells in the A3-A6 segment regions underlying where tergites will form. (H and H’) By the P14-P15(i) stage, Ddc expression persists but at an apparently reduced level. (I and I’) Epidermis expression has become difficult to detect in newly eclosed flies (J and J’). A Ddc sense probe control shows background signal observed in eclosed fly. Red arrowheads indicate stages and segments with conspicuous expression detected in the epidermis. These stages were prioritized for investigations of expression in related fruit fly species. Conserved pale expression is a fixture of evolved pigmentation traits and GRNs

The working model for pigmentation evolution in the Sophophora subgenus places a monomorphic pattern of tergite pigmentation as ancestral, and dimorphic pigmentation being a derived state (Jeong et al., 2006; Rebeiz and Williams, 2017a). Moreover, many species within the obscura species group, including D. pseudoobscura (Figure 2.1A), are darkly pigmented across the entire abdomen. It has been shown that yellow and tan expression correlate with these patterns of pigmentation, and ebony expression has an inverse correlation (Camino et al., 2015;

Jeong et al., 2008a; Johnson et al., 2015; Ordway et al., 2014; Rebeiz et al., 2009). We sought to reveal whether the earliest acting gene in the Drosophila pigmentation pathway, pale, has similarly evolved diverse patterns of expression to shape the visible pigmentation patterns seen among Sophophora (Figure 2.1A).

We focused our attention on the P10, and P15(ii) developmental stages for which we found D. melanogaster pale expression to transition from a mechanosensory-specific expression pattern to a pattern including the abdominal epidermis. We observed that expression at the P10 and P15(ii) stages were conserved between species with dimorphic and monomorphic patterns of pigmentation (Figure 2.3). This outcome suggests that the expression pattern for pale has been a conserved feature of the evolving pigmentation GRNs for abdominal tergite pigmentation.

Figure 2.3 pale abdominal epidermis expression is broadly conserved in Sophophora. (A-H) pale expression in the dorsal abdomens of male and (A’-H’) female pupae. At the P10 stage, pale expression occurs robustly in the mechanosensory bristle cells of (A and A’) D. melanogaster, (B and B’) D. biarmipes, (C and C’) D. pseudoobscura, and (D and D’) D. willistoni. At the P15(ii) stage, pale expression is robust in the abdominal epidermis of (E and E’) D. melanogaster, (F and F’) D. biarmipes, (G and G’) D. pseudoobscura, and (H and H’) D. willistoni.

Ddc is broadly expressed in the abdomen epidermis of highly pigmented species.

The conserved abdominal epidermis pattern of pale expression represents a departure from the evolved patterns observed with yellow, tan, and ebony. We sought to determine whether the second gene in the pigmentation pathway, Ddc, is characterized by a conserved pattern of abdominal epidermis expression. We focused our attention on the P10 and P12 stages of pupal development where we respectively observed a conspicuous pattern of expression in the mechanosensory bristle cells and abdominal epidermis for D. melanogaster (Figure 2.2).

At the P10 stage, robust mechanosensory expression was found in all species evaluated

(Figure 2.4A-D and 2.4A’-D’), consistent with their melanic appearance. At the P12 stage, Ddc expression occurs in a monomorphic pan-abdomen epidermal pattern for D. biarmipes and D. pseudoobscura (Figure 2.4E-2.4G and 2.4E’-2.4G’). D. biarmipes has a male-specific pattern of tergite pigmentation like D. melanogaster, and D. pseudoobscura has a monomorphic melanic pattern that spans the tergites of the A1-A6 segments (Figure 2.1A). Interestingly though, at the

P12 stage we detected little to no Ddc expression in the abdominal epidermis of D. willistoni

(Figure 2.4H and 2.4H’). Expanding our search to a range developmental stages, we failed to detect strong epithelial expression in D. willistoni, despite the clear expression detected in bristle cells (Figure 2.5). Unlike the other species evaluated, D. willistoni lacks notable pigmentation on its abdominal tergites (Figure 2.1A), and this pigmentation pattern is considered to descend from a lineage bearing the ancestral pattern from which dimorphism evolved (Jeong et al., 2006).

These results suggest that the origin of dimorphic pigmentation involved the gain of a robust pattern of Ddc expression in the abdominal epidermis. Thus, we next sought to understand how

Ddc expression is regulated in the abdominal epidermis and elucidate how this expression pattern evolved.

Figure 2.4. Ddc is broadly expressed in the abdominal epidermis of species with elaborate patterns of tergite pigmentation. Ddc expression in the dorsal abdomens of (A-H) male and (A’-H’) female pupae. (A-D and A’-D’) Expression at the P10 and (E-H and E’-H’) P12 developmental stages. At P10, Ddc expression occurs robustly in the mechanosensory bristle cells of (A and A’) D. melanogaster, (B and B’) D. biarmipes, (C and C’), D. pseudoobscura, and (D and D’) D. willistoni. At P12, expression has expanded to include the abdominal epidermis of (E and E’) D. melanogaster, (F and F’) D. biarmipes, and (G and G’), and D. pseudoobscura. (H and H’) For D. willistoni, a similar robust pattern of Ddc expression was not observed at the P12 stage.

Fig. 2.5. D. willistoni exhibits little to no Ddc expression in the abdominal epidermis of various pupal stages through eclosion. In situ hybridization for Ddc expression was performed on the dorsal abdominal epidermis of D. willistoni (A-F) male and (A’-F’) female specimens. (A and A’) Ddc is expressed in the mechansosensory bristle cells as early as the P10 stage of pupal development. (B and B’) By the P11 stage, a low level of Ddc expression can be observed in the epidermis regions underlying where tergites will form. (C and C’) By the P12 stage, little Ddc expression is observed in the epidermis regions underlying where tergites will form. (D and D’) At the P13 and (E and E’) P14-P15(i) stages, little to no expression was observed in the epidermis. (F and F’) A Ddc sense probe control shows background signal observed in eclosed flies.

A multifunctional CRE drives Ddc expression in the abdominal epidermis.

The Ddc locus resides on D. melanogaster chromosome 2, between the genes l(2)37Cc and CG10561 (Figure 2.6A). Ddc has three transcribed exons, although a splice variant exists that includes an additional non-coding exon situated in the first intron. As a first pass at identifying

CREs active in the abdomen during development and pigmentation pattering, we crossed virgin females carrying a UAS-GFP-nls transgene to males of the GMR61H03 and GMR601F07 GAL4 lines (Pfeiffer et al., 2008). These GAL4 lines respectively possess a 1,692 bp piece that spans most of the sequence between the Ddc 1st exon and the last exon of the upstream l(2)37Cc gene, and a 1,115 base pair (bp) sequence spanning the entire Ddc 2nd intron (Figure 2.6A). In pupae inheriting the UAS-GFP-nls and GMR61H03-GAL4 transgenes, we observed GFP expression in the abdominal epidermis of the A1-A6 segments which was noticeably elevated in more posterior segments (Figure 2.6 B). On the other hand, GMR61F07-GAL4 drove GFP expression in the developing mechanosensory bristle cells (Figure 2.6C). Collectively, these patterns of GFP expression recapitulated our observed patterns Ddc mRNA expression detected by in situ hybridization experiments (Figure 2.2), and demonstrates that the two phases of pupal abdomen expression are under the control of multiple modular CREs.

To examine the molecular basis for the evolved patterns of Ddc expression in the abdominal epidermis, we focused on the GMR61H03 fragment. Generating direct fusions of sub- fragments to Enhanced Green Fluorescent Protein (EGFP), we found this Ddc sequence, named

Ddc-MEE, to drive a similar abdominal epidermis pattern of reporter expression (Compare Figure

2.6D to 2.6B). To better resolve which sequences within this Ddc-MEE region drives the pupal abdominal epidermis reporter expression, we created three reporter transgenes that subdivided this region (Figure 2.6A).

We found that the reporter gene containing the promoter-proximal region (Ddc-MEE1) drives EGFP expression in a pattern indistinguishable from that of the Ddc-MEE (compare Figure

2.6E to 2.6B and 2.6D). We found the central (Ddc-MEE3) region to lack any noteworthy pupal

31 abdomen regulatory activity (Figure 2.6G). In contrast, the distal (Ddc-MEE2) region drove strong epidermis activity (Figure 2.6F), which poorly matched expression of the larger fragment or endogenous expression. We interpret this fragment to either be a secondary CRE (Hong et al.,

2008; Lagha et al., 2012), a CRE regulating another gene, or artefactual activity. Since the Ddc-

MEE1 region drives a pattern of expression similar to the evolved pattern of Ddc expression

(Figure 2.4), we next sought to determine how this CRE is regulated and evolved.

Figure 2.6. An abdominal epidermis cis-regulatory element is located 5’ of the endogenous Ddc promoter. (A) To scale representation of the Ddc locus showing regions that were evaluated for CRE activity. (B-I) CRE activity observed by the expression of the EGFP reporter gene in the dorsal abdomens of P14-P15(i) stage transgenic male D. melanogaster pupae. (B and C) UAS- EGFP reporter expression driven by GAL4 expressed under the control of Ddc locus subregions. (B) The entire upstream region of Ddc, named the Ddc-MEE, drove reporter expression in the A2-A6 abdominal regions underlying where tergites develop, a pattern that mimics the endogenous Ddc expression. (C). The 2nd intron region of Ddc drove reporter expression in the mechanosensory bristle cells, a pattern that mimics the endogenous Ddc expression. (D-I) Expression of direct CRE-GFP fusion transgenes. (D) The entire Ddc upstream region drove reporter transgene expression in an abdominal epidermis patter similar to endogenous Ddc expression and that driven by the same region using the GAL4/UAS system. (E) The promoter- proximal Ddc-MEE1 subdivision of the Ddc-MEE retained the patterned abdominal epidermis activity. (F) The Ddc-MEE2 reporter drove expression more broadly in the abdominal epidermis than the Ddc-MEE CRE and endogenous Ddc expression. (G) The Ddc-MEE3 reporter lacks noteworthy abdominal activity. (H and I) Extreme truncated versions of the Ddc-MEE1 region to 364 and 130 base pairs had regulatory activities similar in pattern to that of the Ddc-MEE1 sequence.

The Ddc-MEE1 pigmentation patterning activity is regulated by Grainy head.

The Ddc-MEE1 sequence 5’ of the Ddc first exon possesses binding sites (Figure 2.7A) that contribute to multiple previously described regulatory activities. Within this sequence, a single ecdysone response element (EcRE) is required for the spike in Ddc expression that occurs at pupariation (Chen et al., 2002). For the wound response activity, binding sites for the AP-1,

CREB-A, and Grh transcription factors were necessary (Mace et al., 2005). Hence, we referred to this sequence as the Ddc-Multifunctional Epidermis Element 1 or Ddc-MEE1. We sought to determine whether any of these binding sites were required for the tergite pigmentation patterning activity of Ddc, by introducing the previously characterized mutations that disrupt these sites into the Ddc-MEE1. We observed no noticeable alteration in Ddc-MEE1 activity when the EcRE site was mutated, nor when the two AP-1 and the CREB-A sites were mutated (Figure S2.3 and S2.4).

However, activity of the Ddc-MEE1 was reduced to 49±3% of the activity of the non-mutant element when the two Grh sites were mutated (compare Figure 2.7C to 2.7B).

Figure 2.7. A Grh binding site is a required input in the regulatory logic driving Ddc abdominal epidermis CRE activity. (A) To scale annotation of the Ddc MEE1 region, and the mutant forms of the Ddc-MEE1 that were evaluated for alterations in regulatory activity. (B-E) Regulatory activities of CREs as seen by EGFP reporter gene expression in transgenic D. melanogaster abdomens. (B) Patterned CRE activity driven by the wild type Ddc-MEE1. (C) Compared to the wild type Ddc-MEE1, reporter activity was reduced to 49±3% when the two Grh binding sites were mutated. (D and E) Similarly, the scanning mutant 11 and 12 alterations respectively lowered Ddc-MEE1 reporter activity to 43±5% and 49±7% of the wild type element.

To further validate a role for grh in tergite pigmentation patterning, we used the

GAL4/UAS system (Brand and Perrimon, 1993) to drive a double stranded (ds)RNA specific for

Grh in the dorsal midline region. Compared to the normal tergite pigmentation phenotype observed when RNAi targeted the negative control mCherry gene, pigmentation was greatly reduced upon grh knockdown (compare Figure 2.8A and 2.8A’). Relatedly, when we tested the

Ddc-MEE1 reporter under these conditions, its activity was dramatically (compare Figure 2.9J to

2.9I). Collectively, these data reveal that melanic tergite pigmentation is positively-regulated by

Grh, a result that can be explained by the direct regulation of the Ddc in the pupal abdomen through one or both of the pleiotropic binding sites in the Ddc-MEE1.

Previously, the Ddc wound response element was resolved down to the immediate 472 bp upstream of the gene’s transcriptional start site (Mace et al., 2005). We wanted to determine whether the wound and tergite pigmentation patterning activities shared additional function- driving sequences or whether sharing was limited to the Grh binding sites. We created a set of 14 scanning mutant Ddc-MEE1 sequences, in which a different block of ~100 bp was mutated at every other bp by a non-complementary transversion mutation (figures 2.7A, S5, and S6)

(Camino et al., 2015; Rogers et al., 2013). For twelve of the fourteen mutant CREs, we saw no conspicuous alteration in the expression of the EGFP reporter gene (Figure S2.3).

However, reporter expression observed in the abdominal epidermis for the Ddc-MEE1

CREs with either the SM11 and SM12 mutations were reduced respectively to 43±5% and

49±7% of the activity measured for the wildtype element (compare Figure 2.7D and 2.7E to

2.7B). The sequences disrupted by SM11 and SM12 mutations include sequences necessary for the wound response activity, and the more evolutionarily conserved of the two Grh biding sites

(Mace et al., 2005) in the larger wound element CRE (Figure S7). As the SM11 and SM12 sequences were necessary for the pupal abdomen epidermis regulatory activity, we tested whether these sequences were sufficient in the context of two truncated versions, named the Ddc-MEE364 and Ddc-MEE130 sequences (figures 2.6E, 2.6H and 2.6I). The Ddc-MEE364 contains the non- mutant sequence for the SM11 and SM12 regions. This truncated sequence retained the abdominal epidermis pattern of activity, albeit with modestly-reduced levels of EGFP reporter expression compared to the larger Ddc-MEE1 sequence (82±3%, Figure S2.4). The Ddc-MEE130 sequence contains the non-mutant sequence for the SM11 region and the more conserved Grh binding site, but lacks the adjacent EcRE. Similar to the Ddc-MEE364, the Ddc-MEE130 also

36 had a moderated activity compared to that of the Ddc-MEE1 sequence, though it’s pattern resembled the Ddc-MEE1 and Ddc-MEE364 reporters (86±8%, Figure S2.4). These experiments localize the bulk of the Ddc-MEE1’s patterning and activity to the MEE130 region, though additional sequences elsewhere within the Ddc-MEE1 are required for full activity. We next sought to investigate which transcription factors in addition to Grh shape the pupal abdomen activity of the Ddc-MEE1 CRE.

Figure 2.8. Male-specific pigmentation requires the activity of Grh whose expression has remained conserved. (A) A wild type pattern of pigmentation is exhibited in D. melanogaster males following RNA-interference, RNAi, for the negative control mCherry gene in the dorsal midline pattern of the pnr gene. (A’) Melanic pigmentation was altogether lost in male abdomens following dorsal midline RNAi for the Grh gene. Grh protein is expressed in the abdominal epidermis of (B) P12 and (B’) P14-15(i) stage D. melanogaster. Similar patterns of Grh expression occurs in (C and C’) P12 and (D and D’) P14-15(i) stage D. willistoni.

The abdominal epidermis activity of the Ddc-MEE1 is Hox-regulated.

Previous studies revealed that the D. melanogaster A5 and A6 tergite pigmentation and expression of yellow and tan genes are regulated by Hox transcription factors and their cofactors

(Jeong et al., 2006; Rogers et al., 2014). We were curious whether Ddc-MEE1 regulatory activity is Hox-regulated as well. Abd-B, the Hox gene with the most posterior-limited expression, is present in the A5 segment and more posterior segments (Kopp and Duncan, 2002; Wang and

Yoder, 2012). In the Abd-BMcp mutant background, Abd-B expression additionally includes the

A4 segment. Like the t_MSE, which controls tan expression, ectopic A4 segment reporter expression occurs when the Ddc-MEE1 is assayed in the Abd-BMcp background, indicating that this element is downstream of Abd-B (Compare Figure 2.9B to 2.9A for tan, and 2.9H to 2.9G for

Ddc).

Ddc expression and Ddc-MEE1 activity extends anterior of the A5 segment, and thus other transcription factors must govern expression and CRE activity in these segments. Plausible candidates include the Hox gene abd-A which is expressed up through the A2 segment (Kopp and

Duncan, 2002; Rogers et al., 2014) and the Hox cofactor gene hth which directly represses t_MSE activity in the anterior abdomen segments (Camino et al., 2015). Given the similarities of Ddc expression with tan, we compared the expression of the Ddc-MEE1 with that of the t_MSE under

RNAi conditions for factors that disrupt t_MSE function. The t_MSE activity is reduced upon abd-A RNAi (Figure 2.9D), and shows ectopic expression in the A3 and A4 segments when hth expression is antagonized by RNAi (Figure 2.9E and 2.9F). In contrast, we observed only minor reductions in midline reporter expression of the Ddc-MEE1 when abd-A was reduced (Figure

2.9K). Similarly, RNAi for hth did not increase the A3 and A4 segment reporter expression of the

Ddc-MEE1 (Figure 2.9L). These results suggest that the pupal abdominal epidermis regulatory activity of the D. melanogaster Ddc-MEE1 depends upon a unique set of factors, compared to the t_MSE. The discovery that Ddc expression is Hox-regulated inspired us to examine whether the

38 gain in pupal abdomen Ddc expression for D. melanogaster and its melanic relatives (Figure 2.4) involved cis-regulatory evolution at the Ddc-MEE1.

Figure 2.9. Genetic interactions between pigmentation network transcription factors and CREs regulating tan and Ddc expression. (A-F) EGFP reporter expression driven by the t_MSE was imaged at the P14-15(i) stage. (G-L) EGFP reporter expression driven by the Ddc- MEE1 was imaged at the P14-15(i) stage. The relevant genetic background genotypes are listed at the top of each column. All specimens are hemizygous for the EGFP reporter transgene. Compared to the (A and G) wild type genetic background, (B) t_MSE, and (H) Ddc-MEE1 regulatory activity expands into the A4 segment in which Abd-B is ectopically expressed. Compared to a (C) control genetic background, suppression of (D) grh expression resulted in a stark loss of t_MSE activity. In contrast, the suppression of (E) abd-A and (F) hth expression respectively led to reduced A5 and A6 segment t_MSE activity and an ectopic t_MSE activity in the A4 and A3 segments. Compared to Ddc-MEE1 activity in a (I) control genetic background, suppression of (J) grh expression resulted in a conspicuous loss of Ddc-MEE1 activity in the A5 and A6 segments, whereas suppression of (E) abd-A and (F) hth respectively had little to no effect on A5 and A6 segment Ddc-MEE1 activity, nor any ectopic activity in the A4 and A3 segments. Yellow arrowheads indicate segments where the genetic background alterations resulted in conspicuous ectopic EGFP reporter expression. Red arrowheads indicate segments where the genetic background alterations resulted in a reduced EGFP reporter expression.

The activity of the Ddc-MEE1 was shaped by cis-evolution.

To this point, our results suggested that the transition from an ancestral monomorphic pigmentation to derived melanic patterns involved the gain of a robust pattern of epidermal Ddc expression during the P12 developmental stage (Figure 2.4). One possible explanation for the

39 derived expression pattern is through the gain or modification of a CRE activity driving Ddc expression. To test this possibility, we investigated the regulatory activities of sequences orthologous to the D. melanogaster Ddc-MEE1 from species with dimorphic (D. biarmipes), monomorphic melanic (D. pseudoobscura), and monomorphic non-melanic (D. willistoni) patterns of tergite pigmentation (Figure 2.10A-2.10D). We saw that each of the orthologous sequences were capable of driving reporter expression in the abdominal epidermis of D. melanogaster (Figure 2.10A’-2.10D’), indicating that the origin of the P12 stage epidermal Ddc expression was not due to the evolution of an altogether new CRE activity. Importantly though,

EGFP reporter expression driven by the D. willistoni sequence was consistently more modest in biological replicate specimens (Figure 2.10D’). Quantification of EGFP intensity in the A6 segment revealed that the D. willistoni CRE drove 40±1% of the D. melanogaster Ddc-MEE1 activity (100±7%; compare Figure 2.10D’ to 2.10A’). In contrast, the D. biarmipes and D. pseudoobscura activities respectively were 111±3% and 114±2% of the D. melanogaster CRE’s activity (Figure 2.10B’ and 2.10C’). In a genetic background with ectopic Abd-B expression in the A4 abdomen segment, the activity of the D. melanogaster Ddc-MEE1 increased to 180±14% of the activity in the A4 segment of a Abd-B wild type background (Figure S2.5). In comparison, the activity of D. willistoni sequence orthologous to the D. melanogaster Ddc-MEE1 increased to

148±10% A4 segment activity of the D. willistoni CRE in a wildtype background (Figure S2.5).

These results are consistent with a scenario in which the enhanced expression of Ddc in the pupal abdomen epidermis of D. melanogaster involved cis-evolution. This regulatory evolution seems to have included an increased responsiveness of the Ddc-MEE1 to Hox-regulation compared to the antecedent CRE.

While our results implicate a role for cis-evolution in shaping the activity of the Ddc-

MEE1 CRE, it is also possible that changes to the expressions or activities of trans-regulators were additionally required. One plausible candidate would be spatially and/or temporally expanded expression of Grh, the direct regulator of the Ddc-MEE1. However, we observed a

40 conserved pattern of pan-abdominal epidermis Grh expression in the abdomens of D. melanogaster and the monomorphic D. willistoni (Figure 2.8). Thus, future studies are needed to resolve whether trans-evolution played a role in the derived expression pattern of Ddc.

Figure 2.10. The ancestry of the robust abdominal epidermis regulatory activity of the CRE controlling Ddc expression. (A-D) A schematic representation of the male abdomen pigmentation patterns for several Sophophora species. (A’-D’) EGFP-reporter transgene expression driven by sequences orthologous to the D. melanogaster Ddc-MEE1 in transgenic D. melanogaster male pupae at the P14-15(i) stage. (A’-C’) Sequences from species with ostentatious melanic pigmentation phenotypes drive reporter expression throughout the abdomen, though the activity appears most pronounced in the A5 and A6 segments. (D’) The sequence from the most distantly-related species with monomorphic tergite pigmentation, D. willistoni, has comparatively less abdominal regulatory activity. Regulatory activity measurements are represented as the % of the D. melanogaster Ddc-MEE1 mean A5 intensity plus the Standard Error of the Mean (SEM). Each activity measurement and SEM were derived using images for five biological replicates.

Discussion

Our findings illustrate how an important realizator gene was recruited to a novel trait through the alteration of a presumably ancient CRE that functions in an entirely unrelated task.

By characterizing the expression of two genes whose protein products catalyze early steps of melanin synthesis, we found that strong pale expression is conserved broadly across the

Sophophora subgenus, while high levels of Ddc expression were only observed in species that had derived dark melanic pigments. We uncovered how abdominal Ddc expression in D. melanogaster is driven by a pleiotropic CRE that plays a presumably ancient role in activating

Ddc expression following epidermal wounding (Figure 2.11A). Notably, a Grh binding site required for wound healing is also required for its abdominal function. Comparative analysis of this CRE revealed that its pigment associated activity evolved more recently in the lineage of species with melanic abdomens. These results contribute to a body of literature showing that the origin of dimorphic pigmentation required cis-evolution at several realizator genes in the network

(Figure 2.11B), providing an example in which an entire network was assembled through widespread cis-regulatory modifications.

The novel deployment of a realizator gene pathway by widespread CRE evolution.

The development of most well-studied animal morphological traits relies upon dozens of regulatory genes that specify the spatial and temporal expression of pathways of realizator genes

(Bonn and Furlong, 2008a; Levine and Davidson, 2005b). One of the most difficult challenges in evolutionary biology is to develop molecular explanations of how such vast networks of regulators and realizators emerge to control novel traits. A critical feature that must be resolved is whether these novel traits are built up gradually through evolution at each realizator, or if networks evolve primarily through the co-option of large batteries of realizators from one developmental setting to another by expressing their upstream regulators in new locations

(Monteiro and Gupta, 2016; Rebeiz et al., 2015). To address this distinction, the pigmentation of the Drosophila abdomen is one of the most extensively studied traits for which the evolutionary trajectories of key components of the network have been traced. This study expands a growing perspective of how its network was assembled.

The current favored model for pigment evolution in the Sophophora subgenus places monomorphic, non-melanic pigmentation as the ancestral character state, as exemplified by D.

42 willistoni, and male-specific melanic pigmentation as the derived state, as seen for D. melanogaster and D. biarmipes (Figure 2.1A) (Rebeiz and Williams, 2017a). In the obscura species group, a broadly monomorphic melanic character state evolved. The expression of yellow, tan, and ebony evolved to fit these different pigmentation patterns. For D. willistoni, the dorsal abdominal epidermis experiences little-to-no yellow and tan expression, whereas expression occurs robustly throughout the D. pseudoobscura abdomen and in the male-specific pigmented regions of D. melanogaster (Camino et al., 2015). Thus, the origin of melanic phenotypes required gains in yellow and tan expression. In this study, we show that while pale expression throughout the abdominal epidermis has maintained high levels of ancestral expression, Ddc evolved a robust pattern of expression in the abdomen epidermis in the lineage of melanic species. Thus, a critical question is whether this network evolved its associated expression domains through co-option or independent evolutionary changes.

If the origin of male-specific tergite pigmentation in the D. melanogaster lineage originated via a co-option mechanism we would anticipate two hallmark signs of this event. First, co-option would likely involve a master regulator whose activity is sufficient to deploy all of the underlying regulatory and realizator genes. Second, we would expect these CREs to all share an ancestral activity in a second tissue or developmental setting from which the abdomen melanization function was co-opted (Glassford et al., 2015). The case we presented here of the

Ddc-MEE1 illustrates a clear ancestral function at Ddc that is unique among genes of the pigmentation pathway. To our knowledge, none of the other CREs in the pathway are associated with a wound response function. Further, we have yet to identify a master regulator of the network that would make sense as a source of the co-option event. Of all the transcription factors thus far identified in the abdominal pigmentation network, the most likely master regulator is

Abd-B. CREs of four realizator enzymes of the network respond to Abd-B in trans (Camino et al.,

2015; Jeong et al., 2006), and in the case of yellow, Abd-B is a direct regulator (Jeong et al.,

2006). However, the major problem with considering Abd-B as a master regulator that mediated

43 the redeployment of this network, is that its pattern of expression is likely ancestral, and an Abd-

B dependent ancestral network that drives all of these genes elsewhere in the posterior of the embryo, larvae, or pupae seems unlikely. Indeed, upon discovering the direct binding of Bric-á- brac transcription factors to the upstream enhancer of yellow, we found no-evidence of co-option from an ancestral function of its abdominal CRE (Roeske et al., 2018). Thus, considering the lack of a shared ancestral network or master regulatory gene, we propose that the network of pigmentation realizator CREs was assembled in a piecemeal fashion. The path by which Ddc independently evolved its abdominal activity is a curious one.

Gene expression evolution through the augmentation of a pleiotropic CRE activity.

To understand how GRNs of regulators and realizators emerged to generate novel traits, we must precisely examine how their CRE activities originated. Several mechanisms have been posited, including the gain of a CRE by transposition, the de novo evolution of a CRE activity by gains in transcription binding sites, and the co-option of an existing CRE with an ancestral function (Rebeiz et al., 2015). Problematically though, differences in traits between closely- related species often represent losses, and experimental models for trait gains often require comparisons between more distantly-related taxa whose cis-regulatory regions may be difficult to compare due to extensive sequence divergence.

The gain of a fruit fly male-specific wing pigment spot and abdomen tergite pigmentation have been two productive models for investigating the origins of CREs associated with novel traits. In D. biarmipes, a spot of pigmentation on the wing requires a the expression of yellow that is driven by a CRE that possesses binding sites for the activator Dll and the repressor En (Arnoult et al., 2013; Gompel et al., 2005). The spot CRE resides adjacent to a wing element CRE that drives a low level of yellow expression throughout the developing wing, and comparative analyses of this region suggested that the spot CRE evolved from this weakly active element.

Similarly, the origin of the D. melanogaster abdominal tergite pigmentation required gains of

44 male-specific patterns of yellow and tan expression (Camino et al., 2015; Jeong et al., 2006;

Jeong et al., 2008a). In D. melanogaster, yellow expression is driven by an Abd-B and Bab regulated CRE known as the body element (Jeong et al., 2006; Roeske et al., 2018), and tan is driven by a Hox and Hox cofactor regulated CRE known as the t_MSE (Camino et al., 2015). The origin of these CRE activities remain difficult to resolve, as the surrogate for the ancestral trait,

D. willistoni, lacks these CRE activities and any sequence with noteworthy conservation. Thus, the genetic events and molecular details responsible for the origin of the yellow body element and the tan t_MSE remain nebulous.

In contrast, we found clear evidence for the evolutionary path taken by Ddc, as it gained a robust pattern of abdominal epidermis expression in melanic species. Unlike the CREs of yellow and tan, the orthologous sequence of the Ddc-MEE1 is highly conserved (Figure S7), and the ancestral function can be reasonably inferred as this region is known to drive Ddc expression following epidermal wounding between fruit fly species more distantly-related than those studied here (Mace et al., 2005). Moreover, some of the molecular details regarding the evolution of the

Ddc-MEE1 can be inferred. Both the wound response (Mace et al., 2005) and abdominal epidermis activities require a conserved binding site for the Grh transcription factor (Figure S7).

This indicates that the ancestral wound response CRE was co-opted to play a pleiotropic function in tergite pigmentation (Figure 2.11A). While the precise details on the mechanism of augmentation remains to be elucidated, part of the explanation requires the gain of an increased responsiveness to the Hox gene, Abd-B. Importantly, our work provides an example whereby part of a trait’s origin required the gain of a realizator gene’s expression through the modification of an ancient CRE for an augmentated pleiotropic use. Our ability to discern an evolutionary past to this element highlights how different CREs, even in the same network, will contrast greatly in their rates of divergence. Finally, our findings reinforce how CRE activities can emerge from unexpected developmental contexts that provide a rich molecular explanation for the origin of traits and the CREs that enable them.

Figure 2.11. The evolution of a novel pigmentation trait required cis-regulatory evolution at several realizator loci. (A) In the embryo, Ddc expression (light blue) is upregulated around sites of epidermal wounding as part of a response that forms a melanized plug (black circle). Schematic depicts the anterior embryo. This gene function is conserved, and encoded in the activity of a CRE directly regulated by the Grh transcription factor. In D. willistoni, which serves as a proxy for the ancestrally monomorphic trait, Ddc expression is driven at a low level by the pleiotropic activity of the wound response CRE. This ancestral CRE possesses a direct binding site for Grh and is modestly-responsive to the Hox transcription factor Abd-B. In species with derived melanic abdomen pigmentation phenotypes, Ddc expression is robust throughout the abdomen (dark blue). This expression is in part due to cis-evolution at Ddc, which evolved a stronger, Hox-responsive CRE activity that is apparent for D. melanogaster, and this CRE seemingly evolved to be under the control of some trans-regulator(s) whose identity remains unknown and is represented here as factor “X”. Time scale is depicted in millions of years. Solid lines in cis-regulatory evolution indicate cases where the transcription factor directly binds to the Ddc CRE, and dashed lines indicate cases where the mechanism of transcription factor regulation remains unknown. (B) Model for the origin of the derived dimorphic pigmentation trait of D. melanogaster from an ancestral monomorphic state. Here, abdominal pale expression was ancestral, whereas Ddc, yellow, tan, and ebony each evolved novel expression through changes in CREs controlling their expression. The ancestral states upon which novel CREs evolved for yellow, tan, and ebony remain unknown (question marks). For Ddc, cis-evolution augmented an ancestral wound response CRE to have a robust pleiotropic activity.

Acknowledgements

Species stocks were purchased from the San Diego Drosophila Stock Center or provided by S.B. Carroll. Drosophila melanogaster UAS-RNAi lines were provided by the TRiP at

Harvard Medical School (NIH/NIGMS R01-GM084947). Stocks obtained from the Bloomington

Drosophila Stock Center (NIH P40OD018537) were used in this study. The Grainy head antibody was generously provided by W. Mcginnis.

Supplementary Information

Figure S2.1. Morphological markers used to differentiate the pupal stages of samples subjected to gene expression analyses. Milestones during Drosophila pupal development can be identified by the presence of conspicuous morphological markers. The earliest stage analyzed in this study was P10, which can be identified by the appearance of red-colored eyes, and anteriorly- positioned malpighian tubes on the abdomen. Pupae at the slightly more advanced P12 stage were identified by grey wing pigmentation, and gray bristles on the dorsal abdomen and thorax. Pupae at the even more advanced P14-15(i) stage were identified by black wing pigmentation, and black bristles on the abdomen and thorax, disappearance of the malpighian tubes, and meconium visible at the posterior-tip of the abdomen. Stage P15(ii) is not represented here, but these samples were identified as flies that recently eclosed from their puparium

Figure S2.2. Evaluation of binding site mutations on the activity of the Ddc-MEE1. (A-D) Regulatory activity of Ddc-MEE1 versions as seen by EGFP reporter transgene expression in the dorsal abdominal epidermis of P14-P15(i) stage transgenic D. melanogaster males. (A) Reporter expression driven by the wild type Ddc-MEE1. (B) Reporter expression driven by the Ddc-MEE1 with two mutant Grh binding sites was dramatically reduced. (C) Reporter expression was not noticeably altered when driven by a Ddc-MEE1 possessing mutations in two motifs characteristic of AP-1 binding sites and one motif characteristic of a CREB-A binding sites. (D) Reporter expression was not noticeably altered when driven by a Ddc-MEE1 with mutated version of a motif characteristic of the ecdysone receptor binding.

Figure S2.3. Mapping functional Ddc regulatory sequences through CRE scanning mutagenesis. (A) Name and location of Ddc-MEE1 scanning mutations and the wild type pattern of EGFP expression in transgenic male D. melanogaster pupae. Scanning mutations are indicated as gray blocks when abdomen activity appeared unaltered, and as red blocks when activity was conspicuously reduced. (B-K) The EGFP expression pattern in the male abdomen at the P14- P15(i) developmental stage driven by the Ddc-MEE1 scan mutant sequences that had no apparent effect on abdomen CRE activity.

Figure S2.4. Truncated forms of the Ddc-MEE1 CRE possess most of the pattern and activity of the large CRE version. (A) Reporter expression driven by the Ddc-MEE1 CRE in the 51D attP insertion site. (B and C) Reporter expression driven respectively by the truncated Ddc- MEE364 and Ddc-MEE130 CREs in the 51D attP site. Compared to the larger CRE, the truncated forms drove a similar pattern of expression, yet modestly-reduced activities. Regulatory activity measurements are represented as the % of the D. melanogaster Ddc-MEE1 mean A5 intensity plus the Standard Error of the Mean (SEM). Each activity measurement and SEM were derived using images for five biological replicates.

Figure S2.5. The D. melanogaster Ddc-MEE1 has a more robust response to ectopic Abd-B. (A) EGFP reporter expression for a representative male pupae hemizygous for the D. melanogaster Ddc-MEE1-EGFP reporter transgene in a wild type Abd-B expressing genetic background. (A’) EGFP reporter expression for a representative male pupae hemizygous for the D. melanogaster Ddc-MEE1-EGFP reporter transgene in an Abd-BMcp genetic background with ectopic Abd-B expression in the A4 segment. (B) EGFP reporter expression for a representative male pupae hemizygous for the D. willistoni Ddc-MEE1-EGFP reporter transgene in a wild type Abd-B expressing genetic background. (B’) EGFP reporter expression for a representative male pupae hemizygous for the D. willistoni Ddc-MEE1-EGFP reporter transgene in an Abd-BMcp genetic background with ectopic Abd-B expression in the A4 segment. (A and A’) Regulatory activity measurements are represented as the % of the D. melanogaster Ddc-MEE1 mean A4 intensity plus the Standard Error of the Mean (SEM) in the wild type Abd-B genetic background. (B and B’) Regulatory activity measurements are represented as the % of the D. willistoni Ddc- MEE1 mean A4 intensity plus the Standard Error of the Mean (SEM) in the wild type Abd-B genetic background. Each activity measurement and SEM were derived using images for five biological replicates.

Table S2.1. Primers used to create in situ probes to orthologous Ddc and pale gene sequences.

Species gene Primer F Primer R

D. melanogaster Ddc TTCAGGGCACTGAAGCTCTGGTT ctaatacgactcactatagggCANGAGTACTCCATGTCCTCG

D. melanogaster pale TTGCAGATTAYGGYCTCACCGAGGA ctaatacgactcactatagggTTGGTCATCAGATGGTTGCAGTTRTCC

D. biarmipes Ddc TTCAGGGCACTGAAGCTCTGGTT ctaatacgactcactatagggCANGAGTACTCCATGTCCTCG

D. biarmipes pale TTgCAGATTAYGGYCTcACcGAGGA ctaatacgactcactatagggTTGGTCATCAgaTGgTTGCAgTtRTCC

D. malerkotliana Ddc TTCCGCGCCYTSAAGCTKTGGTTCG ctaatacgactcactatagggTTCCAGGAGTACTCCATGTCCTCG

D. malerkotliana pale TTgCAGATTAYGGYCTcACcGAGGA ctaatacgactcactatagggTTGGTCATCAgaTGgTTGCAgTtRTCC

D. pseudoobscura Ddc TTCCGCGCCCTCAAGCTCTGGTTCG ctaatacgactcactatagggTTCCAAGAGTACACCATGTCTTCG

D. pseudoobscura pale TTgCAGATTAYGGYCTcACcGAGGA ctaatacgactcactatagggTTGGTCATCAgaTGgTTGCAgTtRTCC

D. willistoni Ddc TTCCGTGCCCTGAAGCTTTGGTTCG ctaatacgactcactatagggTTCCATGAGTATTCCATGTCCTCG

D. willistoni pale GGAYaAcTGCAAcCATcTGATGACCAA ctaatacgactcactatagggGAAgGCCARSGARGCRAGGAAGTC Probe1

D. willistoni pale TTgCAGATTAYGGYCTcACcGAGGA ctaatacgactcactatagggCGCACaTACTGKGTGCTCTGGAAGA Probe 2

Note: The ctaatacgactcactataggg sequence in the antisense primers is the introduced T7 promoter that was added for in vitro transcription.

Table S2.2. Primers used to clone D. melanogaster Ddc gene non-coding sequences for use in reporter transgene assays to test for cis-regulatory element (CRE) activity.

Transgene ~Size Primer Sequence

GMR61H03 Fwd TTCCGggcgcgccCACCAAACACCCGCTCGCTTATCTCG Ddc MEE 1700 bp GMR61H03 Rvs TTGCCcctgcaggCTTGTTGCCGAGCTTTACTTCCGTTC

Ddc-MEE1 Fwd TTCCGggcgcgccTTCTCAGTGTATGCGGAACTGC Ddc MEE1 900 bp GMR61H03 Rvs TTGCCcctgcaggCTTGTTGCCGAGCTTTACTTCCGTTC

GMR61H03 Fwd TTCCGggcgcgccCACCAAACACCCGCTCGCTTATCTCG Ddc MEE2 830 bp Ddc-MSE2 Rvs TTGCCcctgcaggGGCAGTTCCGCATACACTGAG

Ddc MEE3 770 bp Ddc-MEE3 Fwd TTCCGggcgcgccAACAGTTGGACCACCCGCAGG

Ddc-MEE3 Rvs TTGCCcctgcaggGAATTCTACTCACGTGCTTCCC

Ddc MEE core 364 364 bp Ddc-MEEcore364F TTCCGggcgcgccGATATCCAGTTACTGATTCAGC

GMR61H03 Rvs TTGCCcctgcaggCTTGTTGCCGAGCTTTACTTCCGTTC

Ddc-MEEcore130F TTCCGggcgcgccGCGGACTGCGATTGAACCGGTC Ddc MEE core 130 130 bp Ddc-MEEcore130R TTGCCcctgcaggTATTTCGGTGCTGATTGCTGTGC

Notes: 1. ‘ggcgcgcc’ and ‘cctgcagg’ are sequences recognized respectively by the AscI and SbfI restriction endonucleases. These restriction enzyme sites were used to clone PCR amplified sequences into the S3aG EGFP reporter vector. 2. The approximate PCR product sizes are reported in base pairs (bp)

Table S2.3. Primers used to create reporter transgenes with sequences orthologous to the D. melanogaster Ddc MEE1 cis-regulatory element.

Transgene ~Size Primer Sequence

Ddc-MEE1 wil Fwd TTCCGggcgcgccTCCAAGAGCGGTTCAGAGCATC D. willistoni 1300 bp Ddc-MEE D. wil. Rvs TTGCCcctgcaggGCTTTTGTTTCCGTTGTATGTACTTG

Ddc-MEE1 pse Fwd TTCCGggcgcgccCACGTCACAGTTGATGCTCTG D. pseudoobscura 800 bp Ddc-MEE D. pse. Rvs TTGCCcctgcaggGCACTTGTCGCGCTTTGTTTCCG

Ddc-MEE1 ana group Fwd TTCCGggcgcgccTCAACGTCACACAYGATGCTC D. malerkotliana 1200 bp Ddc-MEE ana group Rvs TTGCCcctgcaggCACTTGTCAAGCTKTGTTTCCGTTC

Ddc-MEE1 bia F1 TTCCGggcgcgccTTCCCAGTGTAATTCCTATTCACC D. biarmipes 1000 bp Ddc-MEE1 bia R1 TTGCCcctgcaggGCACTTGTCGCGCCTTGTTTCCGCTC

Notes: 1. ‘ggcgcgcc’ and ‘cctgcagg’ are sequences recognized respectively by the AscI and SbfI restriction endonucleases. These restriction enzyme sites were used to clone PCR amplified sequences into the S3aG reporter vector. 2. Degenerate positions included in primer sequences utilize the IUPAC nucleic acid code: K (T or G), R (A or G), Y (C or T), N (A, C, G, or T), and M (C or A).

CHAPTER III

INVESTIGATING THE SIMILARITIES AND DIFFERENCES IN THE GENE

REGULATORY NETWORKS FOR CONVERGENT FRUIT FLY PIGMENTATION TRAITS

Abstract

While there are some mechanistic studies showing the target genes responsible for producing a convergent phenotype, comprehensive understanding of convergent trait evolution is preceded by a big challenge i.e. complexity of trait development. As a result, convergent evolution remains largely unexplored at the level of gene regulatory networks. In this chapter, we addressed the question of convergent evolution by comparing expression profiles for pigmentation enzyme genes and regulatory genes in D. melanogaster and D. funebris. We found out that the five pigmentation genes (yellow, tan, ebony, pale and Ddc) necessary for producing colored pigments are utilized in a similar fashion in both species. At the level of regulatory tier, we have come to appreciate the differences in which these regulatory genes are deployed. Unlike

D. melanogaster, Bab expression in D. funebris is monomorphic and is not responsible for shaping the male-specific pigmentation. Abd-B is expressed in a unique manner in D. funebris and might be regulating the pigmentation gene regulatory network along with some other unidentified factors.

Introduction

In the words of Stephen Jay Gould (a renowned evolutionary biologist); if we were to run the life’s tape again, you would get a completely different outcome. However, if we look into the

56 history of life forms on Earth, there have been numerous occasions when similar traits

(physiological, ecological or morphological) have come into existence as a result of natural selection. Contrary to what was thought by Stephen Jay Gould, it has been found that similar outcomes targeting certain pathways and certain genes are strongly selected. In fact, as per Simon

Conway Morris, the path to evolution is constrained (Stening, 2009) (Stern, 2013)and there are only limited outcomes. While arguments can be made for and against both these cases, what has always puzzled evolutionary developmental biologists is how do similar traits arise? Whether the genetic machinery governing these convergent traits, utilized in a similar or dissimilar fashion?

Before we can address these evolutionary developmental questions, it has to be pointed out that traits are formed by the deployment of orchestrated action of several genes in a complex gene regulatory network (Dawid, 2006). It remains important to understand the nuts and bolts of a gene regulatory network.

To address the question of convergent evolution, we need a trait that’s been recently evolved in the evolutionary history, in which two or more species, starting from a common ancestor have reached to similar outcome. The model we are using here is the male-specific pigmentation (Figure 3.1) in D. melanogaster and D. funebris. It is considered that most recent common ancestor for D. melanogaster and D. funebris was monomorphic and that these two species have evolved independently to a derived dimorphic state. The males from both of these species have elaborate patterns of colored abdomen in posterior segments (Figure 3.1) while females lack this trait.

There has been a wealth of knowledge that has been established from previous studies on the gene regulatory network of abdominal pigmentation in D. melanogaster (Rogers et al., 2014)

(Camino et al., 2015)(Rebeiz and Williams). In a simplistic scenario, there are two tiers of genes in a pigmentation gene regulatory network (Figure 3.2). At the bottom are the pigmentation enzyme genes, namely, yellow, tan, ebony, pale and Ddc. These genes make enzymes as there protein product which metabolizes the biochemical intermediates, leading to ultimate light and

57 dark coloration on the adult abdomen of flies. Above the pigmentation gene tier is the regulatory gene tier which has transcription factor genes, namely bab1, bab2, Abd-B, abd-A, exd, hth and grh whose direct or indirect inputs into the pigmentation gene CRE’s controls the final output of pigmentation genes.

We know that the transcription factor Bab1 and Bab2 regulate pigmentation by repressive input in yellow and tan gene CRE’s. The Hox gene, Abd B (Jeong et al., 2006) acts a direct activator for yellow, while abd A (Camino et al., 2015) acts as a repressor for tan gene activity. The Hox cofactors exd, and hth also have regulatory inputs pigmentation genes tan and ebony. From a previous study (Rogers et al., 2014), a list of 28 novel transcription factors regulating the abdominal pigmentation in some manner was established. While recent progress has been made to understand the function of some of these novel transcription factor genes (grh, vvl), a lot of them (Mi-2, Gug, osa, etc.) remains yet to be understood.

We have a reasonable understanding of the gene regulatory network for D. melanogaster, but there is very little to no knowledge of gene regulatory network for D. funebris. As we make progress in this chapter, an attempt has been made to unravel these details. We speculate that the for D. funebris pigmentation gene regulatory network, some of the genetic machinery is going to be utilized to make the colored patterns on the tergites like that of D. melanogaster. At the same time, these two species are evolved independently for about 60 Mya to the male-specific pigmentation trait and we speculate some dissimilarity in the way the certain genes might be deployed. What remains exciting is to look is most important candidate (Bab) and what it might be doing to shape this derived trait in D. funebris

Figure 3.1: Male specific pigmentation independently evolved in fruit flies D. melanogaster and D. funebris shared their most recent common ancestor approximately 60 million years ago. This ancestor is likely to have possessed a sexually monomorphic pattern of abdominal tergite pigmentation. This ancestral monomorphic state preceded the independent evolution of sexually dimorphic pigmentation in the lineages of D. melanogaster and D. funebris. The tergites covering the A5 and A6 segments of D. melanogaster and the A4-A6 segments of D. funebris are broadly melanized. In females, tergite pigmentation is restricted to posterior stripes.

Figure 3.2: The known tergite pigmentation Gene Regulatory Network for Drosophila melanogaster. The Gene Regulatory Network responsible for the sexually dimorphic tergite pigmentation of D. melanogaster has been well studied. In this network, transcription factor proteins encoded by various regulatory genes drive the expression of yellow, tan, Ddc, and pale genes and turn off the expression of the gene ebony in epidermis cells underlying cuticle that will become melanized. Solid lines represent the direct binding of a transcription factor to cis- regulatory elements of differentiation genes. Dashed lines represent a scenario where a transcription factor’s regulatory control may be direct or indirect. In this network, the Bab1 and Bab2 transcription factor proteins play a necessary role in making the phenotype sexually dimorphic and Abd-B acts as a key input in driving gene expression in the A5 and A6 segments.

Materials and Methods

Fly Stocks

Fly stocks used in this study were maintained at 22oC on a previously published sugar food medium (Salomone et al., 2013b). Species stocks used in this study were D. melanogaster

(14021-0231.04) from the National Drosophila Species Stock Center at Cornell University, and a

D. funebris stock that was obtained from Dr. Sean B. Carroll.

In situ hybridization

A previously described protocol was used in the in situ hybridizations studies (Camino et al., 2015; Jeong et al., 2008b). In brief, digoxigenin-labeled riboprobes for yellow, tan, ebony, pale, Ddc, and Abd-B were prepared through in vitro transcription from PCR templates amplified from each species’ genomic DNA (Table 3.1 for antisense probe primers). PCRs introduced a T7

60 promoter to each template from which antisense transcription is subsequently initiated from.

Abdomens were dissected at the pupal developmental stages P10 (yellow), P14-15(i) (pale, tan,

Ddc and Abd-B) and the newly eclosed adult fly stage P15(i) (ebony). These different stages were identified by inspection for the presence and absence of various morphological markers

(Ashburner et al., 2005) (Chapter 2). Ventral nerve cords were dissected from 3rd instar larvae and used as a positive control for Abd-B expression. Probe hybridizations were made visible with an

HRP conjugated anti-digoxigenin antibody (Roche Diagnostics), whose presence was detected by an alkaline phosphatase reaction using BCIP/NBT (Promega). After development, samples were transferred to glycerol mount, and finally situated between a cover slip and slide for imaging.

Immunohistochemistry

Dorsal abdomens were dissected for immunohistochemistry at the P14-P15(i) stages to detect Bab1 expression for D. melanogaster and D. funebris. Qualitative comparisons of expression patterns were made through immunohistochemical analysis using an antibody specific for Bab1 (Williams et al., 2008b). Abdomens were dissected to isolate the dorsal epidermis.

Samples were fixed for 35 min in PBST (phosphate‐buffered saline with 0.3% Triton X‐100) with

4% paraformaldehyde, and then blocked in blocking buffer (PBST with 1% bovine serum albumin) for 1 hour at room temperature. The abdomens were then incubated overnight with affinity purified rabbit anti-Bab1 primary antibody at a dilution of 1:200 in PBST (Salomone et al., 2013a). After four washes in PBST and then an hour incubation in blocking buffer, specimens were incubated with goat anti-rabbit Alexa Fluor 647 (Invitrogen) secondary antibody at a dilution of 1:500. After four washes in PBST, samples were incubated for 10 minutes in a 1:1 solution of glycerol mount (80% glycerol, 0.1M Tris pH 8.0) and PBST. Samples were transferred to glycerol mount, and finally situated between a cover slip and slide for imaging with a confocal microscope.

For Abd-B, dorsal abdomens were dissected for immunohistochemistry at the P14-P15(i) stages for D. melanogaster. Qualitative comparisons of Abd-B expression patterns were made through immunohistochemical analysis using an antibody specific for Abd-B (anti Abd-B 1A2E9 from Developmental Studies Hybridoma Bank). Abdomens were dissected to isolate the dorsal epidermis. Samples were fixed for 35 min in PBST (phosphate‐buffered saline with 0.3% Triton

X‐100) with 4% paraformaldehyde, and then blocked in blocking buffer (PBST with 1% bovine serum albumin) for 1 hour at room temperature. The abdomens were then incubated overnight with the mouse anti-Abd-B primary antibody at a dilution of 1:10 in PBST. After four washes in

PBST and then an hour incubation in blocking buffer, specimens were incubated with goat anti- mouse Alexa Fluor 647 (Invitrogen) secondary antibody at a dilution of 1:500. After four washes in PBST, samples were incubated for 10 minutes in a 1:1 solution of glycerol mount (80% glycerol, 0.1M Tris pH 8.0) and PBST. Samples were transferred to glycerol mount, and finally situated between a cover slip and slide for imaging with a confocal microscope.

Imaging of fly abdomens

Images of fruit fly abdomen pigmentation patterns and in situ hybridization expression patterns were taken using an Olympus SZX16 Zoom Stereoscope and Olympus DP72 digital camera. For pigmentation patterns, specimens were prepared from 4-day-old flies. Projection images for Bab1 and Abd-B expression patterns were generated with an Olympus Fluoview FV

1000 confocal microscope and software. In all figures where comparisons are made between images, each image was processed through the same sequence of modifications using Photoshop

CS3 (Adobe).

Table 3.1: Primers used to PCR-amplify templates for generating antisense in situ hybridization probes

Species gene Primer F Primer R

D. melanogaster Ddc TTCAGGGCACTGAAGCTCTGGTT ctaatacgactcactatagggCANGAGTACTCCATGTCCTCG

D. melanogaster pale TTGCAGATTAYGGYCTCACCGAGGA ctaatacgactcactatagggTTGGTCATCAGATGGTTGCAGTTRTCC

D. melanogaster yellow AACTTCCAATGGGGCGAGGAGGG ctaatacgactcactatagggAKGCCGTTGTGCTGGTTGAA

D. melanogaster tan GCCACACGGAGGAYGCACTGACGGAG ctaatacgactcactatagggAGCTCSGCRCTGATKGTGTTGATGCT

D. melanogaster ebony TGCAYCGCATCTTCGAGGAGCAGC ctaatacgactcactatagggGCCGGRAAGCTSGGATCGATGGGCA

D. funebris Ddc TGCATHGGATTCACBTGGATHGCSAGTCC ctaatacgactcactatagggACCCARCTGGGRTCCTTNAGCCAC

D. funebris pale GGAYAACTGCAACCATCTGATGACCAA ctaatacgactcactatagggCGCACATACTGKGTGCTCTGGAAGA

D. funebris yellow AACTTCCAATGGGGCGAGGAGGG ctaatacgactcactatagggCGATCSACAATGCCATGRAATTGCGG

D. funebris tan CACATCATCAGYGACAAGCCGCAGGG ctaatacgactcactatagggAGCTCSGCRCTGATKGTGTTGATGCT

D. funebris ebony TGCAYCGCATCTTCGAGGAGCAGC ctaatacgactcactatagggTCGTTGCCRCCGCGCAGCATCTCCTC

D. funebris Abd-B CAAGAAGAACTCRCAGCGMCAGGCC ctaatacgactcactatagggCACTGGTGCATYTTKGYGGCATGGTGAC

Notes: 1) The lowercase letters in the table represent the T7 promoter sequence that was added to the reverse primer for antisense in vitro transcription. 2) Primer pairs lacking the T7 promoter sequences had their PCR product cloned into a pGEM vector. The cloned insert was amplified from the vector with M13F and M13R primers, and this template had an antisense T7 promoter added. Antisense probe synthesis reactions were performed with T7 RNA polymerase.

Results

Spatial-, temporal- and sex-specific expressions of the differentiation genes for the Drosophila melanogaster tergite pigmentation Gene Regulatory Network.

The broad melanic phenotype decorating the A5 and A6 segment tergites of D. melanogaster males requires the expression of pigmentation genes needed to form black pigments, and the absence of expression for pigmentation genes that drive the formation of other

63 pigment colors. The key genes include yellow, tan, ebony, pale, and Ddc (Rebeiz and Williams,

2017a; Wittkopp et al., 2003). The spatial, temporal, and sex-specific nature by which these genes are expressed can be seen by the in situ hybridization technique that uses antisense probes to seek out the endogenously expressed mRNAs. For yellow, a male-specific pattern of expression is seen starting at the P10 stage in the A5 and A6 segments (Figure 3.3A and 3.3A’). A similar male- specific pattern of expression was observed for tan, albeit at the more advanced P14-15ii stage

(Figure 3.3B and 3.3B’). ebony encodes an enzyme that is needed to drive the production of yellow colored cuticle, and its expression occurs in newly eclosed flies (P15 stage) in a reciprocal to those for tan an yellow (Figure 3.3C and 3.3C’). ebony expression is notably absent from the male A5 and A6 epidermis, though this gene is highly expressed in the female A5 and A6 segment epidermis (Figure 3.3C and 3.3C’). The pale and Ddc genes encode enzymes that respectively catalyze the first and second steps in the canonical pigmentation pathway (Wright,

1987), and whose activity are necessary to make both black and yellow pigments. Corresponding with these more general pigment metabolism functions, pale and Ddc expression is seen throughout the abdomen epidermis, though seemingly expression is elevated in the regions where black coloration is widespread (Figure 3.3D-3.3E and 3.3D’-3.3E’). pale and Ddc expression peaks respectively in the P12 and P15 stages.

The expression patterns for these D. melanogaster pigmentation genes provides an overview of the blend of enzyme expressions that shape the formation of this sexually dimorphic phenotype. These expression outcomes inspired me to seek an answer as to whether or not this particular blend of expressions is required to make such a male-specific pigmentation phenotype.

One way to resolve whether pigmentation gene use is constrained or flexible is to see the manner in which they are expressed in a species that independently evolved a similar phenotype.

Figure 3.3: The expression profiles for the differentiation genes responsible for the D. melanogaster tergite pigmentation pattern. (A-E) Male and (A’- E’) female samples for D. melanogaster. yellow (A and A’) is highly expressed in males, most noticeably in A5-A6 segments as compared to lower expression observed in females. tan (B and B’) has high expression in males, most notably in A5-A6 segments as compared to the same segments in females. ebony (C and C’) has high expression in females, most notably in A5-A6 segments with some expression in more anterior segments. The expression of pale (D and D’) and Ddc (E and E’) is generally monomorphic, with expression appearing elevated in the more posterior segments. The yellow, tan, ebony, pale, and Ddc expressions shown are respectively of specimens at the P11, P14-15i, P15i, P15i, and P12 developmental stage.

Spatial-, temporal- and sex-specific expressions of the differentiation genes for the Drosophila funebris tergite pigmentation Gene Regulatory Network.

The male-specific tergite pigmentation of D. funebris has been inferred to be convergent to that of D. melanogaster (Gompel and Carroll, 2003). Thus, this species presents an opportunity to resolve the blend of expressions for the pigmentation enzyme genes making an independently evolved male-specific phenotype. In situ probes were made for the orthologous yellow, tan, ebony, pale, and Ddc genes. yellow and tan were found to be expressed in a male-specific pattern, with robust expression observed in the posterior abdominal segments compared to females

(Compare Figure 3.4A and 3.4B with 3.4A’ and 3.4B’). ebony was found to be expressed in a sex-specific manner too (Figure 3.4C and 3.4C’), with expression reciprocal to yellow and tan, and presaging the cuticle regions that develop to be more yellow/brown in color (Figure 1). In

65 contrast to the sex-limited expressions of yellow, tan, and ebony, pale and Ddc expression were found to be generally the same between males and females (Figure 3.4D-3.4E and 3.4D’-3.4E’).

In addition to the spatial- and sex-specific patterns of expression, these genes were found to be expressed at generally the same developmental stage as their D. melanogaster orthologs.

An interesting question in evolutionary-developmental biology is whether phenotypic evolution is predisposed to utilize certain genes in certain ways. For the independent evolved dimorphic tergite pigmentation of D. melanogaster and D. funebris, my data suggests that there is indeed constraint in how the differentiation gene tier of these gene regulatory networks are temporally, spatially, and sex-specifically deployed. These similarities in differentiation gene use, raised interest in seeing whether similarity also extends to the regulatory tiers of these tergite pigmentation gene regulatory networks.

Figure 3.4: The expression profiles for the differentiation genes responsible for the D. funebris tergite pigmentation pattern. (A-E) Male and (A’- E’) and female samples for D. funebris. yellow (A and A’) is highly expressed in males, most noticeably in A3-A6 segments as compared to lower expression observed in females. tan (B and B’) has high expression in males, most notably in A3-A6 segments as compared to the same segments in females. ebony (C and C’) has high expression underlying tergite regions that will not be melanized. These regions of expression are broader in females than males. The expression of pale (D and D’) and Ddc (E and E’) are generally monomorphic, and widespread throughout the abdominal segments. The yellow, tan, ebony, pale, and Ddc expressions shown are respectively of specimens at the P11, P14-15i, P15i, P15i, and P12 developmental stages.

Dimorphic Bab1 expression is unique to tergite pigmentation Gene Regulatory Network of D. melanogaster.

Critical determinants of the D. melanogaster dimorphic tergite phenotype are the paralogous Bab1 and Bab2 transcription factors. Loss-of-function mutations for the bab1 and bab2 genes result in females with male-like patterns of tergite pigmentation revealing the role these genes naturally play in repressing melanic tergite pigmentation in females (Kopp et al.,

2000; Rogers et al., 2013). Bab1 and Bab2 are expressed throughout the A2-A7 segment pupal epidermis of D. melanogaster females, with expression being absent for the most part from the male abdomen, notably in the A5 and A6 segments (Salomone et al., 2013a) (Figure 3.5A-3.5B and 3.5A’-3.5B’). Since Bab1 and Bab2 play this critical role in differentiating the tergite phenotypes of D. melanogaster, we sought to see whether these paralogs independently evolved to occupy a similar role in the D. funebris tergite pigmentation Gene Regulatory Network. With a cross-reactive antibody to Bab1, we visualized the expression of Bab1 in the pupal abdomen epidermis of D. funebris by an immunohistochemistry protocol (Salomone et al., 2013a). In contrast to the sexually dimorphic expression observed for D. melanogaster, Bab1 expression was present and indistinguishable in the pupal abdomen epidermis of male and female D. funebris

(Figure 3.5C-3.5D and 3.5C’-3.5D’).

The monomorphic expression pattern of Bab1 in D. fuenbris, and presumably for Bab2 as well, does not necessarily demonstrate that the Bab proteins are not a part of this species pigmentation Gene Regulatory Network. However, this expression outcome does indicate that some other transcription factor or transcription factors play the equivalent role to Bab in the regulatory tier of this independently evolved dimorphic pigmentation network. The next natural question to address is which gene or genes plays this critical role. One prime candidate it the Hox gene Abd-B.

Figure 3.5: Bab1 expression differs between D. melanogaster and D. funebris. (A-D) Bab1 protein expression visualized by immunohistochemistry in the dorsal epidermal cell underlying the tergites of developing pupae. In D. melanogaster, (A) Bab1 expression is greatly reduced in the epidermis of the male A5 and A6 segments as compared to the low levels observed in the A2- A4 segments. (B) In females, Bab1 expression extends from the A2 through the A6 segment. For D. funebris, Bab1 is expressed throughout the (C) male and (D) female dorsal abdominal epidermis. Red and yellow dashed lines shown in A and C respectively, represent the zoomed in areas of A-D that are presented in A’-D’. The red arrowheads point to the critical absence of Bab1 expression in the D. melanogaster A5 and A6 segments. Specimens shown are of the P14- 15i developmental stage.

Comparison of Abd-B expression between Drosophila melanogaster and Drosophila funebris.

Abd-B is a key regulatory gene in the abdominal pigmentation GRN of D. melanogaster.

The encoded Abd-B proteins directly activate yellow expression by binding to the body element

CRE (Jeong et al., 2006), indirectly activate tan expression through the t_MSE CRE (Camino et al., 2015), and in some manner activate Ddc expression through the Ddc-MEE1 CRE (Chapter 2).

Because of this known importance to D. melanogaster pigmentation, Abd-B made an excellent candidate gene to investigate for a potential role in the D. funebris GRN. The Abd-B gene resides on D. melanogaster chromosome 3, possessing 5 exons, the fifth that includes the homeobox that encodes the DNA-binding homeodomain (Figure 3.6A). A monoclonal antibody was made to this homeodomain (Celniker et al., 1989), which can be used in immunohistochemistry to visualize

68 the pattern of Abd-B expression during the late pupal time-period when pigmentation gene expression is being spatially and sex-specifically patterned. At late a stage of pupal development,

~90 hours after puparium formation, we observed Abd-B expression in the dorsal abdominal epidermis of the A5 and A6 segments (Figure 3.6D and 3.6D’), the segments in males that are covered by fully melanized tergites. Unfortunately, this antibody is not cross-reactive with the D. funebris Abd-B (data not shown). For this reason, we sought to reveal Abd-B expression as mRNA expression patterns by in situ hybridization. As a test for probe specificity, we explored the pattern of expression in replicate ventral cords during the 3rd instar larvae stage for D. melanogaster (Figure 3.6B and 3.6C). We observed a posterior-limited pattern of expression consistent with the findings in previous studies (Maeda and Karch, 2006), and indicative that our homeobox probe and in situ protocol were effective in revealing the domain of Abd-B expression.

We created an in situ probe to the orthologous gene region for D. funebris, and were able to detect an apparent expression pattern in the dorsal abdominal epidermis at a late stage of pupal development in females (Figure 3.6E and 3.6E’) and males (Figure 3.6F and 3.6F’). Interestingly though, the domain of expression was seen to be expanded into more anterior A3 and A4 abdominal segments in addition to the expected A5 and A6 segment expression. This expansion corresponds with the more anterior segments that are covered with broadly melanic tergites in this species (Figure 3.1). This outcome suggest that Abd-B underwent a radical alteration in expression pattern in the lineage of D. funebris and makes this gene a compelling candidate in the derived sexually dimorphic pigmentation Gene Regulatory Network for this species.

Figure 3.6: Comparison of Abd-B expression between D. melanogaster and D. funebris. (A) Representation of the Abd-B gene and its translated homeobox sequence that were cloned and used as a template for D. melanogaster and D. funebris in situ probes. (B and C) The specificity of the homeobox probe was assessed in the ventral nerve cord of D. melanogaster larvae. (D and D’) Abd-B protein is expressed in the A5 and A6 segment epidermis of D. melanogaster during P14-15i pupal development stage. Abd-B expression seemingly extends from the A3 through the A6 segment epidermis of P14-15i stage (E, E’) female and (F, F’) male D. funebris.

Discussion

In this chapter, we sought to investigate whether the independently evolved male-specific melanic abdomens of two different fruit fly species were achieved with similar or dissimilar Gene

Regulatory Networks. We found that the expression patterns of five key pigmentation genes were strikingly similar between D. melanogaster and D. funebris. At the level of regulatory genes,

Bab1 was found to be similarly expressed in the abdominal epidermis of D. funebris males and

70 females. This pattern contrasts to the female-limited expression in D. melanogaster that is necessary to make D. melanogaster pigmentation sexually dimorphic. This result suggests that another regulatory gene is playing a Bab-like role in the pigmentation Gene Regulatory Network of D. funebris, and raises the possibility that Bab1, and perhaps Bab2 as well, are not a part of this species’ network. In contrast to Bab1, D. funebris Abd-B expression showed some similarities to that seen for D. melanogaster, suggesting that this gene may play a similar role in spatially patterning pigmentation in both species. Abd-B expression has expanded compared to that of D. melanogaster, raising the suspicion that Abd-B might indeed be shaping the greater number of broadly melanic tergites in this species. A suspicion that requires further exploration.

Common deployment of pigmentation genes underlies independently evolved color patterns.

A priori, at least two scenarios can be imagined as to how black melanic pigmentation is spatially and sex-specifically patterned for the species D. melanogaster and D. funebris. One scenario is where the expression patterns known for yellow, tan, ebony, pale, and Ddc in D. melanogaster (Figure 3.3) are not strictly required for the pigmentation phenotype. For example, perhaps yellow, tan, and ebony do not all need to be expressed in a sexually dimorphic manner. A second scenario can be imagined in which these patterns of pigmentation gene expression are a strict requirement for the phenotype. We found that D. funebris utilizes the pigmentation genes

(yellow, tan, ebony, pale and Ddc) in similar spatial-, temporal-, and sex-specific manners (Figure

3.4) to D. melanogaster. These outcomes are consistent with the second scenario, which at the level of differentiation genes, convergent patterns of pigmentation required evolution to find ways to similarly deploy these terminal-acting network genes. This suggest that there is a precise chemistry involved in the making melanic pigments, which is likely to operate in any fruit fly species for which pigmentation is sexually dimorphic. While this is only a single trait of the innumerable number that exist in nature, perhaps in the future it might be found that similarity in

71 differentiation gene expressions might be a pervasive occurrence for the Gene Regulatory

Networks for convergent phenotypes.

The regulatory tiers differ between convergent pigmentation Gene Regulatory Networks.

The next natural question to speculate about regarding the Gene Regulatory Networks for convergent traits is whether the similar patterns of differentiation gene expression is achieved by the directives of the same regulatory genes, or an altogether unique set of genes. In recent years, the regulatory tier of the D. melanogaster tergite pigmentation Gene Regulatory Network has received considerable attention, and is recognized to include at least 28 transcription factor genes

(Rogers et al., 2014). Among these 28, however, two are recognized to be of central importance.

Abd-B is expressed in the A5 and A6 abdomen segments where the overlying tergites are completely melanic in males (Figure 3.6), and acts an essential spatial input for this pattern through its Gene Regulatory Network connections with Bab and several pigmentation genes

(Figure 3.2). The limitation of broad tergite melanism to D. melanogaster males requires the expression of the Bab transcription factors in the female abdomen only, where it can shut down expression of pigmentations genes such as yellow (Roeske et al., 2018) (Figure 3.2).

As a first step in determining whether convergence involved the use of the same regulatory genes, we tested whether or not Abd-B and Bab1 are similarly expressed in D. funebris as in D. melanogaster. In contrast to the pigmentation genes, Bab1 and Abd-B expression differs compared to D. melanogaster. Unlike D. melanogaster, Bab1 is expressed throughout the dorsal abdominal epidermis of male and female D. funebris (Figure 3.5). This rules out Bab1, and seemingly Bab2 as well, as the key determinant for sexually dimorphic pigmentation gene expression. Some other transcription factor or factors must occupy this Gene Regulatory Network role for D. funebris. We surprisingly found that Abd-B expression included the epidermis of more D. funebris abdominal segments (A3-A6) than it does for D. melanogaster (A5-A6) (Figure

3.6). Abd-B expression in D. funebris did not appear to be consistently sex-specific in pattern for

D. funebris, suggesting that it is not the transcription factor responsible for sex-specific pigmentation gene expression. However, this expanded expression is suggestive that it may play a key role in the number of melanic D. funebris tergites.

Hypotheses and implications for the D. funebris Abd-B expression pattern.

The collinear patterns of Hox gene expression along the antero-posterior axis is seen during the development of bilaterian animals as distantly related as humans and fruit flies (Carroll et al., 2004). These nested domain of Hox expression function as selectors for how serially homologous body segments will be individualized. Losses and gains in Hox gene expression are known to result in dramatic homeotic transformation in segment identity (Lewis, 1978), attesting to the powerful roles these genes play in directing development. Although some changes in Hox gene expression have been seen in animals of different orders (Averof and Patel, 1997), Hox expression is thought to be generally conserved at the taxonomic levels of species, genera, and even orders (Carroll, 2008; Yoder and Carroll, 2006). This conservation in embryonic expression patterns is likely due to the deleterious homeotic transformations that occur when domains of expression are changed. However, conservation in expression pattern at earlier embryonic stages of development may not mean that expression is similarly conserved at latter life stages. In fact,

Ubx expression has been shown to differ in pupal stages for water striders and fruit flies, in which these alterations in expression respectively shape leg length and hairiness phentoypes (Khila et al., 2009; Stern, 1998). These differences in expression raised the possibility that Abd-B expression might evolve to drive diversification of fruit fly tergite of pigmentation.

Abd-B expression is known to extend from the posterior most-segments through the A5 segment in larvae (Yoder and Carroll, 2006), early pupae (Kopp and Duncan, 2002; Wang and

Yoder, 2012), and late pupae (Figure 3.6) in D. melanogaster (Figure 3.7). Based on the known pleiotropic roles of Abd-B, our initial expectation was that Abd-B expression would remain conserved in other fruit fly species, including D. funebris (Figure 3.7, hypothesis 1). However, we

73 obtained data that reveals expression extending into additional anterior segments, specifically A4 and A3 (Figure 3.6). First off, the spectacular nature of these results necessitate an additional replication of the data and a comparison to the expression pattern for a negative control sense probe. However, the preliminary results suggest further investigation might validate an impressive expression change that has occurred within the Drosophila genus. A couple explanations can be imagined to explain this drastic expression change. One is where Abd-B expression was altered broadly across developmental time and tissues (Figure 3.7, hypothesis 2).

For this model, we would suspect observing expanded expression not just in the abdominal epidermis of pupae, but additionally in the larval ventral nerve cord. However, such a change would be expected to cause many deleterious effects due to pleiotropy, and thus this is not my favored hypothesis. A second hypothesis for Abd-B expression evolution, is where this anterior expansion is limited to the pupal abdominal epidermis or perhaps pupal developmental stage, thereby limiting the scale of undesired pleiotropy (Figure 3.7, hypothesis 3). One way to differentiate between hypotheses 2 and 3, is to reveal the expression pattern in the D. funebris larval ventral nerve cord, and perhaps in embryos too. Regardless if hypothesis 2 or 3 is supported, this expression change warrants future investigations into how it occurred at the levels of CREs and upstream regulatory genes. Moreover, the role for Abd-B in the D. funebris pigmentation Gene Regulatory Network rests solely on an expression correlation. Future studies should aim to functionally-validate an in vivo role.

Figure 3.7: Hypotheses and their implications for possible patterns of D. funebris Abd-B expression. (A) The established expression pattern of Abd-B in the ventral nerve cord of 3rd. instar larvae and the pupal dorsal abdominal epidermis of D. melanogaster. (B) Abd-B expression could be generally conserved between D. funebris and D. melanogaster, suggesting that evolved Abd-B expression was not responsible for the spatially expanded and sexually dimorphic pattern of D. funebris tergite pigmentation. (C) It is possible that Abd-B expression has expanded in both larvae and pupae, and that this highly pleiotropic change may result in expanded tergite pigmentation in this species among other differences not studied here. (D) It is also conceivable that Abd-B expression evolution was limited to an expansion in the pupal abdominal epidermis, limiting any pleiotropic outcomes to this developmental stage, notably tergite pigmentation.

Putative abdominal pigmentation Gene Regulatory Network for Drosophila funebris.

The gene regulatory network for abdominal pigmentation in D. funebris has many similarities to that of D. melanogaster when it comes to the deployment of genes in the differentiation tier (yellow, tan, ebony, pale and Ddc) that encode proteins involved in pigment metabolism (Wright, 1987). For the network’s regulatory tier there are many differences. First of all, Bab transcription factors were previously thought to act in a similar dimorphic manner due to an early pupa dimorphic pattern of expression in D. funebris (Gompel and Carroll, 2003). Here we show that this dimorphic expression pattern is not seen at the pupal stages when critical pigmentation genes are being expressed (Figure 3.5). Our preliminary data rules out the possibility of Abd-B making melanism male-specific, as Abd-B expression was not observed to be conspicuously dimorphic (Figure 3.6). If not Bab or Abd-B, than what transcription factor gene or genes could be responsible for dimorphism? My studies will Dr. William Rogers revealed many additional transcription factor genes that have loss-of-function tergite pigmentation phenotypes

(Rogers et al., 2014). However, most of these genes did not exhibit apparent sex-limited effects.

There remains one strong candidate gene though, doublesex or dsx. dsx is known to have sex- specific transcripts that results in male and female protein isoforms (An and Wensink, 1995;

Erdman et al., 1996). In D. melanogaster, the female isoform (DSXF) is a direct activator of Bab expression, whereas the male isoform (DSXM) represses Bab expression (Williams et al., 2008a).

Thus, DSX indirectly regulates deployment of the pigmentation genes through the activity of

Bab. Perhaps in D. funebris, DSX bypasses this “middle manager” activity and directly controls genes such as yellow and tan. Future studies should prioritize an analysis of dsx expression in D. funebris. Ultimately though, studies must push beyond revelations of expression patterns, to include gain- and loss-of-function experiments that can definitely indict or exonerate genes as members of this convergent tergite pigmentation Gene Regulatory Network (Figure 3.8).

Furthermore, evidence indicating that such regulatory genes operate within the D. funebris network remains just the beginnings of an investigation. A true understanding of the nature of a

Gene Regulatory Network requires mapping the transcription factors to binding sites in CREs that deploy the target genes of regulation. Importantly though, this chapter supports a conclusion that evolution has some options when it comes to the regulation of the more strictly required differentiation genes by transcription factor genes. This perhaps can be explained by the sheer number of transcription factor genes in an animal genome. For D. melanogaster, this number exceeds 700 genes (Pfreundt et al., 2010).

Figure 3.8: Model for the tergite pigmentation Gene Regulatory Network of Drosophila funebris. The current understanding of D. funebris pigmentation network. Dashed lines represent regulatory scenarios where a direct input between an encoded transcription factor and a pigmentation enzyme gene cis-regulatory element has not been established.

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87 APPENDIX A

Alignment of the Ddc-MEE1 with Binding Site Mutant Versions

AscI

Ddc-MEE1 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT (Grh KO) 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT (AP-1+CREB-A KO) 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT (EcRE KO) 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT

AP-1 site 1 Ddc-MEE1 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT (Grh KO) 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT (AP-1+CREB-A KO) 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT cAaTGAGAGC ATTGGATTAT (EcRE KO) 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT

CREB site 1 Ddc-MEE1 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC (Grh KO) 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC (AP-1+CREB-A KO) 101 TTGAatTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC (EcRE KO) 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC

Grh site 1 Ddc-MEE1 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT (Grh KO) 151 TCTATTccaa GGgTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT (AP-1+CREB-A KO) 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT (EcRE KO) 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT

Ddc-MEE1 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG (Grh KO) 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG (AP-1+CREB-A KO) 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG (EcRE KO) 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG

AP-1 site 2 Ddc-MEE1 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC (Grh KO) 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC (AP-1+CREB-A KO) 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTccCcC (EcRE KO) 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC

Ddc-MEE1 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT (Grh KO) 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT

88 (AP-1+CREB-A KO) 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT (EcRE KO) 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT

Ddc-MEE1 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT (Grh KO) 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT (AP-1+CREB-A KO) 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT (EcRE KO) 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT

Ddc-MEE1 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG (Grh KO) 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG (AP-1+CREB-A KO) 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG (EcRE KO) 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG

Ddc-MEE1 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT (Grh KO) 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT (AP-1+CREB-A KO) 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT (EcRE KO) 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT

Ddc-MEE1 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC (Grh KO) 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC (AP-1+CREB-A KO) 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC (EcRE KO) 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC

Ddc-MEE1 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG (Grh KO) 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG (AP-1+CREB-A KO) 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG (EcRE KO) 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG

-106 Ddc-MEE1 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC (Grh KO) 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC (AP-1+CREB-A KO) 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC (EcRE KO) 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC

Scan Mutant 11 Start (659) Conserved in D. virilis EcRE Grh site 2 -89 -59 Ddc-MEE1 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA (Grh KO) 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGcc aaGGgCCTGC GGAATTGGCA (AP-1+CREB-A KO) 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA (EcRE KO) 651 GtttaaCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA

TATA Box +1 Ddc-MEE1 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA (Grh KO) 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA (AP-1+CREB-A KO) 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA (EcRE KO) 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA

Scan Mutant 11 Start (794) Ddc-MEE1 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA (Grh KO) 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA (AP-1+CREB-A KO) 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA (EcRE KO) 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA

Ddc-MEE1 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC (Grh KO) 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC

89 (AP-1+CREB-A KO) 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC (EcRE KO) 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC

Ddc-MEE1 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA (Grh KO) 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA (AP-1+CREB-A KO) 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA (EcRE KO) 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA

SbfI Ddc-MEE1 901 AGCCTGCAGG (Grh KO) 901 AGCCTGCAGG (AP-1+CREB-A KO) 901 AGCCTGCAGG (EcRE KO) 901 AGCCTGCAGG

APPENDIX B

Sequence Alignment of Ddc-MEE1 with Scanning Mutant Versions

AscI Ddc-MEE1 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM1 1 GGCGCGCCgT aTaAtTtTcT tCtGcAaTgC aCtCgCcAcA tGaTaAcCaT DdcMEE1.SM2 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM3 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM4 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM5 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM6 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM7 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM8 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM9 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM10 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM11 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM12 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM13 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT DdcMEE1.SM14 1 GGCGCGCCTT CTCAGTGTAT GCGGAACTTC CCGCTCAAAA GGCTCAACCT

DdcMEE1 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM1 51 cGaCaAaTgC aCaTcGaAaA cTtCtAcAtT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM2 51 AGCCCACTTC CCCTAGCACA ATGaGcAcGg GcGgGcGcGa AgTtGcTgAg DdcMEE1.SM3 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM4 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM5 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM6 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM7 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM8 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM9 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM10 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM11 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM12 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM13 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT DdcMEE1.SM14 51 AGCCCACTTC CCCTAGCACA ATGCGAAAGT GAGTGAGAGC ATTGGATTAT

DdcMEE1 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM1 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM2 101 TgGcCtTaAa AcTgCaAgGc GaGtTgCcAc AcGaAaGgCc TcTtTGGTGC DdcMEE1.SM3 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTaA gAgGgGtTtC DdcMEE1.SM4 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM5 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM6 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM7 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM8 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM9 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC

91 DdcMEE1.SM10 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM11 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM12 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM13 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC DdcMEE1.SM14 101 TTGACGTCAC AATTCCATGA GCGGTTCAAA AAGCACGTCA TATGTGGTGC

DdcMEE1 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM1 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM2 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM3 151 gCgAgTcAaC tGgTgCaAcG cTtCtCtTcA cGaGgGaCcT gCaAaGtCgT DdcMEE1.SM4 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM5 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM6 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM7 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM8 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM9 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM10 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM11 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM12 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM13 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT DdcMEE1.SM14 151 TCTATTAACC GGTTTCCAAG ATGCGCGTAA AGCGTGCCAT TCCACGGCTT

DdcMEE1 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM1 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM2 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM3 201 cAgCcAgTgC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM4 201 AATaAcTgTa TgGgCgTgCa TcCtAcTcTc AaTgTtTgTc CcTgTgTgTt DdcMEE1.SM5 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM6 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM7 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM8 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM9 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM10 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM11 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM12 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM13 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG DdcMEE1.SM14 201 AATCAATTTC TTGTCTTTCC TACGAATATA ACTTTGTTTA CATTTTTTTG

DdcMEE1 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM1 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM2 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM3 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM4 251 CtTtAgTgTg TaTgCtGtGc GgCaAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM5 251 CGTGATTTTT TCTTCGGGtA tTaCcAtAcA cAaCaTtTgT aGcGgGcCgC DdcMEE1.SM6 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM7 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM8 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM9 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM10 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM11 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM12 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM13 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC DdcMEE1.SM14 251 CGTGATTTTT TCTTCGGGGA GTCCAAGAAA AACCCTGTTT CGAGTGACTC

DdcMEE1 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM1 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM2 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM3 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM4 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT

92 DdcMEE1.SM5 301 cTcAgTtGtG tAgTaCgGcC tAtAgCtCgC gCgTgCaAaA AATTCGAGTT DdcMEE1.SM6 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTgTaCcCc AcTgCtAtTg DdcMEE1.SM7 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM8 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM9 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM10 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM11 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM12 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM13 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT DdcMEE1.SM14 301 ATAATTGGGG GATTCCTGAC GAGATCGCTC TCTTTCCACA AATTCGAGTT

DdcMEE1 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM1 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM2 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM3 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM4 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM5 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM6 351 GtGcAtCcCt TtAtTcGcAg TaAcAcTtTg TgGaTgGaTt TgTgAcAgAg DdcMEE1.SM7 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATcT DdcMEE1.SM8 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM9 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM10 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM11 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM12 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM13 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT DdcMEE1.SM14 351 GGGAAGCACG TGAGTAGAAT TCAAAATGTT TTGCTTGCTG TTTTAAATAT

DdcMEE1 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM1 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM2 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM3 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM4 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM5 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM6 401 CcCgAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM7 401 aAaTcGtTgC gCcAcCgAcT gTaAcAcAgA cTaAcAgTcA tTgCcCcGcG DdcMEE1.SM8 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM9 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM10 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM11 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM12 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM13 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG DdcMEE1.SM14 401 CACTAGGTTC TCAAACTAAT TTCAAAAATA ATCAAATTAA GTTCACAGAG

DdcMEE1 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM1 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM2 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM3 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM4 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM5 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM6 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM7 451 aTtGaAcAgA cAcTtTcAgA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM8 451 CTGGCAAATA AAAgGgAcTc GaTgGaAgGg AgGgAgAgAg AgAgAgTgTg DdcMEE1.SM9 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM10 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM11 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM12 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM13 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT DdcMEE1.SM14 451 CTGGCAAATA AAATGTAATA GCTTGCATGT ATGTATATAT ATATATTTTT

93 DdcMEE1 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM1 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM2 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM3 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM4 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM5 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM6 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM7 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM8 501 TgAcAgTaTc AcTcAcTaCc TtGcAcAgAc AtCaTTTGAT ATCCAGTTAC DdcMEE1.SM9 501 TTAAATTCTA AATAAATCCA TGGAAAATcA cGaCgTgGcT cTaCcGgTcC DdcMEE1.SM10 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM11 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM12 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM13 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC DdcMEE1.SM14 501 TTAAATTCTA AATAAATCCA TGGAAAATAA AGCCTTTGAT ATCCAGTTAC

DdcMEE1 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM1 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM2 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM3 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM4 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM5 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM6 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM7 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM8 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM9 551 gGcTgCcGaG aCaAcTgAcT tCcTtTgCaA cAcAcGgGgC cAcAcAaGgG DdcMEE1.SM10 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAcAcCtTt DdcMEE1.SM11 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM12 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM13 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG DdcMEE1.SM14 551 TGATTCAGCG CCCAATTAAT GCATGTTCCA AAAAAGTGTC AAAAAACGTG

DdcMEE1 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM1 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM2 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM3 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM4 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM5 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM6 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM7 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM8 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM9 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM10 601 CcCcAcTaAc AaGcGcGaTt AcTgTtTgTg TcCtAaAtCt GaTtCtAgTa DdcMEE1.SM11 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM12 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM13 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC DdcMEE1.SM14 601 CACAAATCAA ACGAGAGCTG AATTTGTTTT TACGACAGCG GCTGCGATTC

DdcMEE1 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM1 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM2 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM3 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM4 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM5 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM6 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM7 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM8 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM9 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM10 651 GcAtTgCcGa GtCgGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA

94 DdcMEE1.SM11 651 GAAGTTCAtC tGaTtCtGcC gGaGcTgGcA aCtGgCaTtC tGcAgTtGaA DdcMEE1.SM12 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM13 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA DdcMEE1.SM14 651 GAAGTTCAGC GGCTGCGGAC TGCGATTGAA CCGGTCCTGC GGAATTGGCA

DdcMEE1 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM1 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM2 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM3 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM4 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM5 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM6 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM7 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM8 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM9 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM10 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM11 701 tCtCgGaTtG cCtGtCgTgA cAcGaCcTtG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM12 701 GCGCTGCTGG ACGGGCTTTA AAAtCaAgGt CaAcGcGaGt GaAtCtCgCc DdcMEE1.SM13 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA DdcMEE1.SM14 701 GCGCTGCTGG ACGGGCTTTA AAAGCCATGG CCAAGAGCGG GCAGCGCTCA

DdcMEE1 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM1 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM2 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM3 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM4 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM5 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM6 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM7 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM8 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM9 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM10 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM11 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA DdcMEE1.SM12 751 GgTcAtAtGc GcAaGaCcAt CtCcCcGaAc TaAtCcCaGc AcTcTCAGCA DdcMEE1.SM13 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCtA cAgAgCcGaA DdcMEE1.SM14 751 GTTAAGAGGA GAACGCCAAG CGCACAGCAA TCAGCACCGA AATATCAGCA

DdcMEE1 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM1 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM2 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM3 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM4 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM5 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM6 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM7 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM8 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM9 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM10 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM11 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM12 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC DdcMEE1.SM13 801 gCtAcAgAgC cGaAcAgAcA gAgTcGaTtT gCgAcAaCcG cAtGtCcAcA DdcMEE1.SM14 801 TCGAAATATC AGCAAATAAA TATTAGCTGT TCTAAACCAG AAGGGCAAAC

DdcMEE1 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM1 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM2 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM3 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM4 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM5 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA

95 DdcMEE1.SM6 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM7 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM8 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM9 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM10 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM11 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM12 851 TGAACTTAGA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM13 851 gGcAaTgAtA GCAAAGATTT AGTTCGGAAC GGAAGTAAAG CTCGGCAACA DdcMEE1.SM14 851 TGAcCgTcGc GaAcAtAgTg AtTgCtGcAa GtAcGgAcAt CgCtGaAcCc

SbfI DdcMEE1 901 AGCCTGCAGG DdcMEE1.SM1 901 AGCCTGCAGG DdcMEE1.SM2 901 AGCCTGCAGG DdcMEE1.SM3 901 AGCCTGCAGG DdcMEE1.SM4 901 AGCCTGCAGG DdcMEE1.SM5 901 AGCCTGCAGG DdcMEE1.SM6 901 AGCCTGCAGG DdcMEE1.SM7 901 AGCCTGCAGG DdcMEE1.SM8 901 AGCCTGCAGG DdcMEE1.SM9 901 AGCCTGCAGG DdcMEE1.SM10 901 AGCCTGCAGG DdcMEE1.SM11 901 AGCCTGCAGG DdcMEE1.SM12 901 AGCCTGCAGG DdcMEE1.SM13 901 AGCCTGCAGG DdcMEE1.SM14 901 atCCTGCAGG

96 APPENDIX C

Alignment of Sequence Orthologous to the D. melanogaster Ddc-MEE1

AscI MEE1(mel) 1 GGCGCGCCCT TCTCAGTGTA TGCGGAACTG CCCGTTCAAA AGGCTCAACC MEE1(bia) 1 GGCGCGCC-T TCCCAGTGTA ATTCCTATTC ACCATTCAAA AGGCTCAGCC MEE1(pse) 1 GGCGCGCC------MEE1(Dwil) 1 GGCGCGCC------MEE1(vir) 1 GGCGCGCCgc tgttgttatg ccacataata gagag------

AP-1 site 1 MEE1(mel) 51 TAGCCCACTT CCCCTAGCAC AATGCGAAAG TGAGTGAGAG catTGGATTA MEE1(bia) 50 TAGCCCACTT CCCCTAGCTC AACGGGAACG TGAGTGAGGA tg--GGATTA MEE1(pse) 7 ------MEE1(Dwil) 9 ------MEE1(vir) 36 ------TGAAGCC

CREB site 1 MEE1(mel) 101 TTTGACGTCA CAATTCCATG AGCGGTTCAA AAAGCACGTC ATATGTGGTG MEE1(bia) 98 TTTGACGTCA CAAgcCCATG AGCGGTTCAA AAC-CACGTC ATATATGATG MEE1(pse) 7 ------CACGTC ACAGTTGATG MEE1(Dwil) 9 ------TCCAAG AGCGGTTCAg agcatcaaat tcatttgcga MEE1(vir) 43 TTTGACGTCA TCAAACATTG ATGGAGTCTC GTGCTtgccc gagtatctac

Grh site 1 MEE1(mel) 151 ------CTCTAATA ACCGGTTTCC AAGATGCGCg MEE1(bia) 147 ------CTCGGAGA ACCGGTTCCC CAGAGGCGCG MEE1(pse) 25 ------CTCTGAAA ACCTGTTttc aaaagcgcga MEE1(Dwil) 45 acgtcacaaa aaacatcttt ctCTTTCAAA ACCAGTTTCA AAGATGTGCG MEE1(vir) 93 gagatacaga tacacacacg ttcagctaca gctcgtgcct taatgaattc

MEE1(mel) 179 taAAGCGTGC CATTCcacgg ctTAATCAAT TTCT------TGT MEE1(bia) 175 T-AAGCGTGC CATTCgcagt a-TAATCCAT TTCT------TGc MEE1(pse) 53 aaaacgtacg aatggaataa tgataaatgt ctcgttttct atgaatttca MEE1(Dwil) 95 A-ATGCGTtt ttatTAGGTT CATTAATCTT GTTTTTTTTT CTTtttcTGT MEE1(vir) 143 gcgtgcga------TATGTA AATTAAAGTT GTTTTGTTTT ATTcgaaaat

MEE1(mel) 216 GTTTCCTACG AATTTAACTT TGTTTACATT TTTTtgcgtg att----TTT MEE1(bia) 210 ctcttCTATG AATCTTAATT TGTTTATCTT ATTTgcgtgg ttttttaTTT MEE1(pse) 103 aaaaatcgct gcccggtgtg ac------MEE1(Dwil) 144 CTTCACCATG AATATCAATT TGTTTATgta gtaattctgt gtagtcaact MEE1(vir) 187 a------

AP-1 site 2 MEE1(mel) 262 TCTTCGGGGa gtcCAAGAAA AACCCTGTTT CGAGTGACTC ATGAttGGGG MEE1(bia) 260 TCTTCGGGGc t--CAAGAAA AACCCTGTCT TGAGTGACTC ATTAA-GGGG

97 MEE1(pse) 125 ------GTGACTC ATGAC-GGGG MEE1(Dwil) 194 ggctgagaaa atccctgacc acttgtgatt catcactgt tggggattcc MEE1(vir) 188 ------

MEE1(mel) 312 GATTCCTGAC GAGATCGCTC TCTTTccgca aattcgagTT G------MEE1(bia) 307 GATTCCTGCC GCGATCGCTC TCTTTttct------TT G------MEE1(pse) 141 GATTCCTGga ccgcactcgc acggtcccca taaaacgata cctgggaacg MEE1(Dwil) 244 tttttttctg tggtctttgt aagcaagaac gtatcgatat ggttagaagt MEE1(vir) 188 ------

MEE1(mel) 353 ------GGAA GCACGTGAGT AGAATTCAAA atgttttgct tgctgtttta MEE1(bia) 339 ------GGGA GCACGTGAGC GGAATaaata tcaatctgtt ttaatgggaa MEE1(pse) 191 gcgcttGGAG GATCGTGCGC AGAATTCAAA ccattctgag aggtctttca MEE1(Dwil) 294 taactagttt acgattggtt aaaataatta cagtagcaaa attatgaatt MEE1(vir) 188 ------

MEE1(mel) 397 aatatcacta ggttctcaaa ctaatttcaa aaataatcaa attaagttca MEE1(bia) 383 ataatgtata ttaaacaaag aataaatatt agacattatt ttattcttat MEE1(pse) 241 attttaaatg ggctacta------MEE1(Dwil) 344 catgttttta caaaca------MEE1(vir) 188 ------

MEE1(mel) 447 cagagctggc aaataaaatg taatagcttg catgtatgTA TATATATATA MEE1(bia) 433 tgcaagaatg aaaataaatc aaaatattaa aaatgtgctt ------TA MEE1(pse) 259 ------MEE1(Dwil) 360 ------A TTTTTTTTTA MEE1(vir) 188 ------TA TATATATATA

MEE1(mel) 497 TTTTTTTAAA TTCTAAATAA ATccatggaa aataaagcct ttgatatcca MEE1(bia) 475 TTTTTTTAGC TTAAAAATAC ATaacagtga tattaacgca agtatttaag MEE1(pse) 259 ------MEE1(Dwil) 371 TTTCTTTTCT TTATTTTTta taaattttga atgtattgaa attaaaccgt MEE1(vir) 200 TTTTTTTTAT TCATTTTTgc aacacttttg acttgtatct cttgtgttgt

MEE1(mel) 547 gttactgatt cagcgccc------MEE1(bia) 525 ctatgcactt tttatt------MEE1(pse) 259 ------MEE1(Dwil) 421 taataatagg ctgatttggg taaaattaat ttttaaagta tgctataagc MEE1(vir) 250 atctcattat tgtatctctt ttaacttgta tctcttgtct tgcctgagga

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 259 ------MEE1(Dwil) 471 agtctggtag cttaaagatt gtcaataaac tgttttcgaa ataagattta MEE1(vir) 300 aaccccacgc agcgcgtgag tcatgagctc ggggggaatt cttgtcttga

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 259 ------MEE1(Dwil) 521 tcctttatgt tacagaacat tataaaattt ctcttgagtt tttaaaaaat MEE1(vir) 350 ctgtcgatcc aactgaatag ttaagagctg gttgattatt tctattgcac

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 259 ------

98 MEE1(Dwil) 571 gcatttgttg tctgcatatt gcatatttta gggagccaac tttccttcct MEE1(vir) 400 tttatgcgaa aatgaaacga cgcgtgcaaa atatcaaaca acattgcgct

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 259 ------MEE1(Dwil) 621 ttacatttct aaacaccact tgacacctct taagaagctg aaaaagtaat MEE1(vir) 450 tgtcaac------

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 259 ------MEE1(Dwil) 671 ttatttgtaa acttacagtt tctattatat cgtgtaaaaa gttgaactaa MEE1(vir) 457 ------

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 259 ------TT AATTAATTAT taatataatt MEE1(Dwil) 721 ctttttttga atgaaatgta tacaaattTT AATTAATTAT ccgacttttg MEE1(vir) 457 ------

MEE1(mel) 565 ------MEE1(bia) 541 ------MEE1(pse) 281 gtattaattt attggatttt attggaaaga aaaagtttcc ataaacttct MEE1(Dwil) 771 ttatttcttt tgtgtgaaaa aacattatta aaaaattaga gcaaaacatc MEE1(vir) 457 ------

MEE1(mel) 565 ------MEE1(bia) 541 ------T ATTTTTTATT MEE1(pse) 331 tagatctgcc cattctagtt cgtcagtagc ttccaagaac ataattcaag MEE1(Dwil) 821 taatacagtt ttgattcttg tatgttaaac ttattgtcgT ATTTTTTATT MEE1(vir) 457 ------

MEE1(mel) 565 ------MEE1(bia) 552 Tataaaccga tgtagatcga gttcctaagt tcatgtgatt ttatacagta MEE1(pse) 381 atcgagctgt cccagctctg aaccattcca ttcagagggg tcttcttaca MEE1(Dwil) 871 Ttcgttcaaa gaaatgcaaa ctcgtataaa catgggttaa ttgttaacaa MEE1(vir) 457 ------

MEE1(mel) 565 ------MEE1(bia) 602 cagagcacct gttccggtaa caagtactgt ccagttgACT GAGTCACGCA MEE1(pse) 431 gctttgactg ggcccgagtt taatgtcctg ct-----ACT GAGACACGCG MEE1(Dwil) 921 gtttaatgct ccttataatt tttttttggt tttcataatc aaaatcaaat MEE1(vir) 457 ------

MEE1(mel) 565 --AATTAATG CATGTTCCAA AAaagtgtca aaa------AACGTGCAC MEE1(bia) 652 CAAATgcgtg ttccgtaaaa atcagccaaa accaccttta gAACGTGCGC MEE1(pse) 476 CAAATTAATG CATGTTCCAA AAtaacaacc aaaaaatcac cagaagagcg MEE1(Dwil) 971 ttttaagaaA AAAACAATTT CATTTGAATA ATTATCCAAt caaacctgaa MEE1(vir) 457 ------A AATACAATTT AATATATATA TATATATAAa tacacacaca

MEE1(mel) 605 AAATCaaacg agagctgaat ttgtttttac gacagcggct gcgattCGAA MEE1(bia) 702 AAATCcagtg cagaatgtat tttgcaaccc gcctg------CGGA MEE1(pse) 526 caataacaaa tcaaagagaa tcgcacaaac aagttgagaa tgttgcagtc

99 MEE1(Dwil) 1021 ttacacacac acacaattgc agtatttgta tttttagcga aagagaattt MEE1(vir) 498 cctgcaagtg tttatttatt acaaattaat ttccacagtt tcagttcagc

EcRE Grh site 2 MEE1(mel) 655 GTTCAGCGGC TGCGGACTGC G------AT TGAACCGGTC CTGCGGAatT MEE1(bia) 741 GTTCAGCGGC AGCGGATTTC GGATCGAGGT TGAACCGGTC CTGCGGAgcT MEE1(pse) 576 cggagccacg gccgagcggc ------T TGAACCGGTC CTGCttgggc MEE1(Dwil) 1071 gttattg------TTTC GGCGCAATAT TGAACCGGTC CTGaactgat MEE1(vir) 548 gatgagccga gggtagcagg ------TGAACCGGTC CTGCGGctgc

MEE1(mel) 698 GGCAGCGCTG CTGGAC------GG ----GCTTTA MEE1(bia) 791 GGCAGCGCTG CTGGCC------GG ----GCTTTA MEE1(pse) 617 cgtacccgtg cccgatgcgg tggctgtggg ctgctgctGG ----GCTTTA MEE1(Dwil) 1112 gctcatgccc cccgttttgg agctggtgct ccgctttaa- ----GCTTTA MEE1(vir) 588 tctcgaagct cgctgctcgc agcacgcagc tccaacgagg cgctGCTTTA

MEE1(mel) 722 AAAGCCATGG CC-AAGAGCG GGCAGCGCTC AGTTAAGAGG AGAACGCCAA MEE1(bia) 815 AAAGCCTTGG CC-AAGAGCG AGCAGCGCTC AGTTAGGAGG CGAACGTCGA MEE1(pse) 663 AAAGCCTTGG CC-AAGTGCG AGCAGCGCTC AGTTGACAAG CGAACGCCGA MEE1(Dwil) 1157 AAAGCCTTCG Cattc-TGTG ACTAGCGCTC AGTTCGCAGG CACGCGTCAC MEE1(vir) 638 AAAGCgccgc ccgAAGTGCG ACCAGCACTC AGTTGGCTAA CGgatgccgg

MEE1(mel) 771 GCGCACAGCA Atcagcaccg aaatatcagc atcgaaatat cagcaaataa MEE1(bia) 864 GCGCACAGCA gcccatcgag cgattcaaag cagaagacac ccattgtcgc MEE1(pse) 712 GCtccgaaca ctacaccgaa caccgcacac cgagcaccaa gtgggtccat MEE1(Dwil) 1206 TGTAACAGCA Tctctctatc tctctctctc taatataaac gtgtgttttt MEE1(vir) 688 agcgggcaca gcttgactaa atatagtaag aactaat------

MEE1(mel) 821 atattagctg ttctAAACCA GAAGGGCAAA CTGAACTTAG aGCAAAGATT MEE1(bia) 914 agcta------AAAACA GCAGGGCAAA CTCAAGTTGG -GCACAGATT MEE1(pse) 762 cacagctcag aaagcagcgc aatagacca------AAAGATT MEE1(Dwil) 1256 ctcccgtcgt gtgtgacgaa agattaactt taactaaaat atcaagtaca MEE1(vir) 725 ------AAAACA GCAGGCGAAA Caaaacgaaa cgaaacggaa

SbfI MEE1(mel) 871 TAGTTCGGAA CGGAAGTAAA GCtcggcaac aagCCTGCAG G------MEE1(bia) 954 TAGTTCGGAG CGGAAACAAg GCGCGACAAG TGCCCTGCAG G------MEE1(pse) 798 TAGTTCGAAA CGGAAACAAA GCGCGACAAG TGCCCTGCAG G------MEE1(Dwil) 1306 tac-----AA CGGAAACAAA agc------CCTGCAG G------MEE1(vir) 761 cgcaactaag cgacacattc acggcgaaaa gtgCCTGCAG G------S

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