https://orcid.org/0000-0001-7538-9805
Figures
Abstract
Changes in the control of developmental gene expression patterns have been implicated in the evolution of animal morphology. However, the genetic mechanisms underlying complex morphological traits remain largely unknown. Here we investigated the molecular mechanisms that induce the pigmentation gene yellow in a complex color pattern on the abdomen of Drosophila guttifera. We show that at least five developmental genes may collectively activate one cis-regulatory module of yellow in distinct spot rows and a dark shade to assemble the complete abdominal pigment pattern of Drosophila guttifera. One of these genes, wingless, may play a conserved role in the early phase of spot pattern development in several species of the quinaria group. Our findings shed light on the evolution of complex animal color patterns through modular changes of gene expression patterns.
Citation: Raja KKB, Shittu MO, Nouhan PME, Steenwinkel TE, Bachman EA, Kokate PP, et al. (2022) The regulation of a pigmentation gene in the formation of complex color patterns in Drosophila abdomens. PLoS ONE 17(12): e0279061. https://doi.org/10.1371/journal.pone.0279061
Editor: Barbara Jennings, Oxford Brookes University, UNITED KINGDOM
Received: August 17, 2022; Accepted: November 29, 2022; Published: December 19, 2022
Copyright: © 2022 Raja et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was funded to Thomas Werner by the National Institutes of Health, General Medical Sciences, grant number 1R15GM107801-01A1. https://www.nih.gov/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Pigmentation patterns are among the most beautiful and striking phenotypic features displayed by animals. They are known to play important roles in adaptation (camouflage and mimicry), reproduction (mate choice), and physiology (UV protection and thermoregulation) [1, 2]. Melanin pigments are commonly found in both invertebrates and vertebrates. The biochemical pathway that leads to melanin synthesis is well understood. Several studies have reported that the yellow (y) gene is required for the formation of black melanin in insects [3–9], where it plays a pertinent role in the evolution and diversification of pigmentation patterns. Spatio-temporal changes of y gene expression due to mutations in y cis-regulatory modules (CRMs) as well as changes in the deployment of transcription factors that control y CRMs have contributed to the pigmentation diversity that we see among Drosophila species [3, 10–15]. Prior studies have identified y CRMs and some of the trans-factors (i.e., Abdominal-A (Abd-A), Abdominal-B (Abd-B), Engrailed (En), and Distal-less (Dll)) that regulate y to establish simple pigment patterns on the wings and abdomens of Drosophila species [4, 12, 16–18]. However, how complex color patterns are encoded in DNA, generated during development, and evolve over time, is poorly understood.
To better understand the development and evolution of complex color patterns, we investigated the great variety of abdominal color patterns displayed by members of the quinaria species group within the genus Drosophila, which started diverging ~10 million years ago (MYA), while the common ancestor of Drosophila melanogaster and the quinaria group was dated back to 60 MYA [8, 19–23]. We primarily focused on Drosophila guttifera (D. guttifera), which displays the most complex abdominal color pattern of this group, consisting of four sub-patterns: a dorsal, a median, and a lateral pair of spot rows, as well as a dorsal midline shade (Fig 1A and 1B) [8, 19, 21]. Most other species of the quinaria group lack at least one of the four sub-patterns, providing an intriguing example of pattern modularity among species and thus a good foundation for studying the changes underlying complex pattern formation.
a, Adult, dorsal view showing the dorsal midline shade and the dorsal and median spot rows. b, Adult, lateral view. All three spot rows are visible along with the dorsal midline shade c, yellow mRNA expression pattern in a pupal abdomen at stage P10 showing three pairs of longitudinal spots and a dorsal midline shade. d, Yellow protein expression pattern in a P11 pupal abdomen. dms = dorsal midline shade, d = dorsal, m = median, l = lateral spot rows.
In this study, we show that only one CRM of y controls the entire abdominal spot pattern in D. guttifera. Furthermore, the developmental gene wingless (wg) appears to be the upstream inducer of the spotted pattern. In several members of the quinaria group, both wg and y expression patterns strongly correlate with the spot pattern diversity displayed by this species group.
Results
Gene expression patterns prefigure diverse abdominal spot patterns in species of the quinaria group
We first determined whether the y gene is expressed in developing pupae where black pigment will form on the adult abdomen of D. guttifera. Using in situ hybridization (ISH) and immunohistochemistry in D. guttifera pupae, we found that the y mRNA and Y protein expression patterns accurately prefigure the complex spot and shade pattern on adult flies (Fig 1). In order to identify putative upstream activators of y, we performed an ISH screen for 110 developmental genes (that were likely or proven to play a role in melanin formation as toolkit, immune, or terminal pigmentation genes) to detect those whose expression patterns prefigure that of the y gene (S1 Table) [24]. We found that wg expression precisely foreshadows the six rows of black spots (Fig 2B and S1–S4 Figs). Additionally, decapentaplegic (dpp) expression foreshadowed the dorsal and median pairs of spot rows (Fig 2C and S5A Fig), while abdominal-A (abd-A) expression correlated with the lateral pair of spot rows and the dorsal midline shade (Fig 2D, 2E and S5B Fig). Additionally, hedgehog (hh) and zerknullt (zen) were expressed along the dorsal midline of the abdomen (Fig 2F and 2G, S5C and S5D Fig). Thus, the activation of the D. guttifera color pattern appears to be induced in a modular fashion, which agrees with our observation that abdominal pigmentation patterns within the quinaria group are variations of the D. guttifera pattern ground plan (Fig 3). This situation is reminiscent of the wing pattern ground plan in nymphalid butterflies [25, 26]. Some examples of genes that did not show any detectable spot or shade pattern are shown in S6 and S7 Figs.
a, Adult, lateral view. b-g, ISH in pupal abdomens. b-d, show lateral orientation. e-g, dorsal orientation of the pupal abdomen. b, wg expression is detected in all abdominal spot rows at pupal stage P7. c, P7 pupal abdomen showing dpp expression in the dorsal and median spot rows. d, P9 pupal abdomen showing abd-A in the lateral spot row. e, P9 pupal abdomen showing staining of an abd-A probe along the dorsal midline. f-g, hh and zen expression patterns were detected in P10 pupal abdomens. ISH was performed on pupae at stages P6 –P10 to screen for the mRNA transcripts of each gene. h, Adult, dorsal view. dms = dorsal midline shade, d = dorsal, m = median, l = lateral spot rows.
+ = gain, - = loss of a pattern element. “Repressor” suggests that stripes may be broken into spots by unidentified repressors of pigmentation. The arrows solely illustrate the modularity of these complex patterns and do not imply any evolutionary direction.
We next asked whether the abdominal spot pattern of a species closely related to D. guttifera shares a similar gene expression pattern modularity [22]. We thus performed ISH experiments in Drosophila deflecta (D. deflecta), which displays six longitudinal spot rows on its abdomen but, notably, lacks the dorsal midline shade (Fig 4A and 4B). As in D. guttifera, y mRNA in D. deflecta pupal abdomens was expressed in six rows of spots, but not along the dorsal midline, as we expected (Fig 4C–4E). Similarly, wg foreshadowed all six rows of spots, while dpp expression matched all but the lateral spot rows, just like in D. guttifera (Fig 5B and 5C, S8A Fig). In contrast to the D. guttifera results, abd-A, hh, and zen were absent along the dorsal midline, which agrees with the lack of dark pigment in D. deflecta adults (Fig 5D–5G and S8B). However, abd-A expression was not detectable where the lateral spot rows will form (Fig 5D), suggesting that these particular spots may be controlled by a different gene in D. deflecta.
a, Adult lateral view. b, Adult dorsal view. c-e, y mRNA in the P10 pupal epidermis prefigures the adult spot pattern. d = dorsal, m = median, l = lateral.
a, h, Adult D. deflecta lateral and dorsal view. b, c, Lateral view. Developmental gene expression patterns of wg and dpp at pupal stage P7 in D. deflecta foreshadow distinct subsets of the adult abdominal color pattern. d-g, Dorsal view. abd-A, hh, and zen are not expressed along the dorsal midline of P9 and P10 pupae. ISH was performed on pupae at stages P6 –P10 to screen for the mRNA transcripts of each gene.
We next tested if the developmental genes that prefigured the adult color patterns show different expression patterns in two other closely related species that have fewer spot rows than D. guttifera and D. deflecta. Out of the five developmental genes that are expressed in a matching prepattern, we focused on the wg gene because its expression pattern foreshadowed all six spot rows in both D. deflecta and D. guttifera, while wg is also known to induce the black spots on the wings of D. guttifera [6]. To strengthen the evidence between the wg expression pattern in developing abdomens and the black spots on the adult abdomens in D. guttifera and D. deflecta, we tested if the correlation holds up even in species that display successively fewer rows of black spots—Drosophila palustris (D. palustris) and Drosophila subpalustris (D. subpalustris). These two quinaria group members display partial patterns of the D. guttifera ground plan, i.e., D. palustris and D. subpalustris lack two or four rows of spots, respectively (Fig 6A and 6D). Even in these two additional species, our ISH results showed that wg was expressed only at sites that will develop black spots in the adult (Fig 6B, 6C and 6E), thus strengthening the correlation among four species with varying pigmentation patterns. We have shown earlier that y mRNA also precisely foreshadows the adult abdominal spot patterns of D. palustris and D. subpalustris [27], suggesting that wg induces the abdominal spot patterns by activating the y gene in several members of the quinaria group, like it is true for the D. guttifera wing spot pattern.
a, Lateral view of an adult D. subpalustris showing a lateral spot row. b, c, wg mRNA expression during pupal stage P7 of D. subpalustris. wg expression is observed in the lateral spot row in two different pupal abdomens. d, Lateral view of an adult D. palustris showing lateral and median spot rows. e, wg mRNA expression during pupal stage P7 of D. palustris. The spot rows are designated as lateral (l) and median (m).
The complex abdominal spot pattern of D. guttifera is reproduced by a single y CRM
Several y gene CRMs have been identified in various Drosophila species, and changes in these CRMs and/or in the deployment of trans-factors that regulate y gene expression have been implicated in the diversification of wing and body pigment patterns [6, 12–16]. We hypothesized that the developmental genes wg, dpp, abd-A, hh, and zen activate the y gene through four CRMs, each controlling one sub-pattern to assemble the complete melanin pattern on the abdomen. Hence, we searched for these CRMs by transforming D. guttifera with DsRed reporter constructs containing non-coding fragments of the 42 kb D. guttifera y gene locus (S9 Fig) [6]. Surprisingly, only one 953-bp fragment from the y intron, the gut y spot CRM, drove expression closely resembling all six spot rows and the dorsal midline shade on the developing pupal abdomen (Fig 7 and S10 Fig). To isolate possible sub-pattern-inducing CRMs, we first subdivided the gut y spot CRM into two partially overlapping sub-fragments. Unexpectedly, the 636-bp left sub-fragment displayed stripe expression parallel to the segment boundary (hereafter referred to as “horizontal stripe expression”, when viewing the fly with the head on top and the abdomen on the bottom of the picture) along the posterior edges of each abdominal segment, while the 570-bp right fragment was inactive (#1 & #2, Fig 7). Further dissection of the left sub-fragment revealed a 259-bp sub-fragment, which contained the minimal gut y core stripe CRM with some additional dorsal midline shade activity (#7 Fig 7). We also dissected the right sub-fragment into smaller overlapping fragments (#4 and #5, Fig 7). We observed that a 405-bp sub-fragment #5 drove ubiquitous reporter expression on the abdomen, which could be an artifact resulting from dissecting the y spot regulatory element into smaller sub-fragments. It is well known that disrupting CRMs can lead to spurious expression patterns. Conversely, the entire abdomen of D. guttifera shows light pigmentation in comparison with D. deflecta (Figs 1–5). This sub-fragment #5 may be driving the dark ubiquitous abdominal pigmentation in D. guttifera. The corresponding orthologous fragment of D. deflecta (shown in Fig 8 #5) does not show any such pattern when transformed in D. guttifera.
a, The y gene locus. The red bar indicates the relative position of the gut y spot CRM. b, Subdividing the gut y spot CRM revealed horizontal stripes on each abdominal segment. The rectangular bars (1–8) represent sub-fragments of the gut y spot CRM and the corresponding pupal DsRed expression patterns in transgenic D. guttifera during stage P10 are shown in c. The sub-fragments that drove reporter expression are highlighted in pink (#1, #4, #5, #7, and #8), whereas the constructs that did not show any pattern are shown as rectangular white bars (#2, #3, and #7). For sub-fragments #1, #4 and #7, an additional image focusing on a single stripe is shown below the abdominal images that show horizontal stripes on the abdomen. Sub-fragment #7 shows the minimal y stripe core CRM with reporter expression along the dorsal midline.
a, A schematic of the orthologous def y spot CRM is shown as a solid red bar. The rectangular bars (1–8) under the red bar represent the sub-fragments of the def y spot CRM that were tested for reporter activity. The sub-fragments that drove reporter expression are highlighted in pink, whereas the constructs that did not show any pattern are shown as rectangular white bars. b, The pupal DsRed expression patterns of the sub-fragments are shown in transgenic D. guttifera during stage P10. For sub-fragments #1, #4 and #7, an additional image focusing on a single stripe is shown below the abdominal images that show horizontal stripes on the abdomen. Sub-fragment #7 shows the orthologous minimal def y stripe core CRM. The reporter expression is detected in horizontal stripes but absent along the dorsal midline.
Currently, we cannot offer any direct evidence for specific candidate repressor genes. Neither the ISH experiments nor the bioinformatics analysis, using JASPAR, resulted in putative pigment stripe repressors (S1 File, S11 Fig and S2 Table). Although we identified 24 Engrailed (En)-binding sites and 19 Homothorax (Hth)-binding sites in the gut y spot CRM (both are known repressors of pigmentation in Drosophila) [12, 17], these sites were not enriched in the right half of the CRM, as we would have expected (S11 Fig). However, our transcription factor binding site analysis of the gut y spot CRM sequence revealed putative binding sites for most of the developmental genes that we identified as potential activators in our ISH screen (S1 File and S11 Fig), except for Cubitus interruptus (ci, the gene encoding the transcription factor downstream of hh). This suggests that localized spot activation by some of our identified developmental factors contributes to the formation of the abdominal spot pattern. We also identified other tissue-specific CRMs within the y locus [3, 10]. We found the wing-body CRM approximately 4 kb upstream of the y transcription start site, whereas the CRMs that specify y expression in the head, thorax, bristles, mouth parts, legs, and trachea were found within the y intron. In addition, one 1573-bp fragment located 2 kb upstream of the y transcription start site, as shown in Fig 9A, drove reporter expression on the abdomen in horizontal stripes and resembles the reporter activity of the gut y core stripe CRM (Fig 7C). This fragment (“gut y stripe CRM”) may contain a redundant CRM, and it is possible that the spot pattern output could be due to a combinatorial activity between the two stripe CRMs and the elusive repressors.
a, DsRed reporter assay showing the activity of the gut y stripe CRE at stage P10. This construct drives reporter expression in horizontal stripes across the abdomen. b, ISH signal of the ectopic expression of hh cDNA along the tergites of P10 pupae of D. guttifera showing a stripe pattern. c, ISH signal showing the ectopic expression of wg cDNA along the tergites of P10 pupae of D. guttifera. The probe is detected in horizontal stripes. Negative controls without any significant abdominal staining are shown in S14 Fig.
The core stripe CRM is conserved among D. guttifera and D. deflecta
To understand how changes in CRMs and trans-acting factors play a role in the evolution of the D. guttifera spot pattern, we cloned the 938-bp orthologous abdominal spot CRM from D. deflecta, the def y spot CRM, and transformed it into D. guttifera, using the DsRed reporter assay. The def y spot CRM drove faint dorsal spot row and stripe expression, most strongly where the dorsal spots are located (Fig 8). We further subdivided the def y spot CRM into 8 sub-fragments and identified a minimal def y core stripe CRM (288 bp) (#7 Fig 8). This sub-fragment drove a striped pattern, but without the dorsal midline shade activity that we saw in the D. guttifera minimal gut y core stripe CRM (Fig 7 #7). To test if D. melanogaster trans-factors can activate these two spot CRMs, we further transformed the gut y spot and def y spot CRMs and all sub-fragments into D. melanogaster. As a result, none of the reporter constructs showed any expression in transgenic D. melanogaster pupal abdomens. As the spot CRMs from D. guttifera and D. deflecta are not orthologous to any sequences within the D. melanogaster y locus, changes in cis have contributed to the diversification of pigment patterns between D. melanogaster and the quinaria species group. The y spot CRMs of D. guttifera and D. deflecta show 74% sequence identity, and their sequence alignment is shown in S2 File. The remainder of the sequence has strongly diverged.
Functional studies to provide evidence for the roles of wg, dpp, hh, and abd-A in pigmentation
Although the correlative evidence provided by the mRNA expression data and the transcription factor binding site analysis of the gut y spot CRM sequence are interesting and thought-provoking, they do not provide conclusive evidence for the involvement of the developmental genes in the process of pigmentation. To investigate these correlations further, we performed functional sufficiency experiments by ectopically expressing the cDNAs of wg, hh, and abd-A in transgenic D. guttifera by using the gut y stripe CRM as a driver. The gut y stripe CRM drives expression in the tergites along the intersegmental regions on the D. guttifera abdomen at stage P10 (Fig 9A). All three cDNAs were commercially available and derived from D. melanogaster. Our aim was to change the spot pattern on the abdomen of D. guttifera into stripes. As a result, the adult pigment pattern did not change into stripes. There are at least five possible explanations: 1) The driver does not work, and the developmental genes are not expressed at all. 2) The driver works, but the developmental genes are not sufficient to cause ectopic pigmentation. 3) The developmental genes are sufficient, but the driver acts too late to cause ectopic pigmentation. 4) Multiple developmental genes need to be simultaneously expressed to achieve the desired ectopic expression. 5) The presumptive endogenous repressor(s) is strong enough to suppress the function of the ectopically expressed genes. To investigate this issue further, we tested if and when the gut y stripe CRM drives the D. melanogaster-specific wg, hh, and abd-A transcripts in the transgenic D. guttifera pupae by performing ISH experiments at a variety of pupal stages (P7 –P12). We saw that the wg and hh cDNAs are indeed ectopically expressed in stripes on the tergites around the intersegmental regions of P10 pupae (Fig 9B and 9C), while abd-A did not show any expression, confirming that our overexpression experiment worked. However, as we know from the ISH experiments of the native developmental genes, their expression patterns appear at earlier stages (P7 and P8). Because the gut y stripe CRM is active much later (P10), the ectopic expression of the developmental genes may happen too late and thus fail to induce ectopic pigment. Unfortunately, we currently lack drivers that act earlier in the abdomen. Interestingly, we observed a possible enhancer-trap effect on the wings of two transgenic lines of D. guttifera (transformed with the gut y stripe CRM-hsp-wg cDNA construct) that produced adult flies with dark stripes along the longitudinal veins of their wings (S12 Fig). Although this phenotype was most likely not due to the influence of the gut y stripe CRM activity, the results suggest that the construct produces a functional Wg protein.
In order to test if the developmental genes are necessary for color pattern formation, we further attempted to knock down the mRNAs of the developmental genes wg, hh, and dpp by RNAi, using a ubiquitous heat shock driver that we induced during the P7 –P10 pupal stages. Unfortunately, all flies died immediately after eclosion before their color patterns could fully mature (S13 Fig). This outcome may be due to the vital roles played by these developmental genes during Drosophila development.
Discussion
In this study, we show that five developmental genes (wg, dpp, hh, zen, and abd-A) may collectively induce the y gene through one CRM to orchestrate the complex abdominal spot pattern development in D. guttifera. We further show that wg expression shifted within the quinaria species group, always precisely foreshadowing the diverse abdominal spot patterns of D. guttifera, D. deflecta, D. palustris, and D. subpalustris. These results corroborate an earlier finding that Wg induces the wing spots in D. guttifera [6] and suggest that changes in wg expression may have led to the diversification of abdominal color patterns in the quinaria group.
With the exception of the hh gene (more precisely its downstream transcription factor Ci), the JASPAR binding site analysis suggests that the developmental genes wg, dpp, zen, and abd-A may directly activate the gut y spot CRM. It is worthwhile mentioning that wg, dpp, and hh are homologous to known proto-oncogenes in humans [28], while zen and abdA are Hox genes.
The generation of the complex abdominal spot pattern in D. guttifera through a single CRM of the y gene is similar to our previous finding that the entire spot pattern on the D. guttifera wing is orchestrated by one CRM that responds to an elaborate expression pattern of the wg gene [6]. The underlying mechanism on the wing is thought to be a morphogen gradient, where Wg diffuses from a physical landmark a few cell diameters outwards to induce the y gene. In contrast to the D. guttifera wing spot pattern, the abdominal spots develop in the absence of any visible landmark structures. We speculate that the spots on the abdomen are established in a self-regulating manner, i.e., a Turing pattern [29], with the possible interplay of the morphogens Wg, Dpp, Hh, and perhaps other upstream regulators that co-regulate these developmental genes to assemble the complete pattern. Thus, the abdominal color pattern of D. guttifera may be regulated by multiple developmental pathways consisting of activators and repressors acting in parallel, possibly targeting pigmentation genes other than y as well [14, 15, 27, 30]. We acknowledge that we do not provide any functional evidence and that the regulatory relationships between the identified toolkit genes and the y gene are correlative.
We found that the dissection of the gut y spot CRM reveals a core stripe CRM that drives horizontal stripes on the posterior edges of each abdominal segment. Furthermore, the core stripe CRM appears to be conserved in a closely-related species, D. deflecta, which displays a similar abdominal spot pattern like D. guttifera. These data suggest that the abdominal spot pattern may have been shaped using an ancestral stripe element that activates y in stripes on the abdomen. Over time, the stripe pattern may have been partially repressed leaving isolated spots on the abdomen. Although we did not directly identify repressor genes in this study, there is some visible evidence for the activity of pigmentation repressors found in other species of the quinaria group. As can be seen in Drosophila falleni’s intraspecific pigment variation (Fig 10), spots and stripes seem to be reversible pattern elements. In particular, the production of spots appears to be a result of local stripe repression, resulting in almost triangular spots.
a, Adult thorax and abdomen. b-d, The phenotypic variation of abdominal pigmentation suggests that melanin spot development may be a result of partial stripe repression (arrows). The images shown are taken from wild-collected D. falleni specimens from Houghton, Michigan.
Our multi-pathway model, involving multiple morphogens in the self-establishment of the spot patterns together with yet-to-be-discovered repressor genes fits well with the observation that the abdominal pattern variation presented by quinaria group members is largely due to modular derivations from the D. guttifera ground plan (Fig 3), with many species just displaying a partial pattern of what D. guttifera has to offer. This scenario is quite reminiscent of the modularity found in butterfly wing patterns. Because insects use similar genes for color pattern development [26, 31–34], the quinaria group may serve as a valuable model to understand insect color pattern evolution [22].
Methods
Molecular procedures
ISH was carried out with species-specific RNA probes, as described previously [35]. Immunohistochemistry for the Y protein in abdomens was performed according to [12]. D. guttifera CRMs were identified and tested in D. guttifera according to [6] and in D. melanogaster as described previously [36]. Transgenic experiments were performed as outlined in [37]. At least five independent transgenic lines were tested for DsRed reporter expression patterns for each construct. Pupal stages were identified according to [38]. RNAi was performed using the piggyBac-based vector Pogostick [39].
Supporting information
S1 Fig. D. guttifera pupa stained with a wg probe.
This is the same pupa as in Fig 2B, but with different image manipulations. d = dorsal, m = median, l = lateral row of spots.
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S2 Fig. wg mRNA expression in four different P8 pupae of D. guttifera.
d = dorsal, m = median, l = lateral row of spots.
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S3 Fig. wg mRNA expression in three different P8 pupae of D. guttifera.
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S4 Fig. wg mRNA expression in four different P8 pupae of D. guttifera.
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S5 Fig.
a, D. guttifera P8 pupa stained with a dpp probe. b, D. guttifera P9 pupa (left) and P10 pupa (right) stained with an abd-A probe. c, D. guttifera P10 pupa stained with a hh probe. d, Two different D. guttifera P10 pupae stained with a zen probe. dms = dorsal midline shade, d = dorsal, m = median row of spots.
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S6 Fig. P7 and P8 pupal abdomens of D. guttifera and D. deflecta stained with mRNA anti-sense probes against toolkit genes, Distal-less (Dll), Abdominal-B (Abd-B), bric-a-brac-1 (bab-1), bric-a-brac-2 (bab-2), engrailed (en), scalloped (sd), pannier (pnr).
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S7 Fig. P9 and P10 pupal abdomens of D. guttifera and D. deflecta stained with mRNA anti-sense probes against toolkit genes, snail (sna), invected (inv), spitz (spi), Hairless (H), bric-a-brac-1 (bab-1), pale (ple), spalt major (salm), Abdominal-B (Abd-B), bric-a-brac-2 (bab-2), daughters against dpp (dad), engrailed (en), scalloped (sd), and Distal-less (Dll).
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S8 Fig.
a, Two different D. deflecta P9 pupae stained with a wg probe. b, Two different D. deflecta P10 pupae stained with a hh probe.
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S9 Fig. The y gene locus.
The horizontal bars indicate the DNA fragments of the D. guttifera y gene that were tested in transgenic D. guttifera for regulatory activity. Red: gut y spot CRM.
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S10 Fig. The full D. guttifera y spot CRM showing abdominal spots as well as the dorsal mid-line shade, shown on two different pupal abdomens from independent transgenic lines.
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S11 Fig. A schematic of gut y spot CRM depicting the distribution of putative transcription factor binding sites along the length of the CRM.
The red solid bar is the gut y core stripe CRM.
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S12 Fig. Two transgenic gut y stripe CRM-hsp-wg cDNA lines produced adult D. guttifera with dark stripes along the longitudinal veins of the wings.
a, The wing of an adult D. guttifera, wild type. b, c, Ectopic wing pigmentation of adult D. guttifera expressing the wg cDNA construct.
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S13 Fig. RNAi knockdown of developmental genes in D. guttifera.
a, b, Knockdown of wg mRNA in D. guttifera at pupal stages P7 and P8. c, d, Knockdown of dpp mRNA in D. guttifera at pupal stage P8.
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S14 Fig.
ISH showing the lack of ectopic expression of the D. melanogaster-derived wg (a) and hh (b) genes in wildtype (non-transgenic) D. guttifera pupae at stage P10, acting as the negative controls for the results shown in Fig 9.
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S1 Table. List of selected developmental genes that were screened by ISH for their possible involvement in pigmentation on the abdomen of D. guttifera.
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S2 Table. Numbers of putative binding sites for toolkit transcription factors for the gut y spot CRM by JASPAR analysis.
This table was extrapolated from the data in S9 Fig.
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S1 File. JASPAR analysis showing the numbers of putative binding sites for toolkit transcription factors within the gut y spot CRM.
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S2 File. The sequence of the gut y spot CRM aligned with the orthologous def y spot CRM shows 74% sequence identity.
The gut y core stripe CRM is highlighted in red.
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Acknowledgments
We thank Ryan Bensen, Abigail Meisel, Bridgette Rebbeck, and Jason Hu for technical assistance.
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