The regulation of a pigmentation gene in the formation of complex color patterns inDrosophilaabdomens

Abstract

23Changes in cis-regulatory modules (CRMs) that control developmental gene expression 24 patterns have been implicated in the evolution of animal morphology 1-6 . However, the 25 genetic mechanisms underlying complex morphological traits remain largely unknown. 26 Here we investigated the molecular mechanisms that induce the pigmentation gene yellow 27 (y) in a complex spot and shade pattern on the abdomen of the quinaria group species 28 Drosophila guttifera. We show that the y expression pattern is controlled by only one CRM, 29 which contains a stripe-inducing CRM at its core. We identified several developmental 30 genes that may collectively interact with the CRM to orchestrate the patterning in the 31 pupal abdomen of D. guttifera. We further show that the core CRM is conserved among D. 32 guttifera and the closely related quinaria group species Drosophila deflecta, which displays 33 a similarly spotted abdominal pigment pattern. Our data suggest that besides direct 34 activation of patterns in distinct spots, abdominal spot patterns in Drosophila species may 35 have evolved through partial repression of an ancestral stripe pattern, leaving isolated 36 spots behind. Abdominal pigment patterns of extant quinaria group species support the 37 partial repression hypothesis and further emphasize the modularity of the D. guttifera 38 pattern. 39 40 How complex morphological features develop and evolve is a question of foremost importance 41 in biology. To address this question, we identified genes underlying abdominal pigmentation 42 pattern development in Drosophila guttifera (D. guttifera). The abdomen is decorated with six 43 rows of black spots that run along the anterior-posterior axis, divided by a dark dorsal midline 44shade. This color pattern shows four sub-patterns: a dorsal, median, and lateral pair of spot rows, 45 plus the dorsal midline shade (Fig. 1a, b). D. guttifera belongs to the quinaria species group, 46 whose members display highly diverse abdominal pigmentation patterns 7,8 . While D. guttifera 47 dorsal midline (Extended Data Fig. 2c). Similarly, wg foreshadowed all six rows of spots, while 126 dpp expression matched all but the lateral spot rows (Extended Data Fig. 3b, c). In contrast to the 127 D. guttifera results, abd-A, hh, and zen were absent along the dorsal midline, which is in 128 agreement with the lack of pigment in D. deflecta adults (Extended Data Fig. 3d, e, f, g). 129However, abd-A expression was not detectable where the lateral spot rows will form (Extended 130 Data Fig. 3d), suggesting that these particular spots are controlled differently in D. deflecta. We 131 next cloned the 938 bp orthologous def y spot CRM and transformed it into D. guttifera, using 132 the DsRed reporter assay. The def y spot CRM drove faint dorsal spot row and stripe expression, 133 especially along the dorsal spots (Extended Data Fig. 4). We further subdivided the def y spot 134 CRM into 8 sub-fragments and identified a minimal def y core stripe CRM (288 bp) (#7, 135 Extended Data Fig. 4). This...

RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

“…The images of the gene expression patterns on Drosophila wings and abdomens generated using this protocol are shown in Figure 4 , Figure 5 , Figure 6 and Figure 7 . This protocol has been used to generate quality ISH images for toolkit genes during early pupal stages on wings and abdomens of non-model Drosophila species [ 6 , 20 ] ( Figure 4 and Figure 5 ).…”

“…Here we provide a protocol suitable for model and non-model Drosophila species alike, detailing the steps of probe design and synthesis, pupal tissue dissection, the ISH process, and imaging techniques. We have applied this protocol to investigate the gene-regulatory networks governing the development and evolution of pigmentation patterns in pupal abdomens and wings of a variety of Drosophila species, such as D. melanogaster , D. guttifera , D. deflecta , D. palustris , and D. subpalustris [ 5 , 6 , 20 ]. The users of this protocol should pay close attention to the pupal tissue processing steps, as the outcome of an RNA ISH critically depends on the tissue quality.…”

“…This protocol allows for the detection of gene expression patterns in abdomens and wings of different Drosophila species. We have used this procedure in model and non-model Drosophila species to study toolkit and terminal pigmentation genes involved in color pattern formation, such as D. melanogaster , D. guttifera , D. deflecta , D. palustris , and D. subpalustris [ 5 , 6 , 20 ]. No specialized skills are required to use this protocol; we have had undergraduate students generate high-quality results in our lab.…”

“…The most important advantage of this ISH protocol is that it enables the detection of gene expression patterns in very early pupal abdomens (as soon as the epidermal layer has formed, i.e., from pupal stage P7 onwards), as well as early wings that cannot be dissected without prior fixation (from stage P5ii onwards) ( Figure 2 ). This procedure requires basic laboratory equipment to generate high-quality ISH results [ 5 , 20 ]. However, this method is not without limitations.…”

The Making of Transgenic Drosophila guttifera

“…Previously, a review highlighting strategies for making transgenic D. melanogaster was published [ 16 ]. However, this protocol cannot be directly applied to D. guttifera transgenesis for the following reasons: (1) The egg production rate of D. guttifera is one to two orders of magnitude lower than that of D. melanogaster , and thus, a large fly population is needed; (2) D. guttifera flies die quickly from the ethanol fumes produced by the yeast required for egg-laying, requiring proper ventilation during the egg collection process; (3) D. guttifera females stick their eggs into the substrate, making them virtually impossible to collect from an agar surface; (4) D. guttifera ’s eggs are covered by a thin proteinaceous chorion, which causes rapid embryonic desiccation in this species; (5) there is no white-eyed mutant available for D. guttifera , which is why larvae have to be screened for fluorescent markers; and (6) besides the piggyBac transposon, most other tested transposons do not result in transgenic D. guttifera [ 12 , 14 , 15 , 17 ]. For these reasons, critical steps must be deployed to successfully transform D. guttifera .…”