Changes in <i>cis</i> to the upstream silencer element of <i>ebony</i> are responsible for a difference in <i>ebony</i> expression between strains.

<p>(A) Comparison of the DNA sequence of the light and dark strains of <i>ebony</i>, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.g004" target="_blank">Fig 4A</a>. Schematics of GFP reporter constructs that pinpoint changes to the silencer of <i>ebony</i> are shown above and below the conservation plot. Chimeras appearing below the dark strain are drawn relative to the dark strain due to the presence of a large insertion in this strain. Similarly, chimeras above the light strain are drawn relative to the light strain due to the lack of this repetitive region. A6 midline activity was calculated using the same method described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.g004" target="_blank">Fig 4</a>. The act (D) + sil (<i>serrata</i>) construct in (A) contains the 1kb silencer region of <i>D</i>. <i>serrata</i> placed in the context of the dark <i>D</i>. <i>auraria</i> strain <i>ebony</i> upstream regulatory region (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.g006" target="_blank">Fig 6C</a>). (B) Activity of the dark (“PM”) strain <i>ebony</i> upstream regulatory region, containing both activating enhancer and silencer region. Expression is specifically reduced in the dorsal midline. (C) Activity of the light (“00”) strain <i>ebony</i> upstream regulatory region, orthologous to that used for the dark strain. This reporter construct shows a marked increase in midline activity, recapitulating the <i>ebony</i> expression phenotype of this strain. (D) Chimeric reporter containing the light strain activating enhancer fused to the dark strain upstream silencer element. This reporter construct shows midline repression similar to the dark strain. (E) Chimeric reporter containing the dark strain activating enhancer fused to the light strain upstream silencer region, which recapitulates the light strain activity. (F) The CD3 truncation of the light strain reporter drives expression throughout the A6 segment, similar to the same truncation of the dark strain. (G) Graph comparing the relative activity of the Dark and Light strain Full <i>ebony</i> upstream reporter activity in the A4 body segment, a region unaffected by silencer activity. (H) Graph comparing the relative activity of the Dark and Light strain CD3 truncation constructs in the A4 body segment.</p

Correlation between pigmentation and the gene expression of <i>tan</i> and <i>yellow</i> in the <i>Sophophora</i> subgenus.

<p>(A) Phylogenetic relationship between extant <i>Sophophora</i> species. (B-H, B’-H’, and B”-H’) Each image shown is from a male abdomen. Whole-mount images of dorsal fruit fly abdomens from species representing diverse <i>Sophophora</i> lineages. <i>D</i>. <i>melanogaster</i> bears a derived pigmentation pattern on the A5 and A6 tergites, which arose after it diverged from its most recent common ancestor (MRCA) shared with the monomorphically pigmented <i>D</i>. <i>willistoni</i> (node in phylogeny marked “1”) and perhaps after diverging from the MRCA shared with <i>D</i>. <i>pseudoobscura</i> (node “2”). Node 3 represents the MRCA of the <i>melanogaster</i> species group that includes the Oriental lineage (<i>D</i>. <i>melanogaster</i>), <i>montium</i> subgroup (includes <i>D</i>. <i>auraria</i> and <i>D</i>. <i>kikkawai</i>), and <i>ananassae</i> subgroup (includes <i>D</i>. <i>malerkotliana</i> and <i>D</i>. <i>ananassae</i>). This MRCA is suspected to have possessed male-specific tergite pigmentation that is indicated by the hemi-filled in circle. Since its origin, the number of pigmented male tergites has expanded (<i>D</i>. <i>malerkotliana</i>), retracted (<i>D</i>. <i>auraria</i>) and was independently lost (<i>D</i>. <i>kikkawai</i> and <i>D</i>. <i>ananassae</i>; nodes 4 and 5 that are indicated by the circles with a superimposed X). (B’-H’) Abdominal <i>tan</i> mRNA expression shown by <i>in situ</i> hybridization at a developmental stage equivalent to 85–95 hours after puparium formation (APF) for <i>D</i>. <i>melanogaster</i> pupae. (B”-H”) Abdominal <i>yellow</i> mRNA expression shown by <i>in situ</i> hybridization at a developmental stage equivalent to 75–85 hours APF for <i>D</i>. <i>melanogaster</i> pupae. Species are identified by labels at the top of each column.</p

Model for the parallel loss of the <i>ebony</i> upstream silencer element.

<p>Within the ancestral <i>ebony</i> gene, a silencer element evolved that adopted a repressive role in in the male posterior abdomen. Within the <i>montium</i> subgroup, this silencer’s activity was modified through changes in <i>cis</i> that altered the silencer’s spatial domain of activity. Within <i>D</i>. <i>serrata</i>, this silencer was inactivated, leading to increased <i>ebony</i> expression. In contrast, this silencer was maintained in the species <i>D</i>. <i>auraria</i>, in which intraspecific variation exists for an allele that inactivated this element.</p

Scanning mutagenesis identifies CRE sequences required for <i>yellow</i> and <i>tan</i> expression.

<p>(A) Ten scanning mutant versions, SM1-SM10, of the <i>D</i>. <i>melanogaster</i> yBE0.6 sequence and t_MSE sequence were created. In each mutant, a block of ~70 base pairs was altered such that every other nucleotide was altered by a non-complementary nucleotide transversion. (B-G) EGFP reporter transgene expression in <i>D</i>. <i>melanogaster</i> male pupae at ~85 hours after puparium formation. (B) The yBE0.6 sequence drives reporter expression in the male A5 and A6 segments. (C and D) The SM2 and SM3 mutations resulted in a modest reduction in regulatory activity, whereas the (F and G) SM5 and SM6 mutations resulted in a near-total loss of reporter activity. (E) The SM4 mutation led to a pan-abdomen increase in regulatory activity. (H-J) EGFP reporter transgene expression in <i>D</i>. <i>melanogaster</i> male pupae at ~95 hours after puparium formation. (H) The t_MSE sequence drives expression in the male A5 and A6 segments. (I and J) The SM5 and SM6 mutations resulted in a loss of reporter expression in the male abdomen. Red arrowheads indicate regions where the regulatory activity was greatly reduced due to a scanning mutation and the Orange arrowheads indicate regions where regulatory activity was modestly reduced. Blue arrowheads indicate regions where regulatory activity was gained due to a scanning mutation.</p

<i>D</i>. <i>auraria</i> strains surveyed for the geographic distribution of pigmentation phenotypes.

<p><i>D</i>. <i>auraria</i> strains surveyed for the geographic distribution of pigmentation phenotypes.</p

Intraspecific variation in abdominal pigmentation of <i>D</i>. <i>auraria</i> across Japan.

<p>(A) Map of the Japanese islands, depicting sites (blue dots) from which <i>D</i>. <i>auraria</i> strains in this study were collected. (B-E) The abdominal phenotypes observed in <i>D</i>. <i>auraria</i> range from light (B) to intermediate (C, D), to dark (E).</p

Gene network models for unpigmented and pigment abdominal segments.

<p>Wiring diagram of pigmentation gene networks experienced by the (A) non-melanic male A2-A4 segments and (B) the melanic A5 and A6 segments. (A) Abd-B expression is lacking in the anterior A2-A4 segments and as a result <i>yellow</i> and <i>tan</i> lack the direct and indirect activating input from this transcription factor. In these segments, Abd-A forms direct repressive inputs with <i>tan</i> which are supported (directly or indirectly) by the repressive effects of <i>exd</i> and <i>hth</i>. (B) Abd-B is expressed in the posterior A5 and A6 segments, where it acts as a direct activator of <i>yellow</i> and an indirect activator of <i>tan</i>. In these segments, Abd-A acts as an indirect activator of <i>tan</i> expression as well. In these schematics, inactive genes are indicated in gray coloring, solid connections between genes indicate validated direct interactions between a transcription factor and a pigmentation gene CRE, and dashed connections indicate indirect interactions or those not yet shown to be direct. Connections terminating with an arrowhead indicate connections in which the transcription factor functions as an activator, and connections terminating in a nail head shape indicate a repressive relationship.</p

Parallel inactivation of the <i>ebony</i> upstream silencer in <i>D</i>. <i>serrata</i>.

<p>(A) Expression of <i>ebony</i> in the abdomen of a <i>D</i>. <i>serrata</i> male (UCSD “03” strain). (B) The entire upstream region of <i>D</i>. <i>serrata ebony</i> fused into a GFP reporter construct recapitulates the broad activation of <i>ebony</i> throughout the male posterior abdomen. (C) Chimeric reporter in which a 1 kb segment containing the <i>D</i>. <i>serrata</i> upstream silencer region was placed into the <i>D</i>. <i>auraria</i> dark strain reporter construct. This reporter lacks midline repression, indicating that the <i>D</i>. <i>serrata</i> upstream silencer was inactivated.</p

Localization of a conserved silencer in the <i>D</i>. <i>auraria</i> upstream regulatory region.

<p>(A) Schematic depicting the upstream regulatory region of <i>ebony</i> from a dark <i>D</i>. <i>auraria</i> strain aligned to the orthologous region of <i>D</i>. <i>melanogaster</i>. Gray boxes connecting <i>D</i>. <i>auraria</i> and <i>D</i>. <i>melanogaster</i> sequences indicate the relative position of perfectly conserved stretches. Positions of the minimal abdominal enhancer and silencer are shown above the <i>D</i>. <i>melanogaster</i> sequence are based on [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.ref018" target="_blank">18</a>]. Position of the <i>D</i>. <i>auraria</i> silencer element is based upon the truncation constructs shown below the conservation plot. Regions experimentally determined to be required for silencer activity are listed in black, while the region between the enhancer and silencer are shown in a gray gradient, as they may contribute to silencing activity. “A6 midline activity” was measured for each truncation construct, in which the fluorescent intensity of the midline was expressed as the percentage of the lateral A6 intensity ± S.E.M. (B) Activity of the dark (“PM”) strain full <i>ebony</i> upstream regulatory region, which recapitulates the midline repression of <i>ebony</i> observed by <i>in situ</i> hybridization (dotted lines). Dashed boxes indicate representative midline and lateral patches used to quantify midline activity. (C-D) The CD1 truncation construct (C), and the CD2 construct (D) both show midline repression similar to that observed in the full <i>ebony</i> upstream region. (E) The CD3 truncation construct shows uniform expression of GFP across the A6 body segment, reflecting the elimination of sequences required for repression.</p

Characterization of the direct Hox inputs shaping <i>tan</i> expression.

<p>(A) The SM6 region of the t_MSE possesses five sites, S1-S5, with sequences characteristic of Abd-B (TTAT) and Abd-A (TAAT) binding sites. This CRE region also possesses sites resembling sequences bound by Exd and Hth, though the functionality of the Exd site was not studied here. (B-F) EGFP reporter transgene expression in male pupae at ~95 hours after puparium formation. (B) The t_MSE2 sequence drives robust expression in the male A5 and A6 segments. When all of the (C) TTAT sites and (D) TTAT and TAAT sites depicted in (A) were mutated, regulatory activity in the male A5 segment was reduced to 89±6% and 88±3% respectively. (F) When the entire SM6 region was mutated, regulatory activity decreased to 31±1%. (C) The TTAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 169±4% and 261±6%. (D) The TTAT and TAAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 207±2% and 281±2%. (E) When the Hth site was mutated, regulatory activity in the male A5, A4, and A3 segments respectively increased to 122±7%, 236±6%, and 276±4%.(F) The entire SM6 region mutation resulted in activity decreasing in the A4 and A3 segments respectively to 64±1% and 73±1%. Blue arrowheads indicate segments where activity was notably increased and Red arrowheads indicate segments with notably decreased activity compared to the wild type sequence. Gel shift assays for sequences possessing wild type and mutant site (G) 3, (H) 4, and (I) 5 and the DNA-binding domains for Abd-A and Abd-B. Binding reactions used increasing amounts of the GST-DNA binding domain fusion protein (from left to right: 0 ng, 111 ng, 333 ng, 1000 ng, and 3000 ng). Binding correlated with the amount of input protein for the probes with the non-mutant sequence, whereas binding was dramatically reduced for the probes with a mutant Hox site.</p