20 research outputs found
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
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
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
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
A pigmentation enzyme gene perspective of network evolution.
<p>A pigmentation enzyme gene perspective of network evolution.</p
Tracing the CRE bases for losses in male tergite pigmentation.
<p>(A and B) A schematic representation of the male abdomens for two species with derived losses in male pigmentation. (A’ and B’) EGFP-reporter transgene expression driven by sequences orthologous to the <i>D</i>. <i>melanogaster</i> t_MSE in transgenic male pupae at ~95 hours after puparium formation. (A’) The <i>D</i>. <i>kikkawai</i> sequence possesses robust male-specific regulatory activity in the A5 and A6 segments, (B’) whereas the <i>D</i>. <i>ananassae</i> sequence has little-to-no abdominal regulatory activity. (A” and B”) EGFP-reporter transgene expression driven by sequences orthologous to the <i>D</i>. <i>melanogaster yellow</i> wing/body element in transgenic male pupae at ~85 hours after puparium formation. (A”) The <i>D</i>. <i>kikkawai</i> sequence retains the posterior stripe regulatory activities characteristic of the wing element but lacks the body element’s male-specific activity. (B”) The <i>D</i>. <i>ananassae</i> sequence possesses the regulatory activities characteristic of the wing element and body element. Red arrowheads indicate segments in which the regulatory activity is lacking.</p
Genetic interactions between pigmentation network transcription factors and CREs regulating abdominal <i>tan</i> and <i>yellow</i> expression.
<p>(A-F) EGFP reporter expression driven by the t_MSE was imaged at ~95 hours after puparium formation in male pupae. (A’-F’) EGFP reporter expression driven by the yBE0.6 was imaged at ~85 hours after puparium formation in male pupae. (A”-F”) EGFP reporter expression driven by the <i>yellow</i> 5’ sequence was imaged at ~85 hours after puparium formation in male pupae. Genotypes altering the genetic background are listed at the top of each column. Specimens are (A, A’, and A”‘) homozygous and (B-F, B’-F’, and B”-F”) hemizygous for the EGFP reporter transgene. (B) t_MSE, (B’) yBE0.6, and (B”) <i>yellow</i> 5’ regulatory activity expands into the A3 and A4 segments where <i>Abd-B</i> is ectopically expressed. Compared to a (C) control genetic background, the (D) t_MSE regulatory activity is dramatically reduced in the midline region where <i>abd-A</i> expression is suppressed. Suppression of (E) <i>hth</i> and (F) <i>exd</i> expression results in ectopic t_MSE regulatory activity in the A4 and A3 segments. Suppression of (D’) <i>abd-A</i>, (E’) <i>hth</i>, and (F’) <i>exd</i> expression has little-to-no effect on yBE0.6 regulatory activity compared to the (C’) control genetic background. Suppression of (D”) <i>abd-A</i> and (F”) <i>exd</i> has little-to-effect on the <i>yellow</i> 5’ regulatory activity compared to the (C”) control genetic background. Suppression of (E”) <i>hth</i> results in a mild expansion of regulatory activity into the A3 and A4 segments. Blue arrowheads indicate segments where the genetic background modification resulted in ectopic reporter transgene activity. Red arrowheads indicate segments where the genetic background modification resulted in a loss of reporter transgene activity.</p
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
<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