The Expression of Pigmentation Enzymes Is Highly Dynamic and Modulated by Temperature

<div><p>(A–C) Dynamic expression of <i>ebony-LacZ</i> at 25 °C in the abdomen of pharate adults. Expression is first visible at the base of bristles around 90 h after puparium formation in (A) and then extends progressively from the anterior region of the segments to all epidermal cells within tergites (B and C).</p><p>(D–F) Dynamic expression at 25 °C of the tyrosine hydroxylase using <i>TH-Gal4</i> and <i>UAS-LacZ</i> transgenes. Expression starts earlier than <i>ebony-LacZ,</i> at the base of the large bristles on the posterior border of the segments. In (D), the bristles are not mature yet. The expression is then visible at the base of smaller bristles (E), and eventually in all epidermal cells (F). Note that the most posterior tergite, A7, does not show any strong staining.</p><p>(G–I) Dynamic expression of the tyrosine hydroxylase using <i>TH-Gal4</i> and <i>UAS-LacZ</i> transgenes in <i>Tab/+</i> females at 25 °C at 90 h after puparium formation. Thoraces have an increasing age from left to right, as revealed by the pigmentation at the base of bristles. The abdomen and thoraces shown within the same column do not necessarily have exactly the same age. See text for details.</p><p>(J) Expression of <i>ebony-LacZ</i> in female abdomen just before hatching at 20 °C. No strong expression is visible in A7.</p><p>(K) Expression of the tyrosine hydroxylase visualized using <i>TH-Gal4, UAS-LacZ</i> in female abdomen just before hatching at 20 °C. The expression in A7 is much more visible than at 25 °C. In limiting conditions, such as in females with three doses of <i>Abd-B</i> grown at 25 °C, the melanin almost completely disappears from A7 and remains only at the first sites of expression of <i>TH</i> associated with bristles (L).</p></div

<i>Abd-B</i> Interacts with Temperature in the Regulation of Melanin Production

<p>Shows the effect of variation of <i>Abd-B</i> dosage (1, 2, or 3 copy number) and temperature on melanin production in the lateral region of A6 (A), the median region of A6 (B), and the lateral region of A7 (C).</p

The Pattern of Abdominal Pigmentation Plasticity in Wild-Type Females

<div><p>(A) Abdominal phenotypes of females from the inbred wild-type lines NO1, BV1, and Samarkand grown at 20 °C, 25 °C, and 29 °C. The drawings on the right summarize the plasticity of the different regions of the body according to the color code. A1–A7, abdominal segment number; L, lateral region; D, dorsal region; SAM, Samarkand.</p><p>(B) The pigmentation in wild-type females shows a variegated pattern. (1) Left and right 7th hemitergites of a BV1 female grown at 25 °C. The pigmentation is not perfectly symmetrical. White patches are visible on one side (arrows) where dark pigmentation is observed on the other side. The dark pigmentation follows the insertion of the small bristles in the inner region of the tergite (arrowheads). (2 and 3) Shows the 6th hemitergites of two NO1 females grown at 25 °C. The pigmentation patterns are very similar but not perfectly identical. The limit between the dark and yellowish regions of the tergite is not smooth but variegated. Yellowish patches (arrows) at the base of some bristles are surrounded by dark pigmentation (3, arrowheads).</p></div

Temperature Interacts with a Network of Regulatory Factors and the Chaperone Hsp90 for the Regulation of Melanin Production

<p>Shows the effect of variation of temperature and the alleles <i>bab^AR07</i> and <i>corto⁴²⁰</i> (A–C) and <i>corto⁴²⁰</i> and <i>Hsp83^e6D</i> (D–F) on melanin production in the lateral region of A6 (A and D), the median region of A6 (B and E), and the lateral region of A7 (C and F).</p

Ectopic Expression of <i>Abd-B</i> on the Thorax Is Sufficient to Generate a Sex-Specific Plastic Pigmentation Pattern

<div><p>Effect of temperature on the pigmentation pattern generated by the ectopic thoracic expression of <i>Abd-B</i> in the mutant <i>Transabdominal (Tab)</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030030#pgen-0030030-b017" target="_blank">17</a>] in males (D–F) and females (G–L).</p><p>In wild-type flies (A–C), the pigmentation is homogenous in both sexes except for the trident pattern visible at extreme developmental temperature (A). Ectopic expression of <i>Abd-B</i> on the thorax is sufficient to generate a highly plastic pigmentation pattern in females (G–L).</p></div

Schematic Representation of the Gene Network Involved in Phenotypic Plasticity of Abdominal Pigmentation

<p>One major role of the phenotypic plasticity of abdominal pigmentation is played by <i>Abdominal-B (Abd-B). Abd-B</i> represses <i>ebony (e)</i> and <i>tyrosine hydroxylase (TH),</i> not necessarily directly. The first role is stronger at low temperature and the second role is stronger at high temperature. This correlates with the increase in melanin production at low temperature and the decrease of all pigments at high temperature in the posterior abdomen. <i>Abd-B</i> is known to increase pigmentation in A5 and A6 by repressing <i>bab</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030030#pgen-0030030-b013" target="_blank">13</a>]. Hsp90, BAB, and chromatin regulators, such as Corto and Cramped (CRM), are particularly limiting at intermediate and high temperature for the expression of <i>TH</i>. Thus, at high temperature they do not counteract as strongly as at low temperature the repression of <i>TH</i> by <i>Abd-B</i>. This reduces melanin production in the posterior abdomen. Colocalizations on polytene chromosomes suggest that they directly regulate <i>TH.</i> Some of them may also regulate together the expression of other enzymes, as suggested by the detection of BAB and CRM in the cytological region containing the locus of the <i>ddc.</i> Indeed, we showed that BAB represses <i>ddc,</i> at least indirectly. Furthermore, the detection of BAB in the cytological region containing <i>ebony</i> suggests that it might be also involved in its regulation. The pigment synthesis pathway represented here is a consensus pathway between the models proposed by several authors (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030030#pgen-0030030-sd001" target="_blank">Text S1</a> for details).</p

<i>tan</i> is involved in female abdominal pigmentation plasticity.

<p>(A) Cuticles of control (<i>w</i>^<i>1118</i>) and <i>tan</i> mutant females (<i>t</i>^{<i>d07784</i>}) grown at 18°C, 25°C and 29°C. (B) Reaction norms of the same genotypes (n = 10 per condition). The pigmentation value corresponds to the first component of a principal component analysis of pigmentation in segments A5, A6 and A7 that captures more than 95% of the total variance. There is a significant decrease in thermal plasticity of abdominal pigmentation in <i>tan</i> mutant females. Statistical test: two-way ANOVA. ***: p<0.001.</p

<i>trx</i> is involved in female abdominal pigmentation, whereas <i>trr</i> and <i>Set1</i> are not.

<p>(A, B) When using the <i>y-Gal4</i> driver and <i>UAS-RNAi</i> transgenes at 25°C, <i>trx</i> down-regulation induces abdominal depigmentation, whereas <i>trr</i> or <i>Set1</i> down-regulation does not. In A, the effect of the RNAi transgenes against <i>trr</i>, <i>Set1</i> or <i>trx</i> (<i>VALIUM RNAi</i> lines) was compared to that of an RNAi transgene against <i>GFP</i> inserted at the same site in the same genetic background. In B, the RNAi line against <i>Set1</i> (VDRC line) driven by <i>y-Gal4</i> was compared with females heterozygous for the transgene. (C) <i>trx</i> down-regulation during late pupal stage (<i>pnr-Gal4</i> driver in combination with <i>tub-Gal80</i>^<i>ts</i> transgene) induced abdominal depigmentation. Dashed lines mark left borders of the <i>pnr</i> driver expression domain. The <i>UAS-RNAi-GFP</i> transgene is used as a negative control.</p

Temperature regulates the activity of an abdominal epidermis enhancer of <i>tan</i>, <i>t_MSE</i>.

<p>(A) <i>tan</i> genomic region (after Flybase, <a href="http://flybase.org/" target="_blank">http://flybase.org/</a>) showing the location of <i>t_MSE</i> between the genes <i>CG15370</i> and <i>Gr8a</i>. (B, C) The activity of <i>t_MSE</i> (<i>t_MSE-nEGFP</i> reporter transgene) in abdominal epidermes of young adult females is modulated by temperature. (B) nEGFP fluorescence in abdominal epidermes at 18°C and 29°C. Fluorescence on the left part of the tissue corresponds to the pleura. (C) Quantification of nEGFP fluorescence in A5, A6 and A7 hemi-tergites at 18° and 29°C (n = 10 <i>per</i> temperature). nEGFP intensity is higher at 18°C than at 29°C (t-test; ***: p<0.001).</p

Temperature dramatically modulates the expression of the pigmentation gene <i>tan</i> in posterior abdominal epidermes of females.

<p>(A) Cuticle pigment synthesis pathway [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006218#pgen.1006218.ref028" target="_blank">28</a>]. Enzymes are indicated in red. (B) Quantification of pigmentation gene expression in posterior abdomen epidermes (segments A5, A6 and A7) from female <i>w</i>^<i>1118</i> pharates (left) and young <i>w</i>^<i>1118</i> adult females (right) grown at 18°C or 29°C (pools of 50 epidermes for pharates and 30 epidermes for adults, n = 3, error bars: standard deviations; gene expression at 18°C has been normalized on gene expression at 29°C). The expression of <i>tan</i>, <i>ebony</i>, <i>DDC</i>, <i>yellow</i> and <i>black</i> is moderately modulated by temperature in pharates, whereas only <i>tan</i> is dramatically modulated in young adults (t-test: *: p<0.05; **: p<0.01). The expression of <i>Tyrosine Hydroxylase</i> (<i>TH</i>) and <i>Laccase 2</i> is modulated neither in pharates nor in adults. (C) Analysis of <i>tan</i> expression in abdominal epidermes from young <i>w</i>^<i>1118</i> adult females grown at 18°C or 29°C. Note that <i>tan</i> is more strongly expressed in the posterior abdominal epidermis at 18°C than at 29°C. (D) Adult cuticle (left) and <i>tan</i> expression in abdominal epidermis (right) from females in which <i>tan</i> was down-regulated using the <i>pnr-Gal4</i> driver and a <i>UAS-RNAi-t</i> transgene. The dashed line marks the limit between the <i>pnr</i> driver expression domain (a dorsal strip) and the lateral region used as an internal control. Note the loss of pigmentation (left panel) and the strong decrease in <i>tan</i> expression (right panel) in the dorsal region, showing specificity of <i>tan</i> antisense probe.</p