Phenotypic plasticity in Drosophila pigmentation caused by temperature sensitivity of a chromatin regulator network

Abstract

Phenotypic plasticity is the ability of a genotype to produce contrasting phenotypes in different environments. Although many examples have been described, the responsible mechanisms are poorly understood. In particular, it is not clear how phenotypic plasticity is related to buffering, the maintenance of a constant phenotype against genetic or environmental variation. We investigate here the genetic basis of a particularly well described plastic phenotype: the abdominal pigmentation in female Drosophila melanogaster. Cold temperature induces a dark pigmentation, in particular in posterior segments, while higher temperature has the opposite effect. We show that the homeotic gene Abdominal-B (Abd-B) has a major role in the plasticity of pigmentation in the abdomen. Abd-B plays opposite roles on melanin production through the regulation of several pigmentation enzymes. This makes the control of pigmentation very unstable in the posterior abdomen, and we show that the relative spatio-temporal expression of limiting pigmentation enzymes in this region of the body is thermosensitive. Temperature acts on melanin production by modulating a chromatin regulator network, interacting genetically with the transcription factor bric-à-brac (bab), a target of Abd-B and Hsp83, encoding the chaperone Hsp90. Genetic disruption of this chromatin regulator network increases the effect of temperature and the instability of the pigmentation pattern in the posterior abdomen. Colocalizations on polytene chromosomes suggest that BAB and these chromatin regulators cooperate in the regulation of many targets, including several pigmentation enzymes. We show that they are also involved in sex comb development in males and that genetic destabilization of this network is also strongly modulated by temperature for this phenotype. Thus, we propose that phenotypic plasticity of pigmentation is a side effect reflecting a global impact of temperature on epigenetic mechanisms. Furthermore, the thermosensitivity of this network may be related to the high evolvability of several secondary sexual characters in the genus Drosophila.

Figure 1. The Pattern of Abdominal Pigmentation Plasticity in Wild-Type Females

(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. (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).

Figure 2. Ectopic Expression of Abd-B on the Thorax Is Sufficient to Generate a Sex-Specific Plastic Pigmentation Pattern

Effect of temperature on the pigmentation pattern generated by the ectopic thoracic expression of Abd-B in the mutant Transabdominal (Tab) [17] in males (D–F) and females (G–L). 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 Abd-B on the thorax is sufficient to generate a highly plastic pigmentation pattern in females (G–L).

Figure 3. Abd-B Interacts with Temperature in the Regulation of Melanin Production

Shows the effect of variation of Abd-B 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).

Figure 4. The Expression of Pigmentation Enzymes Is Highly Dynamic and Modulated by Temperature

(A–C) Dynamic expression of ebony-LacZ 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). (D–F) Dynamic expression at 25 °C of the tyrosine hydroxylase using TH-Gal4 and UAS-LacZ transgenes. Expression starts earlier than ebony-LacZ, 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. (G–I) Dynamic expression of the tyrosine hydroxylase using TH-Gal4 and UAS-LacZ transgenes in Tab/+ 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. (J) Expression of ebony-LacZ in female abdomen just before hatching at 20 °C. No strong expression is visible in A7. (K) Expression of the tyrosine hydroxylase visualized using TH-Gal4, UAS-LacZ 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 Abd-B grown at 25 °C, the melanin almost completely disappears from A7 and remains only at the first sites of expression of TH associated with bristles (L).

Figure 5. Temperature Interacts with a Network of Regulatory Factors and the Chaperone Hsp90 for the Regulation of Melanin Production

Shows the effect of variation of temperature and the alleles bab^AR07 and corto⁴²⁰ (A–C) and corto⁴²⁰ and Hsp83^e6D (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).

Figure 6. Chromatin Regulators, Temperature, and Sex Comb Development

The sex comb is a structure made of modified bristles located on the first tarsal segment of the first leg in males. (A) Sex comb phenotype of representative bab^AR07/corto⁴²⁰ males grown at 20 °C, 25 °C, and 29 °C. The normal sex comb is located on the first leg in the first tarsal segment (T1). Ectopic sex comb composed of two and three teeth, respectively, can be seen on the second tarsal segment (T2) in the flies grown at 25 and 29 °C (arrows). (B) Sex comb phenotype of crm⁷/Y; bab^AR07/+ of a fly grown at 29 °C. An ectopic sex comb caused by crm⁷ alone is visible on the first tarsal segment (T1) of the second leg (L2), a typical PcG phenotype as previously reported [35]. A strong synergistic interaction between bab and crm is revealed by ectopic sex comb teeth, not only on the second (T2) but also on the third (T3) tarsal segment of the first leg (L1). Note that the legs are shortened and that the second tarsal segment is inflated, affecting the ectopic sex comb shape. (C) Mean and standard errors of sex comb teeth on the second tarsal segment of the first leg in wild-type (green), bab^AR07/+, crm⁷/Y and crm⁷/Y; bab^AR07/+ (red) males grown at 25 °C. (D) Mean and standard errors of sex comb teeth on the second tarsal segment in bab^AR07/+ single heterozygotes and in combinations with the other mutations indicated on the right grown at 20 °C, 25 °C, and 29 °C.

Figure 7. BAB Colocalizes with Chromatin Regulators on Polytene Chromosomes

Double immunostaining of polytene chromosomes with antibodies against BAB (green; B, D, F, H, J, and L), Corto (red; B, D, and F) and CRM (red; H, J, and L) in the cytological region of ddc (37C; A, B, G, and H), TH (65C; C, D, I, and J), and e (93C; E, F, K, and L). Colocalizations are indicated by yellow arrows. DAPI staining (blue) allows the visualization of euchromatic (lightly stained) and heterochromatic (densely stained) regions. Inverted black and white pictures of the DAPI staining are shown on the left to identify the cytological regions. Note the staining for BAB in the region of all three enzymes (B, D, F, H, J, and L), the colocalizations in the TH region with both Corto (D) and CRM (J), and in the ddc region with CRM (H).

Figure 8. Schematic Representation of the Gene Network Involved in Phenotypic Plasticity of Abdominal Pigmentation

One major role of the phenotypic plasticity of abdominal pigmentation is played by Abdominal-B (Abd-B). Abd-B represses ebony (e) and tyrosine hydroxylase (TH), 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. Abd-B is known to increase pigmentation in A5 and A6 by repressing bab [13]. Hsp90, BAB, and chromatin regulators, such as Corto and Cramped (CRM), are particularly limiting at intermediate and high temperature for the expression of TH. Thus, at high temperature they do not counteract as strongly as at low temperature the repression of TH by Abd-B. This reduces melanin production in the posterior abdomen. Colocalizations on polytene chromosomes suggest that they directly regulate TH. 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 ddc. Indeed, we showed that BAB represses ddc, at least indirectly. Furthermore, the detection of BAB in the cytological region containing ebony 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 Text S1 for details).