The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila

Abstract

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSX(F) activate bab expression whereas in males DSX(M) directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

Figure 1. Bab1 expression in the abdomen is regulated by two CREs

(A and B) Dorsal view of D. melanogaster adult abdomens. Male segments A5 and A6 of are fully pigmented (A). In females, pigmentation of these segments is limited to a posterior stripe (B) (C and D) Expression of Bab1 in male and female pupae at 72 hours APF. Bab1 expression in males is limited to segments A2-A4 (C), but in females, Bab1 expression extends into segments A5 and A6, as well in the female-specific segment A7 (D). (E) Two CREs, the anterior element and dimorphic element, reside in the large 1^st intron of bab1 and govern Bab expression in the abdominal epidermis. (F–I) GFP-reporter expression in dorsal pupal abdomens. (F and G) The anterior element drove GFP-reporter gene activity in segments A2-A4 of both males (F) and females (G). (H) The dimorphic element was inactive in males. (I) The dimorphic element drove reporter expression in female segments A5-A7, with levels increasing from the anterior to posterior.

Figure 2. Genetic regulation of bab CRE activity by the Abd-B and Dsx loci

Images are of dorsal (A–I) and ventral (J–L) abdomens of pupae heterozygous for GFP-reporter constructs. Genotypes are listed at the top of each image. The anterior element is denoted as ‘AE’ and the dimorphic element ‘DE’. Specimens are heterozygous for the AbdB^Mcp (B and E), AbdB^iab9-Tab (C and F), dsx^D+R3 (H and K), and dsx^D (I and L) mutant allele. Red and white arrows respectively indicate regions where reporter activity in the mutant background is decreased or increased, respectively, compared to the wild-type control. (A) The AE drove reporter expression in the anterior segments A2-A4. (B and C) Ectopic expression of Abd-B in segments A4 (B) and A3-A4 (C) resulted in repression of AE activity in these segments. (D, G and J) The DE drove reporter expression in the posterior segments A5-A7 of females. (E and F) Ectopic expression of Abd-B in segments A4 (E) and A3-A4 (F) resulted in ectopic DE activity in these segments. (H and K) In a dsx heterozygous null mutant genetic background, DE activity is indistinguishable from that in a wild-type background. (I and L) In a chromosomal female intersex, where one dsx allele is producing the male transcript instead of the female, DE activity was reduced to 68±2% in A6 (I) and to 36% in A7 (L) of the activity of the DE in wild-type females.

Figure 3. The dimorphic element is directly regulated by ABD-B through multiple binding sites

(A) Schematic of the minimal D. mel. wild-type (mel) CRE sequence conferring robust female-specific activity, with the location of putative ABD-B (yellow and blue diamonds) and DSX (D1 and D2) binding sites indicated. Additional TTAT (non-TTTAT) motifs are indicated by “t”. The spans of the Left, Right, and Middle sub-constructs are indicated below the schematic. (B–M) Comparison of GFP-reporter gene activity in transgenic female pupae at 75 hours APF. Activity measurements are represented as the % of the wild-type (mel) female A6 mean ±SEM. (B) The wild-type dimorphic element drove reporter expression at high levels in A6 and A7. (C–E) Truncation of the dimorphic element into Left (C), Right (D) and Middle (E) sub-fragments resulted in dramatically decreased reporter activity. (F) Reporter activity is reduced to 59±2% in pupae heterozygous for the dimorphic element reporter transgene. (G–L) Activity of dimorphic elements in which subsets of putative ABD-B binding sites have been mutated. (G) Mutation of all fifteen TTAT sites reduced reporter activity to 9±0%. (H) Mutation of all seven TTTAT sites reduced reporter activity to 19±2%. (I) Mutation of all seven TTTAC sites reduced reporter activity to 26±3%. (J) Mutation of ABD-B site 14 reduced reporter activity to 55±0%. (K) Mutation of ABD-B sites 11–13 reduced reporter activity to 79±4%. (L) Mutation of ABD-B sites 9 and 10 had no detectable affect on dimorphic activity. (M) Mutation of ABD-B sites 9–12 and 14 reduced reporter activity to 26±6%.

Figure 4. The dimorphic element is directly regulated by sex-specific isoforms of DSX

(A and B) DNaseI footprinting analysis of the dimorphic element with a GST-DSX DBD fusion protein identified two distinct sites bound by the DSX DNA-binding domain, referred to as Dsx1 (A) and Dsx2 (B). Amounts of each protein used were as follows: lane 2, 1,000 ng GST only; lane 3, no protein; lane 4, 64 ng DSX DBD; lane 5, 160 ng DSX DBD; lane 6, 400 ng DSX DBD; lane 7, 1,000 ng DSX DBD. A G+A sequencing ladder is included in lane 1. Footprinted regions are indicated by a black rectangle with an adjacent number. (C and D) EMSAs on annealed oligonucleotide probes containing wild-type (lanes 1–4) and mutant (lanes 5–8) DSX binding sites. Below are the sequences of the wild-type and mutant Dsx1 (C) and Dsx2 (D) binding sites with mutated bases shown in red. For each probe, binding reactions were performed using increasing amounts of the DSX DBD protein (from left to right: 0 ng, 16 ng, 62 ng, 250 ng, and 1,000 ng). Blue and red arrowheads point to the respective locations on the gel of complexes containing a single or pair of DSX DBD monomers bound to the probe. (E–L) GFP-reporter activity in pupae at 75 hours APF. Activity measurements are represented as the % of the wild-type (mel) female A6 mean ±SEM. (E and I) Activity of the wild-type dimorphic element was much greater in the female (E) than the male (I). (F and J) When the Dsx1 site was mutated, activity in the female was reduced to 23±2% (F), while activity in the male was unchanged (J). (G and K) When the Dsx2 site was mutated, activity in females was reduced to 34±3% (G), while male activity was unchanged (K). (H and L) When both the Dsx1 and Dsx2 sites were mutated, reporter activity in females was reduced to 24±1% (H), and increased to 53±3% in males (L).

Figure 5. The dimorphic element has a deep ancestry

(A) Dorsal views of D. wil. male abdomen. Pigmentation of abdominal tergites on segments A2-A6 is limited to a posterior stripe. (B, C, F, and G) Bab1 expression in pupal abdomens at 65 hours APF (a developmental time point equivalent to 72 hour APF assayed for D. mel.). (B) Bab1 is expressed in segments A2-A6 (A2 not shown) of male pupae. Expression was also observed in longitudinal abdominal muscles. (C) High magnification view of (yellow box; B) dorsal midline showing equivalent levels of Bab1 in segments A4-A6. (D) The D. wil. anterior element drove GFP-reporter expression in segments A2-A6 of males (A2 not shown). (E) Dorsal view of D. wil. female abdomen. The pigmentation pattern is identical to that of the male (compare with A). (F) Bab1 is expressed in segments A2-A7 of female pupae. (G) High magnification view of (red box; F) dorsal midline showing equivalent levels of Bab1 in segments A4-A6. (H) The D. wil. anterior element drove reporter expression in segments A2-A7 of females (A2 and A3 not shown). (I) The D. wil. dimorphic element does not activate reporter expression in posterior segments of males (dorsal view). (J) The D. wil. dimorphic element drove expression in the A7 segment of females (red arrow; dorsal view). (K) Ventral view showing the absence of D. wil. dimorphic element reporter activity in posterior segments of males. (L) Ventral view showing reporter expression driven by the D. wil. dimorphic element in female segment A7 (red arrow), but not in the anterior A6 segment.

Figure 6. bab expression evolved via remodeling of the dimorphic element

In all panels, GFP-reporter expression mediated by D. mel. (A–E and I–L) and D. wil. (F–H) dimorphic elements was assayed in transgenic female pupae at 75 hours APF Activity measurements are represented as the % of the wild-type (mel) female A6 mean ±SEM. Red arrow heads point to dorsal midline regions of A6 where reporter activity was reduced by modification of the D. mel. element. White arrow head points to area of segment A6 where reporter activity was increased by modification of the D. wil. element. (A) Reporter expression driven by the wild-type D. mel. dimorphic element. (B) Mutation of ABD-B site 8 reduced reporter activity to 78±5%. (C) Mutation of ABD-B site 13 had no measurable affect on reporter activity. (D) Reversal of the Dsx1 site polarity in the D. mel. element reduced reporter activity to 87±2%. (E) Reversal of Dsx1 site polarity combined with mutation of ABD-B sites 8 and 13 reduced reporter activity to 66±3%. (F) Reporter expression driven by the D. wil. dimorphic element that includes site 14. (G) Reporter expression driven by the wild-type D. wil. dimorphic element. (H) Reversal of Dsx1 site polarity in the D. wil. element resulted in a dramatic gain of reporter activity in segment A6. Activity increased from 1±1% to 34±3% of the wild-type D. mel element. (I) Insertion of 58 base pairs between ABD-B site 5 and Dsx1 site (region I) reduced reporter expression to 62±3%. (J) Insertion of 98 base pairs between ABD-B sites 8 and 9 (region II) reduced reporter expression to 41±3%. (K) Insertion of 57 base pairs between the Dsx2 site and ABD-B site 11 (region III) increased the activity of the wild-type dimorphic element by 37±3%. (L) Insertion of 58, 98, and 57 base pairs into regions I–III respectively, reduced dimorphic element activity to 44±4%.

Figure 7. Model for the operation and evolution of the dimorphic genetic switch

(A) The operation of the switch. Expression of bab in the posterior abdominal segments A5-A7 of females is mediated by the combined inputs of the segment-specific HOX protein ABD-B and the female-specific isoform DSX^F. Expression of bab results in the repression of full tergite pigmentation in these segments. Expression of bab in male segments A5 and A6 is repressed by the male-specific isoform DSX^M. The absence of bab expression in these segments allows for the development of fully-pigmented tergites. (B) The evolution of the switch. Schematic depiction of the evolution of the dimorphic element from the inferred common ancestor of D. melanogaster and D. willistoni. Yellow boxes indicate binding sites for ABD-B and white boxes indicate DSX binding sites. Yellow and white ovals represent ABD-B and DSX protein monomers respectively. The common ancestral CRE contained two and thirteen orthologous binding sites for DSX and ABD-B, respectively. In the lineage leading to D. wil., ABD-B site 8 was lost, the polarity of Dsx1 was reversed (red arrow) and candidate ABD-B binding sites 12a and 12b were gained (red stars). In the lineage leading to D. mel., inter-binding site spacing was reduced in regions I, II, and III, and ABD-B site 13 was gained (blue star), which collectively contributed to the higher level of gene expression in female segments A5 and A6.