The pattern of trichomes has evolved between Drosophila species due to changes in the enhancers of the svb gene. (a) Lateral view drawing of a first instar larva of D. melanogaster. The dark rectangle indicates the region shown in b and c. (b,c) The pattern of dorso-lateral trichomes on the fourth abdominal segment of D. melanogaster (b) and D. sechellia (c). Some of the dorso-lateral cells differentiate thin “quaternary” trichomes in D. melanogaster and naked cuticle in D. sechellia. (d, e) Pattern of svb RNA expression in stage 14 embryos of D. melanogaster (d) and D. sechellia (e). (f) Diagram illustrating the location of the six enhancers of svb (open boxes). The enhancers 7, E and, A were referred as proximal, medial, and distal, respectively, in ref. 25. Genes in the region are indicated with gray boxes and only the first exon of svb is shown. (g) Summary of the dissection of the E enhancer in D. melanogaster. Boxes indicate the enhancer constructs discussed in the text. (h) The E3 region drives expression in ventral stripes. (i) The E6 region drives expression in quaternary cells.

D. sechellia E6 displays decreased and delayed expression relative to D. melanogaster E6. (a, c, e, g, i) The D. melanogaster E10 construct drives expression that is detected first in the most dorsal cells of stage 12 embryos (a). This expression strengthens and spreads laterally through stages 13 (c), 14 (e), 15 (g) and 16 (i). (b, d, f, h, j) The D. sechellia E10 construct does not drive detectable expression in stage 12 (b) or 13 (d) embryos. Dorsal expression (white arrows) is detected in only some stage 14 embryos (f) and is clearly observable in stage 15 and 16 embryos (h, j). Both the D. melanogaster and D. sechellia E10 constructs drive similar expression in ventral cells (black arrows) (e, f).

Sequence conservation of the E6 region and location of the D. sechellia-specific substitutions. (a) The aligned E6 sequences from D. melanogaster, D. simulans, D. mauritiana, D. sechellia, D. yakuba, and D. erecta are represented as thick horizontal lines, with thin regions indicating gaps in the alignments. (Full alignment is provided as Supp. Fig. 2.) Sequence conservation over a 10 bp sliding window is represented above by the height of the gray bars. The positions of D. sechellia-specific substitutions are indicated with vertical red lines, the seven clusters of substitutions are indicated below the red lines, and the “focal region” is labeled. (b) Sequences of the seven regions containing the D. sechellia-specific substitutions (enclosed in rectangles) with the aligned sequences from D. melanogaster (mel), D. simulans (sim), and D. sechellia (sec). (c, d) Evolutionary trees of the E6 focal region (c) and 9 kb outside of the focal region (d), where branch lengths are proportional to the substitution rate.

Evolutionary engineering of the E10 enhancer reveals the role of evolved substitutions in altering the levels and timing of expression. (a,b) Reporter gene expression driven by the D. melanogaster E10 (a) and D. sechellia E10 (b) constructs in st. 14 D. melanogaster embryos. (c) Introducing all seven clusters of D. sechellia-specific substitutions into a mel_E10 construct (mel_mut_All) strongly reduces dorsal expression in st. 14 embryos. (d) Introducing the respective D. melanogaster nucleotides into a sec_E10 construct (sec_mut_All) almost completely restores dorso-lateral expression in st. 14 embryos. (e) The onset of expression driven by the E10 and mut enhancers was quantified by counting the proportion of embryos showing dorso-lateral expression at each of six embryonic stages. The mel_mut_All and sec_mut_All show strong changes in the onset of expression compared with the respective wild type constructs. Five of the D. melanogaster mut lines also show delayed onset of expression compared with mel_E10 construct. Two of the sec_mut lines show significant differences in the onset of expression compared with sec_E10. The E10 and mut_All comparisons were made at stage 13 and the individual cluster mel_mut and sec_mut comparisons were made at stages 12 and 14, respectively. Sequential Bonferroni test P values: * P < 0.05; ** P < 0.01; n.s.=not significant.

Effect of the engineered substitutions on trichome rescue in dorsal and lateral regions of the sixth abdominal segment of first-instar larvae. (a) Wild type D. melanogaster. (b) svb null. (c) mel_E10 in a svb null background. The dorsal (D) and lateral (L) regions where trichomes were counted are delimited with a dashed line. (d–h) mel_E10 constructs carrying all D. sechellia substitutions (d), or cluster 2 (e), 3 (f), 4 (g), or 5 (h) substitutions in a svb null background. (i) mel_E10 in a svb null background. (j–l) sec_E10 constructs carrying cluster 2 (e), 3 (f), or all D. melanogaster substitutions (l). (m, n) Number of trichomes rescued by the mel (black) and sec (red) constructs in the dorsal (m) and lateral (n) regions. All larvae carrying sec_mut constructs differentiated zero trichomes in the lateral region, and for clarity these data are not shown in n. Open circles represent counts for each individual. Closed circles and lines indicate the means and standard deviations, respectively. Grey shading encompasses the constructs with trichome counts that were significantly different from the E10 construct of the respective species (P < 0.05, Dunnet's test).