The structure and evolution of cis-regulatory regions: the shavenbaby story

Abstract

In this paper, we provide a historical account of the contribution of a single line of research to our current understanding of the structure of cis-regulatory regions and the genetic basis for morphological evolution. We revisit the experiments that shed light on the evolution of larval cuticular patterns within the genus Drosophila and the evolution and structure of the shavenbaby gene. We describe the experiments that led to the discovery that multiple genetic changes in the cis-regulatory region of shavenbaby caused the loss of dorsal cuticular hairs (quaternary trichomes) in first instar larvae of Drosophila sechellia. We also discuss the experiments that showed that the convergent loss of quaternary trichomes in D. sechellia and Drosophila ezoana was generated by parallel genetic changes in orthologous enhancers of shavenbaby. We discuss the observation that multiple shavenbaby enhancers drive overlapping patterns of expression in the embryo and that these apparently redundant enhancers ensure robust shavenbaby expression and trichome morphogenesis under stressful conditions. All together, these data, collected over 13 years, provide a fundamental case study in the fields of gene regulation and morphological evolution, and highlight the importance of prolonged, detailed studies of single genes.

Keywords: cis-regulatory evolution; cis-regulatory structure; evolution of enhancer function; morphological change; shavenbaby gene.

Figure 1.

Trichome pattern variation in first-instar larvae of Drosophila species. (a) Drawing from the lateral perspective of a Drosohphila melanogaster first-instar larva. The black rectangle demarcates the fifth abdominal segment. On the dorsal cuticle, the primary (1°), tertiary (3°) and quaternary (4°) cells (light outline) differentiate trichomes, and the secondary (2°) cells differentiate naked cuticle. A group of stout trichomes (denticles) is present in the ventral cuticle. The grey area within the rectangle indicates the cuticle region shown in (b). (b) Detail of the dorsal cuticle in different species of the genus Drosophila. The quaternary cells of D. sechellia and D. ezoana produce ‘naked’ cuticle. By contrast, D. melanogaster, D. littoralis and D. virilis produce ‘hairy’ cuticles in the quaternary domain (light outline). (c) A svb null first-instar larva lacks dorsal and lateral trichomes and has fewer ventral denticles that are also reduced in size relative to wild-type ventral denticles (arrows). (Online version in colour.)

Figure 2.

Shavenbaby embryonic expression is generated by the activity of multiple transcriptional enhancers. (a) Expression pattern of shavenbaby in the epidermis of a late stage Drosophila melanogaster embryo. (b) Schematic view of the structure of the svb/ovo locus. Black boxes indicate svb embryonic enhancers and grey boxes demarcate exons. The exons present in the svb mRNA are shown below this scheme. Svb protein contains a dominant repressor domain, an activator domain and a DNA-binding domain. The arrow marks the position where Svb is truncated, which converts Svb into a transcriptional activator. (c) Seven transcriptional enhancers generate the expression pattern of shavenbaby in the embryonic epidermis. The black regions within trichome domains schematize the expression patterns of enhancers DG2, DG3, Z, A, E3, E6 and 7. (Online version in colour.)

Figure 3.

Pattern of sequence conservation in the shavenbaby cis-regulatory region. Scheme of a multiple sequence alignment of the shavenbaby upstream region in 12 Drosophila species (bottom). Mel, D. melanogaster; sech, D. sechellia; sim, D. simulans; yak, D. yakuba; ere, D. erecta; ana, D. ananassae; per, D. persimilis; pse, D. pseudoobscura; wil, D. willistoni; moj, D. mojavensis; vir, D. virilis; gri, D. grimshawii. Empty boxes indicate the position of svb embryonic enhancers and triangles indicate coding regions. Z678, A8 and 7H are the ‘minimal’ versions of enhancers Z, A and 7, respectively. The pattern of sequence conservation is shown in grey (top). Sequence identity was calculated in 100 bp windows.

Figure 4.

Genetic changes underlying the evolution of enhancer E6 in D. sechellia. (a) Lateral view of the expression driven by enhancer E6 from D. melanogaster and D. sechellia in stage 15 embryos of D. melanogaster. (b) Selected regions of the full-length alignment of D. melanogaster (mel), D. sechellia (sec) and D. simulans (sim) E6 enhancer that include the seven clusters of sites uniquely derived in D. sechellia E6 (marked in bold). These sites were mutated in the rescue constructs depicted in (c). (c) Scheme of the constructs used to test the effects of mutations (above). Number of trichomes produced by E6 variants in a svb null background (graphs below). Number of trichomes rescued by the D. melanogaster (circles) and D. sechellia (triangles) constructs in the dorsal (left) and lateral (right) regions of the cuticle. Circles and triangles denote the mean number of trichomes. Vertical lines represent ± 1s.d. (n = 10). Larvae carrying D. sechellia rescue constructs produced zero trichomes in the lateral region and are not shown in the figure on the right. WT, wild-type construct. ALL, construct with all clusters mutated. Clusters highlighted in bold (x-axis) caused a significant change in the number of trichomes produced compared with the wild-type construct. (Online version in colour.)

Figure 5.

Conservation of shavenbaby cis-regulatory structure between D. melanogaster and D. virilis. Horizontal lines schematize the svb cis-regulatory region in D. melanogaster (above) and D. virilis (below). Empty boxes indicate the position of transcriptional enhancers. Dotted lines connect orthologous enhancers. Triangles denote coding regions.

Figure 6.

Parallel changes in orthologous enhancers underlie the appearance of convergent morphologies in D. sechellia and D. ezoana. The black regions within trichome domains (figure 1) schematize the expression patterns of shavenbaby enhancers in (a) D. melanogaster, (b) D. sechellia, (c) D. virilis and (d) D. ezoana. Orthologous enhancers are positioned in the same column. Dotted boxes highlight the loss of enhancer activity in D. sechellia and D. ezoana. NT, not tested.