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Gain of Cis-Regulatory Activities Underlies Novel Domains of Wingless Gene Expression in Drosophila

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Gain of Cis-Regulatory Activities Underlies Novel Domains of Wingless Gene Expression in Drosophila Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila Shigeyuki Koshikawaa,b,c, Matt W. Giorgiannia,b, Kathy Vaccaroa,b, Victoria A. Kassnera,b, John H. Yoderd, Thomas Wernere, and Sean B. Carrolla,b,1 aLaboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706; bHoward Hughes Medical Institute, University of Wisconsin, Madison, WI 53706; cThe Hakubi Center for Advanced Research and Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; dDepartment of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487; and eDepartment of Biological Sciences, Michigan Technological University, Houghton, MI 49931 Contributed by Sean B. Carroll, May 10, 2015 (sent for review April 2, 2015; reviewed by Michael Eisen and Gregory A. Wray) Changes in gene expression during animal development are largely In this case, the Dll protein is said to have been co-opted in the responsible for the evolution of morphological diversity. However, evolution of a new morphological trait. the genetic and molecular mechanisms responsible for the origins However, the mechanism underlying the co-option of Dll is of new gene-expression domains have been difficult to elucidate. not known in this case, nor for any other instances of the co- Here, we sought to identify molecular events underlying the origins option of regulatory genes. It is not known, for example, whether of three novel features of wingless (wg) gene expression that new features of gene expression evolve via the de novo origin of are associated with distinct pigmentation patterns in Drosophila enhancers or through the transposition or modification of existing guttifera. We compared the activity of cis-regulatory sequences enhancers. One distinguishing feature shared by most develop- (enhancers) across the wg locus in D. guttifera and Drosophila mental regulatory gene loci is that, like Dll (17, 18), they often melanogaster and found strong functional conservation among the contain vast cis-regulatory regions harboring numerous indepen- enhancers that control similar patterns of wg expression in larval dent enhancers. To complicate matters, some of these enhancers imaginal discs that are essential for appendage development. For may be located far away in other genes. The diversity of enhancers pupal tissues, however, we found three novel wg enhancer activi- belonging to individual regulatory genes is explicit evidence that ties in D. guttifera associated with novel domains of wg expression, gene function has expanded in the course of evolution by accu- including two enhancers located surprisingly far away in an intron mulating additional enhancers, but understanding how this occurs of the distant Wnt10 gene. Detailed analysis of one enhancer (the presents significant experimental challenges. To further our un- vein-tip enhancer) revealed that it overlapped with a region con- derstanding of the molecular basis of gene-expression novelties, it trolling wg expression in wing crossveins (crossvein enhancer) in is necessary both to identify the novel enhancers in the species of D. guttifera and other species. Our results indicate that one novel interest and to ascertain their structural and functional relation- domain of wg expression in D. guttifera wings evolved by co-opting ships to sequences in other species lacking the specific domains of pre-existing regulatory sequences governing gene activity in the gene expression (19). developing wing. We suggest that the modification of existing en- The Wingless (Wg) protein is a secreted signaling molecule that hancers is a common path to the evolution of new gene-expression acts as a morphogen in the development of numerous structures domains and enhancers. and pattern elements in Drosophila and other animals (20–23). Here, we have traced the molecular basis of three novel features enhancers | novelty | gene regulation | development | pigmentation of wingless (wg) gene expression in Drosophila guttifera that are associated with three distinct features of adult pigmentation. By s animals have adapted to diverse habitats they have evolved Amany new and different kinds of body parts. One of the Significance major outstanding questions in evolutionary biology is: What kinds of mechanisms underlie the origin of morphological nov- The origins of novelties pose some of the most difficult ex- elties? It is well established that the regulatory genes responsible perimental challenges in evolutionary biology. Morphological for the formation and patterning of animal bodies and body novelties in animals are generally thought to arise through parts, the so-called “toolkit” genes for animal development, are new domains of expression of so-called “toolkit” regulatory shared and highly conserved among most animal phyla (1–4). Very genes, but how such changes arise has not been explored in different forms are generated by similar sets of developmental depth. Here, we show that three novel features of wingless genes, and a large body of empirical, comparative studies have led gene expression associated with three novel pattern elements to the general consensus that divergence in the expression and are the result of three novel enhancer activities. One of these regulation of toolkit genes and the genes they control largely enhancers is clearly modified from an evolutionarily conserved, underlies morphological diversity (5–9). pre-existing regulatory sequence. We suggest the modification Similarly, several studies have revealed that new features of of extant enhancers is a common path to novelty in gene ex- regulatory gene expression are associated with the evolution of pression and morphology. morphological novelties, such as new color-pattern elements on insect wings (10–14). How new patterns of regulatory gene ex- Author contributions: S.K. and S.B.C. designed research; S.K., M.W.G., K.V., V.A.K., J.H.Y., and T.W. performed research; S.K., K.V., V.A.K., and J.H.Y. contributed new reagents/ pression evolve, however, has been more difficult to elucidate. In analytic tools; S.K., M.W.G., and S.B.C. analyzed data; and S.K., M.W.G., T.W., and S.B.C. principle, new patterns of gene expression may evolve through: wrote the paper. (i) changes in the deployment of upstream trans-acting regulatory Reviewers: M.E., University of California, Berkeley; and G.A.W., Duke University. factors, (ii) changes in the cis-regulatory sequences of the genes The authors declare no conflict of interest. themselves, or (iii) a combination of these mechanisms. For ex- Freely available online through the PNAS open access option. ample, the novel, male-specific wing spot in Drosophila biarmipes Data deposition: The sequences reported in this paper have been deposited in the Gen- and a few close relatives evolved through a combination of changes Bank database (accession nos. 1805591 and KP966547). in the spatial expression of the trans-acting Distal-less (Dll) tran- 1To whom correspondence should be addressed. Email: [email protected]. scription factor and the evolution of Dll and other binding sites in a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cis-regulatory element of at least one pigmentation gene (15, 16). 1073/pnas.1509022112/-/DCSupplemental. 7524–7529 | PNAS | June 16, 2015 | vol. 112 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1509022112 Downloaded by guest on October 1, 2021 searching through the wg and adjacent loci of both D. guttifera and A D. melanogaster 10kb Drosophila melanogaster, we found three novel enhancer activities BCD in D. guttifera. We show that one of these enhancers, the novel wg Wnt6 vein-tip enhancer in D. guttifera, is nestled within a conserved en- hancer in other species. We propose that the new enhancer activity evolved through the modification of the pre-existing enhancer. D. guttifera Results EFG Novel wg Expression Domains in the D. guttifera Pupal Wing. Reg- ulatory genes coordinate important developmental events; thus, BC D their expression patterns are constrained and usually conserved, particularly among closely related species. wg expression patterns in larval imaginal discs (wing disc, eye-antennal disc, and leg disc) of D. melanogaster and D. guttifera adhere to this generality w ea l ea l and are essentially identical (SI Appendix,Fig.S1). In both species, D. melanogaster wg expression was virtually identical in the developing wing EF G pouches and the future nota of wing discs (SI Appendix,Fig.S1A and D), the anterior-ventral parts of antennae, ventral and dorsal sides of eye discs (SI Appendix,Fig.S1B and E), and the anterior- ventral parts of leg discs (SI Appendix,Fig.S1C and F). D. guttifera w ea l ea l In contrast, in the developing pupal wings of D. guttifera, wg is expressed in two domains that are not present in D. melanogaster Fig. 2. Conserved wg cis-regulatory elements control similar gene-expression pupal wings (14, Fig. 1). Whereas in D. melanogaster, wg is patterns in Drosophila imaginal discs. (A) Schematic of enhancers plotted on expressed in cells along the developing wing margin (henceforth the wg locus of D. melanogaster and D. guttifera. Solid vertical lines connected “margin”) and crossveins (Fig. 1A, arrows), in D. guttifera (Fig. 1B) by gray lines represent sequences longer than 40 bp with 100% nucleotide “ ” conservation between species. (B–D) D. melanogaster third-instar imaginal discs wg is also expressed at the tips of longitudinal veins ( vein tip ) showing reporter expression with D. melanogaster enhancer fragments (EGFP, (Fig. 1B, asterisks) and in precursors of the campaniform sensilla green). (E–G) D. guttifera third-instar imaginal discs showing very similar reporter (Fig. 1B, arrowheads). None of the other several species closely expression patterns driven by orthologous D. guttifera enhancer fragments related to D. guttifera within the Drosophila quinaria species group (DsRed, magenta). All discs are oriented with anterior to the left and dorsal on (Drosophila deflecta, Drosophila nigromaculata, Drosophila palustris, top. ea, eye-antennal disc; l, leg disc; w, wing disc.
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