Experimental Approaches to Evaluate the Contributions of Candidate Cis-regulatory Mutations to Phenotypic Evolution

Part of the Methods in Molecular Biology book series (MIMB, volume 772)


Elucidating the molecular bases by which phenotypic traits have evolved provides a glimpse into the past, allowing the characterization of genetic changes that cumulatively contribute to evolutionary innovations. Historically, much of the experimental attention has been focused on changes in protein-coding regions that can readily be identified by the genetic code for translating gene coding sequences into proteins. Resultantly, the role of noncoding sequences in trait evolution has remained more mysterious. In recent years, several studies have reached an unprecedented level of detail in describing how noncoding mutations in gene cis-regulatory elements contribute to morphological evolution. Based on these and other studies, we describe an experimental framework and some of the genetic and molecular methods to connect a particular cis-regulatory mutation to the evolution of any phenotypic trait.

Key words

Cis-regulatory elements CRE Morphological evolution Enhancers Pleiotropy Noncoding sequences Modularity Gene expression 


  1. 1.
    Carroll SB, Grenier JK, Weatherbee SD (2001) From DNA to Diversity. Blackwell Science, MaldenGoogle Scholar
  2. 2.
    Carroll SB (2008) Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134:25–36PubMedCrossRefGoogle Scholar
  3. 3.
    Protas ME, Hersey C, Kochanek D et al (2006) Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism. Nat Genet 38:107–111PubMedCrossRefGoogle Scholar
  4. 4.
    Colosimo PF, Hosemann KE, Balabhadra S et al (2005) Widespread parallel evolution in sticklebacks by repeated fixation of Ectodysplasin alleles. Science 307:1928–1933PubMedCrossRefGoogle Scholar
  5. 5.
    Miller CT, Beleza S, Pollen AA et al (2007) cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans. Cell 131:1179–1189PubMedCrossRefGoogle Scholar
  6. 6.
    Shapiro MD, Marks ME, Peichel CL et al (2004) Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature 428:717–723PubMedCrossRefGoogle Scholar
  7. 7.
    Sucena E, Stern DL (2000) Divergence of larval morphology between Drosophila sechellia and its sibling species caused by cis-regulatory evolution of ovo/shaven-baby. Proc Natl Acad Sci USA 97:4530–4534PubMedCrossRefGoogle Scholar
  8. 8.
    Wittkopp PJ, Williams BL, Selegue JE et al (2003) Drosophila pigmentation evolution: divergent genotypes underlying convergent phenotypes. Proc Natl Acad Sci USA 100:1808–1813PubMedCrossRefGoogle Scholar
  9. 9.
    Sucena E, Delon I, Jones I et al (2003) Regulatory evolution of shavenbaby/ovo underlies multiple cases of morphological parallelism. Nature 424:935–938PubMedCrossRefGoogle Scholar
  10. 10.
    Marcellini S, Simpson P (2006) Two or four bristles: functional evolution of an enhancer of scute in Drosophilidae. PLoS Biol. doi:10.1371/journal.pbio.0040386 PubMedGoogle Scholar
  11. 11.
    Gompel N, Prud’homme B, Wittkopp PJ et al (2005) Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila. Nature 433:481–487PubMedCrossRefGoogle Scholar
  12. 12.
    Cohn MJ, Tickle C (1999) Developmental basis of limblessness and axial patterning in snakes. Nature 399:474–479PubMedCrossRefGoogle Scholar
  13. 13.
    Averof M, Patel NH (1997) Crustacean appendage evolution associated with changes in Hox gene expression. Nature 388:682–686PubMedCrossRefGoogle Scholar
  14. 14.
    Belting HG, Shashikant CS, Ruddle FH (1998) Modification of expression and cis-regulation of Hoxc8 in the evolution of diverged axial morphology. Proc Natl Acad Sci USA 95:2355–2360PubMedCrossRefGoogle Scholar
  15. 15.
    Cretekos CJ, Wang Y, Green ED et al (2008) Regulatory divergence modifies limb length between mammals. Genes Dev 22:141–151PubMedCrossRefGoogle Scholar
  16. 16.
    Abiola O, Angel JM, Avner P et al (2003) The nature and identification of quantitative trait loci: a community’s view. Nat Rev Genet 4:911–916PubMedGoogle Scholar
  17. 17.
    Linnen CR, Kingsley EP, Jensen JD et al (2009) On the origin and spread of an adaptive allele in deer mice. Science 325:1095–1098PubMedCrossRefGoogle Scholar
  18. 18.
    Martin JF, Bradley A, Olson EN (1995) The paired-like homeo box gene MHox is required for early events of skeletogenesis in multiple lineages. Genes Dev 9:1237–1249PubMedCrossRefGoogle Scholar
  19. 19.
    Carroll SB (2005) Evolution at two levels: on genes and form. PLoS Biol. doi:10.1371/journal.pbio.0030245 Google Scholar
  20. 20.
    Baker BS, Burtis K, Goralski T et al (1989) Molecular genetic aspects of sex determination in Drosophila melanogaster. Genome 31:638–645PubMedCrossRefGoogle Scholar
  21. 21.
    Reinhart BJ, Slack FJ, Basson M et al (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901–906PubMedCrossRefGoogle Scholar
  22. 22.
    Vella MC, Choi EY, Lin SY et al (2004) The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR. Genes Dev 18:132–137PubMedCrossRefGoogle Scholar
  23. 23.
    Marin VA, Evans TC (2003) Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1. Development 130:2623–2632PubMedCrossRefGoogle Scholar
  24. 24.
    Pare A, Lemons D, Kosman D et al (2009) Visualization of Individual Scr mRNAs during Drosophila Embryogenesis Yields Evidence for Transcriptional Bursting. Curr Biol 19:2037–2042PubMedCrossRefGoogle Scholar
  25. 25.
    Rebeiz M, Pool JE, Kassner VA et al (2009) Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population. Science 326:1663–1667PubMedCrossRefGoogle Scholar
  26. 26.
    Pool JE, Aquadro CF (2007) The genetic basis of adaptive pigmentation variation in Drosophila melanogaster. Mol Ecol 16:2844–2851PubMedCrossRefGoogle Scholar
  27. 27.
    Williams TM, Selegue JE, Werner T et al (2008) The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila. Cell 134:610–623PubMedCrossRefGoogle Scholar
  28. 28.
    Chan YF, Marks ME, Jones FC et al (2010) Adaptive Evolution of Pelvic Reduction in Sticklebacks by Recurrent Deletion of a Pitx1 Enhancer. Science 327:302–305PubMedCrossRefGoogle Scholar
  29. 29.
    Wittkopp PJ, Haerum BK, Clark AG (2004) Evolutionary changes in cis and trans gene regulation. Nature 430:85–88PubMedCrossRefGoogle Scholar
  30. 30.
    Lettice LA, Heaney SJ, Purdie LA et al (2003) A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 12:1725–1735PubMedCrossRefGoogle Scholar
  31. 31.
    Wang X, Chamberlin HM (2002) Multiple regulatory changes contribute to the evolution of the Caenorhabditis lin-48 ovo gene. Genes Dev 16:2345–2349PubMedCrossRefGoogle Scholar
  32. 32.
    Jeong S, Rokas A, Carroll SB (2006) Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution. Cell 125:1387–1399PubMedCrossRefGoogle Scholar
  33. 33.
    Prud’homme B, Gompel N, Rokas A et al (2006) Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene. Nature 440:1050–1053PubMedCrossRefGoogle Scholar
  34. 34.
    Bischof J, Maeda RK, Hediger M et al (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci USA 104:3312–3317PubMedCrossRefGoogle Scholar
  35. 35.
    Groth AC, Fish M, Nusse R et al (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166:1775–1782PubMedCrossRefGoogle Scholar
  36. 36.
    Shirangi TR, Dufour HD, Williams TM et al (2009) Rapid evolution of sex pheromone-producing enzyme expression in Drosophila. PLoS Biol. doi:10.1371/journal.pbio.1000168 PubMedGoogle Scholar
  37. 37.
    Ludwig MZ, Bergman C, Patel NH et al (2000) Evidence for stabilizing selection in a eukaryotic enhancer element. Nature 403:564–567PubMedCrossRefGoogle Scholar
  38. 38.
    Hare EE, Peterson BK, Iyer VN et al (2008) Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation. PLoS Genet. doi:10.1371/journal.pgen.1000106 Google Scholar
  39. 39.
    McGregor AP, Orgogozo V, Delon I et al (2007) Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448:587–590PubMedCrossRefGoogle Scholar
  40. 40.
    Tomoyasu Y, Wheeler SR, Denell RE et al (2005) Ultrabithorax is required for membranous wing identity in the beetle Tribolium castaneum. Nature 433:643–647PubMedCrossRefGoogle Scholar
  41. 41.
    Tomoyasu Y, Arakane Y, Kramer KJ et al (2009) Repeated Co-options of Exoskeleton Formation during Wing-to-Elytron Evolution in Beetles. Curr Biol 19:2057–2065PubMedCrossRefGoogle Scholar
  42. 42.
    Moczek AP, Rose DJ (2009) Differential recruitment of limb patterning genes during development and diversification of beetle horns. Proc Natl Acad Sci USA 106:8992–8997PubMedCrossRefGoogle Scholar
  43. 43.
    Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210:630–637PubMedCrossRefGoogle Scholar
  44. 44.
    Wray GA (2007) The evolutionary significance of cis-regulatory mutations. Nat Rev Genet 8:206–216PubMedCrossRefGoogle Scholar
  45. 45.
    Stern DL, Orgogozo V (2008) The loci of evolution: how predictable is genetic evolution?. Evolution 62:2155–2177PubMedCrossRefGoogle Scholar
  46. 46.
    Prabhakar S, Visel A, Akiyama JA et al (2008) Human-specific gain of function in a developmental enhancer. Science 321:1346–1350PubMedCrossRefGoogle Scholar
  47. 47.
    Cande J, Goltsev Y, Levine MS et al (2009) Conservation of enhancer location in divergent insects. Proc Natl Acad Sci USA 106:14414–14419PubMedCrossRefGoogle Scholar
  48. 48.
    Zinzen RP, Cande J, Ronshaugen M et al (2006) Evolution of the ventral midline in insect embryos. Dev Cell 11:895–902PubMedCrossRefGoogle Scholar
  49. 49.
    Erives A, Levine M (2004) Coordinate enhancers share common organizational features in the Drosophila genome. Proc Natl Acad Sci USA 101:3851–3856PubMedCrossRefGoogle Scholar
  50. 50.
    Ruvinsky I, Ruvkun G (2003) Functional tests of enhancer conservation between distantly related species. Development 130:5133–5142PubMedCrossRefGoogle Scholar
  51. 51.
    Venken K J, He Y, Hoskins RA et al (2006) a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751PubMedCrossRefGoogle Scholar
  52. 52.
    Lehoczky JA, Innis JW (2008) BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud. Evol Dev 10:421–432PubMedCrossRefGoogle Scholar
  53. 53.
    Oberstein A, Pare A, Kaplan L et al (2005) Site-specific transgenesis by Cre-mediated recombination in Drosophila. Nat Methods 2:583–585PubMedCrossRefGoogle Scholar
  54. 54.
    Sagai T, Amano T, Tamura M et al (2009) A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings. Development 136:1665–16674PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of PittsburghPittsburghUSA
  2. 2.Department of BiologyUniversity of DaytonDaytonUSA

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