Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Morphological evolution caused by many subtle-effect substitutions in regulatory DNA

Abstract

Morphology evolves often through changes in developmental genes, but the causal mutations, and their effects, remain largely unknown. The evolution of naked cuticle on larvae of Drosophila sechellia resulted from changes in five transcriptional enhancers of shavenbaby (svb), a transcript of the ovo locus that encodes a transcription factor that governs morphogenesis of microtrichiae, hereafter called ‘trichomes’. Here we show that the function of one of these enhancers evolved through multiple single-nucleotide substitutions that altered both the timing and level of svb expression. The consequences of these nucleotide substitutions on larval morphology were quantified with a novel functional assay. We found that each substitution had a relatively small phenotypic effect, and that many nucleotide changes account for this large morphological difference. In addition, we observed that the substitutions had non-additive effects. These data provide unprecedented resolution of the phenotypic effects of substitutions and show how individual nucleotide changes in a transcriptional enhancer have caused morphological evolution.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The pattern of trichomes has evolved between Drosophila species owing to changes in the enhancers of the svb gene.
Figure 2: D. sechellia E6 shows decreased and delayed expression relative to D. melanogaster E6.
Figure 3: Sequence conservation of the E6 region and location of the D. sechellia -specific substitutions.
Figure 4: Evolutionary engineering of the E10 enhancer reveals the role of evolved substitutions in altering the levels and timing of expression.
Figure 5: Effect of the engineered substitutions on trichome rescue in dorsal and lateral regions of the sixth abdominal segment of first instar larvae.

References

  1. Monteiro, A. & Podlaha, O. Wings, horns, and butterfly eyespots: how do complex traits evolve? PLoS Biol. 7, e37 (2009)

    Article  Google Scholar 

  2. Stern, D. L. Evolution, Development, & The Predictable Genome (Roberts & Co., 2010)

    Google Scholar 

  3. Stern, D. L. & Orgogozo, V. The loci of evolution: how predictable is genetic evolution? Evolution 62, 2155–2177 (2008)

    Article  Google Scholar 

  4. Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134, 25–36 (2008)

    CAS  Article  Google Scholar 

  5. Erezyilmaz, D. F., Riddiford, L. M. & Truman, J. W. The pupal specifier broad directs progressive morphogenesis in a direct-developing insect. Proc. Natl Acad. Sci. USA 103, 6925–6930 (2006)

    ADS  CAS  Article  Google Scholar 

  6. Davidson, E. H. The Regulatory Genome: Gene Regulatory Networks in Development and Evolution (Academic, 2006)

    Google Scholar 

  7. Carroll, S. B., Grenier, J. K. & Weatherbee, S. D. From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design (Blackwell Science, 2001)

    Google Scholar 

  8. Wilkins, A. S. The Evolution of Developmental Pathways (Sinauer Associates, 2002)

    Google Scholar 

  9. Jeong, S. et al. The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell 132, 783–793 (2008)

    CAS  Article  Google Scholar 

  10. Rebeiz, M., Pool, J. E., Kassner, V. A., Aquadro, C. F. & Carroll, S. B. Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population. Science 326, 1663–1667 (2009)

    ADS  CAS  Article  Google Scholar 

  11. Chan, Y. F. et al. Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science 327, 302–305 (2010)

    ADS  CAS  Article  Google Scholar 

  12. Nadeau, N. J. & Jiggins, C. D. A golden age for evolutionary genetics? Genomic studies of adaptation in natural populations. Trends Genet. 26, 484–492 (2010)

    CAS  Article  Google Scholar 

  13. Phillips, P. C. Epistasis—the essential role of gene interactions in the structure and evolution of genetic systems. Nature Rev. Genet. 9, 855–867 (2008)

    CAS  Article  Google Scholar 

  14. Gerke, J., Lorenz, K. & Cohen, B. Genetic interactions between transcription factors cause natural variation in yeast. Science 323, 498–501 (2009)

    ADS  CAS  Article  Google Scholar 

  15. Weinreich, D. M., Watson, R. A. & Chao, L. Perspective: sign epistasis and genetic constraint on evolutionary trajectories. Evolution 59, 1165–1174 (2005)

    CAS  PubMed  Google Scholar 

  16. Carroll, S. B. Homeotic genes and the evolution of arthropods and chordates. Nature 376, 479–485 (1995)

    ADS  CAS  Article  Google Scholar 

  17. Akam, M. Hox genes, homeosis and the evolution of segment identity: no need for hopeless monsters. Int. J. Dev. Biol. 42, 445–451 (1998)

    CAS  PubMed  Google Scholar 

  18. Stern, D. L. Perspective: evolutionary developmental biology and the problem of variation. Evolution 54, 1079–1091 (2000)

    CAS  Article  Google Scholar 

  19. Wray, G. A. et al. The evolution of transcriptional regulation in eukaryotes. Mol. Biol. Evol. 20, 1377–1419 (2003)

    CAS  Article  Google Scholar 

  20. Ludwig, M. Z. et al. Functional evolution of a cis-regulatory module. PLoS Biol. 3, e93 (2005)

    Article  Google Scholar 

  21. Gompel, N., Prud’homme, B., Wittkopp, P. J., Kassner, V. A. & Carroll, S. B. Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila . Nature 433, 481–487 (2005)

    ADS  CAS  Article  Google Scholar 

  22. Sucena, E. & Stern, D. L. 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, 4530–4534 (2000)

    ADS  CAS  Article  Google Scholar 

  23. Chanut-Delalande, H., Fernandes, I., Roch, F., Payre, F. & Plaza, S. Shavenbaby couples patterning to epidermal cell shape control. PLoS Biol. 4, e290 (2006)

    Article  Google Scholar 

  24. Payre, F., Vincent, A. & Carreno, S. ovo/svb integrates Wingless and DER pathways to control epidermis differentiation. Nature 400, 271–275 (1999)

    ADS  CAS  Article  Google Scholar 

  25. McGregor, A. P. et al. Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448, 587–590 (2007)

    ADS  CAS  Article  Google Scholar 

  26. Frankel, N. et al. Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466, 490–493 (2010)

    ADS  CAS  Article  Google Scholar 

  27. Kliman, R. M. et al. The population genetics of the origin and divergence of the Drosophila simulans complex species. Genetics 156, 1913–1931 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Legrand, D. et al. Species-wide genetic variation and demographic history of Drosophila sechellia, a species lacking population structure. Genetics 182, 1197–1206 (2009)

    CAS  Article  Google Scholar 

  29. Nielsen, R. Molecular signatures of natural selection. Annu. Rev. Genet. 39, 197–218 (2005)

    CAS  Article  Google Scholar 

  30. Tajima, F. Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135, 599–607 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Endo, T., Ikeo, K. & Gojobori, T. Large-scale search for genes on which positive selection may operate. Mol. Biol. Evol. 13, 685–690 (1996)

    CAS  Article  Google Scholar 

  32. Baines, J. F., Chen, Y., Das, A. & Stephan, W. DNA sequence variation at a duplicated gene: excess of replacement polymorphism and extensive haplotype structure in the Drosophila melanogaster bicoid region. Mol. Biol. Evol. 19, 989–998 (2002)

    CAS  Article  Google Scholar 

  33. Andolfatto, P. Adaptive evolution of non-coding DNA in Drosophila . Nature 437, 1149–1152 (2005)

    ADS  CAS  Article  Google Scholar 

  34. Haygood, R., Babbitt, C. C., Fedrigo, O. & Wray, G. A. Contrasts between adaptive coding and noncoding changes during human evolution. Proc. Natl Acad. Sci. USA 107, 7853–7857 (2010)

    ADS  CAS  Article  Google Scholar 

  35. Moses, A. M. Statistical tests for natural selection on regulatory regions based on the strength of transcription factor binding sites. BMC Evol. Biol. 9, 286 (2009)

    Article  Google Scholar 

  36. Orr, H. A. Testing natural selection vs. genetic drift in phenotypic evolution using quantitative trait locus data. Genetics 149, 2099–2104 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Casillas, S., Barbadilla, A. & Bergman, C. M. Purifying selection maintains highly conserved noncoding sequences in Drosophila . Mol. Biol. Evol. 24, 2222–2234 (2007)

    CAS  Article  Google Scholar 

  38. Swanson, C. I., Evans, N. C. & Barolo, S. Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer. Dev. Cell 18, 359–370 (2010)

    CAS  Article  Google Scholar 

  39. Klingler, M., Soong, J., Butler, B. & Gergen, J. P. Disperse versus compact elements for the regulation of runt stripes in Drosophila . Dev. Biol. 177, 73–84 (1996)

    CAS  Article  Google Scholar 

  40. Howard, K. R. & Struhl, G. Decoding positional information: regulation of the pair-rule gene hairy . Development 110, 1223–1231 (1990)

    CAS  PubMed  Google Scholar 

  41. Wittkopp, P. J. Evolution of cis-regulatory sequence and function in Diptera. Heredity 97, 139–147 (2006)

    CAS  Article  Google Scholar 

  42. Williams, T. M. et al. The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila . Cell 134, 610–623 (2008)

    CAS  Article  Google Scholar 

  43. Yuh, C.-H., Bolouri, H. & Davidson, E. H. Genomic cis-regulatory logic: experimental and computational analysis of a sea urchin gene. Science 279, 1896–1902 (1998)

    ADS  CAS  Article  Google Scholar 

  44. Small, S., Blair, A. & Levine, M. Regulation of even-skipped stripe 2 in the Drosophila embryo. EMBO J. 11, 4047–4057 (1992)

    CAS  Article  Google Scholar 

  45. Rasband, W. S. ImageJ 〈http://imagej.nih.gov/ij/〉 (United States National Institutes of Health, 1997–2011

Download references

Acknowledgements

We thank G. Davis, P. Parikh and P. Valenti for assistance with cloning and the Drosophila Species Stock Center for fly stocks. This work was supported by the Pew Charitable Trusts Latin American Fellows Program in the Biomedical Sciences Fellowship to N.F., a Ruth L. Kirschstein National Research Service Award to D.F.E. (F32 GM 83546-02), Agence Nationale de la Recherche (Blanc 2008, Netoshape) to F.P., and NIH (GM063622-06A1) and NSF (IOS-0640339) grants to D.L.S.

Author information

Authors and Affiliations

Authors

Contributions

N.F., D.F.E., A.P.M. and D.L.S. designed the experiments and analysed the data. N.F., D.F.E., A.P.M., S.W. and F.P. performed the experimental work. N.F. and D.L.S. wrote the manuscript. D.F.E., A.P.M. and F.P. commented on the manuscript at all stages.

Corresponding author

Correspondence to David L. Stern.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-2 with legends, Supplementary Methods and Materials, Supplementary Tables 1-3 and additional references. (PDF 813 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Frankel, N., Erezyilmaz, D., McGregor, A. et al. Morphological evolution caused by many subtle-effect substitutions in regulatory DNA. Nature 474, 598–603 (2011). https://doi.org/10.1038/nature10200

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10200

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing