Phenotypic robustness conferred by apparently redundant transcriptional enhancers

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Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns1, 2, 3, 4: primary enhancers located near the basal promoter and secondary, or ‘shadow’, enhancers located at more remote positions. It has been proposed that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness1, 5. We tested this hypothesis by generating a deficiency that removes two newly discovered enhancers of shavenbaby (svb, a transcript of the ovo locus), a gene encoding a transcription factor that directs development of Drosophila larval trichomes6. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning7, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability.

At a glance


  1. The svb cis-regulatory region in D. melanogaster.
    Figure 1: The svb cis-regulatory region in D. melanogaster.

    a, Drawing from the lateral perspective of a D. melanogaster first instar larva. The domain producing quaternary trichomes on the fifth abdominal segment is enclosed in a red outline. b, Diagram of the region upstream of the svb first exon, showing the positions of the five enhancers for this locus: DG2, Z, A, E and 7. The expression driven by these enhancers in quaternary cells is shown in purple in the diagrams below each enhancer. The piggyBac elements used to generate Df(X)svb108 are shown as blue triangles. c, f, Expression pattern driven by D. melanogaster Z::lacZ (c) and DG2::lacZ (f) in the 5th and 6th abdominal segments of a stage-15 embryo (purple). An anti-Dusky-like antibody was used to stain developing trichomes (green). d, g, Expression pattern driven by D. simulans Z::lacZ (d) and DG2::lacZ (g). e, h, Expression pattern driven by D. sechellia Z::lacZ (e) and DG2::lacZ (h). β-galactosidase protein produced by D. melanogaster Z::lacZ is expressed in the cytoplasm; β-galactosidase from all other constructs is localized to the nucleus.

  2. Effect of Df(X)svb108 on the number of quaternary trichomes.
    Figure 2: Effect of Df(X)svb108 on the number of quaternary trichomes.

    a, The lateral patch (green) and dorsal region (blue) in which trichomes were counted. The green and blue boxes correspond to the regions where the Z and DG2 enhancers are expressed strongly. The primary, secondary, tertiary and quaternary cell types are indicated with horizontal lines above the photograph. The arrow marks the spiracle that was used to set the lower boundary for the green box. The blue box was positioned directly above the green box. The red box identifies the stout tertiary trichomes, which were excluded from the counts. b, Number of trichomes in the lateral plus dorsal region (blue and green boxes) of the fifth abdominal segment of the larva. Open circles give trichome numbers for each individual (n = 10); the black symbols and lines show the mean±1s.d. Embryos from each of the two genotypes (C108 and Df(X)svb108) were reared at three different temperatures: 17, 25 and 32°C. c, Cuticle images showing the quaternary trichomes in the lateral patch (below) and dorsal region (above) of Df(X)svb108 first-instar larvae that developed at the three different temperatures. The genotype by temperature interaction term of a two-way ANOVA was highly significant (F = 27.57, P<0.0001).

  3. Rescue of the temperature-dependent trichome loss in the lateral patch by a Z::svb transgene.
    Figure 3: Rescue of the temperature-dependent trichome loss in the lateral patch by a Z::svb transgene.

    a, b, Trichome number in the lateral patch (a) and dorsal region (b) of the fifth abdominal segment of larvae with the genotypes C108, Df(X)svb108 and Df(X)svb108; Z::svb. Open circles represent trichome numbers for each individual (n = 10); the black symbols and lines show the mean±1s.d.

  4. Effect of Df(X)svb108; wg-/+ on the number of quaternary trichomes.
    Figure 4: Effect of Df(X)svb108; wg−/+ on the number of quaternary trichomes.

    C108 and Df(X)svb108 embryos that were heterozygous for a null allele of wingless were reared at 25°C. Quaternary trichomes were counted as described in the legend to Fig. 2. A two-way ANOVA shows a highly significant genotype by temperature interaction (F = 7.79, P = 0.0084), which is caused by a large reduction in the number of trichomes on Df(X)svb108; wg−/+ larvae relative to all other genotypes.


  1. Hong, J. W., Hendrix, D. A. & Levine, M. S. Shadow enhancers as a source of evolutionary novelty. Science 321, 1314 (2008)
  2. Werner, T., Hammer, A., Wahlbuhl, M., Bosl, M. R. & Wegner, M. Multiple conserved regulatory elements with overlapping functions determine Sox10 expression in mouse embryogenesis. Nucleic Acids Res. 35, 65266538 (2007)
  3. Zeitlinger, J. et al. Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Genes Dev. 21, 385390 (2007)
  4. Jeong, Y., El-Jaick, K., Roessler, E., Muenke, M. & Epstein, D. J. A functional screen for sonic hedgehog regulatory elements across a 1Mb interval identifies long-range ventral forebrain enhancers. Development 133, 761772 (2006)
  5. Perry, M. W., Cande, J. D., Boettiger, A. N. & Levine, M. Evolution of insect dorsoventral patterning mechanisms. Cold Spring Harb. Symp. Quant. Biol. advance online publication, doi:10.1101/sqb.2009.74.021 (20 October 2009)
  6. Payre, F., Vincent, A. & Carreno, S. ovo/svb integrates Wingless and DER pathways to control epidermis differentiation. Nature 400, 271275 (1999)
  7. Bokor, P. & DiNardo, S. The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila . Development 122, 10831092 (1996)
  8. Overton, P. M., Chia, W. & Buescher, M. The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity. Development 134, 28072813 (2007)
  9. Chanut-Delalande, H., Fernandes, I., Roch, F., Payre, F. & Plaza, S. Shavenbaby couples patterning to epidermal cell shape control. PLoS Biol. 4, e290 (2006)
  10. Fernandes, I. et al. Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes. Dev. Cell 18, 6476 (2010)
  11. McGregor, A. P. et al. Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448, 587590 (2007)
  12. Sucena, E., Delon, I., Jones, I., Payre, F. & Stern, D. L. Regulatory evolution of shavenbaby/ovo underlies multiple cases of morphological parallelism. Nature 424, 935938 (2003)
  13. Crane-Robinson, C., Dragan, A. I. & Read, C. M. Defining the thermodynamics of protein/DNA complexes and their components using micro-calorimetry. Methods Mol. Biol. 543, 625651 (2009)
  14. Hochachka, P. W. & Somero, G. N. Biochemical Adaptation: Mechanism and Process in Physiological Evolution (Oxford Univ. Press, 2002)
  15. Powsner, L. The effects of temperature on the durations of the developmental stages of Drosophila melanogaster . Physiol. Zool. 8, 474520 (1935)
  16. Delon, I., Chanut-Delalande, H. & Payre, F. The Ovo/Shavenbaby transcription factor specifies actin remodelling during epidermal differentiation in Drosophila . Mech. Dev. 120, 747758 (2003)
  17. Nijhout, H. F. & Davidowitz, G. in Developmental Instability: Causes and Consequences (ed. M. Polak) Ch. 1 (Oxford Univ. Press, 2003)
  18. Boettiger, A. N. & Levine, M. Synchronous and stochastic patterns of gene activation in the Drosophila embryo. Science 325, 471473 (2009)
  19. Cretekos, C. J. et al. Regulatory divergence modifies limb length between mammals. Genes Dev. 22, 141151 (2008)
  20. Xiong, N., Kang, C. & Raulet, D. H. Redundant and unique roles of two enhancer elements in the TCRγ locus in gene regulation and γδ T cell development. Immunity 16, 453463 (2002)
  21. Li, X., Cassidy, J. J., Reinke, C. A., Fischboeck, S. & Carthew, R. W. A microRNA imparts robustness against environmental fluctuation during development. Cell 137, 273282 (2009)
  22. Crickmore, M. A., Ranade, V. & Mann, R. S. Regulation of Ubx expression by epigenetic enhancer silencing in response to Ubx levels and genetic variation. PLoS Genet. 5, e1000633 (2009)
  23. Raser, J. M. & O’Shea, E. K. Noise in gene expression: origins, consequences, and control. Science 309, 20102013 (2005)
  24. van den Heuvel, M., Harryman-Samos, C., Klingensmith, J., Perrimon, N. & Nusse, R. Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein. EMBO J. 12, 52935302 (1993)
  25. Bischof, J., Maeda, R. K., Hediger, M., Karch, F. & Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases. Proc. Natl Acad. Sci. USA 104, 33123317 (2007)
  26. Parks, A. L. et al. Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nature Genet. 36, 288292 (2004)

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  1. Howard Hughes Medical Institute and Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey 08544, USA

    • Nicolás Frankel,
    • Diego Vargas,
    • Shu Wang &
    • David L. Stern
  2. Department of Biology, Bryn Mawr College, 101 N. Merion Ave, Bryn Mawr, Pennsylvania 19010, USA

    • Gregory K. Davis
  3. Université de Toulouse and Centre National de la Recherche Scientifique, Centre de Biologie du Développement, UMR5547, Toulouse, F-31062, France

    • François Payre


N.F., G.K.D. and D.L.S. designed the experiments. N.F., G.K.D., D.V., S.W., F.P. and D.L.S. performed the experimental work. N.F. and D.L.S. wrote the manuscript. G.K.D. and F.P. commented on the manuscript at all stages.

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