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  • Review Article
  • Published:

Limb development: a paradigm of gene regulation

Nature Reviews Genetics volume 18, pages 245–258 (2017)Cite this article

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Key Points

  • The limb is widely used as a model in developmental biology. Although the signalling pathways involved in limb development are largely conserved, striking morphological differences are observed among species, revealing a major role for gene regulation in limb patterning.

  • With recent advancements in genomics, genes and regulatory elements involved in limb development can now be identified in a genome-wide manner. Variations in these regulatory elements cause gene expression changes that can lead to major limb morphological transformations in evolution, the following of which are described in this Review: fin-to-limb transformation, limb loss in snakes, digit reduction in cattle, and wing acquisition in bats.

  • Accumulating evidence indicates that isolated limb malformations are caused by the disruption of gene regulatory elements. Molecular mechanisms include changes in the enhancer sequence or dosage, and rearrangement of the spatial organization of chromatin.

Abstract

The limb is a commonly used model system for developmental biology. Given the need for precise control of complex signalling pathways to achieve proper patterning, the limb is also becoming a model system for gene regulation studies. Recent developments in genomic technologies have enabled the genome-wide identification of regulatory elements that control limb development, yielding insights into the determination of limb morphology and forelimb versus hindlimb identity. The modulation of regulatory interactions — for example, through the modification of regulatory sequences or chromatin architecture — can lead to morphological evolution, acquired regeneration capacity or limb malformations in diverse species, including humans.

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Figure 1: Overview of axes involved in limb development.
Figure 2: Establishment of limb bud identity in mammals.
Figure 3: Pitx1 disruption phenotypes.
Figure 4: Morphological evolution of vertebrate appendages.
Figure 5: Regulatory mechanisms of limb malformations.

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References

  1. Butterfield, N. C., McGlinn, E. & Wicking, C. The molecular regulation of vertebrate limb patterning. Curr. Top. Dev. Biol. 90, 319–341 (2010).

    CAS  PubMed  Google Scholar 

  2. Zeller, R., Lopez-Rios, J. & Zuniga, A. Vertebrate limb bud development: moving towards integrative analysis of organogenesis. Nat. Rev. Genet. 10, 845–858 (2009).

    CAS  PubMed  Google Scholar 

  3. Cotney, J. et al. The evolution of lineage-specific regulatory activities in the human embryonic limb. Cell 154, 185–196 (2013).

    CAS  PubMed Central  PubMed  Google Scholar 

  4. Eckalbar, W. L. et al. Transcriptomic and epigenomic characterization of the developing bat wing. Nat. Genet. 48, 528–536 (2016). By comparing RNA-seq and ChIP–seq analyses in the developing bat forelimb and hindlimb, this study identifies regions in the bat genome that are differentially modified and could contribute to bat wing development.

    CAS  PubMed Central  PubMed  Google Scholar 

  5. Infante, C. R., Park, S., Mihala, A. G., Kingsley, D. M. & Menke, D. B. Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements. Dev. Biol. 374, 234–244 (2013).

    CAS  PubMed  Google Scholar 

  6. Visel, A. et al. ChIP–seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    CAS  PubMed Central  PubMed  Google Scholar 

  7. Vokes, S. A., Ji, H., Wong, W. H. & McMahon, A. P. A genome-scale analysis of the cis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb. Genes Dev. 22, 2651–2663 (2008).

    CAS  PubMed Central  PubMed  Google Scholar 

  8. Cotney, J. et al. Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb. Genome Res. 22, 1069–1080 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  9. VanderMeer, J. E., Smith, R. P., Jones, S. L. & Ahituv, N. Genome-wide identification of signaling center enhancers in the developing limb. Development 141, 4194–4198 (2014). Using H3K27ac ChIP–seq in the AER and ZPA signalling centres of the limb bud, this study identifies novel enhancers that could be important regulators of genes involved in tetrapod limb patterning.

    CAS  PubMed Central  PubMed  Google Scholar 

  10. DeMare, L. E. et al. The genomic landscape of cohesin-associated chromatin interactions. Genome Res. 23, 1224–1234 (2013). This genome-wide scale analysis of cohesin-associated interactions in the developing mouse limb provides insights into the regulatory architecture of limb development.

    CAS  PubMed Central  PubMed  Google Scholar 

  11. Liu, H. et al. Whole transcriptome expression profiling of mouse limb tendon development by using RNA-seq. J. Orthop. Res. 33, 840–848 (2015).

    PubMed Central  PubMed  Google Scholar 

  12. Sears, K. E. et al. The relationship between gene network structure and expression variation among individuals and species. PLoS Genet. 11, e1005398 (2015).

    PubMed Central  PubMed  Google Scholar 

  13. Wang, Z. et al. Unique expression patterns of multiple key genes associated with the evolution of mammalian flight. Proc. Biol. Sci. 281, 20133133 (2014).

    PubMed Central  PubMed  Google Scholar 

  14. Gibson-Brown, J. J. et al. Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56, 93–101 (1996).

    CAS  PubMed  Google Scholar 

  15. Minguillon, C., Gibson-Brown, J. J. & Logan, M. P. Tbx4/5 gene duplication and the origin of vertebrate paired appendages. Proc. Natl Acad. Sci. USA 106, 21726–21730 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Nishimoto, S., Wilde, S. M., Wood, S. & Logan, M. P. RA acts in a coherent feed-forward mechanism with Tbx5 to control limb bud induction and initiation. Cell Rep. 12, 879–891 (2015).

    CAS  PubMed Central  PubMed  Google Scholar 

  17. Agarwal, P. et al. Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. Development 130, 623–633 (2003).

    CAS  PubMed  Google Scholar 

  18. Ng, J. K. et al. The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129, 5161–5170 (2002).

    CAS  PubMed  Google Scholar 

  19. Minguillon, C., Del Buono, J. & Logan, M. P. Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth. Dev. Cell 8, 75–84 (2005).

    CAS  PubMed  Google Scholar 

  20. Ohuchi, H. et al. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development 124, 2235–2244 (1997).

    CAS  PubMed  Google Scholar 

  21. Sekine, K. et al. Fgf10 is essential for limb and lung formation. Nat. Genet. 21, 138–141 (1999).

    CAS  PubMed  Google Scholar 

  22. Xu, X. et al. Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction. Development 125, 753–765 (1998).

    CAS  PubMed  Google Scholar 

  23. Rallis, C. et al. Tbx5 is required for forelimb bud formation and continued outgrowth. Development 130, 2741–2751 (2003).

    CAS  PubMed  Google Scholar 

  24. Ahn, D. G., Kourakis, M. J., Rohde, L. A., Silver, L. M. & Ho, R. K. T-box gene tbx5 is essential for formation of the pectoral limb bud. Nature 417, 754–758 (2002).

    CAS  PubMed  Google Scholar 

  25. Don, E. K. et al. Genetic basis of hindlimb loss in a naturally occurring vertebrate model. Biol. Open 5, 359–366 (2016).

    CAS  PubMed Central  PubMed  Google Scholar 

  26. Naiche, L. A. & Papaioannou, V. E. Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development 130, 2681–2693 (2003).

    CAS  PubMed  Google Scholar 

  27. Szeto, D. P., Ryan, A. K., O'Connell, S. M. & Rosenfeld, M. G. P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development. Proc. Natl Acad. Sci. USA 93, 7706–7710 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Lonfat, N. & Duboule, D. Structure, function and evolution of topologically associating domains (TADs) at HOX loci. FEBS Lett. 589, 2869–2876 (2015).

    CAS  PubMed  Google Scholar 

  29. Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012). This study uses Hi-C to determine the topological domains of the human and mouse genomes in embryonic stem cells and terminally differentiated cells, providing major insights into the 3D spatial organization of mammalian genomes.

    CAS  PubMed Central  PubMed  Google Scholar 

  30. Andrey, G. et al. A switch between topological domains underlies HoxD genes collinearity in mouse limbs. Science 340, 1234167 (2013).

    PubMed  Google Scholar 

  31. Acemel, R. D. et al. A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation. Nat. Genet. 48, 336–341 (2016).

    CAS  PubMed  Google Scholar 

  32. Minguillon, C. et al. Hox genes regulate the onset of Tbx5 expression in the forelimb. Development 139, 3180–3188 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  33. Nishimoto, S., Minguillon, C., Wood, S. & Logan, M. P. A combination of activation and repression by a colinear Hox code controls forelimb-restricted expression of Tbx5 and reveals Hox protein specificity. PLoS Genet. 10, e1004245 (2014).

    PubMed Central  PubMed  Google Scholar 

  34. Adachi, N., Robinson, M., Goolsbee, A. & Shubin, N. H. Regulatory evolution of Tbx5 and the origin of paired appendages. Proc. Natl Acad. Sci. USA 113, 10115–10120 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Menke, D. B., Guenther, C. & Kingsley, D. M. Dual hindlimb control elements in the Tbx4 gene and region-specific control of bone size in vertebrate limbs. Development 135, 2543–2553 (2008).

    CAS  PubMed  Google Scholar 

  36. Infante, C. R. et al. Shared enhancer activity in the limbs and phallus and functional divergence of a limb-genital cis-regulatory element in snakes. Dev. Cell 35, 107–119 (2015). This paper shows that numerous limb enhancers are retained in snake genomes, and functional enhancer assays show how their function could have changed.

    CAS  PubMed Central  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  38. Rodriguez-Esteban, C. et al. The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398, 814–818 (1999).

    CAS  PubMed  Google Scholar 

  39. Takeuchi, J. K. et al. Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature 398, 810–814 (1999).

    CAS  PubMed  Google Scholar 

  40. Logan, M. & Tabin, C. J. Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity. Science 283, 1736–1739 (1999).

    CAS  PubMed  Google Scholar 

  41. DeLaurier, A., Schweitzer, R. & Logan, M. Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb. Dev. Biol. 299, 22–34 (2006).

    CAS  PubMed  Google Scholar 

  42. Ouimette, J. F., Jolin, M. L., L'Honore, A., Gifuni, A. & Drouin, J. Divergent transcriptional activities determine limb identity. Nat. Commun. 1, 35 (2010).

    PubMed  Google Scholar 

  43. Lanctot, C., Moreau, A., Chamberland, M., Tremblay, M. L. & Drouin, J. Hindlimb patterning and mandible development require the Ptx1 gene. Development 126, 1805–1810 (1999).

    CAS  PubMed  Google Scholar 

  44. Szeto, D. P. et al. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development. Genes Dev. 13, 484–494 (1999).

    CAS  PubMed Central  PubMed  Google Scholar 

  45. Duboc, V. & Logan, M. P. Pitx1 is necessary for normal initiation of hindlimb outgrowth through regulation of Tbx4 expression and shapes hindlimb morphologies via targeted growth control. Development 138, 5301–5309 (2011).

    CAS  PubMed Central  PubMed  Google Scholar 

  46. Al-Qattan, M. M., Al-Thunayan, A., Alabdulkareem, I. & Al Balwi, M. Liebenberg syndrome is caused by a deletion upstream to the PITX1 gene resulting in transformation of the upper limbs to reflect lower limb characteristics. Gene 524, 65–71 (2013).

    CAS  PubMed  Google Scholar 

  47. Spielmann, M. et al. Homeotic arm-to-leg transformation associated with genomic rearrangements at the PITX1 locus. Am. J. Hum. Genet. 91, 629–635 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  48. Seoighe, D. M. et al. A chromosomal 5q31.1 gain involving PITX1 causes Liebenberg syndrome. Am. J. Med. Genet. A 164A, 2958–2960 (2014).

    PubMed  Google Scholar 

  49. Domyan, E. T. et al. Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species. eLife 5, e12115 (2016).

    PubMed Central  PubMed  Google Scholar 

  50. Sears, K. E., Behringer, R. R., Rasweiler, J. J. IV & Niswander, L. A. Development of bat flight: morphologic and molecular evolution of bat wing digits. Proc. Natl Acad. Sci. USA 103, 6581–6586 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Booker, B. M. et al. Bat accelerated regions identify a bat forelimb specific enhancer in the HoxD locus. PLoS Genet. 12, e1005738 (2016).

    PubMed Central  PubMed  Google Scholar 

  52. Hockman, D. et al. A second wave of Sonic hedgehog expression during the development of the bat limb. Proc. Natl Acad. Sci. USA 105, 16982–16987 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Cretekos, C. J. et al. Regulatory divergence modifies limb length between mammals. Genes Dev. 22, 141–151 (2008). By replacing a mouse limb enhancer sequence with a bat sequence, this article shows how changes in regulatory elements can have a functional consequence.

    CAS  PubMed Central  PubMed  Google Scholar 

  54. Cretekos, C. J. et al. Isolation, genomic structure and developmental expression of Fgf8 in the short-tailed fruit bat, Carollia perspicillata. Int. J. Dev. Biol. 51, 333–338 (2007).

    CAS  PubMed  Google Scholar 

  55. Chen, C. H., Cretekos, C. J., Rasweiler, J. J. IV & Behringer, R. R. Hoxd13 expression in the developing limbs of the short-tailed fruit bat, Carollia perspicillata. Evol. Dev. 7, 130–141 (2005).

    CAS  PubMed  Google Scholar 

  56. Cooper, K. L. et al. Patterning and post-patterning modes of evolutionary digit loss in mammals. Nature 511, 41–45 (2014).

    CAS  PubMed Central  PubMed  Google Scholar 

  57. Sears, K. E. et al. Developmental basis of mammalian digit reduction: a case study in pigs. Evol. Dev. 13, 533–541 (2011).

    PubMed  Google Scholar 

  58. Cooper, K. L. et al. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions. Nature 495, 375–378 (2013).

    CAS  PubMed Central  PubMed  Google Scholar 

  59. Lopez-Rios, J. et al. Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature 511, 46–51 (2014). This article shows that digit reduction in cattle is associated with changes in SHH signalling that are due to evolutionary alterations of a PTCH1 regulatory module.

    CAS  PubMed  Google Scholar 

  60. Thewissen, J. G. M. et al. Developmental basis for hind-limb loss in dolphins and origin of the cetacean bodyplan. Proc. Natl Acad. Sci. USA 103, 8414–8418 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Keyte, A. L. & Smith, K. K. Developmental origins of precocial forelimbs in marsupial neonates. Development 137, 4283–4294 (2010).

    CAS  PubMed  Google Scholar 

  62. Sears, K. E. et al. Disparate Igf1 expression and growth in the fore- and hind limbs of a marsupial (Monodelphis domestica). J. Exp. Zool. B Mol. Dev. Evol. 318, 279–293 (2012).

    CAS  PubMed  Google Scholar 

  63. Chew, K. Y., Yu, H., Pask, A. J., Shaw, G. & Renfree, M. B. HOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii. BMC Dev. Biol. 12, 2 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  64. Sanger, T. J., Revell, L. J., Gibson-Brown, J. J. & Losos, J. B. Repeated modification of early limb morphogenesis programmes underlies the convergence of relative limb length in Anolis lizards. Proc. Biol. Sci. 279, 739–748 (2012).

    PubMed  Google Scholar 

  65. Cohn, M. J. & Tickle, C. Developmental basis of limblessness and axial patterning in snakes. Nature 399, 474–479 (1999).

    CAS  PubMed  Google Scholar 

  66. Shapiro, M. D., Hanken, J. & Rosenthal, N. Developmental basis of evolutionary digit loss in the Australian lizard Hemiergis. J. Exp. Zool. B Mol. Dev. Evol. 297, 48–56 (2003).

    PubMed  Google Scholar 

  67. Leal, F. & Cohn, M. J. Loss and re-emergence of legs in snakes by modular evolution of Sonic hedgehog and HOXD enhancers. Curr. Biol. 26, 2966–2973 (2016).

    CAS  PubMed  Google Scholar 

  68. Kvon, E. Z. et al. Progressive loss of function in a limb enhancer during snake evolution. Cell. 167, 633–642 (2016).

    CAS  PubMed Central  PubMed  Google Scholar 

  69. Guerreiro, I. et al. Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan. eLife 5, e16087 (2016).

    PubMed Central  PubMed  Google Scholar 

  70. Butterfield, N. C. et al. Patched 1 is a crucial determinant of asymmetry and digit number in the vertebrate limb. Development 136, 3515–3524 (2009).

    CAS  PubMed  Google Scholar 

  71. Dai, M. et al. Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals. PLoS ONE 9, e106100 (2014).

    PubMed Central  PubMed  Google Scholar 

  72. Sugiura, T., Wang, H., Barsacchi, R., Simon, A. & Tanaka, E. M. MARCKS-like protein is an initiating molecule in axolotl appendage regeneration. Nature 531, 237–240 (2016).

    CAS  PubMed Central  PubMed  Google Scholar 

  73. Endo, T., Bryant, S. V. & Gardiner, D. M. A stepwise model system for limb regeneration. Dev. Biol. 270, 135–145 (2004).

    CAS  PubMed  Google Scholar 

  74. Nacu, E., Gromberg, E., Oliveira, C. R., Drechsel, D. & Tanaka, E. M. FGF8 and SHH substitute for anterior–posterior tissue interactions to induce limb regeneration. Nature 533, 407–410 (2016).

    CAS  PubMed  Google Scholar 

  75. Muneoka, K., Holler-Dinsmore, G. & Bryant, S. Regeneration from discontinuous circumferences in axolotl limbs. Prog. Clin. Biol. Res. 217A, 61–65 (1986).

    CAS  PubMed  Google Scholar 

  76. Yakushiji, N. et al. Correlation between Shh expression and DNA methylation status of the limb-specific Shh enhancer region during limb regeneration in amphibians. Dev. Biol. 312, 171–182 (2007).

    CAS  PubMed  Google Scholar 

  77. Hayashi, S. et al. Epigenetic modification maintains intrinsic limb-cell identity in Xenopus limb bud regeneration. Dev. Biol. 406, 271–282 (2015).

    CAS  PubMed  Google Scholar 

  78. Kang, J. et al. Modulation of tissue repair by regeneration enhancer elements. Nature 532, 201–206 (2016). Using epigenetic profiling in the regenerating hearts and fins of zebrafish, this study provides evidence for TREEs that trigger gene expression in injury sites.

    CAS  PubMed Central  PubMed  Google Scholar 

  79. VanderMeer, J. E. & Ahituv, N. Cis-regulatory mutations are a genetic cause of human limb malformations. Dev. Dyn. 240, 920–930 (2011).

    CAS  PubMed Central  PubMed  Google Scholar 

  80. Spielmann, M. & Mundlos, S. Looking beyond the genes: the role of non-coding variants in human disease. Hum. Mol. Genet. 25, R157–R165 (2016).

    CAS  PubMed  Google Scholar 

  81. Lettice, L. A. et al. Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly. Proc. Natl Acad. Sci. USA 99, 7548–7553 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Williamson, I., Lettice, L. A., Hill, R. E. & Bickmore, W. A. Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity. Development 143, 2994–3001 (2016).

    CAS  PubMed Central  PubMed  Google Scholar 

  83. Amano, T. et al. Chromosomal dynamics at the Shh locus: limb bud-specific differential regulation of competence and active transcription. Dev. Cell 16, 47–57 (2009).

    CAS  PubMed  Google Scholar 

  84. Lettice, L. A. et al. Development of five digits is controlled by a bipartite long-range cis-regulator. Development 141, 1715–1725 (2014).

    CAS  PubMed Central  PubMed  Google Scholar 

  85. Galli, A. et al. Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet. 6, e1000901 (2010).

    PubMed Central  PubMed  Google Scholar 

  86. Capellini, T. D. et al. Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression. Development 133, 2263–2273 (2006).

    CAS  PubMed  Google Scholar 

  87. Lettice, L. A. et al. Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly. Dev. Cell 22, 459–467 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  88. Kozhemyakina, E., Ionescu, A. & Lassar, A. B. GATA6 is a crucial regulator of Shh in the limb bud. PLoS Genet. 10, e1004072 (2014).

    PubMed Central  PubMed  Google Scholar 

  89. Harfe, B. D. et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, 517–528 (2004).

    CAS  PubMed  Google Scholar 

  90. Riddle, R. D., Johnson, R. L., Laufer, E. & Tabin, C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401–1416 (1993).

    CAS  PubMed  Google Scholar 

  91. Sagai, T., Hosoya, M., Mizushina, Y., Tamura, M. & Shiroishi, T. Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. Development 132, 797–803 (2005).

    CAS  PubMed  Google Scholar 

  92. Sharpe, J. et al. Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch. Curr. Biol. 9, 97–100 (1999).

    CAS  PubMed  Google Scholar 

  93. Lettice, L. A., Hill, A. E., Devenney, P. S. & Hill, R. E. Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly. Hum. Mol. Genet. 17, 978–985 (2008).

    CAS  PubMed  Google Scholar 

  94. Dorshorst, B., Okimoto, R. & Ashwell, C. Genomic regions associated with dermal hyperpigmentation, polydactyly and other morphological traits in the Silkie chicken. J. Hered. 101, 339–350 (2010).

    CAS  PubMed  Google Scholar 

  95. Shamseldin, H. E., Faden, M. A., Alashram, W. & Alkuraya, F. S. Identification of a novel DLX5 mutation in a family with autosomal recessive split hand and foot malformation. J. Med. Genet. 49, 16–20 (2012).

    PubMed  Google Scholar 

  96. Sowinska-Seidler, A., Badura-Stronka, M., Latos-Bielenska, A., Stronka, M. & Jamsheer, A. Heterozygous DLX5 nonsense mutation associated with isolated split-hand/foot malformation with reduced penetrance and variable expressivity in two unrelated families. Birth Defects Res. A Clin. Mol. Teratol. 100, 764–771 (2014).

    CAS  PubMed  Google Scholar 

  97. Wang, X. et al. Exome sequencing reveals a heterozygous DLX5 mutation in a Chinese family with autosomal-dominant split-hand/foot malformation. Eur. J. Hum. Genet. 22, 1105–1110 (2014).

    CAS  PubMed Central  PubMed  Google Scholar 

  98. Ullah, A., Ullah, M. F., Khalid, Z. M. & Ahmad, W. A novel heterozygous frameshift mutation in DLX5 gene underlies isolated split hand foot malformation type 1. Pediatr. Int. 58, 1348–1350 (2016).

    CAS  PubMed  Google Scholar 

  99. Birnbaum, R. Y. et al. Functional characterization of tissue-specific enhancers in the DLX5/6 locus. Hum. Mol. Genet. 21, 4930–4938 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  100. Rasmussen, M. B. et al. Phenotypic subregions within the split-hand/foot malformation 1 locus. Hum. Genet. 135, 345–357 (2016).

    PubMed  Google Scholar 

  101. Birnbaum, R. Y. et al. Coding exons function as tissue-specific enhancers of nearby genes. Genome Res. 22, 1059–1068 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  102. Marinic, M., Aktas, T., Ruf, S. & Spitz, F. An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. Dev. Cell 24, 530–542 (2013).

    CAS  PubMed  Google Scholar 

  103. Sheth, R. et al. Decoupling the function of Hox and Shh in developing limb reveals multiple inputs of Hox genes on limb growth. Development 140, 2130–2138 (2013).

    CAS  PubMed  Google Scholar 

  104. Goodman, F. R., Majewski, F., Collins, A. L. & Scambler, P. J. A 117-kb microdeletion removing HOXD9–HOXD13 and EVX2 causes synpolydactyly. Am. J. Hum. Genet. 70, 547–555 (2002).

    CAS  PubMed Central  PubMed  Google Scholar 

  105. Spitz, F., Gonzalez, F. & Duboule, D. A global control region defines a chromosomal regulatory landscape containing the HoxD cluster. Cell 113, 405–417 (2003).

    CAS  PubMed  Google Scholar 

  106. Klopocki, E. et al. Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion. J. Med. Genet. 49, 119–125 (2012).

    CAS  PubMed  Google Scholar 

  107. Schatz, O., Langer, E. & Ben-Arie, N. Gene dosage of the transcription factor Fingerin (bHLHA9) affects digit development and links syndactyly to ectrodactyly. Hum. Mol. Genet. 23, 5394–5401 (2014).

    CAS  PubMed  Google Scholar 

  108. Malik, S. et al. Mutations affecting the BHLHA9 DNA-binding domain cause MSSD, mesoaxial synostotic syndactyly with phalangeal reduction, Malik–Percin type. Am. J. Hum. Genet. 95, 649–659 (2014).

    CAS  PubMed Central  PubMed  Google Scholar 

  109. Lettice, L. A. et al. Enhancer-adoption as a mechanism of human developmental disease. Hum. Mutat. 32, 1492–1499 (2011).

    CAS  PubMed  Google Scholar 

  110. Matharu, N. & Ahituv, N. Minor loops in major folds: enhancer-promoter looping, chromatin restructuring, and their association with transcriptional regulation and disease. PLoS Genet. 11, e1005640 (2015).

    PubMed Central  PubMed  Google Scholar 

  111. Lupianez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161, 1012–1025 (2015). This article describes distinct human limb malformations caused by chromosomal rearrangements that disrupt TAD boundaries, providing criteria for predicting the pathogenicity of structural variants, particularly in non-coding regions of the genome.

    CAS  PubMed Central  PubMed  Google Scholar 

  112. Franke, M. et al. Formation of new chromatin domains determines pathogenicity of genomic duplications. Nature 538, 265–269 (2016).

    CAS  PubMed  Google Scholar 

  113. Inoue, F. & Ahituv, N. Decoding enhancers using massively parallel reporter assays. Genomics 106, 159–164 (2015).

    CAS  PubMed  Google Scholar 

  114. Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR–Cas9. Nat. Rev. Genet. 16, 299–311 (2015).

    CAS  PubMed Central  PubMed  Google Scholar 

  115. Ritter, D. I., Dong, Z., Guo, S. & Chuang, J. H. Transcriptional enhancers in protein-coding exons of vertebrate developmental genes. PLoS ONE 7, e35202 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  116. Zhou, V. W., Goren, A. & Bernstein, B. E. Charting histone modifications and the functional organization of mammalian genomes. Nat. Rev. Genet. 12, 7–18 (2011).

    PubMed  Google Scholar 

  117. Koch, F. et al. Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters. Nat. Struct. Mol. Biol. 18, 956–963 (2011).

    CAS  PubMed  Google Scholar 

  118. van Arensbergen, J., van Steensel, B. & Bussemaker, H. J. In search of the determinants of enhancer–promoter interaction specificity. Trends Cell Biol. 24, 695–702 (2014).

    CAS  PubMed Central  PubMed  Google Scholar 

  119. Merika, M., Williams, A. J., Chen, G., Collins, T. & Thanos, D. Recruitment of CBP/p300 by the IFNβ enhanceosome is required for synergistic activation of transcription. Mol. Cell 1, 277–287 (1998).

    CAS  PubMed  Google Scholar 

  120. Lai, F. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013).

    CAS  PubMed Central  PubMed  Google Scholar 

  121. Boisvert, C. A., Mark-Kurik, E. & Ahlberg, P. E. The pectoral fin of Panderichthys and the origin of digits. Nature 456, 636–638 (2008).

    CAS  PubMed  Google Scholar 

  122. Sordino, P., van der Hoeven, F. & Duboule, D. Hox gene expression in teleost fins and the origin of vertebrate digits. Nature 375, 678–681 (1995).

    CAS  PubMed  Google Scholar 

  123. Spitz, F., Gonzales, F. & Duboule, D. A global control region defines a chromosomal regulatory landscape containing the HoxD cluster. Cell 113, 405–417 (2003).

    CAS  PubMed  Google Scholar 

  124. Shubin, N., Tabin, C. & Carroll, S. Fossils, genes and the evolution of animal limbs. Nature 388, 639–648 (1997).

    CAS  PubMed  Google Scholar 

  125. Dolle, P. et al. Disruption of the Hoxd13 gene induces localized heterochrony leading to mice with neotenic limbs. Cell 75, 431–441 (1993).

    CAS  PubMed  Google Scholar 

  126. Kherdjemil, Y. et al. Evolution of Hoxa11 regulation in vertebrates is linked to the pentadactyl state. Nature 539, 89–92 (2016).

    CAS  PubMed Central  PubMed  Google Scholar 

  127. Freitas, R., Zhang, G. & Cohn, M. J. Biphasic Hoxd gene expression in shark paired fins reveals an ancient origin of the distal limb domain. PLoS ONE 2, e754 (2007).

    PubMed Central  PubMed  Google Scholar 

  128. Woltering, J. M., Noordermeer, D., Leleu, M. & Duboule, D. Conservation and divergence of regulatory strategies at Hox loci and the origin of tetrapod digits. PLoS Biol. 12, e1001773 (2014).

    PubMed Central  PubMed  Google Scholar 

  129. Schneider, I. & Shubin, N. H. The origin of the tetrapod limb: from expeditions to enhancers. Trends Genet. 29, 419–426 (2013).

    CAS  PubMed  Google Scholar 

  130. Davis, M. C., Dahn, R. D. & Shubin, N. H. An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish. Nature 447, 473–476 (2007).

    CAS  PubMed  Google Scholar 

  131. Shubin, N. H., Daeschler, E. B. & Jenkins, F. A. Jr. The pectoral fin of Tiktaalik roseae and the origin of the tetrapod limb. Nature 440, 764–771 (2006).

    CAS  PubMed  Google Scholar 

  132. Gehrke, A. R. et al. Deep conservation of wrist and digit enhancers in fish. Proc. Natl Acad. Sci. USA 112, 803–808 (2015).

    CAS  PubMed  Google Scholar 

  133. Johanson, Z. et al. Fish fingers: digit homologues in sarcopterygian fish fins. J. Exp. Zool. B Mol. Dev. Evol. 308B, 757–768 (2007).

    Google Scholar 

  134. Ahn, D. & Ho, R. K. Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages. Dev. Biol. 322, 220–233 (2008).

    CAS  PubMed  Google Scholar 

  135. Nakamura, T., Gehrke, A. R., Lemberg, J., Szymaszek, J. & Shubin, N. H. Digits and fin rays share common developmental histories. Nature 537, 225–228 (2016).

    CAS  PubMed Central  PubMed  Google Scholar 

  136. Schneider, I. et al. Appendage expression driven by the Hoxd Global Control Region is an ancient gnathostome feature. Proc. Natl Acad. Sci. USA 108, 12782–12786 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Kawakami, Y. et al. WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104, 891–900 (2001).

    CAS  PubMed  Google Scholar 

  138. Kengaku, M. et al. Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud. Science 280, 1274–1277 (1998).

    CAS  PubMed  Google Scholar 

  139. Yang, A. et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718 (1999).

    CAS  PubMed  Google Scholar 

  140. Ianakiev, P. et al. Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am. J. Hum. Genet. 67, 59–66 (2000).

    CAS  PubMed Central  PubMed  Google Scholar 

  141. Robledo, R. F., Rajan, L., Li, X. & Lufkin, T. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev. 16, 1089–1101 (2002).

    CAS  PubMed Central  PubMed  Google Scholar 

  142. Lo Iacono, N. et al. Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects. Development 135, 1377–1388 (2008).

    CAS  PubMed  Google Scholar 

  143. Kouwenhoven, E. N. et al. Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus. PLoS Genet. 6, e1001065 (2010).

    PubMed Central  PubMed  Google Scholar 

  144. Guerrini, L., Costanzo, A. & Merlo, G. R. A symphony of regulations centered on p63 to control development of ectoderm-derived structures. J. Biomed. Biotechnol. 2011, 864904 (2011).

    PubMed Central  PubMed  Google Scholar 

  145. Haro, E. et al. Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development. PLoS Genet. 10, e1004468 (2014).

    PubMed Central  PubMed  Google Scholar 

  146. Lango Allen, H. et al. Next generation sequencing of chromosomal rearrangements in patients with split-hand/split-foot malformation provides evidence for DYNC1I1 exonic enhancers of DLX5/6 expression in humans. J. Med. Genet. 51, 264–267 (2014).

    PubMed  Google Scholar 

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Acknowledgements

The authors apologize to those colleagues whose important contributions could not be discussed and/or referenced owing to space limitations. N.A. is supported in part by the National Human Genome Research Institute and the National Cancer Institute (grant number: 1R01CA197139), by the National Institute of Mental Health (grant number: 1R01MH109907) and by the National Institute of Child Health and Human Development (grant number: 1P01HD084387). K.E.S. is supported in part by the National Institutes of Health, Office of the Director (grant number: 1R21OD022988), by the National Science Foundation, Division of Environmental Biology (grant number: 1442314) and by the National Science Foundation, Division of Integrative Organismal Biology (grant number: 1257873). F.P. is supported in part by the Fulbright Research Scholar Program.

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Authors and Affiliations

  1. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 94158, California, USA

    Florence Petit & Nadav Ahituv

  2. University of Lille, CHU Lille, EA 7364-RADEME, Lille, F-59000, France

    Florence Petit

  3. School of Integrative Biology, Institute for Genomic Biology, University of Illinois, Urbana, 61801, Illinois, USA

    Karen E. Sears

  4. Institute for Human Genetics, University of California, San Francisco, 94158, California, USA

    Nadav Ahituv

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  1. Florence Petit
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  2. Karen E. Sears
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  3. Nadav Ahituv
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Correspondence to Karen E. Sears or Nadav Ahituv.

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PowerPoint slides

Glossary

Promoters

Cis-regulatory DNA sequences immediately upstream of transcription start sites at which RNA polymerases and transcription factors bind to initiate gene transcription.

Enhancers

Cis-regulatory DNA sequences that, when bound by specific transcription factors, enhance the transcription of an associated gene. Enhancers can be located upstream or downstream of the gene, and at variable distances.

Silencers

Cis-regulatory DNA sequences that, when bound by specific transcription factors, repress the transcription of an associated gene. Silencers can be located upstream or downstream of the gene, and at variable distances.

Insulators

DNA sequence elements that protect genes from inappropriate regulatory signals emanating from their surrounding environment. Insulators act like barriers, blocking the effect of a regulatory element on a promoter, or preventing the advance of chromatin condensation.

ChIA–PET

A method that combines chromatin conformation capture and DNA paired-end high-throughput sequencing to analyse chromatin interaction (ChIA) across the genome.

RNA-seq

A technique that combines high-throughput sequencing of cDNA molecules obtained by reverse transcription within a biological sample to determine the sequence and relative abundance of each RNA molecule.

Topologically associating domains

(TADs). Conserved megabase-sized sub-orders of chromosome organization, that are delineated by boundaries enriched in architectural proteins (CCCTC-binding factor and cohesin). Within a TAD, chromatin interactions occur at a high frequency and allow enhancer–promoter contacts.

Accelerated regions

Highly conserved sequences that have experienced a marked increase of substitution rates in a particular lineage. These regions are good candidates for the identification of regulatory DNA sequences that could have contributed to specific morphological differences in that lineage.

Chromatin conformation capture

A technique that is used to quantify DNA–DNA contacts in the nucleus, within a single locus or on a genome-wide scale, to study the 3D organization of the genome.

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Petit, F., Sears, K. & Ahituv, N. Limb development: a paradigm of gene regulation. Nat Rev Genet 18, 245–258 (2017). https://doi.org/10.1038/nrg.2016.167

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