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When the Snake Lost Its Limbs, What Did the Mouse and Lizard Say?
Developmental Cell
Previews When the Snake Lost Its Limbs, What Did the Mouse and Lizard Say? Diego Villar1,* and Duncan T. Odom1,2,* 1University
of Cambridge, Cancer Research UK Cambridge Institute, Robinson Way, Cambridge CB2 0RE, UK Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK *Correspondence: [email protected] (D.V.), [email protected] (D.T.O.) http://dx.doi.org/10.1016/j.devcel.2015.09.022 2Wellcome
In this issue of Developmental Cell, Infante et al. (2015) compare regulatory DNA sequences in mice, lizards, and limbless snakes to reveal widespread sharing of enhancer activity in developing limbs and genitalia. Genetic deletion of a limb-genital enhancer demonstrates that common regulatory elements affect development of both appendages. The transition of vertebrates from an aquatic to a terrestrial environment required major adaptations in locomotion and reproduction and resulted in the evolution of limbs and external genitalia. Commonalities in the development of these anatomical structures include shared signaling pathways and gene expression similarities (Tschopp et al., 2014), suggesting an evolutionary link between the two appendages. In this issue of Developmental Cell, Infante et al. (2015) provide compelling evidence for shared cis-regulatory activities between developing limbs and genitalia. Many limb-specific enhancers have been identified in mammals (Cotney et al., 2013). Here, Infante et al. (2015) use limbless snakes to show that the sequences of many of these enhancers can be retained, even when the anatomical structures where they are active have vanished. These results further highlight the plasticity of amniote gene-regulatory activities and demonstrate that shared regulatory architectures can contribute to the development of limbs and external genitalia. Despite their somewhat disparate morphologies, outgrowth of both limbs and genitalia during embryonic development can depend on common genes, such as the posterior HOXD and HOXA clusters (Kondo et al., 1997), and mutations in these developmental regulators lead to disorders such as the human hand-foot-genital syndrome (Mortlock and Innis, 1997). More recently, it was revealed that these loci have similar regulatory topologies in developing limb buds and genital tubercle (GT) (Lonfat et al., 2014). The chromatin architecture involved promoter-enhancer interactions that were shared in the two
appendages, as well as some specific to either tissue. Further analysis of the shared interactions suggested that some limb enhancers could also function in developing genitalia. Moreover, a recent comparison of genital development in mice and squamates reported co-expression of many genes in limbs and genitalia and suggested ancestral commonalities in the developmental mechanisms leading to the two structures (Tschopp et al., 2014). Infante and colleagues (2015) set out to determine the extent to which genital and limb transcriptional networks may deploy common cis-regulatory elements (Figure 1). They reasoned that shared regulatory activity between limbs and genital enhancers could lead to retention of limb regulatory sequences in limbless species. Specifically, testing the sequence conservation of a set of mouse enhancers with known limb activity in squamate genomes showed that DNA sequences of mouse limb enhancers are often retained in snakes—in fact, sequence conservation of these enhancers was similar in the genomes of limbed and non-limbed squamates. This result prompted the authors to re-evaluate the tissue specificity of limb enhancers in developmentally matched forelimb, hindlimb, and GT of mice by profiling genomic locations where lysine 27 on histone H3 is acetylated (H3K27ac). This histone mark identifies active regulatory regions, such as promoters and enhancers (Creyghton et al., 2010). Previously validated limb enhancers provided a convincing set of candidate regions, where enrichment of H3K27ac was commonly shared in limb and GT samples but largely absent in those of non-appendage tissues.
The maps of enhancer activity in mouse limbs and GT show that almost half of either forelimb or hindlimb regulatory elements can be active in genitalia. By profiling the same tissues in Anolis lizard embryos, the authors further identified whether tissue specificity of enhancer activity is conserved in orthologous squamate regions; this analysis showed significantly lower conservation for enhancer activity restricted to developing genitalia. These results suggest a higher evolutionary turnover of GT-specific regulatory elements versus those active in limb or limb-GT. The authors continue in more detail by focusing on HLA and HLB, a pair of enhancers targeting the hindlimb developmental gene TBX4 (Naiche and Papaioannou, 2003), which is also expressed in the GT. The activity of both HLA and HLB is conserved in Anolis and mouse, but whereas HLA activity is restricted to the limb, HLB is active in both limbs and genitalia. Infante and colleagues (2015) employ transgene expression in mouse embryos to show that HLB snake sequences do not drive limb expression, but have retained partial activity in the GT. Consistent with this result, engineering of a HLB deletion in mice leads to defects in the development of both limbs and genitalia. Detailed functional analyses further demonstrate pleiotropic roles of this enhancer, including defects in nonappendage tissues such as the kidney. The analysis by Infante et al. (2015) raises a number of questions for follow-up investigation. The interesting observations on the transgenic activity of snake HLB sequences suggest that limb enhancers can be repurposed in species
Developmental Cell 35, October 12, 2015 ª2015 Elsevier Inc. 3
Developmental Cell
Previews
whether regulatory elements in the GT may be evolving faster than limb enhancers. In sum, Infante et al. (2015) add to the growing evidence for dynamic regulatory activity in vertebrate evolution (Vierstra et al., 2014), where the frequency and mechanisms dictating repurposing of tissue-specific regulatory regions are an increasingly exciting area of investigation. REFERENCES Cotney, J., Leng, J., Yin, J., Reilly, S.K., DeMare, L.E., Emera, D., Ayoub, A.E., Rakic, P., and Noonan, J.P. (2013). Cell 154, 185–196.
Figure 1. Regulatory Commonalities between Developing Limbs and Genitalia Inspired by the sequence conservation of limb enhancers in snake genomes, Infante et al. asked whether regulatory activities in developing limbs and genitalia may have more in common than previously thought. Profiling of enhancer activity in limbs and genital tubercle (GT) from mice and lizards revealed shared regulatory elements in the two appendages (limb-GT enhancers). Analysis of a TBX4 enhancer sequence in snakes demonstrates that such limb-genital enhancers can be repurposed in limbless species by maintaining activity in developing genitalia. Mya, million years ago.
that have evolved limb loss, and future work should analyze the activity of these sequences in snakes (or other limbless vertebrates) to determine how often this repurposing occurs in the GT as opposed to other tissues. Moreover, subsequent studies exploiting the unique anatomy of squamates to understand genetic evolution would benefit from a strategy that includes Anolis (instead of mouse) as the primary reference species. First, snake developmental biology and subsequent anatomy is much more related to lizards than mammals (Tschopp et al., 2014).
Second, the evolutionary distances of mouse-Anolis-Serpentes (Figure 1) are approximately the same as mouseopossum-chicken, which would facilitate comparative functional genomics using commonly deployed tools and approaches (Cotney et al., 2013). These reasons are part of why comparing data from a query species to data from both in- and out-group species is a powerful and longutilized method in comparative genomics (Necsulea and Kaessmann, 2014). Carefully designed cross-species analyses of enhancer activity should also clarify
4 Developmental Cell 35, October 12, 2015 ª2015 Elsevier Inc.
Creyghton, M.P., Cheng, A.W., Welstead, G.G., Kooistra, T., Carey, B.W., Steine, E.J., Hanna, J., Lodato, M.A., Frampton, G.M., Sharp, P.A., et al. (2010). Proc. Natl. Acad. Sci. USA 107, 21931– 21936. Infante, C.R., Mihala, A.G., Park, S., Wang, J.S., Johnson, K.K., Lauderdale, J.D., and Menke, D.B. (2015). Dev. Cell 35, this issue, 107–119. Kondo, T., Za´ka´ny, J., Innis, J.W., and Duboule, D. (1997). Nature 390, 29. Lonfat, N., Montavon, T., Darbellay, F., Gitto, S., and Duboule, D. (2014). Science 346, 1004–1006. Mortlock, D.P., and Innis, J.W. (1997). Nat. Genet. 15, 179–180. Naiche, L.A., and Papaioannou, V.E. (2003). Development 130, 2681–2693. Necsulea, A., and Kaessmann, H. (2014). Nat. Rev. Genet. 15, 734–748. Tschopp, P., Sherratt, E., Sanger, T.J., Groner, A.C., Aspiras, A.C., Hu, J.K., Pourquie´, O., Gros, J., and Tabin, C.J. (2014). Nature 516, 391–394. Vierstra, J., Rynes, E., Sandstrom, R., Zhang, M., Canfield, T., Hansen, R.S., Stehling-Sun, S., Sabo, P.J., Byron, R., Humbert, R., et al. (2014). Science 346, 1007–1012.
Previews When the Snake Lost Its Limbs, What Did the Mouse and Lizard Say? Diego Villar1,* and Duncan T. Odom1,2,* 1University
of Cambridge, Cancer Research UK Cambridge Institute, Robinson Way, Cambridge CB2 0RE, UK Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK *Correspondence: [email protected] (D.V.), [email protected] (D.T.O.) http://dx.doi.org/10.1016/j.devcel.2015.09.022 2Wellcome
In this issue of Developmental Cell, Infante et al. (2015) compare regulatory DNA sequences in mice, lizards, and limbless snakes to reveal widespread sharing of enhancer activity in developing limbs and genitalia. Genetic deletion of a limb-genital enhancer demonstrates that common regulatory elements affect development of both appendages. The transition of vertebrates from an aquatic to a terrestrial environment required major adaptations in locomotion and reproduction and resulted in the evolution of limbs and external genitalia. Commonalities in the development of these anatomical structures include shared signaling pathways and gene expression similarities (Tschopp et al., 2014), suggesting an evolutionary link between the two appendages. In this issue of Developmental Cell, Infante et al. (2015) provide compelling evidence for shared cis-regulatory activities between developing limbs and genitalia. Many limb-specific enhancers have been identified in mammals (Cotney et al., 2013). Here, Infante et al. (2015) use limbless snakes to show that the sequences of many of these enhancers can be retained, even when the anatomical structures where they are active have vanished. These results further highlight the plasticity of amniote gene-regulatory activities and demonstrate that shared regulatory architectures can contribute to the development of limbs and external genitalia. Despite their somewhat disparate morphologies, outgrowth of both limbs and genitalia during embryonic development can depend on common genes, such as the posterior HOXD and HOXA clusters (Kondo et al., 1997), and mutations in these developmental regulators lead to disorders such as the human hand-foot-genital syndrome (Mortlock and Innis, 1997). More recently, it was revealed that these loci have similar regulatory topologies in developing limb buds and genital tubercle (GT) (Lonfat et al., 2014). The chromatin architecture involved promoter-enhancer interactions that were shared in the two
appendages, as well as some specific to either tissue. Further analysis of the shared interactions suggested that some limb enhancers could also function in developing genitalia. Moreover, a recent comparison of genital development in mice and squamates reported co-expression of many genes in limbs and genitalia and suggested ancestral commonalities in the developmental mechanisms leading to the two structures (Tschopp et al., 2014). Infante and colleagues (2015) set out to determine the extent to which genital and limb transcriptional networks may deploy common cis-regulatory elements (Figure 1). They reasoned that shared regulatory activity between limbs and genital enhancers could lead to retention of limb regulatory sequences in limbless species. Specifically, testing the sequence conservation of a set of mouse enhancers with known limb activity in squamate genomes showed that DNA sequences of mouse limb enhancers are often retained in snakes—in fact, sequence conservation of these enhancers was similar in the genomes of limbed and non-limbed squamates. This result prompted the authors to re-evaluate the tissue specificity of limb enhancers in developmentally matched forelimb, hindlimb, and GT of mice by profiling genomic locations where lysine 27 on histone H3 is acetylated (H3K27ac). This histone mark identifies active regulatory regions, such as promoters and enhancers (Creyghton et al., 2010). Previously validated limb enhancers provided a convincing set of candidate regions, where enrichment of H3K27ac was commonly shared in limb and GT samples but largely absent in those of non-appendage tissues.
The maps of enhancer activity in mouse limbs and GT show that almost half of either forelimb or hindlimb regulatory elements can be active in genitalia. By profiling the same tissues in Anolis lizard embryos, the authors further identified whether tissue specificity of enhancer activity is conserved in orthologous squamate regions; this analysis showed significantly lower conservation for enhancer activity restricted to developing genitalia. These results suggest a higher evolutionary turnover of GT-specific regulatory elements versus those active in limb or limb-GT. The authors continue in more detail by focusing on HLA and HLB, a pair of enhancers targeting the hindlimb developmental gene TBX4 (Naiche and Papaioannou, 2003), which is also expressed in the GT. The activity of both HLA and HLB is conserved in Anolis and mouse, but whereas HLA activity is restricted to the limb, HLB is active in both limbs and genitalia. Infante and colleagues (2015) employ transgene expression in mouse embryos to show that HLB snake sequences do not drive limb expression, but have retained partial activity in the GT. Consistent with this result, engineering of a HLB deletion in mice leads to defects in the development of both limbs and genitalia. Detailed functional analyses further demonstrate pleiotropic roles of this enhancer, including defects in nonappendage tissues such as the kidney. The analysis by Infante et al. (2015) raises a number of questions for follow-up investigation. The interesting observations on the transgenic activity of snake HLB sequences suggest that limb enhancers can be repurposed in species
Developmental Cell 35, October 12, 2015 ª2015 Elsevier Inc. 3
Developmental Cell
Previews
whether regulatory elements in the GT may be evolving faster than limb enhancers. In sum, Infante et al. (2015) add to the growing evidence for dynamic regulatory activity in vertebrate evolution (Vierstra et al., 2014), where the frequency and mechanisms dictating repurposing of tissue-specific regulatory regions are an increasingly exciting area of investigation. REFERENCES Cotney, J., Leng, J., Yin, J., Reilly, S.K., DeMare, L.E., Emera, D., Ayoub, A.E., Rakic, P., and Noonan, J.P. (2013). Cell 154, 185–196.
Figure 1. Regulatory Commonalities between Developing Limbs and Genitalia Inspired by the sequence conservation of limb enhancers in snake genomes, Infante et al. asked whether regulatory activities in developing limbs and genitalia may have more in common than previously thought. Profiling of enhancer activity in limbs and genital tubercle (GT) from mice and lizards revealed shared regulatory elements in the two appendages (limb-GT enhancers). Analysis of a TBX4 enhancer sequence in snakes demonstrates that such limb-genital enhancers can be repurposed in limbless species by maintaining activity in developing genitalia. Mya, million years ago.
that have evolved limb loss, and future work should analyze the activity of these sequences in snakes (or other limbless vertebrates) to determine how often this repurposing occurs in the GT as opposed to other tissues. Moreover, subsequent studies exploiting the unique anatomy of squamates to understand genetic evolution would benefit from a strategy that includes Anolis (instead of mouse) as the primary reference species. First, snake developmental biology and subsequent anatomy is much more related to lizards than mammals (Tschopp et al., 2014).
Second, the evolutionary distances of mouse-Anolis-Serpentes (Figure 1) are approximately the same as mouseopossum-chicken, which would facilitate comparative functional genomics using commonly deployed tools and approaches (Cotney et al., 2013). These reasons are part of why comparing data from a query species to data from both in- and out-group species is a powerful and longutilized method in comparative genomics (Necsulea and Kaessmann, 2014). Carefully designed cross-species analyses of enhancer activity should also clarify
4 Developmental Cell 35, October 12, 2015 ª2015 Elsevier Inc.
Creyghton, M.P., Cheng, A.W., Welstead, G.G., Kooistra, T., Carey, B.W., Steine, E.J., Hanna, J., Lodato, M.A., Frampton, G.M., Sharp, P.A., et al. (2010). Proc. Natl. Acad. Sci. USA 107, 21931– 21936. Infante, C.R., Mihala, A.G., Park, S., Wang, J.S., Johnson, K.K., Lauderdale, J.D., and Menke, D.B. (2015). Dev. Cell 35, this issue, 107–119. Kondo, T., Za´ka´ny, J., Innis, J.W., and Duboule, D. (1997). Nature 390, 29. Lonfat, N., Montavon, T., Darbellay, F., Gitto, S., and Duboule, D. (2014). Science 346, 1004–1006. Mortlock, D.P., and Innis, J.W. (1997). Nat. Genet. 15, 179–180. Naiche, L.A., and Papaioannou, V.E. (2003). Development 130, 2681–2693. Necsulea, A., and Kaessmann, H. (2014). Nat. Rev. Genet. 15, 734–748. Tschopp, P., Sherratt, E., Sanger, T.J., Groner, A.C., Aspiras, A.C., Hu, J.K., Pourquie´, O., Gros, J., and Tabin, C.J. (2014). Nature 516, 391–394. Vierstra, J., Rynes, E., Sandstrom, R., Zhang, M., Canfield, T., Hansen, R.S., Stehling-Sun, S., Sabo, P.J., Byron, R., Humbert, R., et al. (2014). Science 346, 1007–1012.