The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention1,2, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals3,4,5,6; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Shubin, N., Tabin, C. & Carroll, S. Fossils, genes and the evolution of animal limbs. Nature 388, 639–648 (1997)
Wagner, G. P. & Chiu, C. H. The tetrapod limb: a hypothesis on its origin. J. Exp. Zool. 291, 226–240 (2001)
Kondo, T., Zakany, J., Innis, J. W. & Duboule, D. Of fingers, toes and penises. Nature 390, 29 (1997)
Yamada, G. et al. Molecular genetic cascades for external genitalia formation: an emerging organogenesis program. Dev. Dyn. 235, 1738–1752 (2006)
Cohn, M. J. Development of the external genitalia: conserved and divergent mechanisms of appendage patterning. Dev. Dyn. 240, 1108–1115 (2011)
Lin, C. et al. Delineating a conserved genetic cassette promoting outgrowth of body appendages. PLoS Genet. 9, e1003231 (2013)
Greer, A. E. Limb reduction in squamates: identification of the lineages and discussion of the trends. J. Herpetol. 25, 166–173 (1991)
Cope, E. D. On the hemipenes of the Sauria. Proc. Acad. Nat. Sci. Philadelphia. 48, 461–467 (1896)
Haraguchi, R. et al. Unique functions of sonic hedgehog signaling during external genitalia development. Development 128, 4241–4250 (2001)
Perriton, C. L., Powles, N., Chiang, C., Maconochie, M. K. & Cohn, M. J. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Dev. Biol. 247, 26–46 (2002)
Gros, J. & Tabin, C. J. Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition. Science 343, 1253–1256 (2014)
Ohta, S., Suzuki, K., Tachibana, K., Tanaka, H. & Yamada, G. Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge. Development 134, 4315–4324 (2007)
Suzuki, K., Economides, A., Yanagita, M., Graf, D. & Yamada, G. New horizons at the caudal embryos: coordinated urogenital/reproductive organ formation by growth factor signaling. Curr. Opin. Genet. Dev. 19, 491–496 (2009)
Matsumaru, D. et al. Genetic analysis of the role of Alx4 in the coordination of lower body and external genitalia formation. Eur. J. Hum. Genet. 22, 350–357 (2014)
Naiche, L. A. Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development 130, 2681–2693 (2003)
Chapman, D. L., Garvey, N., Hancock, S. & Alexiou, M. Expression of the T-box family genes, Tbx1–Tbx5, during early mouse development. Dev. Dyn. 206, 379–390 (1996)
Raynaud, A. & Pieau, C. in Biology of the Reptilia, Development B (eds Gans, C. & Billett, F. S. ) Vol. 15 149–300 (John Wiley & Sons, 1985)
Arendt, D. Genes and homology in nervous system evolution: comparing gene functions, expression patterns, and cell type molecular fingerprints. Theory Biosci. 124, 185–197 (2005)
Wagner, G. P. The developmental genetics of homology. Nature Rev. Genet. 8, 473–479 (2007)
Shubin, N., Tabin, C. & Carroll, S. Deep homology and the origins of evolutionary novelty. Nature 457, 818–823 (2009)
Wang, Z., Young, R. L., Xue, H. & Wagner, G. P. Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds. Nature 477, 583–586 (2011)
Merkin, J., Russell, C., Chen, P. & Burge, C. B. Evolutionary dynamics of gene and isoform regulation in mammalian tissues. Science 338, 1593–1599 (2012)
Ashburner, M. et al. Gene ontology: tool for the unification of biology. Nature Genet. 25, 25–29 (2000)
Haraguchi, R. et al. Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development 127, 2471–2479 (2000)
Miyagawa, S. et al. Dosage-dependent hedgehog signals integrated with Wnt/β-catenin signaling regulate external genitalia formation as an appendicular program. Development 136, 3969–3978 (2009)
Suzuki, K. et al. Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia. PLoS ONE 7, e43453 (2012)
Larkins, C. E. & Cohn, M. J. Phallus development in the turtle Trachemys scripta. Sex Dev. http://dx.doi.org/10.1159/000363631 (2014)
Griffiths, M. in The Biology of the Monotremes (Academic, 1978)
De Barros, M. A. et al. Marsupial morphology of reproduction: South America opossum male model. Microsc. Res. Tech. 76, 388–397 (2013)
Jurberg, A. D., Aires, R., Varela-Lasheras, I., Novoa, A. & Mallo, M. Switching axial progenitors from producing trunk to tail tissues in vertebrate embryos. Dev. Cell 25, 451–462 (2013)
Herrera, A. M. & Cohn, M. J. Embryonic origin and compartmental organization of the external genitalia. Sci. Rep. 4, 6896 (2014)
Sanger, T. J., Losos, J. B. & Gibson-Brown, J. J. A developmental staging series for the lizard genus Anolis: a new system for the integration of evolution, development, and ecology. J. Morphol. 269, 129–137 (2008)
Gomez, C. et al. Control of segment number in vertebrate embryos. Nature 454, 335–339 (2008)
Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92 (1951)
Metscher, B. D. MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions. Dev. Dyn. 238, 632–640 (2009)
McGlinn, E. & Mansfield, J. H. Detection of gene expression in mouse embryos and tissue sections. Methods Mol. Biol. 770, 259–292 (2011)
Barde, I., Salmon, P. & Trono, D. Production and Titration of Lentiviral Vectors (John Wiley & Sons, 2001)
Punzo, C. & Cepko, C. L. Ultrasound-guided in utero injections allow studies of the development and function of the eye. Dev. Dyn. 237, 1034–1042 (2008)
Buchtová, M. et al. Initiation and patterning of the snake dentition are dependent on Sonic Hedgehog signaling. Dev. Biol. 319, 132–145 (2008)
Grant, G. R. et al. Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq Unified Mapper (RUM). Bioinformatics 27, 2518–2528 (2011)
Eckalbar, W. L. et al. Genome reannotation of the lizard Anolis carolinensis based on 14 adult and embryonic deep transcriptomes. BMC Genomics 14, 49 (2013)
Brawand, D. et al. The evolution of gene expression levels in mammalian organs. Nature 478, 343–348 (2011)
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)
Wagner, G. P., Koryu, K. & Lynch, V. J. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 131, 281–285 (2012)
Suzuki, R. & Shimodaira, H. Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22, 1540–1542 (2006)
Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010)
Chen, H. & Boutros, P. C. VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinformatics 12, 35 (2011)
McGrew, M. J. et al. Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity. Development 135, 2289–2299 (2008)
The authors thank D. Duboule, H. Kaessmann, A. Necsulea, B. Okaty, G. Rey and G. P. Wagner for discussions, M. A. de Bakker for the snake Tbx5 probe and A. M. Herrera and M. J. Cohn for discussing and sharing unpublished results. μCT scans were performed at the Center for Nanoscale Systems, Harvard University (supported by National Science Foundation award ECS-0335765) and at the Museum of Comparative Zoology. Next-generation sequencing was performed at the HMS Biopolymers Facility and computational analyses were run on the Orchestra Cluster, HMS Research Computing. P.T. was supported by post-doctoral fellowships from the Swiss National Science Foundation, EMBO and the Human Frontiers Science Program. A.C.G. was supported by a post-doctoral fellowship from the Swiss National Science Foundation. This work was supported by National Institutes of Health grant R37-HD032443 to C.J.T.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Two separable ventral cell populations give rise to the murine genital tubercle.
a, b, Injection into the most distal ventral part of the embryo, the tail bud, marks cells posterior/ventral to the phallic part of the urethra (a, arrow; n = 7), whereas injection closer to the allantois, into the infra-umbilical mesenchyme, labels cells anterior/dorsal to the phallic part of the urethra (b, arrow; n = 4). Cells lining the peritoneal cavity are also marked (arrowheads), owing to accidental piercing of the coelom. gt, genital tubercle; ur, urethra. Scale bars, 200 μm.
Extended Data Figure 2 The squamate hemipenis mesenchyme initiates with limb-like cellular dynamics from the coelomic epithelium through an EMT.
a, Injection of GFP-expressing lentiviruses into the coelom of chicken embryos at HH14 labels cells emerging from the epithelium that contribute to the hindlimb mesenchyme (arrowhead). b, In lizards, labelled cells leaving the coelomic epithelium contribute to the hemipenis mesenchyme (arrowheads). c, Dorsal view of the hindlimb region of an E10.0 mouse embryo. d, Transversal section of a limb bud, showing EMT of the coelomic epithelium (diffuse laminin staining, open arrowhead), as cells contribute to the limb-bud mesenchyme. e, Dorsal view of the budding hemipenis of a snake embryo, 1 day after egg deposition. f, Transversal section of the hemipenis region. The basement membrane of the coelomic epithelium is breaking down (open arrowhead), while it is intact for both the nephric duct and the surface ectoderm (arrowheads). g–o, Expression of genitalia and limb genes during hemipenis initation. g–i, Tbx4 is expressed early (h, arrow) and late during hemipenis initiation, in both the coelomic epithelium (i, arrowhead) and the hemipenis mesenchyme (i, arrow). j–l, Tbx5 is only expressed later, in the mesenchyme (l, arrow), but is absent from the coelomic epithelium (l, open arrowhead). m–o, Limb marker gene Lhx9 (see also Fig. 4e) is absent from both epithelium (o, open arrowhead) and mesenchyme (o, open arrow), but can be detected in dI1 neurons (o, asterisk). All gene expression was assessed in at least n = 3 samples. cl, cloaca; co, coelom; hp, hemipenis; lb, limb; nd, nephric duct. Scale bars, 50 μm.
Extended Data Figure 3 Heat maps of Pearson’s and Spearman’s rank correlation coefficients and cluster analysis of whole-transcriptome data.
a, b, Hierarchical clustering on pairwise correlation coefficients for whole-transcriptome data from anole (a) and mouse (b) samples. Numbers at nodes represent approximately unbiased P values obtained by multiscale bootstrap resampling. Sample identifiers: a, anole; m, mouse; GT, genital tubercle; HP, hemipenis; LB, limb; e, early; l, late.
Extended Data Figure 4 Heat maps of Pearson’s and Spearman’s rank correlation coefficients and cluster analysis of transcription factor and signalling pathway data.
a, b, Hierarchical clustering on pairwise correlation coefficients of transcription factor (TF) and signalling pathway data from anole (a) and mouse (b) samples. Numbers at nodes represent approximately unbiased P values obtained by multiscale bootstrap resampling. Sample identifiers: a, anole; m, mouse; GT, genital tubercle; HP, hemipenis; LB, limb; e, early; l, late.
Extended Data Figure 5 Heterotopic grafting of the cloacal signalling centre leads to ectopic outgrowths and genitalia-like transcriptional changes.
a–c, Schematics and close-up images of the cloacal grafting procedure. a, The cloaca of a stage HH17–19 GFP-transgenic chicken embryo (red rectangle) is transplanted into the proximal-ventral portion of the limb of a wild-type embryo. b, c, Only the ventral-most part of the cloaca, including the cloacal membrane, is dissected out (b, red box), and subsequently cleared of excess mesenchymal cells attached to the SHH-expressing endoderm (c, red outline). d–g, Grafting of beads soaked in SHH and FGF can induce ectopic outgrowths on both limbs (e; n = 6/48) and tail (g; n = 3/31). h–k, Ectopic expression of genital markers GATA2 (h, i, arrowheads) and RUNX1 (j, k, arrowhead) in limb buds, following cloaca-to-limb grafts. l–n, Ectopic expression of genital marker GATA2 (m, arrowheads) and RUNX1 (n, arrowheads) in the tail region, following cloaca-to-tail grafts. All gene expression was assessed in at least n = 3 samples. al, allantois; cl, cloaca; lb, limb. Scale bar, 200 μm.
Extended Data Figure 6 Pairwise differential expression analysis of limb and genitalia transcriptomes.
a–d, Smear plot visualization of differential expression analyses of early anole (a), late anole (b), early mouse (c) and late mouse (d) limb versus genitalia transcriptomes. Genes used for the Venn diagram in Fig. 4d (|log2(fold change)| > 1.5; P value < 0.05) are highlighted in red, core 25 marker genes (see Fig. 4e and text) are highlighted and labelled in blue. CPM, counts per million; FC, fold change. e, f, Heat map of Z-score-normalized expression values for all genes fulfilling Venn diagram criteria (n = 2,003), for anole (e) and mouse (f) data. Row-based hierarchical clustering was used; core 25 marker genes are indicated on the right.
a–r, Genitalia markers Isl1 (a–d), Runx1 (e, f), Gata2 (g–j), Eya4 (k, l), Tbx5 (m–p) and Dkk2 (q, r). Gata2 only becomes visibly expressed at the later stages of house snake hemipenis development (j, inset). s–a′, Limb markers Lhx9 (s–v), Tbx18 (w, x) and Lmx1b (y–a′). All gene expression was assessed in at least n = 3 samples. cl: cloaca; gt: genital tubercle; hp: hemipenis; lb: limb. Scale bar, 200 μm.
Extended Data Figure 8 Pairwise differential expression analysis of tail bud and genitalia transcriptomes.
a, b, Smear plot visualization of differential expression analyses of early anole (a) and early mouse (b) tail bud versus genitalia transcriptomes. Genes used as input for the Venn diagram in c (|log2(fold change)| > 1.5; P value < 0.05) are highlighted in red, overlapping 257 marker genes are highlighted in blue. Top 25 genes in the two species, based on logCPM (counts per million) and logFC (fold change), are labelled. c, Venn diagram showing overlap of pairwise differential expression analysis results (log(fold change) > 1.5, P value < 0.05) of tail bud versus genital tissues for early budding stages in both anole and mouse.
This file contains a list of orthologous genes between mouse and anole employed for GO-term analysis. Genes are ordered according to their absolute loading value from principal component analysis (see Fig. 3e), for principal component 2 (PC2), and top 500 genes (in bold, see Fig. 3f) were used for GO-term enrichment analysis. (PDF 3748 kb)
About this article
Cite this article
Tschopp, P., Sherratt, E., Sanger, T. et al. A relative shift in cloacal location repositions external genitalia in amniote evolution. Nature 516, 391–394 (2014). https://doi.org/10.1038/nature13819
International Journal of Molecular Sciences (2020)
Development of the chick wing and leg neuromuscular systems and their plasticity in response to changes in digit numbers
Developmental Biology (2020)
Developmental Biology (2019)
Trends in Genetics (2019)