The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.
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European Nucleotide Archive
Gene Expression Omnibus
All 4C-seq data sets are deposited in the Gene Expression Omnibus repository under accession number GSE52988. The GLI3 ChIP-seq data relevant to this study are deposited in the Gene Expression Omnibus repository under accession number GSE52939. All the raw data for sequencing of the Ptch1 locus are deposited in the European Nucleotide Archive (study accession number PRJEB5056).
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We thank K. Cooper and C. Tabin for sharing their results before publication and providing camel genomic DNA. Dolphin samples were received from C. Frere and D. Duffield. Pigmy hippopotamus mouth swabs for DNA extraction were provided by S. Furrer and B. Zimmerman from the Zoo of Zurich, whereas all other artiodactyl blood samples were donated from available frozen stocks by S. and M. Hoby from the Zoo of Basel. B. Wainwright provided the Ptch1 conditional mouse line. We are grateful to E. Terszowska and A. Offinger for mouse care; V. Metis and M. Rondon for assistance in colony maintenance and collection of Prx1-Cre Ptch1Δc/Δc mouse embryos; and N. Dumesnil for assistance in collecting bovine embryos. All lentivector-mediated transgenic mouse embryos were produced by the EPFL transgenic platform, whereas conventional mouse transgenic embryos were produced commercially by Cyagen Biosciences Inc. We thank S. Beck-Cormier, A. Gritli-Linde, D. Haag-Wackernagel, Y. Lallemand, P. Zimmermann, G. Nusspaumer and A. Zuniga for technical advice and input and M. Bertaud, I. Ginez, D. Jardet and M. Moroldo for technical assistance. We are grateful to V. Taylor and members of our research groups for critical discussions and input on the manuscript. This research was supported by SNF grants 31003A_130803/146248 and the University of Basel (to R.Z.), EU reintegration grant PERG-GA-2009-246576 (to J.L.-R.), SystemsX.ch iPhD Grant 20101078 (to D.I. and R.Z.), INRA (to A.D.), ANR grant No. 06-MRAR-027-01 (to B.R.), the EPFL and ERC grant SystemsHox.ch (to D.D.), NIH grant no. NS 033642 (to A.P.M.) and Australian National Health and Medical Research Council grant no. 569713 (to C.W.).
The authors declare no competing financial interests.
Extended data figures and tables
a, Table listing the developmentally equivalent bovine fore- and hindlimb bud stages in comparison to mouse forelimb buds. CRL, approximate crown-rump length as previously defined49. b, False-coloured CT scan of a bovine fetus (at 5 months of gestation) to detect the ossified skeletal bones. Left limbs were coloured blue and right limbs orange. Radius and ulna form as two distinct skeletal elements, while the metacarpals/metatarsals fuse during ossification to give rise to the cannon bone, a convergent trait in many artiodactyls2. Lower panels illustrate the similarities of the forelimb (FL) and hindlimb (HL) autopod skeletal bones. Arrowheads indicate the plane of fusion of the metacarpal/metatarsal bones. Two independent fetuses were analysed. s, scapula; h, humerus; r, radius; u, ulna; c*, region corresponding to the not yet ossified carpus; cb, cannon bone; p, phalanges. III, IV, digits.
Extended Data Figure 2 Apoptosis in bovine forelimb buds during digit formation and gene expression during the onset of limb bud development.
a, The SOX9 transcriptional regulator (red immunofluorescence) marks all chondrogenic progenitors of the forming digit primordia in the bovine forelimb autopod. Apoptotic cells (TUNEL; green fluorescence) are only detected in the interdigit region that is being eliminated at these developmental stages (arrowhead in top panel). No apoptosis is detected in the digit-forming territories, except in the forming joints (arrowhead in bottom panel). Sections were counterstained with Hoechst-33258 to label cell nuclei. b, During initiation of limb bud outgrowth, the complementary expression of Gli3 and Hand2 marks the establishment of anterior–posterior asymmetry and is required to activate Shh expression. Mesenchymal cells responding to SHH signalling upregulate the expression of the SHH receptor Ptch1. Msx2 is a transcriptional sensor of BMP activity and its distribution marks territories of active BMP signalling. The expression of these genes is comparable in early mouse and bovine forelimb buds. In all Extended Data figures, limb buds are oriented with anterior to the top. Per marker and species, the distribution was reproduced in at least n = 3 independent limb bud samples in independent experiments. Scale bar, 0.25 mm.
Extended Data Figure 3 Validation of the whole-mount in situ hybridization probes and techniques in bovine embryos.
a, b, OPT analysis of Hoxd13 transcripts in the mesenchyme of mouse (a) and bovine (b) forelimbs. White arrowheads indicate the planes of the virtual OPT sections shown in the right panels. This analysis establishes that mesenchymal transcripts are equally well detected in mouse and bovine limb buds. Note the distal shift and more symmetric expression of Hoxd13 transcripts in bovine limb buds. c, Bovine and mouse embryos hybridized with the riboprobes for Shh, Ptch1, Gli1 and Hoxd13. No apparent changes in their expression in tissues other than limb buds were detected by comparing bovine and mouse embryos. Per gene and species, the transcript distribution was reproduced in at least n = 3 independent samples in independent experiments. Scale bar, 0.25 mm.
a, OPT analysis of Ptch1 expression in mouse and bovine limb buds. White arrowheads indicate the levels of optical sections shown in the middle panels. Right panels show enlargements of the posterior part. b, Ptch2 expression in mouse and bovine limb buds. White arrowheads indicate the levels of optical sections shown in the right panels. Per gene and species, the transcript distribution was reproduced in at least n = 3 independent limb bud samples in independent experiments. Scale bars, 0.25 mm.
Extended Data Figure 5 Mathematical simulation of the mesenchymal response to SHH signal transduction with intact (mouse) and absent (bovine) Ptch1 upregulation in limb buds.
a, Simulations in wild-type mouse limb buds using the following minimal regulatory network (P. Germann and D. Iber, unpublished data). In the absence of Shh, Ptch1 promotes the formation of Gli repressor (predominantly Gli3R, indicated in blue) and antagonizes (in red) the formation of Gli activators (Gli3A, but also representing contributions by Gli2). Ptch1 is also genetically required for Gli3 expression in limb buds. Binding of Shh to Ptch1 (PS) relieves the tonic inhibition of Smo by Ptch1 (not shown), which promotes activator and inhibits repressor formation. Both Gli3A and Gli3R isoforms impact on Ptch1 and Gli1 expression, the expression kinetics of which were simulated (middle and right panels). For details of network construction and all references see Supplementary Note. In wild-type mouse limb buds, Ptch1 and Gli1 are upregulated in the posterior mesenchyme. b, Simulation of Ptch1 and Gli1 expression in bovine limb buds. The impact of Gli3A and Gli3R on Ptch1 transcription was removed from the network and the rate of Ptch1 production fixed at constant low levels. Simulations using this altered network result in progressive anterior expansion of Gli1 expression. c, Simulations of hypothetical bovine limb buds with an intact network results in posterior upregulation of both Ptch1 and Gli1 expression. d, Increasing Shh diffusion rates by tenfold (green dot in scheme) in an otherwise intact network leads to upregulation and anterior expansion of both Ptch1 and Gli1 expression, that is, fails to reproduce the real gene expression patterns. Broken lines in all panels indicate the simulated Ptch1 and Gli1 transcriptional activities at the indicated time points and the grey shaded area bordered by a solid line corresponds to the overall transcript levels. Red dotted line marks the position of peak expression. Gene symbols correspond to the computational variables used for simulations. A, anterior; P, posterior; h, hours; d, days. For detailed description of all simulations see Supplementary Note.
Extended Data Figure 6 Functional analysis of the conserved mouse and bovine core regions A and B using lentiviral transgenesis.
a, A variable and artiodactyl-specific microsatellite expansion (CA/CG2-27, red shading) in region A is located just upstream of exon 17 (Fig. 5a). b, Expression of a lacZ reporter inserted into the Ptch1 locus. c, d, Expression of lentivector ßlacZ reporter constructs controlled by the mouse and bovine conserved regions A and B (Fig. 5a) in transgenic mouse embryos (mouse: n = 12 of 41; bovine: n = 12 of 20 embryos). FL, forelimb. Scale bar, 0.25 mm.
a, Expression of the mouse and bovine LRM in mouse hindlimb buds (E11.5). b, Transgenic mouse embryos collected at different stages reveal the temporal kinetics of activation and upregulation of the mouse LRM-ßlacZ transgenic reporter in forelimb buds (n = 3 independent embryos per stage). c, Forelimb buds of n = 6 independent transgenic embryos expressing the lacZ reporter under control of the bovine LRM region. The variable expression is in all cases restricted to the core mesenchyme (compare to Fig. 5d). Scale bar, 0.25 mm.
Extended Data Figure 8 Prx1-Cre-mediated conditional inactivation of Ptch1 in the mouse limb mesenchyme phenocopies the loss of asymmetry and alterations in digit primordia similar to bovine handplates.
a, b, Detection of Hoxd13 (a) and Sox9 (b) transcripts in mouse (left), bovine (middle) and Ptch1Δc/Δc (right panels) forelimb buds. Per gene, species and mouse genotype, the transcript distribution was reproduced in at least n = 3 independent limb bud samples in independent experiments. Vestigial digit primordia are indicated in red. Asterisks indicate digit primordia with uncertain identities. Scale bars, 0.25 mm.
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Lopez-Rios, J., Duchesne, A., Speziale, D. et al. Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature 511, 46–51 (2014). https://doi.org/10.1038/nature13289
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