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Patterning and post-patterning modes of evolutionary digit loss in mammals

Nature volume 511, pages 41–45 (2014)Cite this article

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Abstract

A reduction in the number of digits has evolved many times in tetrapods, particularly in cursorial mammals that travel over deserts and plains, yet the underlying developmental mechanisms have remained elusive. Here we show that digit loss can occur both during early limb patterning and at later post-patterning stages of chondrogenesis. In the ‘odd-toed’ jerboa (Dipus sagitta) and horse and the ‘even-toed’ camel, extensive cell death sculpts the tissue around the remaining toes. In contrast, digit loss in the pig is orchestrated by earlier limb patterning mechanisms including downregulation of Ptch1 expression but no increase in cell death. Together these data demonstrate remarkable plasticity in the mechanisms of vertebrate limb evolution and shed light on the complexity of morphological convergence, particularly within the artiodactyl lineage.

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Figure 1: Convergent evolution of the embryonic limb skeleton in multiple mammal species.
Figure 2: Expression of early patterning genes: Shh, Ptch1, Gli1 and HoxD13.
Figure 3: Patterns of cell death in each mammalian limb.
Figure 4: Expression of Msx2 at the start of digit chondrogenesis.
Figure 5: Fgf8 expression is restricted to the AER overlying nascent digits.

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Accession codes

Data deposits

The probe sequence data for all genes and species has been deposited in the NCBI Probes database with the following accession numbers: CAMELBMP4, Pr032067180; CAMELFGF8, Pr032067181; CAMELGLI1, Pr032067182; CAMELHOXD13, Pr032067183; CAMELPTCH1, Pr032067184; HORSEBMP4, Pr032067185; HORSEFGF8, Pr032067186; HORSEGLI1, Pr032067187; HORSEHOXD13, Pr032067188; HORSEMSX2, Pr032067189; HORSEPTCH1, Pr032067190; HORSESHH, Pr032067191; JERBOABMP4, Pr032067192; JERBOAFGF8, Pr032067193; JERBOAGLI1, Pr032067194; JERBOAHOXD13, Pr032067195; JERBOAMSX2, Pr032067196; JERBOAPTCH1, Pr032067197; JERBOASHH, Pr032067198; MOUSEBMP4, Pr032067199; MOUSEFGF8, Pr032067200; MOUSEGLI1, Pr032067201; MOUSEHOXD13, Pr032067202; MOUSEMSX2, Pr032067203; MOUSEPTCH1, Pr032067204; MOUSESHH, Pr032067205; PIGFGF8, Pr032067206; PIGPTCH1, Pr032067207; PIGSHH, Pr032067208.

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Acknowledgements

We thank J. Lopez-Rios and R. Zeller (University of Basel, Switzerland) for generously providing data and discussion before publication. We also thank J. Carlos Izpisua Belmonte and A. Aguirre for sharing space and materials to complete experiments subsequent to review. Jerboa embryos were harvested with the assistance of S. Wu and colleagues in Xinjiang, China. Pig embryos were harvested with the assistance of D. Urban. Additional horse embryos were provided by R. Turner and H. Galatino-Homer (University of Pennsylvania) and by R. Fritsche and S. Lyle (Louisiana State University). Mouse Gli1 probe plasmid, used in the pig in situ, was provided by A. Joyner. This work was supported by NIH grant R37HD032443 to C.J.T., and NSF IOS grant 1257873 to K.E.S.

Author information

Author notes

  1. Kimberly L. Cooper

    Present address: Present address: Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, USA.,

  2. Kimberly L. Cooper, Karen E. Sears and Aysu Uygur: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Kimberly L. Cooper, Aysu Uygur & Clifford J. Tabin

  2. Department of Animal Biology, University of Illinois Urbana-Champaign, Urbana, 61801, Illinois, USA

    Karen E. Sears & Jennifer Maier

  3. École Normale Supérieure de Lyon, 69007 Lyon, France,

    Karl-Stephan Baczkowski

  4. Department of Molecular Biology and Genetics, Cornell University, Ithaca, 14853, New York, USA

    Margaret Brosnahan & Doug Antczak

  5. The Camel Reproduction Centre, Dubai, United Arab Emirates

    Julian A. Skidmore

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Contributions

K.L.C., K.E.S. and C.J.T. conceived of and initiated the project. K.L.C. and C.J.T. wrote the manuscript. K.L.C performed the mouse, three- and five-toed jerboa, horse and camel in situ hybridizations, PH3 IHC, and skeletal stains. A.U. performed TUNEL and Sox9 IHC. J.M. and K.E.S. performed the pig in situ hybridizations. K.-S.B. cloned the pig probes. M.B. and D.A. provided most of the horse embryos and material for cloning the horse probes. J.A.S. provided the camel embryos and material for cloning the camel probes.

Corresponding authors

Correspondence to Kimberly L. Cooper or Karen E. Sears.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The proximal remnants of truncated skeletal elements in D. sagitta are correctly patterned.

Alcian blue and alizarin red stained skeletons of postnatal day 0 mouse (left, n = 4 animals) and three-toed jerboa, D. sagitta (right, n = 4 animals) with proximal (ankle) at the top. a, Anterior view highlights the first metatarsal (arrowhead). b, Posterior view highlights the fifth metatarsal (arrow). c, Dissected first tarsal-metatarsal elements demonstrate the morphology of the truncated first metatarsal of D. sagitta (right, arrowhead) compared to mouse (left). Joint interzone indicated by white dashed line. Scale bars, 0.5 mm.

Extended Data Figure 2 The shape of the three-toed jerboa hindlimb differs from the mouse as early as 11.5 dpc.

Trace outlines of limb buds of the mouse (black) and three-toed jerboa, D. sagitta (green) over a developmental time series. Outlines are of embryos most representative of 3–4 individuals per stage.

Extended Data Figure 3 Proliferation is unchanged in the D. sagitta hindlimb bud.

Phospho-histone H3 detection in sections of mouse and three-toed jerboa, D. sagitta, limb buds. a, Forelimbs. b, Hindlimbs. n = 2 embryos per stage and species.

Extended Data Figure 4 Developmental time course and species comparisons of Bmp4 expression.

a, b, Forelimb buds (FL) (a) and hindlimb buds (HL) (b) of mouse and the three-toed jerboa, Dipus sagitta, at 10.5, 11, 11.5, 12 and 12.5 dpc (n = 2 embryos per stage). c, Forelimb and hindlimb of the five-toed jerboa, A. elater, at approximately 12.25 dpc (n = 1 embryo). d, Forelimb and hindlimb of the horse at 30 dpc (approximately equivalent to mouse 12 dpc) (n = 2 embryos). e, Forelimb and hindlimb of the camel at 38 dpc (approximately equivalent to mouse 12.5 dpc) (n = 1 embryo). Scale bar, 1 mm for D. sagitta, A. elater, horse and camel. Scale bar, 0.8 mm for mouse limbs.

Extended Data Figure 5 Developmental time course and species comparisons of Msx2 expression.

a, b, Forelimb buds (FL) (a) and hindlimb buds (HL) (b) of mouse and the three-toed jerboa, D. sagitta, at 10.5, 11, 11.5, 12 and 12.5 dpc (n = 2 embryos per stage). c, d, Msx2 expression in the mouse (c) and D. sagitta (d) embryo at 10.5 dpc. e, Forelimb and hindlimb of the five-toed jerboa, A. elater, at approximately 12.25 dpc (n = 1 embryo). f, Forelimb and hindlimb of the horse at 30 dpc (approximately equivalent to mouse 12 dpc) (n = 2 embryos). g, Forelimb and hindlimb of the camel at 38 dpc (approximately equivalent to mouse 12.5 dpc) (n = 1 embryo). Scale bar, 1 mm for D. sagitta, A. elater, horse and camel. Scale bar, 0.8 mm for mouse limbs.

Extended Data Figure 6 Developmental time course of Fgf8 expression and early TUNEL in the jerboa hindlimb.

a, Time series of Fgf8 expression in the mouse and three-toed jerboa, D. sagitta, hindlimb (n = 2 embryos per stage). b, Fgf8 expression in the pig (25 dpc) and camel (42 dpc) hindlimbs of embryos in Fig. 5. TUNEL labelling of cell death in the 12.5 dpc D. sagitta hindlimb (n = 2 embryos). Limbs in b are aligned with the closest stage matched embryos in a.

Extended Data Table 1 Whole mount in situ hybridization probe information
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Cooper, K., Sears, K., Uygur, A. et al. Patterning and post-patterning modes of evolutionary digit loss in mammals. Nature 511, 41–45 (2014). https://doi.org/10.1038/nature13496

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