INTRODUCTION The specification of the digits in the vertebrate limb is an excellent example of patterning in vertebrate embryos. The chick wing has three digits, designated 2, 3 and 4. These arise at different positions across the anteroposterior axis (digit 2 being most anterior and digit 4 most posterior) and are morphologically distinct. Anteroposterior digit pattern is controlled by the polarising region, a small region of mesenchyme cells at the posterior margin of the limb bud (Saunders and Gasseling, 1968). When the polarising region is grafted from one wing bud to the anterior margin of a second wing bud, a pattern of duplicated digits results, with an additional 432 in mirror-image symmetry with the normal set of digits 234 (Saunders and Gasseling, 1968; Tickle et al., 1975). A gradient model has been proposed to account for signalling by the polarising region (Tickle et al., 1975). According to this model, the polarising region produces a diffusible morphogen that sets up a concentration gradient across the limb bud. Thus cells at different positions across the limb bud will be exposed to different concentrations of morphogen and can read the local morphogen concentration to find their position. Cells exposed to high concentrations of morphogen will form digit 4, while cells exposed to a low concentration will form digit 2. The essential features of this model are that the morphogen acts in a dose-dependent manner and is long range (Tickle et al., 1975). A number of signalling molecules have been found to be associated with the polarising region. Retinoic acid was the first defined chemical to be discovered that could mimic the activity of the polarising region (Tickle et al., 1982, 1985). Retinoic acid is now known to induce expression of a gene encoding another signalling molecule, Sonic Hedgehog (Shh) (Riddle et al., 1993) and Shh can reproduce the effects of retinoic acid on digit patterning (Riddle et al., 1993; Lopez- Martinez et al., 1995; Yang et al., 1997). Genes encoding Bone Morphogenetic Proteins (Bmps) have also shown to be expressed in the polarising region (Francis et al., 1994) and their expression is induced by Shh (Laufer et al., 1994; Yang et al., 1997), but Bmps cannot reproduce the effects of Shh on digit patterning (Duprez et al., 1996). Thus Shh appears to be a good candidate for the polarising morphogen that specifies digit identity. 1337 Development 127, 1337-1348 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 DEV2527 It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity. Key words: Shh, Bmp, Limb development, Patched, Signalling, Chick SUMMARY A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling G. Drossopoulou 1,2 , K. E. Lewis 3 , J. J. Sanz-Ezquerro 1,2 , N. Nikbakht 1 , A. P. McMahon 4 , C. Hofmann 5 and C. Tickle 1,2 1 Department of Anatomy and Physiology, The Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK 2 Department of Anatomy and Developmental Biology, University College London, WC1E 6BT, UK 3 Developmental Genetics Programme, Krebs Institute, University of Sheffield, Sheffield, S10 2TN, UK 4 Department of Molecular and Cellular Biology, The Biolabs, Harvard University, Cambridge Massachusetts, USA 5 GSF-Forschungszentrum fur Umwelt und Gesundheit, Institut fur Saugetiergenetik, Neuherberg, Germany Accepted 17 January; published on WWW 7 March 2000