Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK. e-mail: c.a.tickle@dundee.ac.uk doi:10.1038/nrm1830 Published online 21 December 2005 Mesenchyme A loose meshwork of cells found in vertebrate embryos, which is usually derived from the mesoderm, the middle of the three germ layers. Ectoderm The epithelium that is derived from the outer of the three germ layers of the embryo and will give rise to the epidermis of the skin. Positional information The instructions that are interpreted by cells to determine their differentiation with respect to their position within the embryo. Morphogen A diffusible chemical substance that carries information in embryos, for example, cell position. Making digit patterns in the vertebrate limb Cheryll Tickle Abstract | The vertebrate limb has been a premier model for studying pattern formation ��� a striking digit pattern is formed in human hands, with a thumb forming at one edge and a little finger at the other. Classic embryological studies in different model organisms combined with new sophisticated techniques that integrate gene-expression patterns and cell behaviour have begun to shed light on the mechanisms that control digit patterning, and stimulate re-evaluation of the current models. A fundamental biological question is how the body plan is laid down during embryonic development and how pre- cise arrangements of specialized cells and tissues arise. The vertebrate limb has a complex anatomy and is an excel- lent model in which to address this question. Vertebrate limbs develop from small buds of apparently homogene- ous unspecialized mesenchyme cells that are encased in the ectoderm. As the buds grow out from the body wall, these unspecialized cells begin to differentiate into various tissues of the limb ��� including the cartilage and, later in development, the bone ��� that make up the skeleton. The limb skeleton consists of a defined number of bones of characteristic size and shape that are arranged in a specific pattern. The anatomy of the limb can be described with respect to three orthogonal axes, proximo���distal, from shoulder to finger tips, antero���posterior, from thumb to little finger, and dorso���ventral, from the back of the hand to the palm (FIG. 1). The processes that regulate limb forma- tion are highly coordinated for example, each digit forms in a specific place. But how is this pattern of cell differentia- tion controlled? For the last 40 years or so, this problem and, in partic- ular, how digit pattern arises, has been tackled by apply- ing the concepts of positional information1,2. According to these concepts, cells are first informed of their position in the limb bud and, as a result, they acquire a positional value that encodes this information. In a second step, these values are interpreted, leading to the formation of the appropriate structure at that position. Positional information across the antero���posterior limb axis is pro- vided by signalling of the polarizing region at the poste- rior margin of the early limb bud. Classical experiments on chick embryos3,4 (BOX 1) led to the identification of this region and the proposal that the polarizing region produces a morphogen which diffuses, over time, into adjacent limb tissue to give a concentration gradient. Cells at different positions across the limb bud would be exposed to different concentrations of the morphogen ��� cells nearest the polarizing region, at the posterior of the limb, would be exposed to high concentrations of the morphogen, whereas cells further away, at the anterior of the limb bud, would be exposed to low concentrations. Therefore, the local concentration of the morphogen could provide information about position across the antero���posterior axis. These experiments also indicated that a ratchet-type mechanism is in operation, such that the anterior positional values can be promoted irrevers- ibly to more posterior positional values, and the most posterior positional values are then remembered. In the last 15 years, the molecular basis of limb devel- opment has begun to be unravelled through the identifi- cation of the signals that are generated in the polarizing region and the discovery of molecules that are produced in response to these signals. These molecular advances come mainly from genetic studies in mice (Mus muscu- lus), although many originated in fruitflies (Drosophila melanogaster). Recent sophisticated analyses in mouse embryos, which link gene expression and cell fate in developing limbs, have stimulated the re-evaluation of the mechanisms of digit patterning and have high- lighted the particular problem of providing positional information in a growing organ. In this article, I begin by outlining the embryological studies that revealed the signalling properties of the polarizing region and then discuss the molecular basis of polarizing signalling in the developing limb bud, with particular emphasis on the secreted molecule, sonic hedgehog (SHH). I conclude by discussing the outstanding challenges in identifying how antero���posterior values are encoded molecularly and how they are ultimately translated into digit anatomy. 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Anterior Anterior Posterior Posterior Proximal Proximal Distal Distal Dorsal Ventral Tbx family Related transcription factors that contain a T-Box. Apical ectodermal ridge Thickening of the ectoderm rim at the tip of a developing limb bud in a vertebrate embryo. It is required for bud outgrowth. Polarizing activity The ability of cells, tissue or defined chemicals to induce the formation of extra digits from the anterior region of a chick limb bud. Fate map A diagram that is obtained experimentally by tracing marked cells and shows the structures that derive from cells in different regions of an embryo. Digital plate Broad region that forms late during limb development at the distal end of the bud and contains the digit primordia. Embryology of digit patterning Digits form at relatively late stages of vertebrate limb development. By this time, the small bud has grown substantially and changed shape so that the main regions of the future limb can be made out (BOX 2). However, experiments on chick embryos have shown that cells are specified to form limbs, long before any buds are visible, and that both the antero���posterior and the dorso���ventral polarity of the limb are already estab- lished (reviewed in REF. 5). Fibroblast growth factors (FGFs) and Wnt-signalling molecules are involved in limb initiation and budding and control the limb- specific expression of genes that encode members of the Tbx family of transcription factors6. An early event in bud formation is the development of the apical ectodermal ridge. The apical ridge forms at a compartment boundary between the dorsal ectoderm and the ventral ectoderm ��� this ensures that the limb buds form at the sides of the body7. Continued out- growth of the bud depends on FGF signalling by the apical ridge8. Another important event in limb-bud development is the formation of the polarizing region that controls the antero���posterior pattern of distal structures. Once equipped with an apical ectodermal ridge and a polarizing region, the limb bud can develop autonomously. Insights from chick embryos. The normal chick wing has three digits known as 2, 3 and 4 (BOX 2), and it has been proposed that digit patterning involves signalling between the cells of the polarizing region and the adja- cent limb-bud cells3. Grafting the polarizing region from one chick wing bud to the anterior (opposite) side of a second bud results in a dramatic change in digit pattern six digits develop instead of three, with the extra set of digits in mirror-image symmetry with the normal set, giving rise to the pattern 4 3 2 2 3 4 (BOX 1). Grafts of the posterior margin of mammalian limb buds to chick wing buds have also been shown to have polarizing activity (for example, see REF. 9). The extra digits induced in this case are, nevertheless, chick digits, which indicates that, although the signal is the same, the interpretation differs. But how does the polarizing region produce such a pattern? The results of many chick embryological experiments are consistent with the idea that the polar- izing region produces a long-range morphogen that specifies antero���posterior positional values. Polarizing activity can be detected in the posterior region of the chick wing bud from early stages until the stage that the digits start to form10. Long-range signalling by the polarizing region operates over a few hundred ��m (about 10���30 cells BOX 1). Therefore, it is likely that positional values are specified in the early limb bud. Indeed fate maps show that digits arise from the poste- rior region of the early limb bud, which then expands to fill the digital plate11,12. Fate maps do not provide information about commitment, but, because there is experimental evidence for positional memory (BOX 1), the prevailing model has been that polarizing-region signalling sets up a morphogen gradient, which speci- fies antero���posterior positional values in the early bud (FIG. 2a). This set of initially tightly packed positional values then becomes distributed across the limb bud as the bud grows, and later dictates the development of each digit primordium. Polarizing-region grafts change digit number in addi- tion to pattern. One of the consequences of grafting a polarizing region to the anterior margin of the wing bud is an increase in the width of the bud to accommodate the extra digits4. This is accompanied by an increase in length of the apical ectodermal ridge. It was postulated a long time ago that the polarizing region regulates production of an apical-ridge maintenance factor by the mesenchyme cells in the posterior region of the bud13. It has also been suggested that signalling through the polarizing region might have a direct effect on cell prolif- eration14, because changes in cell proliferation have been detected prior to changes in ridge length. Morphogen gradient mechanism The vitamin-A derivative, retinoic acid, was the first defined signalling molecule to be identified as having the ability to induce mirror-image duplications in the chick wing15 (BOX 1). Although it was shown that retinoic acid is readily diffusible and functions in a concentra- tion-dependent fashion15, the main role of endogenous retinoic acid in polarizing signalling is now thought to be the induction of the expression of the SHH gene16. It should be noted that retinoic acid also has other roles in limb-bud initiation17 and in patterning of the proximal part of the limb18. Identification of candidate morphogens. SHH is expressed in the polarizing region of both the chick16 and the mouse limb buds, and immunohistochemical studies19 and a biological assay ��� based on the ability of SHH to induce differentiation in a cell line20 ��� indicate that the SHH protein diffuses some distance away into the limb bud. Beads soaked in SHH protein cause con- centration-dependent changes in digit pattern and the Figure 1 | The three main axes of the human hand. The diagram shows the three main axes, proximo���distal, antero���posterior and dorso���ventral, of a human hand. Tightly regulated processes during embryonic development ensure that the thumb arises at one edge of the hand, whereas the little finger arises at the other. REVIEWS 46 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/molcellbio