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WIREs Dev Biol
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The Sonic hedgehog gradient in the developing limb

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Abstract A gradient of Sonic hedgehog (Shh) plays a major role in specifying the antero‐posterior pattern of structures that develop in the distal part of the vertebrate limb, in particular, the antero‐posterior pattern of the digits. Classical embryological experiments identified the polarizing region (or zone of polarizing activity, ZPA), a signaling region at the posterior margin of the early chick wing bud and, consistent with a model in which production of a diffusible morphogen specifies antero‐posterior positional information, polarizing region signaling was shown to be dose dependent and long range. It is now well established that the vertebrate hedgehog gene, Sonic hedgehog (Shh), which encodes a secreted protein, is expressed in the polarizing region of the chick wing and that Shh signaling has the same characteristics as polarizing region signaling. Shh expression at the posterior of the early limb bud and the mechanism of Shh signal transduction are conserved among vertebrates including mammals. However, it is unlikely that a simple Shh gradient is responsible for digit pattern formation in mammalian limbs and there is still little understanding of how positional information specified by Shh signaling is encoded and translated into digit anatomy. Alterations in Shh signaling underlie some congenital limb abnormalities and also changes in timing and extent of Shh signaling appear to be related to the evolution of morphological diversity of vertebrate limbs. WIREs Dev Biol 2013, 2:275–290. doi: 10.1002/wdev.70 This article is categorized under: Establishment of Spatial and Temporal Patterns > Gradients Vertebrate Organogenesis > Musculoskeletal and Vascular

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Diagram showing promotion of digit identity over time in chick wing development. Sonic hedgehog (Shh) diffusing from polarizing region first specifies cells next to the polarizing region to form a digit 2, then as the concentration of Shh to which these cells are exposed increases over time, the threshold level to specify a digit 3 is exceeded and these cells are now promoted to form a digit 3; at the same time cells further away from the polarizing region are now exposed to low levels of Shh and are specified as digit 2. At a longer time point, there is further promotion of the identity of the cells near the polarizing region to form a digit 4, cells that were specified to form digit 2 are now promoted to form digit 3 and cells still further away from the polarizing region are specified to form digit 2. Experiments in which cyclopamine was added to chick wing buds to inhibit Shh signaling suggest that this process is completed in 12 h. Digits color coded to represent the Shh concentration.

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Sonic hedgehog (Shh) expression in chick embryos and comparison of distribution of Shh transcripts and Shh protein in chick wing buds, together with Ptch1 and Gas1 transcripts. (a) Image of a chick embryo (Hamilton–Hamburger stage 23) following whole in situ hybridization protocol to highlight localization of Shh transcripts in the limb buds. Shh transcripts at posterior margin of wing buds and right leg bud indicated by red lines (left leg bud not in view). Image by Hannah Brunsdon. (b–e) Diagrams comparing distribution of Shh transcripts (b) Shh protein (c) Ptch1 transcripts (d) and Gas1 transcripts (e) in early chick wing bud (Hamilton–Hamburger stage 20).

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Discovery of the polarizing region in the chick wing bud and morphogen gradient model for specifying digit pattern. (a) Diagram of the early chick wing bud showing the polarizing region (purple dots) at the posterior margin and the apical ectodermal ridge rimming the distal tip. Red arrows indicate reciprocal interactions that sustain signaling by both the polarizing region and the apical ectodermal ridge. Axes indicated at top right, Pr, proximal; Po, posterior; A, anterior; D, distal. (b) Images of whole mounts of skeletons of the wings of a 10‐day chick embryo stained with alcian green; left wing (control; upper skeleton) and right wing that received polarizing graft (lower skeleton). Sketch showing a polarizing region grafted to anterior margin of host wing bud, taken from research note book; 19+ = Hamilton–Hamburger stage of host wing bud. Note each of the three digits (2, 3, 4 ) is morphologically distinct and in the right wing, morphological changes can be seen in the proximal end of the humerus, the anterior fore‐arm element appears to be an ulna, and there is a mirror‐image symmetrical pattern of digits (4, 3, 2, 2, 3, 4). (c) Morphogen model for specifying digit pattern of chick wing. The cells of the polarizing region at the posterior margin produce a morphogen that diffuses across the antero‐posterior axis to set up a concentration gradient. Cells that are exposed to a high morphogen concentration form digit 4, while cells exposed to lower concentrations, digit 3 and then digit 2. Threshold concentrations that specify each digit are indicated and spacing reflects the relative numbers of polarizing region cells required to specify an additional digit of each type.

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Comparison of cellular origins of the digits in chick wing, chick leg, and mouse limb and implications for mechanisms of Sonic hedgehog (Shh) signaling that specify positional information. In the chick wing, the three digits develop from cells responding to a gradient of Shh (pink spots and arrow) diffusing from the polarizing region (purple spots); paracrine signaling. In the chick leg, the three more anterior digits (I, II, and III‐graded shades of pink) also arise in response to paracrine Shh, while the most posterior digit (IV‐ purple) develops from the polarizing region itself (autocrine signaling). In the mouse limb, the three more anterior digits have similar origin to the three more anterior digits in the chick leg (specified by paracrine signaling‐ graded shades of pink), while the two posterior digits arise from the polarizing region (autocrine signaling‐ purple). Some polarizing cells may also contribute to digit 3. Note that the most anterior digit in the chick leg and mouse leg can develop independently of Shh signaling. The specification of digit 4 versus digit 5, in the mouse limb, is proposed to be controlled by the length of time the cells spend in the polarizing region exposed to the highest concentrations of Shh signaling. Cells that form digit 4 spend a shorter time before being displaced by growth than cells than form digit 5. Comparisons of these fate maps suggest that the three digits of the chick wing are equivalent to 1, 2, 3 as indicated.

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Diagram illustrating gradient of GliA:GliR in developing chick wing buds. Processing of full‐length Gli transcription factors to function as transcriptional activators (GliA) occurs at high levels of Shh signaling in the posterior region of the wing bud, resulting in the activation of expression of genes involved in negatively regulating levels of Shh signaling, e.g., Ptch1. Where Shh levels are low, in more anterior regions of the wing bud, Gli proteins are processed to short forms that function as transcriptional repressors (GliR) resulting in repression of genes involved in digit formation, e.g., Hoxd13 and genes involved in growth, e.g., CyclinD1. The evidence that Gli3R represses Hoxd13 and CyclinD1 is that the expression of these genes extends anteriorly in wing buds of talpid3 mutant embryos in which Gli3 processing fails due to lack of cilia. Thus in the normal wing bud, Hoxd13 and CyclinD1 expression is localized at the posterior with digits forming from the posterior part of the bud which also grows more than the anterior. In addition, the gradient in the ratio of GliA/GliR across the antero‐posterior axis of the wing bud reflects the Shh gradient.

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Sonic hedgehog (Shh) signal transduction in vertebrate cells. Diagram showing processing of Gli proteins on a primary cilium and passage into the nucleus. (a) In the absence of Shh ligand–GliR (transcriptional repressor) predominates. (b) In the presence of Shh ligand–GliA (transcriptional activator) predominates.

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