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Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

We analysed spatio-temporal expression of dorso-ventral genes - Wnt-7a, En-1, Lmx-1 and Fgf-8 - during both normal and ectopic limb formation following fibroblast growth factor (FGF) application to the flank. We confirm that Wnt-7a is the first of these genes to be expressed in dorsal ectoderm in limb-forming regions. We also noticed patterns and kinetics of gene expression specific to chick that could account for differences observed in ridge formation between chick and mouse. We find that Wnt-7a expression, in dorsal ectoderm, is rapidly and locally induced by FGF application. In contrast, ectopic induction of Lmx-1 expression, in dorsal mesoderm, is much slower, occurs first at a distance from the FGF-2 bead and seems initially independent of direct Wnt-7a signalling during FGF-2 limb induction. Finally, we show that there is no contribution to extra-limb mesoderm from normal limb mesoderm and confirm that flank cells give rise to the extra limb. Furthermore, we suggest that an inhibitor present in the flank normally prevents Lmx-1 expression in this region and restricts its expression to limb-forming regions.

SnR, twist and Fgf10 are expressed in presumptive limb territories of early chick embryos. When FGF-2/FGF-8 beads are implanted in chick flank, an ectopic limb develops and SnR is irreversibly activated as early as 1 h. Ectopic Fgf10 and twist expression are activated much later at 17 and 20 h, respectively. FGF-10 can also induce SnR, but much later, and in this case activation occurs simultaneously with that of twist and Fgf10 via the Fgf8- expressing ridge. Tbx-4 and Tbx-5 are expressed in leg and wing forming regions, respectively, in a similar pattern to SnR and twist. FGF-2 leads to ectopic expression of Tbx-4 and Tbx-5 as rapidly as ectopic expression of SnR, but the patterns of ectopic transcripts suggest that induction of SnR and Tbx gene expression occur via different pathways.

We examined systematically the ability of chick limb bud stumps to regenerate distal structures when fibroblast growth factor (FGF)-4 is applied. When amputations were made within 600 mu m of the tip and FGF-4 applied either posteriorly or both apically and posteriorly, outgrowth of stump tissues occurred and a virtually complete skeleton developed. 'Regeneration' of distal structures was correlated with reactivation of Msx-1 and Shh expression. At proximal amputation levels where FGF-4 did not lead to 'regeneration', neither Msx-1 nor Shh expression was induced. We also grafted cells from progressively more proximal levels of mouse limb buds to chick wing bud tips beneath the apical ridge and the pattern of reactivation of Msx-1 expression along the proximal distal axis of the limb buds was similar to that found in chick limb buds.

Limb development endures as an excellent model for pattern formation in vertebrates. We have identified Gnot1 as a member of a new homeobox gene subfamily. Gnot1 is expressed in a dynamic temporospatial distribution in the developing limb, initially correlating with regions destined to form distal structures and then becoming progressively more restricted to specific regions determined to give rise to wrist and ankle. Micro-surgical alteration of the developmental program of the limb reveals that Gnot1 is expressed in a position- and fate-dependent manner, responding both to signals from the apical ridge and the polarizing zone. Furthermore, Gnot1 activation by polarizing signals occurs temporally downstream of Hoxd gene activation, but well before the first appearance of condensations that will give rise to the carpus of the wrist. The features of Gnot1 expression suggest a role for this gene in regulating pattern formation during limb development.