Fig. 1. Analysis of the spatial competence to activate and the temporal kinetics to upregulate the expression of SHH target signals in the limb bud mesenchyme. Left panels: contralateral control limb buds (not grafted). Right panels: contralateral, wild-type (A-D) or Shh^-/- mutant (E-H) forelimb buds received posterior grafts of SHH-producing cell aggregates at E10.25 (31-33 somites). The red circles indicate the positions and approximate sizes of the grafts after culturing. White arrowheads indicate the endogenous gene expression domains. Blue arrowheads indicate the induced gene expression in response to a SHH graft. Limb buds are oriented with anterior towards the top. (A-D) Analysis of the temporal kinetics of the differential transcriptional response to SHH signalling in wild-type forelimb buds. (A) A ring of mesenchymal cells responds to SHH signalling by activating expression of the downstream target Gli1 within 6 hours of grafting. (B) Within 9 hours, Grem1 expression is expanded distoanteriorly in response to SHH signalling. (C,D) Jag1 expression is activated in the distal anterior mesenchyme after 9 hours (C), but upregulation of its expression in the distoanterior mesenchyme requires at least 15 hours (D). The small white arrowheads indicate localized endogenous Jag1 expression in the anterior limb bud mesenchyme. (E-H) Shh-deficient limb buds were cultured for 15 hours after receiving a graft of SHH-producing cells to detect strong expression of the gene of interest in all cases. Asterisks indicate the posteroproximal margins of the limb buds after culture. (E) SHH signalling induces transcriptional response in a large area of mesenchymal cells surrounding the graft as assessed by Gli1 expression. (F) Grem1 expression is upregulated in mesenchymal cells that are always located distally to the SHH graft. (G) Jag1 expression is activated in mesenchymal cells located also always distally to the SHH graft. The weak and speckled AER staining corresponds to crossreactivity that appears in a fraction of cultured limb buds. (H) A posterior SHH graft upregulates Bmp2 expression in mesenchymal cells located proximal to the graft (blue arrowhead). Upregulation of Bmp2 expression can be seen in the AER (striped arrowhead).