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Fig. 6. Anterior expansion of Grem1 expression in the distal mesenchyme and propagation of Jag1 expression do not require continuous SHH signal transduction. T₀: non-cultured control forelimb bud at the stage indicated. Wt: wild-type control forelimb bud cultured for 15 hours. Cyc: wild-type forelimb bud cultured for 15 hours in the presence of 10 µM cyclopamine. E10.25, 31-33 somites; E10.5, 34-36 somites; E10.75, 37-39 somites. All limb buds are oriented with anterior towards the top and posterior towards the bottom. Blue broken lines indicate the approximate anterior limits of Grem1 expression when necessary in A-C. (A) Inhibition of SHH signal transduction at E10.25 does not significantly alter Grem1 expression. (B) Inhibition of SHH signal transduction at E10.5 reduces Grem1 levels in the distal mesenchyme (indicated by bracket), but does not significantly alter its anterior expression limit. (C) Grem1 expression in Shh^-/- forelimb buds is rapidly downregulated, but is clearly detectable in the distoanterior mesenchyme at E9.75 (28 somites) and at E10.25 (32 somites). (D-F) Effects of cyclopamine treatment on Jag1 expression. (D) Inhibition of SHH signal transduction at E10.25 interferes to some extent with upregulation of Jag1 expression in the posterior limb bud mesenchyme. (E) Inhibition of SHH signal transduction at E10.5 does not significantly alter the onset of anterior expansion of Jag1 expression in the distal mesenchyme, albeit expression levels remain slightly lower. (F) Similarly, cyclopamine treatment at E10.75 does not interfere with continued anterior expansion of Jag1 expression in the distal limb bud mesenchyme.

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