Fig. 4. GREM1 is required to time the mesenchymal response to SHH signalling. Forelimb buds of E10.25 (31-33 somites) embryos received anterior grafts of SHH cell aggregates (red circles indicate approximate positions and sizes of grafts after culture) and the temporal kinetics of inducing/expanding the distal expression domains of Jag1 and Hoxd13 were assessed. Anterior grafts were used to clearly distinguish endogenous (white arrowheads) from exogenous SHH induced gene expression (blue arrowheads). All whole-mount in situ results were developed for the same amount of time to clearly reveal the weak ectopic Jag1 and Hoxd13 expression in Grem1^In2/In2 limb buds after 22 hours. (A) Wild-type limb buds. SHH induces ectopic Jag1 expression (blue arrowhead) within 22 hours. (B) Grem1-deficient limb buds. Left panel: only low levels of SHH induced ectopic Jag1 expression (blue arrowhead) are detected after 22 hours. Right panel: significant ectopic Jag1 expression (blue arrowhead) is detected only after 32 hours. (C) Wild-type limb buds. SHH induces significant anterior expansion of the Hoxd13 expression domain (blue arrowhead) within 22 hours. (D) Grem1-deficient limb buds. Left panel: only low levels of ectopic Hoxd13 expression (blue arrowhead) are detected after 22 hours. Right panel: significant levels of SHH induced anterior ectopic Hoxd13 expression (blue arrowhead) are detected after 32 hours.