Figure 5.

The limb bud regulatory region 10.24 is responsive to SHH signaling. SHH-expressing cells were grafted to the anterior limb bud mesenchyme (E10.25, 32–34 somites) and trunks cultured for 16–20 h prior to analysis. (A) Nongrafted limb bud of an Fmn^Δ10 heterozygous embryo (control). (B) Ectopic LacZ in the contralateral limb bud having received an anterior graft of SHH expressing cells. (C) Control Fmn^Δ10.24 heterozygous limb bud. (D) Failure to induce LacZ expression in response to SHH-expressing cells in an Fmn^Δ10.24 heterozygous limb bud. (E) Control limb bud of an ld^In2 homozygous embryo. (F) Induction of Fmn expression in response to ectopic SHH signaling in an ld^In2 homozygous embryo. In A–F, a green arrow indicates the position of SHH expressing cells, a black arrowhead and an asterisk indicate ectopic gene expression, and an open arrowhead indicates endogenous expression. (G–L) Gremlin and Formin expression are maintained in limb buds in which FGF signaling transduction has been blocked by the inhibitor SU5402. Forelimb buds of Fmn^Δ10/Δ10 embryos (E10.0, 29–32 somites) were cultured in the presence of 10 μM SU5402 (+SU5402; stock dissolved in DMSO) for 14–16 h prior to analysis. Controls were cultured in the presence of an equal concentration of DMSO (+DMSO; 0.03% final concentration in medium). (G) LacZ detection in an untreated limb bud. (H) LacZ remains in a limb bud cultured in the presence of SU5402. Note the down-regulation of LacZ in the AER due to flattening in the absence of FGF signal transduction. (I) Gremlin expression in an untreated limb bud. (J) Gremlin remains in a limb bud cultured in the presence of SU5402. (K) Detection of Shh (arrowhead) and Fgf8 transcripts (AER) in an untreated limb bud. (L) Loss of both Shh and Fgf8 expression in a limb bud cultured in the presence of SU5402.