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Fig. S1. FGF8 is unable to induce hairy2 in the AND. Representative results of hairy2 expression obtained upon implantation of FGF8-beads in the AND of HH23-24 forelimb buds. hairy2 expression is never induced in the AND even upon higher dosage (iv) and longer incubation periods (vi). Dorsal view; anterior to the top. *Beads.
Fig. S2. Both SHH signaling and hairy2 expression are abolished upon cyclopamine treatment. (A) In situ hybridization for fgf8 and patched1 revealing unaffected AER/fgf8 expression and impaired SHH signaling, shown by patched1 down-regulation in the treated limb. (B) hairy2 expression over time in response to cyclopamine treatment, evidencing a long-term effect, since only mild hairy2 down-regulation is observed at earlier time points (arrows). Dorsal view; anterior to the top.
Fig. S3. hairy2 expression requires cooperative AER/FGF and ZPA/SHH signaling. (i–iv) SHH is unable to induce hairy2 in the DCD (n = 4/4) and PPD (n = 3/3) in the absence of AER/FGF. (v,vi) FGF8-beads implanted in the PND following 6 hours of ZPA ablation, no longer induce hairy2 (n = 6/6). Dorsal view; anterior to the top. *Beads.
Fig. S4. ZPA/SHH and AER/FGF signaling are jointly required for hairy2 expression. FGF8 beads have been described to expand shh expression proximally when implanted near the ZPA (Yang and Niswander, 1995). This region coincides with the PND, where FGF8-beads induced hairy2 expression (Fig. 1Cvi). A detailed time-lapse study performed upon FGF8-bead implantation shows that after 30 minutes, neither shh nor hairy2 expression is obtained (i,ii,v,vi; shh: n = 2/2; hairy2: n = 2/2) and, 15 minutes later, ectopic hairy2 and shh expression are simultaneously induced (iii,iv,vii,viii; shh: n = 3/3; hairy2: n = 4/4). These observations strongly support the requirement of a cooperative action of FGF8 and SHH signaling for hairy2 induction in the limb. Furthermore, they suggest that hairy2 induction mechanism does not involve a relay of FGF and SHH signals, but results from a parallel convergence of signaling pathways, in both time and space, i.e. both signals are required at the same time, in the same tissue.
Fig. S5. Down-regulation of hairy2 expression observed after AER or ZPA ablation is not due to cell death. Cell death was assessed by performing TUNEL after AER- or ZPA-ablation, in transversal and longitudinal limb sections, respectively. (i,iv) Limb buds treated with DNase display apoptosis in the entire limb mesenchyme and ectoderm. (ii,v) Contralateral control limbs, showing normal levels of apoptosis. (iii) Stage HH22 limb didn't show elevated cell death in the distal limb mesenchyme after 2 hours of AER ablation. (vi) Unaltered levels of cell death observed after 6 hours of ZPA ablation in stage HH23 limb. Distal towards the right and anterior to the top.
