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. 2021 Jun 9;148(11):dev199158. doi: 10.1242/dev.199158

Fig. 5.

Fig. 5.

FGF10 impacts chondrogenesis and operates upstream of NOGGIN. (A) Anti-NOGGIN antibody application to distal amputations improves regeneration in regeneration-restricted and -incompetent tadpoles. Regeneration-restricted and -incompetent tadpole right and left hindlimbs were amputated, and beads containing anti-IGG antibody or anti-NOGGIN antibody were placed on the right hindlimbs. Formed digits and digit-like structures were quantified in the right and left hindlimbs, and the difference calculated. Anti-IGG antibody total, n=17 from three biological replicates; anti-NOGGIN antibody total, n=28 from four biological replicates. (B) The effect of FGF10 on chondrogenesis is assessed by measuring the chondrogenic outgrowth at the proximal sites of regeneration-restricted explants at 3 dpa. Red lines show measured proximal chondrogenesis. Implanting 0.1% BSA/PBS beads at the proximal site or supplying 0.1% BSA/PBS to the media had no significant effect on chondrogenesis, while implanting Fgf10 beads at the proximal site or supplying FGF10 in media reduced chondrogenesis. Contralateral limbs were used as controls and are labelled as empty. From left to right, empty and PBS beads total, n≥7 from at least two biological replicates; empty and FGF10 bead total, n≥14 from at least four biological replicates; empty and 0.1% BSA/PBS in media total, n=10 from three biological replicates; empty and FGF10 in media, n≥14 from at least three biological replicates. ns, not significant; *P<0.05 and **P<0.001. Data are mean±s.e.m. Scale bar: 50 µm. (C) Left: example images of SU5402-treated explants showing extensive chondrogenesis at the proximal site. Red lines show measured proximal chondrogenesis. Right: blocking FGFR via the small molecule inhibitor SU5402 extends chondrogenesis in 3 days for regeneration-competent and -restricted explants. Contralateral limbs were used as controls and treated with DMSO. DMSO total, n=29 from seven biological replicates; SU5402 total, n=25 from seen biological replicates. **P<0.001. Data are mean±s.e.m. Scale bar: 200 µm. (D) Representative sectioned histology images for 3 dpa explants treated with SU5402. The outgrowing structures are Alcian Blue rich, which is indicative of chondrogenic cells. (E) Regeneration-competent explants were treated with a combination of FGF10 and recombinant BMP4, or with recombinant NOGGIN, with LDN193189 or with anti-NOGGIN antibody. 0.1% BSA/PBS and anti-IGG antibody were used as controls. From left to right: BSA total, n=8 from two biological replicates; recombinant FGF10 and recombinant BMP4 total, n=8 from two biological replicates; DMSO and BSA total, n=8 from two biological replicates; FGF10 and LDN total, n=8 from two biological replicates; BSA and anti-IGG antibody total, n=12 from three biological replicates; FGF10 and anti-NOGGIN antibody total, n=10 from three biological replicates; BSA total, n=8 from two biological replicates; recombinant FGF10 and recombinant NOGGIN total, n=8 from two biological replicates. **P<0.001. Data are mean±s.e.m. (F) Representative whole-mount stereomicroscope image of rFGF10 and anti-NOGGIN antibody treated explants can show a substantial Fgf8.L expression at different sites of the explant (n=5/9 from two biological replicates, compared with n=0/121 in controls, P<0.0001). Scale bar: 200 μm. (G) Recombinant FGF10 application to distal amputations restore regeneration in -regeneration-restricted and -incompetent tadpoles. Regeneration-restricted and -incompetent tadpole right and left hindlimbs were amputated, and beads containing 0.1% BSA/PBS or recombinant FGF10 or recombinant FGF10 and NOGGIN were placed on the right hindlimbs. Formed digits and digit-like structures were quantified in the right and left hindlimbs and the difference calculated. Box and whisker plots show minimum to maximum for all data points. Red dots represent individual data points. Where applicable, boxes show interquartile range (25-75%). Mean for No bead: 0.15; PBS: −0.5; rFGF10: 1.04; rFGF10 + rNOGGIN: 0. Empty total, n=19 from two biological replicates; 0.1%/PBS bead total, n=17 from five biological replicates; recombinant FGF10 bead total, n=25 from five biological replicates; recombinant FGF10 and NOGGIN bead total, n=25 from four biological replicates. ns, not significant; **P<0.001.