Fig. 6

Re-evaluation of cellular contribution in the axolotl intercalary response. a Schematic diagram of the experimental design. b Hand grafting 10 days after surgery. The line indicates the approximate border of the graft. The scale bar is 3 mm. c Day 30. The elongated portion is recognizable between the grafted hand and the stump (double-headed arrows). d, e Skeletal pattern was revealed by Alcian blue staining. d Fgf2 + Fgf8 bead-grafted sample. Arrowheads indicate the two induced cartilages between the graft and the stump. (D’) Higher magnification view of D. e PBS-soaked bead grafting resulted in no intercalary responses (E’). f Histological observation of the Fgf2 + Fgf8 bead-grafted limb. Cartilage formation is readily evident between the hand graft and the stump. The scale bar indicates 1 mm. g–o The identical section underwent in situ hybridization and immunofluorescence. (G–I) Type II collagen (Col2A) expression pattern in the intercalary induced cartilage. (J–L) GFP expression pattern in the intercalary induced cartilage. The host was a GFP transgenic axolotl. Proximally derived cells were GFP-positive. m–o Hoechst staining for nuclei. g, j, and m are the distal regions of the induced zeugopod, and i, l, and o are the proximal. H, K, and N are the higher magnification views of g, j, and m, respectively. The scale bars in G and H are 500 and 200 μm, respectively. p–s The same experiment was performed with a normal host animal and a graft derived from a GFP transgenic axolotl. p, r) Type II collagen (Col2A) expression pattern in the intercalary induced cartilage. q, s GFP expression pattern in the intercalary induced cartilage. R and S are higher magnification views