Figure 3.

Wnt and BMP signaling controls AEC formation during fin/limb regeneration. (A–F) SEM images of zebrafish pectoral fin (A–D) and axolotl larval limb (E,F) injected with Ad-eGFP (A,C,E) and Ad-Dkk1 (B,D,F). (A,C) Surface of the control pectoral fin with Ad-eGFP at 24 h post-amputation shows the characteristic smooth ectoderm (arrowheads). (B,D) Injection of Ad-Dkk1 caused thickening and alterations in the shape of ectodermal cells (arrowheads). C and D are close-ups of A and B, respectively, showing a single ray. (E) Axolotl larvae limb at 2 d post-amputation and Ad-eGFP-injected shows a smooth surface covering the amputation site (arrowhead). (F) Ad-Dkk1-injected larvae limb shows a rough and disorganized surface at the amputation site 2 d post-amputation (arrowhead). (G–J) Difference in ectoderm stratification during Xenopus hindlimb regeneration as revealed by immunoreactivity for p63 (brown color). Sections were counterstained with hematoxylin-eosin. (G) A Xenopus hindlimb, amputated at stage 50, formed stratified ectoderm 7 d after amputation (arrow). (H) Amputation of a stage 50 hindlimb, followed by injection of Ad-Dkk1, resulted in a single layer of ectoderm (red arrow). (I) Amputation at stage 53 resulted in the formation of a single layer of ectoderm after 15 d (red arrow). (J) Injection of Ad-CA-β-catenin to stage 53 amputated hindlimb induced ectoderm stratification after 15 d, similar to the one observed in G (arrow). (K–P) BMP/Smad signaling is necessary for pectoral fin regeneration and CA-β-catenin can restore BMP-inhibited regeneration. Gross morphology of pectoral fin (K,N) at 7 d post-amputation and expression of lef1 (L,O) and msxb (M,P) at 2 d post-amputation Ad-Smad7-injected (K–M) and coinjection of Ad-Smad7 + Ad-CA-β-catenin (N–P). Inhibition of BMP/Smad signaling by overexpression of Smad7 inhibited fin regeneration (K), lef1 (L), and msxb expression (M). Coinjection of Ad-CA-β-catenin restored the defects in regeneration caused by Smad7 (N) and lef1 (O) msxb expression (P).