Fig. 5

Blastema Formation Involves Wnt Target Gene Activation and Dkk1b Attenuation

(A and B) Female TCFsiam; dkk1b:EGFP caudal fins at 2 dpa (A) and 4 dpa (B), shown as whole-mount images. TCFsiam is expressed in distal regions during regeneration. dkk1b:EGFP is not detectable at 2 dpa but is evident in regenerates 4 dpa. Arrowheads indicate amputation plane. Scale bar in (A), 500 µm.

(C) Section images of female TCFsiam caudal fins at 2 (top) or 4 dpa (bottom). TCFsiam is expressed in a subset of cells in the distal portion of the blastema, some of which are BrdU-positive 30 min after treatment. Arrows indicate BrdU⁺ and TCFsiam⁺ cells. Amputation site in 4 dpa is proximal to imaged area. Scale bar for (C) through (E), 50 μm.

(D) Section images of female dkk1b:EGFP caudal fins at 2 dpa (top) or 4 dpa (bottom). dkk1b:EGFP is not detectable at 2 dpa. At 4 dpa, it is expressed in a very small domain at the distal tip of the regenerate (arrow) and in more proximal areas of osteoblast patterning. Expression domains are adjacent to areas of low BrdU incorporation (red), but not to more proliferative blastemal mesenchyme.

(E) Section images of female TCFsiam; dkk1b:EGFP caudal fins at 2 (top) or 4 dpa (bottom). dkk1b:EGFP⁺ cells at the distal tip of the regenerate at 4 dpa colocalize with TCFsiam (arrow).

(F) Experimental design for experiments testing effects of heat-induced overexpression of dkk1b during regeneration.

(G) Whole-mount images of 4 dpa wild-type and hsp70:dkk1b female pectoral fin regenerates, indicating that overexpression of dkk1b inhibits regeneration. n = 6. Scale bar, 500 μm.

(H and I) Whole-mount images of wild-type (H) and hsp70:dkk1b (I) female pectoral fin regenerates at 34 hpa, indicating that dkk1b overexpression blocks induction of TCFsiam in the newly formed blastema. n = 6. Scale bar in (H), 500 μm.