actβA Is Expressed in an Interray Domain and in the Blastema (A and C–H) In situ hybridization of fins with actβA mRNA antisense probe (blue staining). Scale bars in (A) represent 100 μm. (B) Real-time RT-PCR quantification of actβA expression in fins at 1, 3, 6, and 24 hpa (n = 4) relative to control fins at 0 hpa (n = 3), which as a calibrator sample were normalized to 1.00. Error bars represent the standar error of the mean (SEM). p < 0.001 indicates a significant difference from the control. n refers to the number of biological samples; each sample was prepared from 15–20 fins. (A, C, E, and G) Wild-type fins at different stages of regeneration. Each panel shows a longitudinal section (left) and a fragment of the whole-mount fin (right). At 6 hpa, actβA is detected in scattered mesenchymal cells along the wound epidermis of the interrays (A). The ray contains dermal bones; the interray spans soft tissue between rays. At 12 hpa, the expression expands in the mesenchyme of the interray pockets at the cut edge (C). At 24 hpa, actβA is additionally induced in the mesenchyme underlying the wound epidermis of the rays, where the blastema is formed (arrows) (E). At 72 hpa, the expression is strongly detected in the blastema (arrows) (G). (D, F, and H) actβA expression is independent of FGF20a signaling. dob homozygous mutant fins were incubated at a restrictive temperature of 33°C. actβA mRNA is expressed in the interray pockets of dob mutant fins at 6 hpa (D), 24 hpa (F), and 72 hpa (H). No or little activin A expression is detected in the ray mesenchyme (arrows) because the blastema fails to form in dob mutants (n = 6).
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