actβA Is Markedly Increased during Fin Regeneration in Comparison to Other Candidate Genes (A) Quantification of expression levels of alk4 and Alk4/5/7 ligands normalized to ubiquitously expressed β-actin1 and β-actin2 reference genes in amputated fins at 0 hpa (n = 3), 6 hpa (n = 4), and 24 hpa (n = 4). The normalized gene expression (NGE) values were calculated with the Pfaffl method and multiplied by a factor of 10,000. squint, cyclops, southpaw, TGFβ2, GDF8, and GDF11 have very low NGE values, indicating that they are not significantly expressed in fins. Error bars indicate the SEM, and n refers to the number of biological samples; each sample was prepared from 15–20 fins. (B) n-fold change in NGE in regenerating fins at 6 hpa and 24 hpa (test samples) relative to cut fins at 0 hpa, which as a calibrator sample were normalized to 1.00. alk4 displays nearly 2-fold induction in fins at 6 and 24 hpa; actβA is the only Alk4/5/7 ligand significantly upregulated during fin regeneration. Error bars indicate the SEM. (C–E) In situ hybridization of fins at 24 hpa. Each panel shows a fragment of a whole-mount (left) and a longitudinal section through the ray (right). In this and subsequent figures, the fins are oriented with the distal edge up. alk4 mRNA (purple) is weakly ubiquitously expressed with upregulation in the blastema (arrows) and the disorganized mesenchyme; "b" indicates bones, "e" indicates epidermis, "m" indicates mesenchyme, "we" indicates wound epidermis, and "bl" indicates blastema (C). actβB mRNA is detected in the entire epidermis (D). No labeling was detected with a squ mRNA antisense probe (E).