A TREE linked to cx43 directs blastemal gene expression. (A) RNA-seq and ATAC-seq browser tracks indicating the cx43E24 TREE near the upregulated cx43 transcript in regenerating fibroblast and whole-fin tissue. Tracks represent normalized combined replicates. (B) The cx43E24-fos:EGFP enhancer reporter construct. The 945 bp candidate TREE (blue arrow) was placed upstream of a 96 bp c-fos minimal promoter (yellow arrow) to direct fluorescent EGFP expression (green arrow). (C) Larval cx43E24:EGFP animals at 5 days post fertilization (dpf) displaying EGFP expression in trunk muscle (mus) and early pectoral fins. High magnification view of a larval pectoral fin (arrowhead) (C′). Scale bars: 500 μm. (D) The three dorsal-most caudal fin rays in some strongly expressing cx43E24:EGFP animals express EGFP in uninjured tissue, although this is not present in all lines. Scale bar: 300 μm. (E-G′) Longitudinal sections indicate cx43 expression (violet) in fibroblasts (white asterisks) and joint osteoblasts in wild-type fish (E) and cx43E24Δ/Δ fish (F) by in situ hybridization. Expression in forming joints is marked by black arrowheads. cx43E24:EGFP reporter fish exhibit a similar EGFP immunofluorescence pattern to endogenous cx43, although EGFP expression is detected in most blastemal osteoblasts (white arrowheads) in addition to blastemal fibroblasts (white asterisk) (G,G′). Scale bars: 100 μm. (H-J) cx43E24:EGFP fish display mesenchymal EGFP expression by 4 dpa, fading by 21 dpa. Arrowheads indicate amputation plane. Scale bars: 300 μm. (K,L) qPCR analysis of cx43 transcript levels in cx43E24Δ/Δ and wild-type fins. Expression is induced at 4 dpa but to a lesser extent than in wild-type fins. Each biological replicate pool of three fins each was performed in technical triplicate, and paired two-tailed t-tests were performed to indicate significance (n=8). (M) Lengths of regenerating fins are similar in cx43E24Δ/Δ and wild-type fish (n=14). Paired two-tailed t-tests were performed to indicate significance. (N) Fin ray segment length at 7 dpa was decreased by 6.2% distal to the amputation plane and 5.2% shorter proximal to the amputation plane in regenerating cx43E24Δ/Δ fins compared with wild-type fins (n=14). Paired two-tailed t-tests were performed to indicate significance. Data are mean±s.d.
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