|
Fig. 1 Generation of Cre Tg lines with itgb4 or krt18 promoters. (A) Whole-mount ISH analysis of itgb4 and krt18 expressions in adult zebrafish fin. dpa, days post amputation. Respective longitudinal sections are shown on the right side. itgb4 is expressed in the basal layer of amputated and uncut fin, whereas krt18 is only expressed in the regenerating fin. Arrowheads show the basal layer of the epidermis. Note that the ISH signal of krt18 is also seen in the blastema (bl). Scale bars: 100 µm (whole mount); 30 µm (section). (B) Diagram of the Tol2 transposon-based vector used to generate the Tgs. cryaa, crystalline alpha a promoter. (C) Scheme illustrating the Cre-mediated lineage tracking. The double Tgs of Cre-expressing line and the loxP reporter line, Tg(Olactb:loxP-dsred2-loxP-egfp) (RSG), were treated with tamoxifen (TAM) or fulvestrant (ICI) to induce the recombination at loxP sites. The resulting EGFP expression persists in all progeny cells. pA, polyadenylation signal sequence. (D,E) EGFP expression in amputated and uncut fins of Tg(krt18:creERt2) (D) and Tg(itgb4:creERt2) (E). EGFP expression is also seen in a fraction of mesenchymal cells within the fin ray of Tg(krt18:creERt2). TAM was added at 3 dpa (Amputated) and all images were taken at 4 days post labelling (dpl). Dotted line, amputation plane. Dashed line, basement membrane. Scale bars: 300 µm (left); 50 µm (right). White arrows indicate the posterior side (P). Experimental procedures are schematically shown below each image.