Analysis of the actinotrichia-specific components in the regenerative outgrowth. (A-F) In-situ hybridization with probes against four actinodin genes on longitudinal fin sections at 3 dpa. N ≥ 4 fins; ≥ 3 sections per fin. (A, B) and1 mRNA is detected in the mesenchyme (m) of the blastema and in the basal wound epithelium (bwe). No expression is detected in the basal wound epithelium adjacent to osteoblasts (ob) in the ray. (C, D) and2 (E) and3 (F) and4 genes are co-expressed in the mesenchyme of the blastema, but not in the epidermis (e). (G-J) Immunofluorescence staining of longitudinal fin sections of ray (G, H) and interray (I, J) at 3 dpa. And1 (green) and And2 (red) proteins are co-localized in actinotrichia fibers. Rays and interrays are identified by the presence versus absence of Zns5-positive osteoblasts (H, J, blue). Actinotrichia fibers are predominantly found in the subepithelial position with the exception of osteoblast-containing regions. N = 4 fins; 3 sections per fin. Higher magnifications of framed areas are labeled with the same letter with a prime symbol. Fin amputation planes are shown with a dashed line. The same rules apply to all subsequent figures.

Regenerating lepidotrichia replace actinotrichia in the subepidermal space. (A-D) In-situ hybridization on cross sections of distal-most and distal fin outgrowth at 3 dpa. N = 4 fins. (A, B) In the distal blastema, and1 is differentially expressed in the basal wound epithelium of the rays and interrays. Arrowheads indicate the positions of the interrupted and1 expression in the basal epithelium. m, mesenchyme; bwe, basal wound epithelium. (C, D) and2 is absent from the wound epidermis. (E, F) In-situ hybridization against and1 on transversal (E) and longitudinal sections (F) of osterix:nlsGFP transgenic fins. A lack of and1 expression in the basal wound epithelium of rays is associated with the presence of underlying osteoblasts (green). Arrowheads indicate the positions of the interrupted and1 expression in the basal epithelium. N = 6 fins, 2 sections per fin. (G-J) Immunofluorescence staining for And1 (green), Zns5 (red) and Chondroitin sulfate (H, J, blue) at 5 dpa. (G, H) In the ray, the leading edge of osteoblasts (arrow) replaces actinotrichia in the subepidermal compartment. In the mesenchyme, actinotrichia are disrupted and misaligned. (I, J) In the interrays, no Zns5 and chondroitin sulfate is detected. Actinotrichia remain longitudinally organized in the junctional region between the epidermis and the mesenchyme. The green signal at the base of the outgrowth corresponds to blood autofluorescence. N = 4 fins; 2 sections per fin.

Dynamics of actinotrichia regeneration correlate with lepidotrichia regrowth. (A-P) Immunofluorescence staining for And1 (green) in osterix:nlsGFP fish (osteoblasts; red) at different time points after amputation (dpa). Bone matrix/tissue autofluorescence shown in blue. (Q) Quantification of the length of And1-positive regions at different time points during regeneration. Each fin was represented by the measurements of the second, third, and fourth ray relative to the edge of both lobes. N ≥ 4 fins for each time point. 6 rays per fin. Error bars represent SEM. (J′) Magnification of a single ray at 5 dpa, showing inverse correlation between And1 deposition and osterix:nlsGFP. The V-shaped tip of the regenerating lepidotrichium indicates an initiated ray bifurcation.

Modulation of and1 and and2 expression in response to pulse inhibition of TGFβ/Activin-βA and IGF signaling. (A) Experimental design for inhibition of TGFβ/Activin-βA signaling by SB431542 and of IGF signaling by AEW541. (B) Quantification of length of regenerate as the average length of the second, third, and fourth rays from the lateral edge of the fin. N ≥ 4 for each group. Error bar indicates SEM, *P<0.05, ** P<0.01. (C-H) Whole-mount in-situ hybridization for and1 and and2 on 4 dpa fins. Inhibition of TGFβ/Activin-βA (SB431542) blocked expression of both genes, whereas inhibition of IGF (AEW541) had no effect. (I-K) Whole-mount immunofluorescence staining for And1 (green) on 4 dpa fins. TGFβ/Activin-βA inhibition (K) caused a loss of actinotrichia as compared to control (I) and IGF inhibition (J). (L) qRT-PCR analysis of and1 expression after 6 h or 16 h of treatment with the pharmacological inhibitors AEW541 (IGF pathway) and SB431542 (TGFβ/Activin-βA pathway). Treatments started at 2 dpa. The relative expression was normalized to control fins at 2 dpa. N = 3 (9 fins each). Error bars represent SEM, *** P<0.001.

FGF signaling is essential for actinotrichia formation and maintenance. (A-C) In-situ hybridization for and1 on 3 dpa sections in control, hsp70:dnfgfr1-egfp and dob fish. Control and dnfgfr1 fish underwent heat shock (hs) at 2 dpa. N = 4 fins per group. (D-F) Immunofluorescence for And1 (green) on longitudinal sections at 3 dpa in control, hsp70:dnfgfr1-egfp, and dob fish. N = 4 fins per group. (G-I) Whole-mount immunofluorescence staining for And1 (green) overlaid with bright field images for all experimental groups. N = 3 fins per group. Both, transgenic expression of dnfgfr1 and dob mutation cause a drastic decrease in and1 expression (B-C) and And1 deposition (E-F, H-I). (J, K) Immunofluorescence staining with And1 (green) and Zns5 (red) on 3 dpa longitudinal sections of fins of control fish and fish treated with PD173074 for 1 day. One day of treatment with the inhibitor of FGF signaling starting at 2 dpa is sufficient to disrupt actinotrichia in the blastema. N = 4 fins. (L) qRT-PCR analysis of and1 expression after 6 h of treatment with 10 μM of the pharmacological inhibitor of the FGF pathway, PD173074. Treatment started at 2 dpa. The relative expression was normalized to control fins at 2 dpa. N = 3 (9 fins each). Error bars represent SEM. *** P<0.001.

Assembly of actinotrichia detected by electron microscopy of the distal-most blastema.

(A-B) Electron microscopy of the distal-most blastema at 3 dpa was performed after peroxidase- DAB immunohistochemical staining following no primary antibody (control; A) or in combination with And1 antibody (darker immunolabeled actinotrichia; B). Thin fibrils of a constant width of ≈ 20 nm (arrowheads; A’’) assemble into higher order striated fibers (A’). (A’) The main periodic striation of ≈ 60 nm is further subdivided into finer bands. (A’’) An accretion of fibrils on the extremities and surface of the actinotrichia are observed, so that the units may grow in width and in length.

Dynamics of bone matrix formation during regeneration.

(A-H’) Dynamics of bone matrix formation during fin regeneration visualized by Alcian blue and Alizarin red histological staining. Calcified lepidotrichia/bone in pink, non-calcified lepidotrichia in blue. Actinotrichia seen in contrast in the magnified panels. Black dashed lines represent plane of amputation. N ≥ 4 fins for each time point.

Actinotrichia fibers form palisades on both sides of the distal blastemal mesenchyme.

(A-D) 3D projections of actinotrichia (And1; green) in uninjured (A, B) and at 3 dpa (C, D) fins. Images extracted from S1 Movie. Side views (B, D) show that actinotrichia fibers form two-sided palisades.

Actinotrichia resume regeneration after a pulse inhibition of signaling pathways.

(A-C) Pulse exposure to the inhibitors of the IGF (AEW541), TGFβ/Activin-βA (SB431542) and FGF (PD173074) signaling pathways. (A) Experimental design. (B) Live-imaging at 4 and 10 dpa. Fins resumed regeneration without major morphological defects after interruption of treatments. N = 4 fish per treatment. (C) Whole-mount immunofluorescent staining for And1 (green) and Zns5 (red) of 10 dpa fins shows recovery of actinotrichia fibers after arrest of treatment. Bone autofluorescence in blue. N = 4 fish per treatment. (D-F) Pulse inhibition of the FGF pathway through heat-shock activation of the dominant negative FGF receptor type 1 (hsp70:dnfgfr1-egfp fish). (D) Experimental design (E) Live-imaging at 2, 3 and 10 dpa shows recovery after pulse inhibition of the FGF pathway. Persistent morphological defects in 2 of 4 transgenic fins. (F) Whole-mount immunofluorescent staining for And1 (green) and Zns5 (red) of 2, 3 and 10 dpa fins. Bone autofluorescence in blue. After heat-shock actinotrichia fibers decreased in the blastema (3 dpa). At 10 dpa, actinotrichia recovered. N ≥ 3 fish per group per time point. hs: heat shock.