Actinotrichia formation during fin development and regeneration. Immunofluorescence with JAS′96 antibody against actinotrichia and AFCs (green) and hoechst nuclei staining (blue). (A–C) Fin fold sections at 30 (A), 36 (B) and 72 hpf (C). (D–E) Longitudinal sections of regenerating fin at 2 dpa (D) and 3 dpa (E). Transversal section at 5 dpa (F). Bars represent 10 (A–C) and 25 μm (D–F). Arrowhead is actinotrichia. Arrow is actinotrichia forming cell (AFC). e is epidermis. b is basal epidermal layer. c is connective tissue. n is notochord. Connective tissue and basal epidermal layer are separated by white lines in F.

Colocalization of Collagen type I and Type II in actinotrichia during development and regeneration. Immunofluorescence against Collagen I (red) and Collagen II (green). Sequential confocal sections were obtained in the Z-axis of the whole thick of the samples. Projections of the Z-stack are shown for the red (A), the green (B) and the merged channels (C) of the fin fold of a doubly immunostained 72 hpf embryo. A confocal plane of the merged red and green channels, at a higher magnification, shows the actinotrichia fibers in detail (D). Z-stack projections of all confocal sections .of a 4 dpa longitudinal fin section doubly immunostained and with nuclei counterstained in blue (E–G). E: Red and blue channels. F: Green and blue channels. G: All-merged channels. H: A detail of all merged channels shows colocalization of the green and red labels. Bars represent 100 μm in A–C and E–G; and 25 μm in D and H. Arrowhead is actinotrichia. e is epidermis. b is basal epidermal layer. c is connective tissue. n is notochord.

col2a1b expression. (A) Semiquantitative RT-PCR of col2a1a and col2a1b in the whole body of 1 month postfertilization (left) and regenerating fin (rigth) tissues. b-actin is used as a control of constitutive expression. (B–D) In situ hybridization of a zebrafish embryo at 48 h stage. B. Whole mount in situ hybridization of col2a1b gene. Arrows point at the pectoral and tail fin folds. C. Detail of col2a1b gene expression at the tail bud. Note the expression at neuromast cells along the prospective lateral line. Arrow points at the distal portion of the tail fin fold. D. Dorsal view of col2a1b gene expression. Note the expression at the forebrain and midbrain. Arrow points to a labeled left pectoral fin bud. Bars represent 0.5 mm (B and D) and 0.2 mm (B).

Collagens type II alpha 1 b and type I alpha 1 are constituents of the fin skeleton. (A–F) In situ hybridization of col1a1 (A, C and E) and col2a1b (B, D and F) in longitudinal (A–D) and transversal (E–F) sections of regenerated fins. A–B: Ray blastema at 2.5 dpa. C–D: Ray blastema at 3 dpa. E–F: Ray blastema at 7 dpa. Bars represent 25 μm (E–F) and 50 μm (A–D). Arrowhead is actinotrichia. Asterisk is lepidotrichia. e is epidermis. b is basal epidermal layer. c is connective tissue. (G–H) Expression analysis of col1a1a and col2a1b. Total RNA was extracted from different stages of fin regeneration and the relative transcript levels of col1a1a and col2a1b were determined by qRT-PCR. col1a1a (G), col2a1b (H) in different regeneration stages: Fin, non regenerating fin; 1 dpa, 1 days post amputation; 2 dpa, 2 days post amputation; 3 dpa, 3 days post amputation; 4 dpa, 4 days post amputation; 7 dpa, 7 days post amputation. Bars represent the mean of two independent biological samples ± SE. Different letters indicate a significant difference between samples according to the corresponding ANOVA (P < 0.05).

col2α1b and col1α1a knockdown. Morpholino knockdown of col1a1a (A–D), col2a1b (F–H). Morphants of these genes (B, F, D and H) are compared with their corresponding 5-mismatch morphant control (A, E, C and G). Bars represent 0.5 mm (A, B, E and F) and 0.2 mm (C, D, G and H).

col2α1b and col1α1a knockdown in fin development. Morpholino knockdown of col2a1b (A–C), col2a1a (D–F) and col1a1 (G–I). Morphants of these genes (B, E and H) are compared with their corresponding 5-mismatch morphant controls (A, D and G). Fin fold area (in pixels) of each gene experiment were statistically compared (C, F and I). Asterisks represent significant differences after Mann–Whitney u-test. Note: E shows the strongest phenotype induced by col2a1aMO.

col1a1a dominant chi/+ mutant shows aberrant fin exoskeleton. Actinotrichia and lepidotrichia pattern in mature (A–B, E–F) and regenerating fin at 4 dpa (C–D). Wild type (A, C and E) and heterozygous chihuahua mutant (col1a1a^dcl24/+; B, D and F). Arrowhead represents actinotrichia. (G–H) Statistical comparisons of length and number of actinotrichia per fin ray between chi mutant (black column) and wild type (gray column). All images were obtained under polarized optics following staining with Picrosirius red. Asterisks represent significant difference by Mann–Whitney u-test.

lh1 is implicated in actinotrichia synthesis. (A–D) In situ hybridization of lh1 in fin sections. Longitudinal sections of ray blastema at 3 (A) and 7 dpa (B). (C–D) Transversal sections of a 7-day regenerated fin at the positions outlined in (B). Arrowhead is actinotrichia. Asterisk is lepidotrichia. e is epidermis. c is connective tissue. (E–F) Morpholino knockdown of lh1 at 3 dpf (F) and 5 mismatch morphant control at 3 dpf (E). Fin fold areas (in pixels) are statistically compared following by Mann–Whitney U-test (G). Asterisk represents significant difference. Bars represent 25 (C and D) or 50 μm (A and B).