Vasculature in Tg(fli1a:EGFP) pectoral fins. (A-C) Blood vessels (EGFP, green) in female zebrafish. (D-F) BT clusters in males are highly vascularized (boxed in D), but not in surrounding areas (arrow in D). (B,C,E,F) Higher magnification of the boxed regions in A,D. (C,F) Merged brightfield and fluorescent images. (G-R) Confocal micrographs. (G-I) Female with a single artery (red arrowheads), two veins (blue arrowheads) and intervessel commissures (white arrowhead). (J-L) Male with vascularized BTs, an artery (red arrowhead), intervessel commissures (white arrowhead), interray vessels (yellow arrowhead) and veins (blue arrowhead). (M-R) Longitudinal sections. (M-O) Female showing blood vessels that lie between the two hemirays. (P-R) Male showing blood vessels present in a web-like pattern (pink arrowhead) in the epidermis and above the artery (red arrowhead). fr, fin ray; hr, hemiray. Scale bars: 200 μm in A,D; 100 μm in B,C,E,F; 50 μm in G-L; 25 μm in M-O; 50 μm in P-R.

Male pectoral fin BT regeneration. Mallory stain of intact (A) and regenerating (B-F) pectoral fin longitudinal sections. (A,B) Keratinized caps (red) and underlying epidermis (blue) are observed on intact fins (A) but are lost in 3-dpa regenerates (B). (C,D) At 4-5 dpa, a layer of keratin is deposited in the proximal regenerate (black arrows). (E) At 7 dpa, BTs (black arrow) are observed in the fin regenerate. (F) Females do not possess BTs. Yellow arrows indicate blood cells. Black lines (B-F) indicate the amputation plane. dpa, days post-amputation. hr, hemiray. Scale bar: 50 μm.

Male and female Tg(fli1a:EGFP) blood vessel regeneration. (Aa-h,Ba-h,Da-h) Blood vessel regeneration follows similar patterns in the female (A,B) and posterior (D) male rays. (Ca-h) Plexus formation (Cb) is delayed in the male anterior ray compared with females (Ab) and appears irregular in shape. (Cd-h) At 5 dpa, a second wave of neo-angiogenesis occurs underneath new BT formation in the male anterior ray and proceeds in a proximal-to-distal manner (red arrowheads). Anterior, fin ray 4; posterior, fin ray 7. White lines indicate the plane of amputation. Scale bars: 100 μm.

BT cluster regeneration occurs alongside a second wave of neo-angiogenesis. Confocal images of longitudinal sections of regenerating male and female pectoral fins. (A,C,E,G,I) Female blood vessels regenerate along the center of the ray. (E) Arrowhead indicates the distal ray folded back on itself. (B) The first wave of male blood vessel regeneration occurs down the center of the ray. (D,F,H,J) At 5-8 dpa, a second wave travels in a proximal-to-distal fashion along the base of the epidermis (red arrowhead). (J) New BTs (white arrowheads) and blood vessels (yellow arrowhead) are observed in the ventral fin regenerate. Blue arrowhead indicates blood vessels along the base of BTs located in the stump. Black vertical lines indicate the amputation plane. Scale bars: 100 μm.

Androgens induce and estrogens inhibit pectoral fin BT formation. BTs are observed in male regenerate and intact fins treated with testosterone (T) (1 μg/l) (A,B), 5α-dihydrotestosterone (DHT) (1 μg/l) (M,N) and fadrozole (50 μg/l) (Q,R). Treatment with testosterone (T) (1 μg/l) (C,D), 5α-dihydrotestosterone (DHT) (1 μg/l) (O,P) and fadrozole (50 μg/l) (S,T) for 15 days induces BT formation in the regenerating and intact female fin. Flutamide treatment (2 mg/l) (23 dot) (I,J) and 17β-estradiol (E2) (1 μg/l) (16 dot) (U,V) inhibit and reduce BT formation in the male regenerate and intact fin, respectively. Flutamide (K,L) and E2 (W,X) treatments do not affect females. Controls (E-H). dot, days of treatment. Scale bar: 100 μm.

Pectoral fin BTs regress following cessation of chemical treatments in females. (A-F) Regenerating (A) and intact (D) fins treated for 18 days with fadrozole. (B,E) At 30 dpt, BTs appear reduced. (C,F) At 58 dpt, BTs have disappeared. (G,J) Female regenerate (G) and intact fins (J) following 14 dot with DHT. (H,K) At 29 dpt, BTs appear smaller. (I,L) At 88 dpt, BTs are barely visible. White vertical line indicates the amputation plane. dpt, days post-treatment. Scale bar: 100 μm.

Androgens induce neo-angiogenesis in intact and regenerating pectoral fins. (A-F,I,J) Intact and regenerating fins of T-treated (B,B′,D,D′) and DHT-treated (F,J) females (14 dot) develop vascularized BTs not observed at 0 dot (A,C,E,I). (G,H,K,L) Blood vessels of flutamide-treated (14 dot) male intact and regenerating fins acquire a female-like appearance. (M-T) Control untreated females (M,N,Q,R) and males (O,S,P,T). White lines indicate the amputation plane. Scale bars: 100 μm, except 50 μm in B′,D′.

Pectoral fin endothelial and epidermal cells proliferate upon testosterone treatment. Pcna (red) and DAPI (blue) staining of Tg(fli1a:EGFP) intact fin longitudinal sections. (A) Females display few proliferating cells. (B) Males present many proliferating cells deep in the epidermis. (C) T-treated females (5 dot) develop a proliferation profile similar to that of males in the epidermis. Endothelial cell proliferation is induced (arrows). Scale bar: 50 μm.

Inhibition of neo-angiogenesis affects T-induced BT growth in the female pectoral fin. (Aa-Ja) A single row of BTs on the intact fin and stump after 6-14 dot with PTK787 and 4-12 dot with T. Small BTs appear in the fin regenerate after 8 dot with PTK787 and 6 dot with T (Da,Ea). A small BT cluster appears on the regenerate after 14 dot with PTK787 and 12 dot with T (Ga,Ha). (Ab-Jb) After 4 dot with T, BTs develop on the regenerate, stump and intact fin and continue to grow from 6-12 dot. (Jb) After 12 dot, the blood vessel network of T-treated females is similar to that of males. (Ac-Jc) PTK787-treated females do not possess BTs. (Aa-Ja,Ac-Jc) Neo-angiogenesis and regenerative outgrowth are inhibited in all PTK787-treated females. (Ad-Jd) System water controls regenerate normally and do not grow BTs. B.F., brightfield. Fli, fli1a:EGFP (green). KR21 (red) outlines the BTs. White vertical lines indicate the amputation plane. Scale bar: 100 μm.