Bifurcation of the regenerating ray depends on the presence of surrounding interray blastemas. (A) Detail of the central rays (R9) of the dorsal (R9d) and ventral (R9v) lobes of the tail fin of D. rerio at the level of first bifurcations. Note that they are segmented and bifurcated. I9, interray 9. (B) Detail of R1 and R2 distal to the first bifurcation of R2. Note that R1 does not bifurcate and R2 (arrowheads) shows an asymmetrical pattern of bifurcations in which the closest branch to R1 (R2a) does not secondarily bifurcate in contrast to the second branch of R2 (R2b). 2bif indicates the second bifurcation in only one of the branches of R2. (C–F) Examples of regenerates obtained following single ray ablation. (C) After 4 days of regeneration, in 57% of the fins, the distal ray blastema is in contact with the distal epidermis. (D) In these cases, the regenerates later form a normal bifurcation with an internal interray space as in the control (χ², f.d.: 2, α: 0.05). (E) In 43% of the fins, 2 or 3 days after single ray ablation (space shown by the arrow), the epithelial tissue fills the entire space between R2 and R4. In these cases, ray regeneration is delayed. (F). These rays do not bifurcate (asterisk) or they seldom do it distally. b, blastema; e, epidermis. All scale bars represent 100 μm.

Regenerate of nonbifurcating ray R1 can form bifurcation when grafted in the interray tissue between bifurcating rays R9. (A) Detail of the R1 fragment 30 days after proximal grafting between the R9 rays. (B) Hematoxilin–eosin staining of a cross-section of a fin at the level of the graft (h), 30 days after grafting. e, epidermis; h, hemiray. (C) Picrosirius–hematoxilin staining of a cross-section of the graft showing that the epidermis of R1 (arrow) is still attached to the R1 lepidotrichia (l) 2 days after grafting. (D) Distal region of a segmented regenerate growing out of the fin (arrow) 1 month postoperation. (E) Transverse section of one hemiray of an R1–I9 heterotopical graft which has regenerated inside the interray tissue. The interray epidermis covering the graft (arrowhead) becomes thinner than the normal interray epidermis (asterisk) and resembles the epidermis of a ray. (F) Transverse section through the blastema of a 4 days regenerate developing from the grafted piece of R1 that was labeled with DiI prior grafting. DiI labeling is restricted to the blastema cells (b). In contrast, epithelial cells (e) and interray tissue are not labeled. (G). White cells (arrows) are restricted to the distal portion of the lateral long rays (R1–R4) of each lobe of the tail fin. After mounting the fins, these cells become orange and are easily distinguished from the more abundant melanocytes, which are black (arrowhead). R1 and R2, original first and second rays. (H) Detail of a regenerate obtained following amputation at the level of the R1–I9 graft. The regenerate is surrounded by the central interray tissue (I9) and presents white cells (arrow) characteristic of R1 and melanocytes (arrowhead), which are found in most of rays. (I) Detail of the distal margin a regenerated R1–I9 heterotopical graft. The graft regenerate shows a bifurcation and segmentation pattern similar to neighbor rays but it slightly overgrows the neighboring rays and presents the characteristic white cells of R1. Arrowheads show segmentation process in the bifurcated ray. Scale bars represent 25 (F), 33 (C), 50 (E), 100 (A, G, H), and 200 μm (B, D, I).

Regeneration following operations (c–f). (A) Detail of a regenerate obtained after making a hole in the first long ray (R1) of the tail fin. A regenerate forms from the proximal part of the hole and grows outwards (arrows) without healing with the distal stump (asterisk) of R1 generating an ectopic R1 (eR1) and an ectopic first interray (eI1) (see Fig. 1 C′); R2, original second ray; I1, original first interray. (B) Distal portion of R1, ectopic R1 (eR1), and R2 regenerates obtained after operation (c). Under this condition, R1 regenerates surrounded by two interrays. (C) Origin of an aborted bifurcation observed in an ectopic R1 regenerate (eR1) following cut of the fin. Similar transient bifurcations are observed in R1 after induction of an eR1. (D) Fused branches (arrows) of aborted bifurcations in R1 and eR1 (brackets). The rays continue to regenerate as in control. (E) Distal margin of a R9 to R3 graft regenerate. When a fragment of R9 is grafted in place of R3, its regenerate (arrow) is three to six segments longer than the original R9 (only 3–4 segments shorter than R2 and R4). (F) Distal margin of a regenerate obtained from a R3 fragment grafted in R3 position (R3*) reaching its original size. (G) Distal margin of a regenerate from a R8 fragment grafted in I9. Grafted R8 regenerates nearly as long as neighboring rays (R9) following cut of the fin at the level of the graft. (H) The patterns of the regenerate obtained from a grafted R8 and of the neighboring R9, following R8–I9 graftings, are similar. eR1, ectopic first ray; R1, original first ray; R2, original second ray; R4, original fourth ray. Scale bars represent 100 (A, C–D, G), 200 (B), and 250 μm(E–F, H).

(A) Normal expression pattern of msxD in the epidermis distal to the blastema in control fins. Note that msxD is expressed in the epidermis covering the ray and the interray (asterisks). (B) Following single ray ablation, msxD expression is restricted to the epidermis overlying the distal blastema of the regenerating ray. (C) Cross-section of a fin similar to that shown in (B) at the level of the ray blastema, showing msxD expression in the epithelial tissue surrounding the blastema (asterisk). The lateral epidermis (e) does not express this gene at this level. (D) msxA expression is similar to that of msxD under the same experimental conditions. (E) When ablation of R3 is preceded by the amputation of R1 and R2 a few days before, the epidermis covering R3 (e) remains in contact with neighboring R2 blastema. (F). Expression of msxD under this experimental condition (h) when the ray blastema has reached the distal epidermis (arrow). Regeneration of tissue without ray is faster than normal regeneration but stops at the level of the neighboring blastemas (arrowhead) because it needs two neighboring rays to occur. Control neighboring ray blastemas do not induce msxD expression in the interray (asterisk). (G) During growth of the R1 grafted piece to the I9 interray, msxD expression in the overlying epidermis of the host is only induced at the level of the graft blastema (arrowhead). (H) During normal regeneration, bmp4 is expressed in the distal part of the regenerate (arrow). No expression is observed in the interray region (asterisk). (I) Longitudinal sections of ray shows that bmp4 expression is restricted to the distal blastema (arrow). (J) During single ray regeneration, bmp4 expression is induced in the region occupied by the distal ray blastema (arrow). (K) Cross-section of a single ray regenerate showing that bmp4 is expressed in the distal ray blastema (arrow). There is no expression in the interray blastema and covering epidermis (e). (L) During single ray regeneration, shh is expressed in the basal layer of the epidermis covering the proximal blastema (arrow). Note that shh is expressed in two subdomains (asterisks) in one of the hemirays, suggesting that a bifurcation is about to occur. Scale bars represent 25 (I), 50 (C, K), 100 (A–B, D, F–G, J, L), 200 (E), and 250 μm (H). R1, R2, R3, R4, and R5, original first, second, third, fourth, and fifth ray, respectively; e, epidermis; b, ray blastema.