sparse-like (kitlga^tc244b) caudal fins fail to regenerate melanocytes during the initial 7 days post amputation (dpa).

A, B, C. Wild-type, sparse-like (kitlga^tc244b), and sparse (kita^b5) fins were amputated and allowed to regenerate at 25°C. A′, B′, C′. No new melanocytes are observed during the first 3 dpa, irrespective of genotype. A′′. By 5 dpa, wild-type fins have newly differentiated kit-dependent melanocytes (grey arrowheads) in the regenerate. A′′′. By 7 dpa, more melanocytes emerge and organize into stripes. B′′-B′′′. kitlga^tc244b fins fail to produce new melanocytes during the first 7 dpa. C′′-C′′′. kita^b5 fins fail to produce new melanocytes during the first 7 dpa. The few melanocytes near the amputation plane (dashed-line) in the regenerate of kitlga^tc244b and kita^b5 fins are previously differentiated melanocytes (black arrowheads) that are drawn from the stump into the regenerated tissue as it grows. Scale bar = 500 mm.

Multiple clone lineages are competent to support melanocyte rescue in the regenerating fins of kitlga^tc244b mutants following expression of kitlga.

A-A′′′. Skin clone that has not been subjected to heat shock shows no rescued melanocytes associated with the kitlga clone. B-B′′′. Dermal fibroblasts robustly support melanocyte rescue as a result of expressing kitlga. C-E. Skin (C-C′′′), osteoblasts (D-D′′′), and vasculature (E-E′′′) can support melanocyte rescue, but with a greater degree of variability of the strength of rescue as compared to dermal fibroblasts. F-F′′′. Lateral line clones (black arrow) could be distinguished from intra-ray glia by their neuromasts (asterisk) and were never able to rescue melanocytes. Strong rescue is shown in B′′′, C′′′, and E′′′. Weak rescue is shown in D′′′. Red dashed lines indicate the amputation plane. Yellow rectangles in A and A′ indicate insets magnified in greater detail in A′′ and A′′′, respectively. Grey arrowheads indicate newly differentiated kit-dependent melanocytes. Black arrowheads indicate previously differentiated melanocytes drawn into the regenerate from the stump. Scale bar = 250 mm.

Expression of kitlga promotes differentiation of new melanocytes in the stump.

A-A′. At the time of amputation and prior to induction of kitlga, melanocytes are well organized and form the proximal portion of the dorsal melanocyte stripe of the caudal fin (yellow trapezoid). B-B′. Following 7 days of kitlga expression, new melanocytes are visible both in the regenerate (brackets) as well as in the stump (grey arrowheads) in association with the dermal fibroblast clone. Red dashed lines indicate the amputation plane. Scale bar = 250 mm.

Skin clones show diminished ability to rescue melanocytes in xanthophores stripe regions of the regenerate.

A-A′. Dermal fibroblasts strongly rescue melanocytes irrespective of where the clone occurs in the fin. B-C. Skin clones within the melanocyte stripe (B-B′) show more robust melanocyte rescue than skin clones that occur in a xanthophores stripe (C-C′). D. Dermal fibroblast, osteoblast, and skin clones were scored for the quality of melanocyte regeneration relative to their occurrence in a presumptive melanocyte stripe or xanthophore stripe. A single clone (as summarized in figure 4) may be scored as two clones if it occurs in both a xanthophore and melanocyte stripe region. Only skin clones showed a statistically significant difference in strength of rescue relative to the region in which the clone was regenerated (chi-squared 3×2 test, p value = 0.035.) Red dashed lines indicate the amputation plane. Grey arrowheads indicate newly differentiated kit-dependent melanocytes. Black arrowheads indicate previously differentiated kit-independent melanocytes. Scale bar = 250 mm.

Melanocyte rescue occasionally occurs at a distance from the clone boundary in skin clones.

A-A′′. While the majority of clones expressing kitlga only cause melanocyte rescue within the clone boundary (yellow line), some skin clones promote additional melanocyte rescue at a distance >1 fin ray (individual grey arrowheads. A′. Rogue labeled epidermal cells (arrows) along the edges of clonal boundaries may separate from the bulk of the clone mass and intercalate with unlabeled epidermal populations, supporting limited melanocyte regeneration at a distance. B. Quantification of the ability of different clone lineages to rescue melanocytes at significant distances from the clonal boundary. Brackets show the proximal-distal extent of melanocyte rescue within the clone. Black arrowheads indicate previously differentiated kit-independent melanocytes from the stump. Grey arrowheads indicate newly differentiated melanocytes. White arrows indicate GFP+ cells that are outside of the contiguous regenerated skin clone. Red dashed lines indicate the amputation plane. Scale bar = 250 mm.

Variability of melanocyte regeneration from skin clones expressing kitlga. A-A′. Representative skin clone categorized as strongly supporting melanocyte regeneration. B-B′. Representative skin clone categorized as weakly supporting melanocyte regeneration. C. Summary of the percentage of clonal area that contained melanocytes within skin clones. Thresholds were set using ImageJ to mask dark pixels (melanocytes) and the percentage of the clonal region masked was calculated. Averages and standard deviations for the two subgroups (strong and weak) are shown. Specific calculated percentages are shown for A′ and B′.