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

<p>A-A’’. While the majority of clones expressing <i>kitlga</i> 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.</p

Summary of melanocyte rescue by pt2-hsp70l><i>kitlga</i> clones.

<p>A. The majority of clones failed to rescue <i>kit</i>-dependent melanocytes in the absence of heat shock induced <i>kitlga</i> expression. Strong rescue was never observed and only 16% of uninduced clones showed weak rescue. B. Following heat shock induction of <i>kitlga</i>, variable levels of melanocyte rescue were observed depending on the labeled lineage. Dermal fibroblast showed the clearest effect of strongest rescue. Skin, osteoblast, vascualure, xanthophore/melanocyte, and resident blood showed more variable and weaker rescue. Lateral line and iridiphore clones were unable to rescue melanocytes during regeneration.</p

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

<p>A-A’’’. Skin clone that has not been subjected to heat shock shows no rescued melanocytes associated with the <i>kitlga</i> clone. B-B’’’. Dermal fibroblasts robustly support melanocyte rescue as a result of expressing <i>kitlga.</i> 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 <i>kit</i>-dependent melanocytes. Black arrowheads indicate previously differentiated melanocytes drawn into the regenerate from the stump. Scale bar  =  250 mm.</p

Expression of <i>kitlga</i> promotes differentiation of new melanocytes in the stump.

<p>A-A’. At the time of amputation and prior to induction of <i>kitlga</i>, 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 <i>kitlga</i> 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.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102317#pone-0102317-g004" target="_blank">figure 4</a>) 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 <i>kit</i>-dependent melanocytes. Black arrowheads indicate previously differentiated <i>kit</i>-independent melanocytes. Scale bar  =  250 mm.</p

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

<p>A, B, C. Wild-type, <i>sparse-like</i> (<i>kitlga</i>^tc244b), and sparse (<i>kita</i>^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 <i>kit</i>-dependent melanocytes (grey arrowheads) in the regenerate. A’’’. By 7 dpa, more melanocytes emerge and organize into stripes. B’’-B’’’. <i>kitlga</i>^tc244b fins fail to produce new melanocytes during the first 7 dpa. C’’-C’’’. <i>kita</i>^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 <i>kitlga</i>^tc244b<i>and kita</i>^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.</p

Experimental procedure for clonal gene expression analysis of <i>kitlga.</i>

<p>A. The pt2-hsp70l><i>kitlga</i> transposon consists of a <i>Xenopus</i> ef1a promoter driving GFP for labeling and identification of clone lineages and a <i>Danio rerio</i> hsp70l promoter driving <i>kitlga</i> to allow heat inducible expression of <i>kitlga.</i> B. Clones are generated by co-injecting pT2-hsp70l><i>kitlga</i> DNA with transposase mRNA into 1 cell <i>sparse-like</i> (<i>kitlga</i>^tc244b) embryos. C. Larvae are screened initially at 3dpf to select for GFP+ integrants. D. GFP+ larvae are subsequently grown to adult stages when they are re-screened for caudal fin clones, shown in green. E. Fins are amputated, leaving behind labeled cells from the clone in the stump to regenerate Fish are then placed in a heat shock cabinet in order to provide daily heat shock pulses of 38°C. F. After 7 days of regeneration and heat shock induction of <i>kitlga</i>, fins are observed for rescue of melanocyte regeneration.</p

Iridophore enriched genes.

<p>RPKM values for genes expressed in iridophores at least 30-fold greater than melanocytes and RPE, and 100-fold greater than embryos.</p

Candidate control genes are differentially expressed.

<p>Selected genes indicative of pigment cell identity or shared functions are shown.</p

The guanine synthesis cycle is highly enriched in iridophores.

<p>Shown is a model for guanine production based on transcriptome data as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067801#pone-0067801-t006" target="_blank">Table 6</a>. Genes that are statistically enriched compared to melanocytes are shown in bold, those not statistically different are in normal font. The arrow thicknesses correspond to the fold changes in iridophores relative to whole embryos. Chemical structures are from the KEGG Compound database.</p