Contribution of <i>sox9b</i> to pigment cell formation in medaka fish

SoxE-type transcription factors, Sox10 and Sox9, are key regulators of the development of neural crest cells. Sox10 specifies pigment cell, glial, and neuronal lineages, whereas Sox9 is reportedly closely associated with skeletogenic lineages in the head, but its involvement in pigment cell formation has not been investigated genetically. Thus, it is not fully understood whether or how distinctly these genes as well as their paralogs in teleosts are subfunctionalized. We have previously shown using the medaka fish Oryzias latipes that pigment cell formation is severely affected by the loss of sox10a, yet unaffected by the loss of sox10b. Here we aimed to determine whether Sox9 is involved in the specification of pigment cell lineage. The sox9b homozygous mutation did not affect pigment cell formation, despite lethality at the early larval stages. By using sox10a, sox10b, and sox9b mutations, compound mutants were established for the sox9b and sox10 genes and pigment cell phenotypes were analyzed. Simultaneous loss of sox9b and sox10a resulted in the complete absence of melanophores and xanthophores from hatchlings and severely defective iridophore formation, as has been previously shown for sox10a −/−; sox10b −/− double mutants, indicating that Sox9b as well as Sox10b functions redundantly with Sox10a in pigment cell development. Notably, leucophores were present in sox9b −/−; sox10a −/− and sox10a −/−; sox10b −/− double mutants, but their numbers were significantly reduced in the sox9b −/−; sox10a −/− mutants. These findings highlight that Sox9b is involved in pigment cell formation, and plays a more critical role in leucophore development than Sox10b.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish.

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type

Leucophore formation does not require Sox10 function but is repressed by Sox10 and Sox5 (A-H) 9 dpf.

<p>Dorsal views. Leucophores (orange) are distributed along the dorsal surface throughout the anterioposterior axis of WT, as scattered individual cells in the head, and along the midline in the body (A). As the number of functional <i>sox10</i> alleles decreases, leucophore numbers on the head are progressively increased (A-D, I, <i>p</i><0.05 by Kruskal-Wallis test), whereas in the body they are progressively decreased (J, <i>p</i><0.05 by Kruskal-Wallis test). (I, J) Comparison between genotypes was performed by Kruskal-Wallis test with SDCF post hoc test. Statistical significance (<i>p</i><0.05) was detected between the groups shown by letters (a, b or c) above each scatter plots. <i>sox10a</i>^<i>-/-</i> mutants have some ectopic leucophores on dorsal trunk (B, arrowheads). In <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>+/-</i> mutants, trunk leucophores are restricted to the anterior dorsal region (C). In <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>-/-</i> mutants, leucophores are largely restricted to the head (D). Intriguingly, the total number of leucophores in the whole body is not statistically different among these genotypes (K, <i>p</i> = 0.51 by Kruskal-Wallis test), although numbers do become much more variable after loss of <i>sox10a</i>. (I-K) WT, n = 19; <i>sox10b</i>^<i>-/-</i>, n = 16; <i>sox10a</i>^<i>-/-</i>, n = 27; <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>+/-</i>, n = 22; <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>-/-</i>, n = 18. In <i>sox5</i>^<i>-/-</i> mutants, leucophores are formed in excess (L) and scatter more laterally (E). In <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutants (F), there is a further increase in leucophores, which are scattered laterally like in <i>sox5</i>^<i>-/-</i> mutants (E). Abundant leucophores (greater than in <i>sox5</i>^<i>-/-</i> mutants; L) are observed in <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>+/-</i><i>;sox5</i>^<i>-/-</i> mutants, but these are more scarce in the body from posterior trunk backwards (G). In <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutants, most leucophores are located on the head, but with some in the anterior trunk (H). (L) WT, n = 12; <i>sox5</i>^<i>-/-</i>, n = 20; <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i>, n = 32; <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>+/-</i><i>;sox5</i>^<i>-/-</i>, n = 23; <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i>, n = 13. Comparison between the genotypes was performed by Kruskal-Wallis test with SDCF post hoc test. Statistical difference (<i>p</i><0.05) was detected between the groups shown by letters (a, b or c) above each scatter plots. (I-L) Bars show mean and error bar (s.d.). Scale bar: (A) 200 μm.</p

Cooperative function of Sox10 and Sox5 is required for xanthophore formation (A-H) 9 dpf hatchlings.

<p>Lateral views. UV images. (I-M) 5 dpf embryos. In WT (A), xanthophores (white arrows) are widely scattered under the skin, concentrated in the dorsal body. Xanthophores are reduced in <i>sox10a</i>^<i>-/-</i> mutants (B), and further reduced in <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>+/-</i> mutants while a few residual cells remain in the anterior body (C). <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>-/-</i> mutants completely lack xanthophores (D). Loss of <i>sox5</i> function results in complete absence of xanthophores, regardless of <i>sox10</i> mutant status (E-H). Xanthoblasts immunostained with anti-Sox5 antibody (I-L) were quantified in the body. (M) Specific signals cannot be detected in <i>sox5</i>^<i>-/-</i> embryos. <i>sox5</i>^<i>+/-</i> embryos (J) have significantly fewer Sox5-positive xanthoblasts than WT (I) (N, WT, n = 12; <i>sox5</i>^<i>+/-</i>, n = 8, *<i>p</i><0.05). Further reduction is observed in <i>sox10a</i>^<i>-/-</i> mutants (K) (N, n = 26). The number in <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>+/-</i> mutants (L) (N, n = 19) is comparable to <i>sox10a</i>^<i>-/-</i> embryos (<i>p</i> = 0.288). Comparison between the genotypes was performed by Kruskal-Wallis test with Steel-Dwass-Critchlow-Fligner (SDCF) post hoc test. The compound mutant of <i>sox10a</i>^<i>+/-</i><i>;sox10b</i>^<i>-/-</i> results in reduced numbers of Sox5-positive xanthoblasts compared to WT (O, WT, n = 16; <i>sox10a</i>^<i>+/-</i><i>;sox10b</i>^<i>-/-</i>, n = 16). In <i>sox10a</i>^<i>+/-</i><i>;sox10b</i>^<i>-/-</i><i>;sox5</i>^<i>+/-</i> mutants (n = 13), the number of Sox5-positive cells is reduced compared to both <i>sox5</i>^<i>+/-</i> (n = 15) and <i>sox10a</i>^<i>+/-</i><i>;sox10b</i>^<i>-/-</i> mutants (O). Comparison between the genotypes was performed by Kruskal-Wallis test with SDCF post hoc test. **, <i>p</i><0.05. (N, O) Bars show mean and error bar (s.d.). Scale bars: (A, I) 200 μm.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 1

<p><b>Medaka <i>sox10a</i> and <i>sox10b</i> are expressed in neural crest and their loss of function affects pigment cell development</b>. (A-E) <i>sox10a</i>. (F-J) <i>sox10b</i>. (E, J) Transverse sections. (A, B, C, D, F, G, H, I) Lateral views. (A’, F’) Dorsal views. (K-O) 9 dpf. Lateral views. (A, F) At 6-somite stage (6s, 34 hpf), both <i>sox10a</i> and <i>sox10b</i> are expressed in premigratory neural crest cells (black arrows). (B-E, G-J) At 24-somite stage (24s, 58 hpf), <i>sox10a-</i> and <i>sox10b-</i> expressing cells are found in glial precursors on the posterior lateral line nerve (black arrowheads in C, E, H and J) and migrating neural crest cells between neural tube and somite (medial pathway, white arrows in D, E, I and J) and between epidermis and somite (lateral pathway, white arrowheads in C, E, H and J). (B, G) In head, both <i>sox10a</i> and <i>sox10b</i> are expressed in otic vesicle (ov) and cranial ganglia (g). (C, H) Close-up images of lateral trunk surface. (D, I) Close-up images of migrating neural crest through medial pathway. (K, L) WT and <i>sox10b</i>^<i>-/-</i> hatchlings show a normal pigment pattern, composed of dorsal stripe (DS), lateral stripe (LS), ventral stripe (VS) and yolk sac cluster (YSC). (M) In <i>sox10a</i>^<i>-/-</i> mutants, melanocytes in LS (*) and in posterior side of VS (from caudal end of yolk sac to tail, **) are severely reduced. (N) <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>+/-</i> mutants retain melanocytes only in the head and the anterior part of the body. (O) <i>sox10a</i>^<i>-/-</i><i>;sox10b</i>^<i>-/-</i> mutants lack all chromatophores, except that leucophores on the head are retained (red arrows, see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.g004" target="_blank">Fig 4D</a>). Scale Bars: (A) 200 μm, also applied to A’, F and F’; (B) 200 μm, also applied to G; (E) 20 μm, also applied to J; (K) 250μm, also applied to L, M, N and O.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 2

<p><b>Reductions in melanoblast and iridoblast specification in <i>sox10a</i> mutants were partially rescued by loss of <i>sox5</i></b>. (A-D) <i>mitfa</i>. (E-H) <i>dct</i>. (I-L) <i>ltk</i>. (A, E, I) WT. (B, F, J) <i>sox5</i>^<i>-/-</i>. (C, G, K) <i>sox10a</i>^<i>-/-</i>. (D, H, L) <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i>. (M-O) Quantitation of number of cells expressing each fate marker in the whole embryonic body (M, N) and in the lateral patches (O). (A-H) Lateral views. (I-L) Dorsal views. (A-H) 30-somite stage (30s, 64 hpf). (I-L) 34-somite stage (34s, 74 hpf). The box in (A) indicates the enlarged region (anterior trunk) in insets. Melanoblasts, defined by expression of <i>mitfa</i> and <i>dct</i>, are unaltered in the <i>sox5</i>^-/- mutant (B, F) as compared with WT (A, E). Expression of <i>mitfa</i> and <i>dct</i> are both dramatically reduced in <i>sox10a</i>^<i>-/-</i> mutants (C, G), but substantially recovered in <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutants (D, H). The number of <i>mitfa</i> (M, <i>mitfa</i>^<i>+</i>)- or <i>dct</i> (N, <i>dct</i>^<i>+</i>)- expressing cells is not different between WT (<i>mitfa</i>, n = 9; <i>dct</i>, n = 14) and <i>sox5</i>^<i>-/-</i> mutants (<i>mitfa</i>, n = 15; <i>dct</i>, n = 15). <i>p-</i>values are <i>p</i> = 0.78 (<i>mitfa</i>) and <i>p</i> = 0.52 (<i>dct</i>). <i>sox10a</i>^<i>-/-</i> mutants have significantly fewer of those cells (<i>mitfa</i>, n = 13; <i>dct</i>, n = 12) than WT (<i>p</i><0.05 (m<i>itfa</i>) and <i>p</i><0.05 (<i>dct</i>)). In the <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> double mutants (<i>mitfa</i>, n = 10; <i>dct</i>, n = 13), cell counts are significantly increased as compared with <i>sox10a</i>^<i>-/-</i> single mutant (*, <i>p</i><0.05). <i>p</i>-values were calculated by Mann-Whitney test. Iridoblasts, as evidenced by <i>ltk</i> expression, in WT (I), <i>sox5</i>^<i>-/-</i> mutant (J), <i>sox10a</i>^<i>-/-</i> mutant (K) and <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutant (L) are shown for the lateral patches, a region of concentrated iridophores dorsolateral to the developing gut in the anterior trunk. The number of <i>ltk-</i>expressing cells (O, <i>ltk</i>^<i>+</i>) is indistinguishable between WT (I, n = 20) and the <i>sox5</i>^<i>-/-</i> mutant (J, n = 10) (<i>p =</i> 0.98). In the <i>sox10a</i>^<i>-/-</i> mutant (K, n = 18), the number is significantly decreased as compared with WT (I). Again, iridoblasts cell counts between the <i>sox10a</i>^<i>-/-</i> mutant and the <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutant (L, n = 23) is statistically different (*, <i>p</i><0.05), showing partial rescue in the double homozygote. <i>p</i>-values were calculated by Mann-Whitney test. (M-O) Bars show mean and error bar (s.d.). Scale bars: (A) 200 μm, (I) 40 μm.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 8

<p>Based on a new working model for pigment cell development in medaka and zebrafish based on our previous work in zebrafish [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.ref046" target="_blank">46</a>], we propose that a chromatoblast, a multipotent common progenitor of chromatophores, gives rise to three pigment cell types, melanocytes (M), iridophores (I) and xanthophores (X) in zebrafish, development of which is all dependent on SoxE with antagonistic modulation by Sox5. Likewise in medaka, Sox5 counteracts SoxE in the melanocyte and iridophore lineages and, by extension, presumably in formation of xanthophore/leucophore progenitor (XL). In an additional step, an evolutionary novelty of medaka, Sox5 likely functions in collaboration with Sox10 to promote xanthophore specification, but to repress leucophore (L) formation, from a novel shared progenitor.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 6

<p><b>Role of Sox5 in adult xanthophore development in medaka remains opposite to that in zebrafish</b>. (A, B) Medaka. 6 mpf. (D, E) Zebrafish. 2 mpf. (D’, E’) Schematics of stripe pattern. (D”, E”) Enlarged images of X0 interstripe. (F, G) Zebrafish. 1.5 years old. In WT medaka (A), melanocytes, xanthophores (yellow arrowhead) and leucophores (white arrowhead) are scattered on the body surface. <i>sox5</i>^<i>-/-</i> mutant medaka (B) have fewer xanthophores and more leucophores than WT (C, Xan and Leu, *<i>p</i><0.05 by Mann-Whitney test; WT, n = 5; <i>sox5</i>^<i>-/-</i>, n = 5; Bars show mean and error bar (s.d.)). Pigment cells were counted from a 1 mm² area on the dorsal body surface. The melanocyte numbers were not significantly different between WT and <i>sox5</i>^<i>-/-</i> (C, Mel, <i>p</i> = 0.1 by Mann-Whitney test). The adult pigment pattern of WT zebrafish is composed of 5 melanocyte stripes (2D, 1D, 1V, 2V, 3V) and xanthophore interstripes (X1D, X0, X1V, X2V) (D, D’). Zebrafish <i>sox5</i>^<i>-/-</i> mutants lack two ventral interstripes (2V and 3V) (E, E’), and thus have fewer stripes than WT. This is the case regardless of sex (F, female; G, male) after the mutant fish get larger and older than 1.5 years. The <i>sox5</i>^<i>-/-</i> zebrafish mutants have wider X1D and X0 (two-way arrow in D” and E”) interstripes and larger numbers of xanthophores in these interstripes than WT. (H) Scatter plot of stripe width or pigment cell numbers in each stripe, comparing <i>sox5</i>^<i>-/-</i> and their WT siblings. X axis shows the standard body length of zebrafish examined. Analysis of covariance was performed to examine the differences in width or cell numbers between WT and <i>sox5</i>^<i>-/-</i> mutants, by using standard length as a covariate. The <i>p</i> values are as follows; width X1D (<i>p</i><0.05), 1D (<i>p</i><0.05), X0 (<i>p</i><0.05), cell number X1D (<i>p</i><0.05), 1D (<i>p</i> = 0.985), X0 (<i>p</i><0.05). The width and number of xanthophore in the xanthophore stripes (X0, X1D) in <i>sox5</i>^<i>-/-</i> mutants (black boxes) show significant increase compared with WT siblings (white boxes). The number of melanocyte in the 1D stripe is comparable between WT (white boxes) and <i>sox5</i>^<i>-/-</i> mutant (black boxes), but the width is slightly but significantly different (<i>p</i><0.05). Scale bar: (A) 200 μm, (D) 3 mm, (D”) 200 μm, (F) 3 mm.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 5

<p><b>Sox10-mediated pigment cell formation is modulated by Sox5 in zebrafish</b>. (A, B, C, E) Swim bladder inflation stage (10 dpf). Lateral views. (C, E) UV images. (D, F) 24 dpf. Lateral views. (G, I-O) 4 dpf. (G) Dorsolateral view. (I) Lateral view. (J-O) Fluorescing xanthophores. Dorsal views anterior to the left. The <i>sox5</i> mutant is indistinguishable from WT, exhibiting four stripes of melanocytes (A, B) and having fluorescing xanthophores (C, E) and <i>gch</i>-expressing xanthoblasts (D, F). Melanocytes are almost completely absent from <i>sox10</i>^{<i>baz1/baz1</i>} mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8B Fig</a>), but are partially recovered in <i>sox10</i>^{<i>baz1/baz1</i>} mutants that have also lost <i>sox5</i> WT allele(s) (G, H). The ratio of the embryos without melanocytes or with more than one melanocytes was compared among genotypes by Chi-squared test (*<i>p</i><0.05). A few xanthophores develop on surface of the head in <i>sox10</i>^{<i>baz1/baz1</i>} mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8E, S8I and S8J Fig</a>), and reduction of <i>sox5</i> is likely to elevate xanthophore formation (K, L). Whereas <i>sox10</i>^<i>t3/t3</i> mutant almost completely lacks xanthophores (M), a few xanthophores are rescued as the <i>sox5</i> WT allele(s) are reduced (N, O). The counts are shown for xanthophores on the <i>t3</i> background (P). Comparison between the genotypes was performed by Kruskal-Wallis test with SDCF post hoc test. **<i>p</i><0.05. A substantial number of iridophores are formed in <i>sox10</i>^{<i>baz1/baz1</i>} mutants (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8G and S8H Fig</a>), and the counts are not significantly altered with reduction of the <i>sox5</i> WT allele(s) (Q, <i>p</i> = 0.775 by Kruskal-Wallis test). Iridophores are almost completely lost in <i>sox10</i>^<i>t3/t3</i> mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8I Fig</a>), but are partially recovered in <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/-</i> and <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>-/-</i> mutants (R **<i>p</i><0.05 by Kruskal-Wallis test with SDCF post hoc test). (H) <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/+</i>, n = 22; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/-</i>, n = 34; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>-/-</i>, n = 33. (Q) <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/+</i>, n = 46; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/-</i>, n = 73; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>-/-</i>, n = 47. (P) <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/+</i>, n = 7; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/-</i>, n = 19; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>-/-</i>, n = 16. (R) <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/+</i>, n = 24; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/-</i>, n = 48; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>-/-</i>, n = 40. (H, P-R) Bars show mean and error bar (s.d.). Arrowheads point to weakly melanised cells (G) and at the corresponding position of the head (I-O). Scale bars: (A, D) 200 μm.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 7

<p><b>Misexpression of Sox5 can reverse the xanthophore vs leucophore fate choice in medaka</b>. (A) Transgenic construct. (B-C”) 3 dpf. (B, C) Bright field. (B’, C’) Dsred. (B”, C”) Venus. (D-G) 7 dpf. Dorsal views. (D’, E’) lateral views. UV images. (F’, G’) Magnified images of the head in (F, G). (B, D) <i>sox5</i>^<i>-/-</i>;<i>Tg(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i><i>)</i>. (C, E) <i>sox5</i>^<i>-/-</i><i>;Tg(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i><i>)</i> injected with <i>cre</i> mRNA. (F) WT(<i>sox5</i>^<i>+/+</i>);<i>Tg(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i><i>);Tg(hsp70</i>:<i>cre)</i> without heat shock. (G) WT;<i>Tg(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i><i>);Tg(hsp70</i>:<i>cre)</i> with heat shock. A transgenic fish in which the <i>pax7a</i> promoter drives <i>sox5-2A-venus</i> expression once the upstream <i>dsred</i> region is excised at the flanking loxp sites by Cre recombinase <i>TgBAC(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i>) was used for Sox5 misexpression in a shared xanthophore/leucophore progenitor. In the absence of <i>cre</i> mRNA, <i>sox5</i>^<i>-/-</i><i>;Tg</i> embryos (<i>sox5</i>^<i>-/-</i>;<i>TgBAC(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i>)) were strongly expressing Dsred (B-B”, n = 8). When <i>cre</i> mRNA was injected in the 1-cell <i>sox5</i>^<i>-/-</i><i>;Tg</i> embryos, they became positive for Venus (C-C”, n = 7). Venus-expressing animals showed rescued xanthophore and leucophore phenotypes in <i>sox5</i>^<i>-/-</i> mutants (D, E). When <i>sox5</i>^<i>+/+</i> WT embryos obtained from crossing the transgenic fish Nagoya (<i>sox5</i>^<i>+/+</i>);<i>TgBAC(pax7a</i>:<i>loxp-dsred-loxp-sox5</i>^<i>WT</i><i>)</i> with <i>Tg(hsp70</i>:<i>cre)</i> were heat shocked, Dsred became barely detectable. Leucophores (Leu) were significantly reduced in the hatchlings treated with heat shock (+) compared with those without heat shock (-) whereas melanocyte (Mel) formation was not altered by heat shock (F-H). (H) Bars show mean and error bar (s.d.). Comparison between with and without heat shock was performed by paired t-test (*<i>p</i><0.0001).</p