Fig. 6

Fig. 6

a 1 dpf CRISPR/Cas9 injected embryos with sgRNAs targeting the sox10 minimal promoter show DNA laddering (arrowheads) as compared to control injected embryos. b CRISPR/Cas9 injected embryos with sgRNAs targeting peak5 show complete disruption of the wild-type locus as evidenced by DNA laddering (arrowheads) compared to Cas9 injected embryo. c The stl792 allele contains a 200 bp deletion at the sox10 minimal promoter locus, which contains the TSS of sox10. d The stl538 allele contains 369 bp deletion at the peak5 locus, mainly disrupting the conserved region of peak5. e WISH for sox10 mRNA shows robust expression in wild-type 1 dpf embryos and f absent expression in stl729 homozygous mutant 1 dpf embryos. g Quantification of WISH expressing the percent of embryos displaying either “strong, “reduced,” or “absent” sox10 expression. p < 0.0001, Fisher’s exact test. h WISH for sox10 mRNA shows robust expression in wild-type 1 dpf embryos and i reduced expression in stl538 homozygous mutant 1 dpf embryos. j Quantification of WISH expressing the percent of embryos displaying either “strong, “reduced,” or “strongly reduced” sox10 expression. p = 0.0019, Fisher’s exact test. k Lateral view of one side of a wild-type Tg(BRAF^V600E);p53^−/− adult fish with 1 stripe break (arrowhead) compared to l a stl538 homozygous adult fish with 3 stripe breaks (arrowheads). m Quantification of the number of stripe breaks per fish. Each dot represents one fish. Wild-type fish exhibit a median of 1 stripe break per fish, IQR 0–2. stl538 homozygous fish exhibit a median of 4 stripe breaks per fish, IQR 3–8. *****p < 0.0001, Welch’s t-test. n Lateral view of one side of a wild-type Tg(BRAF^V600E);p53^−/− adult fish with 1 stripe break (arrowhead) compared to o a peak5^stl538 heterozygote with two stripe breaks (arrowheads), p a sox10min^stl792 heterozygote with 3 stripe breaks (arrowheads) and q a peak5^stl538;sox10min^stl792 trans-heterozygote with 5 stripe breaks (arrowheads). r Quantification of the number of stripe breaks per fish. Each dot represents one fish. Wild-type fish exhibit a median of 0 stripe breaks, IQR 0–3.25. peak5^stl538 heterozygotes exhibit a median of 3 stripe breaks, IQR 1.5–4. sox10min^stl792 heterozygotes exhibit a median of 5.5 stripe breaks per fish, IQR 3.75–8.25. peak5^stl538/+;sox10min^stl792/+ trans-heterozygotes exhibit a median of 9.5 stripe breaks per fish, IQR 7.75–12.75. *p < 0.05, **p = 0.001, ***p < 0.0002, Welch’s t-test. s In our model, peak5 activates sox10 expression in NCCs during development and then again in melanoma precursor cells, likely through dimeric SoxE TFBS. This leads to upregulation of neural crest-related genes and melanoma onset. tpeak5 is most likely not the only sox10 enhancer that regulates sox10 expression. Multiple elements within the sox10 super-enhancer may coordinate together to upregulate sox10 expression during melanoma initiation.