Fig. 2

Melanogenesis in cls mutants resulting from expression of lamprey SoxE sequences inserted into pCS2+ vectors. (A-E) 96 hpf zebrafish larvae. (A) wildtype. (B) cls; RPE, retinal pigment epithelium. Expression of PmSoxE1 (C), PmSoxE2 (D), and PmSoxE3 (E) in cls mutants. (F) RT-PCR of PmSoxE2-injected cls mutants. mitfa expression in a cls embryo resulting from PmSoxE2 injection (lane 1) is absent in the control cls embryo (lane 2). β-actin internal control (lanes 3-4). (G) box and whisker plot indicating the number of melanophores in zebrafish injected with Drsox10 (positive control), lamprey PmSoxE1, PmSoxE2, and PmSoxE3, and cls embryos mock-injected with empty pCS2 vector as a negative control. Whiskers represent standard error. Each dot above the positive whiskers represents individual data points of the first quartile. Sample sizes are indicated under each bar along the X-axis. Comparison of means among samples all resulted in statistical significance (ANOVA, p-value<0.05). (H-J) in situ hybridization using lamprey SoxE probes in zebrafish wildtype embryos injected with either lamprey SoxE1 (H), SoxE2 (I), or SoxE3 (J) reveals similar mRNA expression, suggesting lamprey SoxE constructs injected into zebrafish embryos yields a comparable pattern and expression level irrespective of the lamprey SoxE gene sequence. Results shown are representative of 19/58 (PmSoxE1), 45/229 (PmSoxE2), and 90/356 (PmSoxE3) injections of lamprey constructs into early cleavage stage wildtype zebrafish embryos. vh, ventral horn; ys, yolk sac; ye, yolk extension; ds, dorsal stripe; ls, lateral stripe; vs, ventral stripe. Arrows point to melanophores, arrowheads point to iridophores. Orientation: anterior facing left.