A combined zebrafish and yeast approach to probe chemical-genetic interactions in melanocyte pigmentation. (A) Overview of work flow. Phenotypic screening in zebrafish informs as to the developmental and physiological target pathway. Small molecules that promote similar phenotypes in zebrafish are then tested in genome-wide chemical-genetic screens in yeast to reveal potential target genetic pathways. Conserved target pathways are then directly re-tested for chemical-genetic interactions in the zebrafish system using genetic mutant lines, morpholino oligonucleotides or small molecules known to impinge on the same target. (B) Phenotypic screening of a >1500-compound yeast-bioactive library (S.D., J.W., M. Spitzer and M.T., unpublished data) and the Sigma LOPAC library for altered pigmentation in zebrafish embryos revealed a copper-metabolism phenotype characterized by hypopigmentation, an expanded hindbrain and an undulating notochord (Mendelsohn et al., 2006). Representative phenotype caused by 10 μM JFD00692 was rescued by treatment with exogenous copper chloride (5 μM or 15 μM) during development. (C) Examples of the copper-metabolism phenotype induced by some of the compounds identified in the screen.

Yeast chemical-genetic profiles of small molecules that affect copper metabolism. (A) Copper-pathway mutant strains that were specifically sensitive or resistant to U0126, neocuproine or the CM subset compounds. At least two concentrations for each compound were used for analysis. Genes with copper-dependent functions were compiled from Saccharomyces Genome Database (SGD, http://www.yeastgenome.org); this list includes copper-containing proteins and known factors in copper and/or iron transport and homeostasis. Color scale indicates degrees of sensitivity (red) or resistance (green); white indicates no data available in a given experiment. (B) Yeast chemical-genetic profiles reveal shared and distinct modes of action for CM subset compounds. A heatmap of GO biological process categories that correlated with sensitivity (red) or resistance (green) to different compounds is shown. Two different concentrations of each compound were screened. Scores for each GO category that contained at least four genes were calculated as the average z-score of all mutants in the category. GO categories contained in the heatmap represent the top 2% of affected categories. GO categories with overlapping gene sets are clustered (see also Table 2).

Phenotype elicited by U0126 treatment in zebrafish embryos. (A) U0126 phenocopy of neocuproine (neo) in 2-day-old zebrafish embryos. As a control, the more selective MEK inhibitor PD0325901 did not affect pigmentation but instead prevented development of posterior features. (B) U0126 caused blood loss as detected by o-dianisidine (red) staining. (C) U0126 and neocuproine complex with copper, as indicated by color change upon addition to aqueous copper chloride solution. (D) Inhibition of MEK activity in vivo. Lysates from 12-hpf zebrafish embryos were assessed for phospho-ERK and phospho-MEK levels after treatment with PD0325901 (PD) and U0126 (U0). Note inhibitor treatment is known to increase phospho-MEK (Pratilas et al., 2009). A short (2-hour) treatment (asterisk) with PD0325901 also reduced phospho-ERK levels in zebrafish embryos, demonstrating the elevated potency of PD0325901. (E) U0126 (10 μM) treatments prevent pigmentation when administered before 21 hpf. When administered at 21 hpf or later, the embryos develop pigment at about 283 hpf, similar to untreated zebrafish embryos. U0126 treatments are each represented by a barline, and the color of the barline represents the color of the zebrafish melanocytes. The effects on pigmentation are reversible, and pigmentation can rapidly recover from U0126 treatment. For example, U0126-treated unpigmented 33 hpf zebrafish embryos recover pigmentation 4 hours after shifting to fresh embryo medium (asterisk). These phenotypes are consistent with a role for U0126 in blocking the copper-dependent pigmentation enzyme, tyrosinase (Rawls and Johnson, 2000). (F) Electron microscopy of 2-day-old untreated and U0126-treated (5 μM) zebrafish embryos reveals smaller and less densely pigmented melanosomes in the latter.

Novel target pathways for the MEK inhibtior U0126. (A) Heat map of deletion strains that are sensitive (red) or resistant (green) to U0126, neocuproine and CI-1040. U0126 shares a chemical profile with neocuproine and with CI-1040, whereas neocuproine and CI-1040 do not share a common profile. (B) Neocuproine (yellow), U0126 (green) and CI-1040 (blue) treatment affect common and distinct biological processes. GO categories were examined for each of the deletion mutants that were affected by each compound (see Table 2). The genes in a specific GO category were compared with the number of strains actually affected by each compound and the P values determined. Only GO categories with more than four genes were examined and only P values <0.05 were considered.

Identification of two new gene-nutrient interactions in melanocyte pigmentation. Morpholino oligonucleotides (1–3 pg) directed against ap1s1 and ap3s2 result in morphants with normal development and pigmentation. Higher concentrations of ap1s1-MO have been reported to cause pigmentation phenotypes (Montpetit et al., 2008). Treatment with 5 μM neocuproine caused a copper-deficiency phenotype in all animals that could be rescued with the addition of 15 μM copper chloride. At a concentration of 1.0 μM neocuproine, copper limitation caused a loss of pigmentation in the ap3s2 morphants compared with control-treated embryos, and a severe loss of pigmentation at 2 μM neocuproine, compared with mild (1 μM) to moderate (2 μM) loss of pigmentation in control animals. A milder pigmentation phenotype was also visible in the ap1s1 morphants treated with 1 μM neocuproine. Experiments were repeated multiple times (more than three times for each morpholino; n>20 per treatment), and similar results could be obtained with an additional splice-site MO designed to target each gene (see Methods). (B) Simplified schematic of gene-copper interactions in a zebrafish melanocyte. Partial reduction of AP3 complex activity (such as in the ap3s2 morphant; represented by a dotted line) retains sufficient AP3 activity for melanosome pigmentation in optimal-copper nutrient conditions, and the melanocyte is darkly pigmented. Likewise, melanocytes with normal AP3 activity in suboptimal-copper nutrient conditions retain the ability to generate pigment. By contrast, in low-copper nutrient conditions, reduced AP3 function is no longer sufficient for promoting proper melanosome maturation, and the melanocytes are hypopigmented. Cu, copper; E, endosomal bodies; G, trans-Golgi network; M, melanosomes.