The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.

Phakomatosis pigmentovascularis is a diagnosis that denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement, including CNS disease, asymmetrical growth, and a predisposition to malignancy. Using a tight phenotypic group and high-depth nextgeneration sequencing of affected tissues, we discover here clonal mosaic variants in gene PTPN11 encoding SHP2 phosphatase as a cause of phakomatosis pigmentovascularis type III or spilorosea. Within an individual, the same variant is found in distinct pigmentary and vascular birthmarks and is undetectable in blood. We go on to show that the same variants can cause either the pigmentary or vascular phenotypes alone, and drive melanoma development within pigmentary lesions. Protein structure modeling highlights that although variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modeling of the missense variants confirms downstream MAPK pathway overactivation and widespread disruption of human endothelial cell angiogenesis. Importantly, patients with PTPN11 mosaicism theoretically risk passing on the variant to their children as the germline RASopathy Noonan syndrome with lentigines. These findings improve our understanding of the pathogenesis and biology of nevus spilus and capillary malformation syndromes, paving the way for better clinical management.

Melanocytes, the pigment-producing cells, are replenished from multiple stem cell niches in adult tissue. Although pigmentation traits are known risk factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs and lack key lineage markers with which to identify McSCs and study their function. Here we find that Tfap2b and a select set of target genes specify an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and is essential for McSC-dependent melanocyte regeneration. Fate mapping data reveal that tfap2b+ McSCs have multifate potential, and are the cells of origin for large patches of adult melanocytes, two other pigment cell types (iridophores and xanthophores), and nerve-associated cells. Hence, Tfap2b confers McSC identity in early development, distinguishing McSCs from other neural crest and pigment cell lineages, and retains multifate potential in the adult zebrafish.

Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models.

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.

SUMMARY Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical rele-vance for the activity of PRL3 in regenerating tissue and cancer. used. The starting translation and orientation of the ligand and the torsion angles of all rotatable bonds were set to random. The Autogrid grid point spacing was set at 0.2 A ˚ . The Autodock parameter file specified 50 Lamarckian genetic algorithm runs, 7,625,700 energy evaluations and a population size of 300. The lowest energy conformation of the most populous cluster was predicted to bind with a Ki of 878 nM (± 2.5 kcal/mol).

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.

Zebrafish have become an increasingly important model organism in the field of wound healing and regenerative medicine, due to their high regenerative capacity coupled with high-resolution imaging in living animals. In a recent study, we described multiple physical and chemical methods to induce notochord injury that led to highly specific transcriptional responses in notochord cellular subpopulations. The notochord is a critical embryonic structure that functions to shape and pattern the vertebrae and spinal column. Here, we describe precision needle injury, tail-notochord amputation, and chemical inhibition of caveolin that trigger a wound-specific wt 1b expression response in the notochord sheath cell subpopulation. We propose that these procedures can be used to study distinct cell populations that make up the cellular processes of notochord repair.

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.

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BACKGROUND. Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS. To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS. We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION. Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies. FUNDING. This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L’Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).

Background Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited and multi-disciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). Project To investigate the pathogenesis of 160 sporadic VMs in which known genetic causes had been excluded, sequencing of affected tissue DNA was undertaken using deep next generation sequencing with analysis optimised for detection of low mutant allele frequency. Results Mosaic activating variants in KRAS, NRAS, BRAF and MAP2K1 were identified, most commonly in AVM, both intracranial and extracranial. Transgenic zebrafish expressing BRAFV600E only in the vasculature recapitulated the human phenotype. Treatment of zebrafish with the BRAF inihibitor, vemurafinib, restored blood flow in AVM. Conclusions These findings reveal an important unifying cause of sporadic vascular malformations of different clinical types, and offer the potential of personalised medical treatment for affected individuals by repurposing of existing licensed cancer therapies.

Melanoma is the most lethal form of skin cancer with high mortality rates. Most melanoma cases have activating mutations in BRAF (V600E) and the selective inhibitors of BRAF(V600E) have been successfully used in patients. However, after initial tumor regression, the majority of patients develop drug resistance resulting in tumor regrowth. It is therefore important to understand the mechanisms underlying these processes. We have recently described the role of the master melanocyte transcription factor MITF in tumor growth, regression, and recurrence. Here, we describe protocols to study regression and recurrence in vivo, as well as for histology and immunohistochemistry, using a temperature-sensitive zebrafish model of human melanoma.

Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Ga subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11(R183C) and GNA11(Q209L) in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11(R183C) under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities.

s of papers presented at the 26th Genetics Society’s Mammalian Genetics and Development Workshop held at the Institute of Child Health, University College London on 20th November 2015. Edited by: NICHOLAS D.E. GREENE AND ANDREW J. COPP UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH Exploring the Role of FGFR2c in the Pathogenesis of Craniofacial Birth Defects KEVIN K.L LEE, EMMA PESKETT, PHILIP STANIER AND ERWIN PAUWS Developmental Biology & Cancer Programme Genetics & Genomics Medicine Programme, UCL Institute of Child Health, 30 Guilford Street, London

Dear Editor, Temperature-sensitive (ts) mutations have provided fundamental insight into our understanding of gene function in vivo. Although they are rare in higher organisms, temperature-sensitive mutations in the pigmentation pathways include tyrosinase ts mutations found in oculocutaneous albinism (OMIM: 606952), in Siamese cats and the Himalayan mouse (Giebel et al., 1991), and in kit in zebrafish (Rawls and Johnson, 2001). Microphthalmia-associated transcription factor (MITF) is the master melanocyte transcription factor that has critical functions in melanocyte development, melanocyte stem cell renewal, and the tanning response, and is an important drug target(Hsiao and Fisher, 2014). Given the importance of MITF, it is critical to develop laboratory animals that enable conditional control of MITF activity in different temporal, cellular, developmental, and disease contexts. Conditional or ts alleles in MITF have not been described in mammals. We (SLJ, JAL) have previously identified the first conditional MITF mutation (mitfavc7) in an animal in an ENU-based genetic screen in zebrafish, but the molecular mechanism underlying this mutation was unknown (Johnson et al., 2011). Here, we explain the molecular mechanism that enables the mitfavc7 mutation to confer conditional temperature-dependent control of MITF activity in zebrafish, and describe an unusual intron mutation that leads to aberrant splicing of wild-type and dominant negative splice variants at the restrictive temperatures. Most temperature-sensitive mutations affect exon sequences, but the mitfavc7 mutation is unusual because it is in an intron splice donor (t>a at position 39638 in mitfa genomic locus or position 2 in intron 6; Johnson et al., 2011). We collected wild-type and mitfavc7 mutant zebrafish embryos and grew the animals at 24, 26, 28.5, and 32°C. At higher temperatures, zebrafish embryos grow more rapidly, and to compensate for this, for all experiments, zebrafish embryos were carefully stage-matched. At 32°C, mitfavc7 zebrafish embryos lack all neural crest-derived melanocytes due to loss of mitfa activity (Figure 1A). At 24°C, wild-type and mitfavc7 zebrafish embryos develop similar numbers of clearly visible and pigmented melanocytes (Johnson et al., 2011), albeit with a delay in differentiation and/or cell size in the mitfavc7 mutant embryos at 2 days post-fertilization (Figure 1A). We examined the mitfa RNA encompassing exons 4–7 and found correct splicing occurs at low (permissive) temperatures along with aberrant splicing, while aberrant splice forms predominate with increasing temperature (Figure 1B; as described in the Appendix S1). Temperature-sensitive splicing at this locus had not previously been detected (Johnson et al., 2011), possibly due to PCR conditions. We cloned each of the splice forms (labeled a–c) and found that the aberrant splice forms include those that skip exons or retain introns (Figure 1C). Splice form 'a' includes introns 5 and 6 (mitfa+in5,6), form 'b' includes an in-frame intron 6 (mitfa+in6), and splice form 'c' leads to an in-frame deletion of exon 6 (mitfa∆ex6). At the highest temperature (32°C), almost no wild-type mitfa or mitfa+in6 was detectable, while there was strong expression of mitfa∆ex6. These results demonstrate that aberrant of the mitfa RNA correlates with the temperature sensitivity of the mitfavc7 mutation. Aberrant splice variants can have partial or neomorphic function. To establish whether the splice variants had activity, we cloned the splice variants under the control of the mitfa promoter and microinjected them into zebrafish null mitfa (the mitfaw2 (nacre) mutation, a premature stop in exon 3, in which there are no neural crest-derived melanocytes; Lister et al. 1999; Figure 1C). Expression of wild-type mitfa rescued the nacre mutation, and melanocytes were clearly visible at 5 dpf. In contrast, the mitfa∆ex6 isoform was unable to stimulate melanocyte development in nacre mutants. The mitfa+in6 splice variant was functional in this assay, albeit with significantly reduced activity compared to wild-type mitfa. In addition, the mitfa+in5,6 isoform demonstrated significantly reduced activity compared to mitfa and compared to mitfa+in6. At 32°C, mitfa+in6 was significantly reduced in its activity and mitfa+in5,6 had no function at all, demonstrating the temperature sensitivity of the splice products. Given the activity of mitfa+in6, and the very weak activity of mitfa+in5,6, we hypothesized that some mitfa pre-RNA species can be correctly spliced in these transcripts to wild-type mitfa and promote melanocyte development. To explore this idea, we examined the mitfa transcripts in the mitfa+in6 embryos at 24 and 28°C and found transcripts that were the same size as wild-type mitfa. However, the interpretation of this was complicated by the presence of mitfanacre transcripts that are also the same size as wild-type mitfa (502 bp; Figure 2A). Sequencing the 502-bp species in the mitfa+in6-expressing nacre mutant embryos revealed both the nacre and wild-type mitfa transcripts indicating that mitfa+in6 splice variants could be correctly spliced to the wild-type form in the nacre embryos (Figure 2B). While not tested, we anticipate a similar mechanism explains the few melanocytes that develop in the mitfa+in5,6-expressing nacre animals, rather than functional Mitfa activity of the protein product of the splice variant, because the encoded protein for the mitfa∆ex6–9 lacks the DNA binding and dimerization domains (Figure 1C, D). Subsequent PCR analysis suggests that the reduction of the mitfanacre transcript in the mitfa+in6-injected embryos (Figure 2A) is due to PCR template competition in favor of the ectopically expressed mitfa+in6, rather than regulation of mitfanacre expression by Mitfa+In6 (data not shown). Next, we altered the splice acceptor and donor sites to test whether the mitfa+in6 splice variants can have activity independently of splicing to the wild-type form (Figure 2C–E). Restoration of the splice donor site (ga>gt) in the mitfa+in6 cDNA was sufficient to restore Mitf activity in the transgene, while change to another nucleotide (ga>gg) resulted in further reduced activity (Figure 2D). In contrast, when we maintained the mitfa+in6 mutation, but destroyed the 3′ splice acceptor site (ag>tg), it was non-functional in the nacre mutants (Figure 2E). Taken together, these experiments indicate that the mitfa+in6 can be spliced to the wild-type mitfa form and that the activity of mitfa+in6 is due to the minor accumulation of the wild-type species. The particular mutation (t>a) appears to be crucial to the unique temperature-sensitive splicing. MITF binds to DNA as a homodimer, and the aberrant splice variants could interfere with wild-type MITF activity. Deletion of exon 6 is predicted to truncate the basic region (Figure 1C). The dominant negative alleles in mouse cluster in the basic region, which is necessary for DNA binding, while retaining dimerization capability (reviewed in Steingrímsson et al., 2004). Heterozygous mitfavc7 mutants appear similar to wild-type zebrafish at 5 dpf (Johnson et al., 2011), suggesting that the dominant negative activity of the vc7 mutation is not sufficient to produce a robust phenotype by interfering with wild-type protein. However, when combined with the weakly active mitfaz25 mutation (a substitution, I219F, in the first helix of the HLH domain; Johnson et al., 2011), the mitfaz25/vc7 mutant embryos failed to develop melanocytes at 32°C, while the mitfaz25/w2 mutant embryos were able to develop a few and weakly pigmented melanocytes (Figure 2F). These results indicate that the mitfavc7 splice variants have some weak dominant interfering activity that may contribute to complete loss of mitfa activity at 32°C. Temperature-sensitive mutations are classic genetic tools that enable functional and temporal control of gene action. Our zebrafish mitfavc7 temperature-sensitive mutant is the only conditional MITF mutation in vertebrates and has already provided insight into the function of MITF in melanocyte stem cells (Johnson et al., 2011), melanocyte development and differentiation (Johnson et al., 2011; Taylor et al., 2011), and in melanoma (Lister et al., 2014). Unusually, rather than increasing temperature affecting the protein function directly as is the case with most temperature-sensitive mutations, mitfavc7 is an intron mutation that leads to aberrant splice forms. Few examples of temperature-sensitive splicing due to intron mutations are found in the literature. In human disease, temperature-dependent splicing in ß-globin pre-mRNA of patients with thalassemia is caused by a mutation in intron 2 (Gemignani et al., 2002), and temperature-sensitive aberrant splicing of type III procollagen transcripts is caused by mutations in splice donor sites within introns in patients with Ehler-Danlos syndrome type IV (Lee et al., 1991; and references therein). In Arabidopsis, an exon mutation close to a 5′-splice site confers temperature sensitivity of RNA splicing in the floral homeotic gene APETALA3 (called the ap3-1 mutant) that controls stamen and petal development (Sablowski and Meyerowitz, 1998). Conditional mutants have been cleverly engineered by the addition of temperature-sensitive DEGRON (Dohmen et al., 1994) and self-excising intein (excising protein) sequences (Tan et al., 2009; Zeidler et al., 2004). We have tested whether the intron 6 can confer temperature sensitivity to GFP in zebrafish but have thus far been unsuccessful: additional exonic sequences may be required to enable temperature-sensitive splicing to be engineered into other genes. To conclude, our work explains the temperature sensitivity of the mitfavc7 mutation to be due to an unusual intron 6 mutation that leads to reduced levels of wild-type mitfa RNA. The production of interfering variants may also ensure that no melanocytes develop at 32°C. We suggest that the mitfavc7 mutation compromises base pairing with the small nuclear RNAs of the spliceosome and that this interaction becomes destabilized at the restrictive temperatures. Alternative splicing is an integral feature of MITF pre-RNA processing and gives rise to multiple MITF spice variants that are both melanocyte specific and relevant in melanocyte development and melanoma (Bharti et al., 2010; Cronin et al., 2009; Debbache et al., 2012; Simmons et al., 2014). Given the importance of MITF in melanocytes, the mitfavc7 allele enables careful examination of MITF activity at multiple stages of melanocyte development, stem cells and melanoma, and is a unique means to explore the function of aberrant pre-RNA splicing in zebrafish. We are grateful to Professor Ian Jackson for many helpful discussions and to Dr. Karthika Paranthaman and Wei Qing for zebrafish husbandry. This work was funded by the NIH (to SLJ, Grant Number RO1GM056988), the MRC (to ZZ, EEP), and Concern Foundation for Cancer Research (to JAL). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Altered phosphodiesterase (PDE)-cyclicAMP(cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanismunderlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP.

The microphthalmia-associated transcription factor (MITF) is the "master melanocyte transcription factor" with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAF(V600E) to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAF(V600E)mitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment.