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The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF- or NRAS-activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAFV600E); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAFV600E); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAFV600E-mutant human melanoma cell line. We found that pathway stimulation upstream of BRAFV600E could paradoxically reduce signaling downstream of BRAFV600E, and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAFV600E signaling. In vivo, expression of wild-type BRAF delayed melanoma onset, but only in a kit-dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAFV600E-driven melanoma formation. Cancer Res; 77(21); 5820–30. ©2017 AACR.

This file contains Supplementary Figures S1-S9 with accompanying legends. Supplementary Fig. S1 examines the relationship between KIT expression and BRAFV600E mutations in TCGA human melanoma samples. Supplementary Fig. S2 shows the rescue of melanocytes in kit(lf)-mutant zebrafish by BRAFV600E. Supplementary Fig. S3 shows tumor onset for an additional kit loss-of-function allele as well as tumor invasion images representative of a mosaic analysis. Supplementary Fig. S4 portrays the levels of Mitfa and pAkt in zebrafish melanomas. Supplementary Fig. S5 shows RAF dimerization and signaling experiments performed with V-BRAFV600E stimulated by NRASQ61K. Supplementary Fig. S6 shows RAF dimerization and signaling experiments performed with V-BRAFWT stimulated by KIT and SCF. Supplemental Fig. S7 shows results of experiments overexpressing KIT in A375 and UACC257 human melanoma cells with accompanying growth and signaling changes. Supplementary Fig. S8 shows survival curves for TCGA patients with melanomas expressing high or low KIT. Lastly, Supplementary Fig. S9 displays a measurement of BRAFV600E levels in zebrafish melanomas overexpressing BRAFWT.

Supplementary Table S1 and S2 show the BRET50 data for Supplementary Fig. S5A and S6B, respectively. Supplementary Table S3 and S4 show further analyses of the relationship between KIT expression and several clinical parameters of melanoma progression or tumor suppressor gene alteration.

Summary There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

Tissue-resident stem cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells self-renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem cells (McSCs) are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified adult McSCs in the zebrafish. To elucidate mechanisms governing McSC self-renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for McSCs, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of McSC direct differentiation. Analysis of the scRNAseq dataset also revealed a population of mitfa/aox5 co-expressing cells that divides following melanocyte destruction, likely corresponding to cells that undergo self-renewal. Our findings show how activation of different subpopulations of mitfa-positive cells underlies self-renewal and differentiation to properly reconstitute the melanocyte pigmentary system following injury.

Skin diseases affect nearly one third of the world's population. Disease types range from oncologic to inflammatory, and outcomes can be as severe as death and disfigurement. Although many skin diseases have been modeled in murine models, the advantages of zebrafish models have led to recent increasing use in modeling human disease. Their rapid development, comparable skin architecture, tractable genetics, unparalleled optical properties, and straightforward drug screens make them an excellent model to study skin disease. In this review, we discuss the attributes of the zebrafish model system as well as current zebrafish models for dermatologic diseases, including melanoma, squamous cell carcinoma, vitiligo, epidermal bullosa, psoriasis, and wounding.

Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAFV600E, the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in vitro and in a zebrafish melanoma model. Mechanistically, BRAFV600E causes decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAFV600E activity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAFV600E activity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.Statement of SignificanceWhole-genome doubling is prevalent in many types of solid tumors and important in shaping tumor genomes, yet the causes of whole-genome doubling are not well understood. Here, we discover that oncogenic BRAFV600E can induce whole-genome doubling through suppression of cytokinesis, and BRAFV600E-induced whole-genome doubling can occur in melanocytes and be present in nascent melanoma cells upon tumorigenesis.

Human melanomas are commonly driven by activating mutations in BRAF, which promote melanocyte proliferation through constitutive stimulation of the MAPK pathway. However, oncogenic BRAF alone is insufficient to promote melanoma; instead, its expression merely induces a transient burst of proliferation that ultimately ceases with the development of benign nevi (i.e. moles) comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that induce this melanocytic growth arrest remain poorly understood. Recent modeling studies have suggested that the growth arrest of nevus melanocytes is not solely due to oncogene activation in individual cells, but rather due to cells sensing and responding to their collective overgrowth, similar to what occurs in normal tissues. This cell growth arrest is reminiscent of the arrest induced by activation of the Hippo tumor suppressor pathway, which is an evolutionarily conserved pathway known to regulate organ size. Herein, we demonstrate that oncogenic BRAF signaling activates the Hippo pathway in vitro, which leads to inhibition of the pro-growth transcriptional co-activators YAP and TAZ, ultimately promoting the growth arrest of melanocytes. We also provide evidence that the Hippo tumor suppressor pathway is activated in growth-arrested nevus melanocytes in vivo, both from single-cell sequencing of mouse models of nevogenesis and human tissue samples. Mechanistically, we observe that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome-doubling events, and that these two effects independently promote Hippo pathway activation. Lastly, we demonstrate that abrogation of the Hippo pathway, via melanocyte-specific deletion of the Hippo kinases Lats1/2, enables oncogenic BRAF-expressing melanocytes to bypass nevus formation, thus leading to the rapid onset of melanoma with 100% penetrance. This model is clinically relevant, as co-heterozygous loss of LATS1/2 is observed in ∼15% of human melanomas. Collectively, our data reveal that the Hippo pathway enforces the stable growth arrest of nevus melanocytes and therefore represents a critical and previously unappreciated barrier to melanoma development.

Melanoma arises from the melanocyte lineage and is the most aggressive and lethal form of skin cancer. There are several genetic, genomic, and cellular changes associated with melanoma initiation. Here, we discuss these alterations and the melanoma cells of origin in which they are proposed to promote melanomagenesis.

Melanoma is the deadliest form of skin cancer and one of few cancers with a growing incidence. A thorough understanding of its pathogenesis is fundamental to developing new strategies to combat mortality and morbidity. Zebrafish—due in large part to their tractable genetics, conserved pathways, and optical properties—have emerged as an excellent system to model melanoma. Zebrafish have been used to study melanoma from a single tumor initiating cell, through metastasis, remission, and finally into relapse. In this review, we examine seminal zebrafish studies that have advanced our understanding of melanoma.

Melanoma is an aggressive and deadly skin cancer that develops from melanocytes, a neural crest cell derivative. Melanoma cells and neural crest cells share similar gene expression, behaviors, and cellular mechanisms. Using cross-species oncogenomics, we identified genes recurrently deleted in both human and zebrafish melanomas, which includes PRDM1/prdm1a , a neural crest developmental regulator. We investigated its role in melanoma tumor formation using datasets, human tissue arrays, and transgenic zebrafish lines. High PRDM1 expression in melanoma patients is correlated with better patient survival. We used a stable transgenic zebrafish line, Tg( mitfa :BRAF V600E );p53−/−, and found that when one copy of prdm1a was mutated, melanoma onset occurred more quickly and the resulting tumors were more invasive. We then analyzed expression of a downstream target and well-established melanoma marker, SOX10, and found in the human and zebrafish data, when PRDM1 expression is low, SOX10 is correspondingly high. Thus, the mechanism by which PRDM1 functions as a tumor suppressor in melanoma is likely through regulation of SOX10 expression. SIGNIFICANCE Melanoma cells behave similarly to neural crest cells in that they express many of the same genes, share a common regulatory network, and activate similar genes for self-renewal and migration. Here we show that PRDM1/Prdm1a, a neural crest developmental regulator, also acts as a tumor suppressor in melanoma. Low PRDM1 expression is correlated with worse patient survival in humans and leads to quicker melanoma onset and more invasive tumors in zebrafish. Moreover, in both species, low PRDM1 corresponds to high expression of SOX10 , another neural crest regulator and clinical melanoma marker, suggesting that PRDM1 regulates SOX10 expression.

Preventing terminal differentiation is important in the development and progression of many cancers including melanoma. Recent identification of the BMP ligand GDF6 as a novel melanoma oncogene showed GDF6-activated BMP signaling suppresses differentiation of melanoma cells. Previous studies have identified roles for GDF6 orthologs during early embryonic and neural crest development, but have not identified direct regulation of melanocyte development by GDF6. Here, we investigate the BMP ligand gdf6a, a zebrafish ortholog of human GDF6, during the development of melanocytes from the neural crest. We establish that the loss of gdf6a or inhibition of BMP signaling during neural crest development disrupts normal pigment cell development, leading to an increase in the number of melanocytes and a corresponding decrease in iridophores, another neural crest-derived pigment cell type in zebrafish. This shift occurs as pigment cells arise from the neural crest and depends on mitfa, an ortholog of MITF, a key regulator of melanocyte development that is also targeted by oncogenic BMP signaling. Together, these results indicate that the oncogenic role ligand-dependent BMP signaling plays in suppressing differentiation in melanoma is a reiteration of its physiological roles during melanocyte development.

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers.

The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF- or NRAS-activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAFV600E); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAFV600E); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAFV600E-mutant human melanoma cell line. We found that pathway stimulation upstream of BRAFV600E could paradoxically reduce signaling downstream of BRAFV600E, and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAFV600E signaling. In vivo, expression of wild-type BRAF delayed melanoma onset, but only in a kit-dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAFV600E-driven melanoma formation. Cancer Res; 77(21); 5820–30. ©2017 AACR.

The work by Tang et al. provides a comprehensive, single-cell, transcriptomic analysis of kidney and blood cells from the adult zebrafish, identifying novel cell types, including two classes of NK immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells., Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.

Kasheta et al. report the identification of T reg–like cells in zebrafish, a means to track and live-image these cells, and foxp3a-deficient mutants that display lymphoproliferation, severe inflammation, and other hallmarks of T reg deficiency syndromes., Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg–deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

Preventing terminal differentiation is important in the development and progression of many cancers including melanoma. Recent identification of the BMP ligandGDF6as a novel melanoma oncogene showedGDF6-activated BMP signaling suppresses differentiation of melanoma cells. Previous studies have identified roles forGDF6orthologs during early embryonic and neural crest development, but have not identified direct regulation of melanocyte development by GDF6. Here, we investigate the BMP ligandgdf6a, a zebrafish ortholog of humanGDF6, during the development of melanocytes from the neural crest. We establish that the loss ofgdf6aor inhibition of BMP signaling during neural crest development disrupts normal pigment cell development, leading to an increase in the number of melanocytes and a corresponding decrease in iridophores, another neural crest-derived pigment cell type in zebrafish. This shift occurs as pigment cells arise from the neural crest and depends onmitfa, an ortholog ofMITF, a key regulator of melanocyte development that is also targeted by oncogenic BMP signaling. Together, these results indicate that the oncogenic role ligand-dependent BMP signaling plays in suppressing differentiation in melanoma is a reiteration of its physiological roles during melanocyte development.

Moore et al. reports the first single-cell gene expression analysis in zebrafish blood to distinguish major blood lineages, identify new cell types, and delineate heterogeneity in T cell leukemia., Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.

SummaryEfficient regeneration following injury is critical for maintaining tissue function and enabling organismal survival. Cells reconstituting damaged tissue are often generated from resident stem or progenitor cells or from cells that have dedifferentiated and become proliferative. While lineage-tracing studies have defined cellular sources of regeneration in many tissues, the process by which these cells execute the regenerative process is largely obscure. Here, we have identified tissue-resident progenitor cells that mediate regeneration of zebrafish stripe melanocytes and defined how these cells reconstitute pigmentation. Nearly all regeneration melanocytes arise through direct differentiation of progenitor cells. Wnt signaling is activated prior to differentiation, and inhibition of Wnt signaling impairs regeneration. Additional progenitors divide symmetrically to sustain the pool of progenitor cells. Combining direct differentiation with symmetric progenitor divisions may serve as a means to rapidly repair injured tissue while preserving the capacity to regenerate.

The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF or NRAS activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAFV600E); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAFV600E); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAFV600E-mutant human melanoma cell line. We found that pathway stimulation upstream of BRAFV600E could paradoxically reduce signaling downstream of BRAFV600E, and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAFV600E signaling. In vivo, expression of wild-type BRAF delayed melanoma onset, but only in a kit-dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAFV600E-driven melanoma formation.

Large-scale sequencing studies have revealed several genes that are recurrently mutated in melanomas. To annotate the melanoma genome, we have expressed tumor-associated variants of these genes in zebrafish and characterized their effects on melanocyte development and function. Here, we describe expression of tumor-associated variants of the recurrently mutated metabotropic glutamate receptor 3 (GRM3) gene. Unlike wild-type GRM3, tumor-associated GRM3 variants disrupted trafficking of melanosomes, causing their aggregation in the cell body. Melanosomes are trafficked in a cAMP-dependent manner, and drugs that directly or indirectly increased cAMP levels were able to suppress melanosome aggregation in mutant GRM3-expressing melanocytes. Our data show that oncogenic GRM3 variants dysregulate cAMP signaling, a heretofore unknown role for these oncogenes. cAMP signaling has been implicated in melanoma progression and drug resistance, and our data show that oncogenic properties of GRM3 could be mediated, at least in part, by alterations in cAMP signaling.

Embryonic development involves high levels of proliferation, migration, and differentiation to form all the tissues and organs of an organism. Under normal circumstances, cell–cell signaling in an embryo is coordinated both temporally and spatially to allow development to occur without error. Embryogenesis is a once-in-a-lifetime event, and many developmental signaling pathways and processes are not physiologically active in adult tissues, with notable exceptions in the maintenance of certain stem cell populations (Goldstein and Horsley 2012). However, in disease states such as cancer, malignant cells adopt similar characteristics to those seen in embryonic cells – proliferation, migration, and lack of differentiation. Over recent decades, investigation into various stages of melanocyte development has given great insight into specific characteristics of melanoma, leading to a deeper understanding of melanomagenesis. In this chapter, we review key literature describing connections between embryonic development and melanoma, including aspects of embryonic patterning and melanocyte lineage specification that are co-opted to promote melanoma progression.

Over a decade has elapsed since the first genetically-engineered zebrafish cancer model was described. During this time remarkable progress has been made. Sophisticated genetic tools have been built to generate oncogene expressing cancers and characterize multiple models of solid and blood tumors. These models have led to unique insights into mechanisms of tumor initiation and progression. New drug targets have been identified, particularly through the functional analysis of cancer genomes. Now in the second decade, zebrafish cancer models are poised for even faster growth as they are used in high-throughput genetic analyses to elucidate key mechanisms underlying critical cancer phenotypes.

Over a decade has elapsed since the first genetically-engineered zebrafish cancer model was described. During this time remarkable progress has been made. Sophisticated genetic tools have been built to generate oncogene expressing cancers and characterize multiple models of solid and blood tumors. These models have led to unique insights into mechanisms of tumor initiation and progression. New drug targets have been identified, particularly through the functional analysis of cancer genomes. Now in the second decade, zebrafish cancer models are poised for even faster growth as they are used in high-throughput genetic analyses to elucidate key mechanisms underlying critical cancer phenotypes.

The rapid emergence of the zebrafish as a cancer model has been aided by advances in genetic, chemical, and imaging technologies. Melanoma in particular highlights both the power and challenges associated with cancer modeling in zebrafish. This chapter focuses on the lessons that have emerged from the melanoma models as paradigmatic of what will apply to nearly all cancer models in the zebrafish system. We specifically focus on methodologies related to germline and mosaic transgenic melanoma generation, and how these can be used to deeply interrogate additional cooperating oncogenes or tumor suppressors. These transgenic tumors can in turn be used to generate zebrafish-specific, stable melanoma cell lines which can be fluorescently labeled, modified by cDNA/CRISPR techniques, and used for detailed in vivo imaging of cancer progression in real time. These zebrafish melanoma models are beginning to elucidate both cell intrinsic and microenvironmental factors in melanoma that have broader implications for human disease. We envision that nearly all of the techniques described here can be applied to other zebrafish cancer models, and likely expanded beyond what we describe here.

Dear Editor, Nevi are senescent and benign tumors of melanocytes, some of which can progress to melanoma (Gray-Schopfer et al., 2007). BRAFV600E is the most frequent mutation in human nevi and melanoma, and promotes senescence in human melanocytes (Gray-Schopfer et al., 2007). The functional activity of BRAFV600E has been validated in both zebrafish and mouse animal models (Damsky and Bosenberg, 2010; Patton et al., 2010). Both models display nevus-like melanocytic hyperplasia; however, the focus has been on the malignant transformation of these nevi to melanoma and not the nevi themselves. In zebrafish, the transgenic expression of BRAFV600E from the mitfa promoter can promote fish-nevus development, but an additional genetic mutation, for example, in p53 is required to promote progression to malignancy (Patton et al., 2005). BRAFV600E nevi develop in the young adult fish, and once formed remain static, and do not continue to grow for the remainder of the life of the fish. Thus, like in humans, fish-nevi appear to have limited growth potential, most likely due to oncogenic BRAF-induced senescence pathways. Even in BRAFV600E animals that are deficient for p53, only some fish-nevi progress to melanoma (Patton et al., 2005), suggesting that the constraints on fish-nevus growth are robust and that multiple cellular changes are required to promote transformation to melanoma. We were able to exploit the regenerative capacity of the zebrafish to explore the self-renewal potential of the fish-nevus. The zebrafish pigmentation pattern consists of three pigment cell types: the melanocytes, the iridophores, and the xanthophores (Kelsh et al., 2009). Partial amputation of the fin tissue has previously been studied to dissect the genetic pathways responsible for melanocyte regeneration (Rawls and Johnson, 2000, 2001). Following microinjection of the BRAFV600E transgene into the single-cell embryo (mosaic transgenics), nevi occur randomly, with a proportion in the caudal fin (Appendix S1). Zebrafish fin pigmentation patterns are highly stereotyped, and zebrafish-nevi are clearly distinguishable from normal patterning by ectopic dark, diffusely pigmented and often larger melanocytes. This allowed us to ask whether the constraints on fish-nevus growth are maintained in the context of the regenerating fin tissue. The distal portion of the caudal fin was amputated, removing between one-quarter and one-half of the nevus, and the regrowth of the tail and fish-nevus was recorded. We reasoned that there could be four possible outcomes: nevus regrowth with the regenerating tail fin, enhanced nevus regrowth with the regenerating tail fin, no nevus growth with the regenerating tail fin, or regression of the remaining nevus. Thirty-four zebrafish displaying fish-nevi within the caudal fin underwent partial amputation (Figure 1B and Table 1). Zebrafish were imaged initially as a reference image, immediately following partial nevus removal (Figure 1A, B) and subsequently at 1-week intervals for at least 3 weeks post-surgery (Figure 1C–G). Four different outcomes were observed (Table 1). The most frequent outcome was complete regrowth of the nevus (regenerate; n = 32; Figure 1C–G). Regenerating fin nevus tissue carried the mitfa-BRAFV600E transgene cells, as confirmed by genotyping of the regenerate fin tissue (data not shown). However, because the fish are mosaic transgenics, non-nevus tail fin tissue also carried and expressed the transgene preventing us from determining the origin of the repopulating fish-nevus melanocytes. Fin regeneration without regrowth of the nevus was also observed (n = 1; non-regenerate; Figure S1), as was one case of nevus regression in which the remaining segment of nevus appeared to regress leaving the original stripe pattern evident (regression; Figure S2). Rates of fish-nevus regrowth could vary, but all fish-nevi could be clearly seen to begin recurrence within 1 week. Most fish-nevi had recurred by 3 weeks, although one fish required up to 10 weeks for complete regrowth. Three fish showed enhanced regrowth along the length of the tail fin (Figure S3). Thus, we find that most fish-nevi have the potential for recurrence within the context of the regenerating tail fin tissue. Table 1 Summary of BRAFV600E zebrafish nevus response Figure 1 Regeneration of a partially amputated fish-nevus. (A) Mosaic integration of mitf-BRAFV600E promotes fish-nevus development in wild-type zebrafish. Ectopic melanocytes form a fish-nevus in the tail fin (black tissue, ventral portion of tail fin). The red ... Fish-nevi recurrence was not altered by different wild-type background (AB or TE strains). We also tested recurrence in zebrafish disrupted for cell-to-cell contact between pigment cells in the mutant line leopard (mutation in connexin 41.8; (Watanabe et al., 2006) and in zebrafish that have fin overgrowth (longfin; mutation in kcnh2l; S. Johnson, personal communication). Fish-nevus recurrence was recorded in all fish, regardless of background (Table 1). Notably, fish-nevi could recur after multiple and successive fin amputations (up to at least three times). We then addressed the cell population that generates the recurrent nevus. One possibility is that the differentiated melanocytes in the remaining fish-nevus undergo migration and/or proliferation to repopulate the regenerating fin. Previous studies have shown that regeneration of the pigment stripes following fin amputation involves unpigmented precursor cells (Lee et al., 2010; Rawls and Johnson, 2000, 2001). Ten fish with regenerative fish-nevi were selected, and recurrence was observed in the presence of N-phenylthiourea (PTU) to block de novo melanin synthesis (Figure 2). In this way, melanin is used as a lineage tracer that pigments the new melanocytes derived from division of original nevus cells or allows for the visualization of migrating nevus cells into the regenerating tail. We found that the tail fins regenerated in PTU lack pigmentation of either the regenerating stripes or nevus cells. Unpigmented cells did repopulate the tail fin; however, because upon the initiation of melanin synthesis (24 h after removal of PTU), we observed pigmented melanocytes in the tail fin and the regenerated fish-nevus. This indicates that the melanocytes responsible for repopulating the fish-nevus, at least in the initial stages, are primarily derived from an unpigmented precursor cell type and not from significant migration or proliferation of differentiated pigmented fish-nevus melanocytes. Figure 2 Regenerating fish-nevi develop from an undifferentiated precursor. (A) A zebrafish tail fin with a fish-nevus (asterisks) was partially amputated and regrown in the presence of phenylthiourea (PTU) for 11 days (B). PTU blocks new melanin synthesis (Rawls ... Tail fin regeneration did not promote melanoma in any of the recurring fish-nevi. Notably, two nevi fish had p53+/− mutations and also did not develop melanoma (up to 3 months post-caudal fin amputation) at the regenerating nevus, despite developing tumors from additional nevi elsewhere on the body (Table 1). Following this, we wondered whether tail regeneration might stimulate tumor formation in tumor prone BRAFV600Ep53 lines in which all melanocytes carry the BRAFV600E transgene. We repeated our tail regeneration assay in five stable transgenic BRAFV600E/V600Ep53−/− zebrafish and found no progression to melanoma at the tail fin (followed up to 4 weeks post-amputation; data not shown). Likewise, amputation of the tail fins in the highly tumor prone RASV12 stable lines (Santoriello et al., 2010) also did not stimulate tumorigenesis (followed up to 3 weeks post-amputation; n = 24; data not shown). Thus, in the proliferative environment of the regenerating tail fin, sufficient cellular controls are maintained to prevent tumorigenesis in BRAFV600E- and RASV12-expressing melanocytes. In conclusion, otherwise growth-restricted zebrafish fish-nevi have the potential to repopulate large portions of a nevus from an unpigmented precursor cell type, without promoting tumorigenicity. UV light exposure and BRAF mutations contribute to nevus initiation in humans but the maintenance, recurrence, and regression of nevi are not well understood. Nevi often regress in older people (Tucker et al., 2002), and a proportion of patients display nevi recurrence following removal by surgical curettage or dermabrasion (King et al., 2009). These recurrent nevi are not tumorigenic but can often resemble a dysplastic nevus or melanoma (pseudomelanoma). The source of melanocytes that repopulate a recurrent nevus is unknown, but it has been postulated that the melanocytes may be derived from nearby melanocyte stem cells or residual nevus melanocytes at the site of removal (King et al., 2009). In our model, the regenerative nevi appear to be derived from an unpigmented precursor population, at least during the first 11 days of regeneration. The potential for differing regenerative outcomes of the fish-nevi suggests that fish-nevi may actively regulate and sustain their growth. While we do not know how human nevus maintenance compares with the zebrafish-observed nevus outcomes, this model provides a novel platform to study fundamental questions about nevus maintenance, regrowth, and regression.

The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome-wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5-Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end-sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development.

Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC(+/-) embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC(-/-) embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC(+/-) zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC(Min/+) mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration.

The Caenorhabditis elegans synthetic multivulva (synMuv) genes act redundantly to antagonize the specification of vulval cell fates, which are promoted by an RTK/Ras pathway. At least 26 synMuv genes have been genetically identified, several of which encode proteins with homologs that act in chromatin remodeling or transcriptional repression. Here we report the molecular characterization of two synMuv genes, lin-37 and lin-54 . We show that lin-37 and lin-54 encode proteins in a complex with at least seven synMuv proteins, including LIN-35, the only C. elegans homolog of the mammalian tumor suppressor Rb. Biochemical analyses of mutants suggest that LIN-9, LIN-53, and LIN-54 are required for the stable formation of this complex. This complex is distinct from a second complex of synMuv proteins with a composition similar to that of the mammalian Nucleosome Remodeling and Deacetylase complex. The class B synMuv complex we identified is evolutionarily conserved and likely functions in transcriptional repression and developmental regulation.

The class A and class B synMuv genes are functionally redundant negative regulators of a Ras signaling pathway that induces C. elegans vulval development. A number of class B synMuv genes encode components of an Rb and histone deacetylase complex that likely acts to repress transcription of genes required for vulval induction. We discovered a new class of synMuv genes that acts redundantly with both the A and B classes of genes in vulval cell-fate determination. These new class C synMuv genes encode TRRAP, MYST family histone acetyltransferase, and Enhancer of Polycomb homologs, which form a putative C. elegans Tip60/NuA4-like histone acetyltransferase complex. A fourth gene with partial class C synMuv properties encodes a homolog of the mammalian SWI/SNF family ATPase p400. Our findings indicate that the coordinated action of two chromatin-modifying complexes, one with histone deacetylase and the other with histone acetyltransferase activity, is important in regulating Ras signaling and specifying cell fates during C. elegans development.

Previous studies have shown that a synthetic multivulva phenotype results from mutations in genes that antagonize the ras-mediated intercellular signaling system responsible for vulval induction in Caenorhabditis elegans. Synthetic multivulva mutations define two classes of genes, A and B, and a mutation in a gene of each class is required to produce the multivulva phenotype. The ectopic vulval tissue in multivulva animals is generated by vulval precursor cells that in the wild type do not generate vulval tissue. One of the class B synthetic multivulva genes, lin-35, encodes a protein similar to the retinoblastoma (Rb) protein. In this article, we describe the isolation and characterization of 50 synthetic multivulva mutations, the identification of new components of both the class A and class B lin-35 Rb pathways, and the cloning of lin-52, a class B gene that may have a conserved role in Rb-mediated signaling.

The zebrafish has emerged as a powerful model system to study human diseases, including a variety of neoplasms. Principal components that have contributed to the rise in use of this vertebrate model system are its high fecundity, ease of genetic manipulation, and low cost of maintenance. Vital imaging of the zebrafish is possible from the transparent embryonic stage through adulthood, the latter enabled by a number of mutant lines that ablate pigmentation. As a result, high-resolution analyses of tumor progression can be accomplished in vivo. Straightforward transgenesis of zebrafish has been employed to develop numerous tumor models that recapitulate many aspects of human neoplastic disease, both in terms of pathologic and molecular conservation. The small size of zebrafish embryos has enabled screens for novel chemotherapeutic agents. Its facile genetics have been exploited in studies that extend beyond modeling cancer to investigations that define new cancer genes and mechanisms of cancer progression. Together, these attributes have established the zebrafish as a robust and versatile model system for investigating cancer. In this chapter we describe methods that are used to study a gene's impact on melanoma progression. We detail methods for making transgenic animals and screening for tumor onset as well as methods to investigate tumor invasion and propagation.

Melanoma is the most aggressive and lethal form of skin cancer. In 2013 over 75,000 Americans were diagnosed with melanoma, and nearly 10,000 died from this disease. It has been known for over a decade that mutations that overactivate the BRAF and NRAS genes promote melanoma formation. At the same time it has also become clear that these mutations are not sufficient for melanoma formation and other genes are involved. Using genomic studies and cross-species comparisons, we identified the BMP factor GDF6 as a gene that may cooperate with mutant BRAF to promote melanoma. The aims of this grant are to determine if GDF6 does in fact cooperate with mutant BRAF and uncover the mechanisms by which GDF6 acts in melanomas and normal melanocytes. Toward these aims, we have used our zebrafish model to demonstrate cooperativity between GDF6 and mutant BRAF in accelerating melanoma onset. Furthermore, we have knocked down GDF6 in human melanoma cells, finding that loss of GDF6 causes cells to cease proliferating. These and other data suggest that GDF6 promotes melanoma progression and its withdrawal is detrimental to melanoma cell growth. We are currently investigating whether blocking GDF6 function is a viable therapeutic strategy.

The zebrafish has emerged as a powerful model system to study human diseases, including a variety of neoplasms. Principal components that have contributed to the rise in use of this vertebrate model system are its high fecundity, ease of genetic manipulation, and low cost of maintenance. Vital imaging of the zebrafish is possible from the transparent embryonic stage through adulthood, the latter enabled by a number of mutant lines that ablate pigmentation. As a result, high-resolution analyses of tumor progression can be accomplished in vivo. Straightforward transgenesis of zebrafish has been employed to develop numerous tumor models that recapitulate many aspects of human neoplastic disease, both in terms of pathologic and molecular conservation. The small size of zebrafish embryos has enabled screens for novel chemotherapeutic agents. Its facile genetics have been exploited in studies that extend beyond modeling cancer to investigations that define new cancer genes and mechanisms of cancer progression. Together, these attributes have established the zebrafish as a robust and versatile model system for investigating cancer. In this chapter we describe methods that are used to study a gene's impact on melanoma progression. We detail methods for making transgenic animals and screening for tumor onset as well as methods to investigate tumor invasion and propagation.

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

SummaryDNA methylation at the 5 position of cytosine (5-mC) is a key epigenetic mark that is critical for various biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases. Here, we report that “loss of 5-hmC” is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Genome-wide mapping of 5-hmC reveals loss of the 5-hmC landscape in the melanoma epigenome. We show that downregulation of isocitrate dehydrogenase 2 (IDH2) and TET family enzymes is likely one of the mechanisms underlying 5-hmC loss in melanoma. Rebuilding the 5-hmC landscape in melanoma cells by reintroducing active TET2 or IDH2 suppresses melanoma growth and increases tumor-free survival in animal models. Thus, our study reveals a critical function of 5-hmC in melanoma development and directly links the IDH and TET activity-dependent epigenetic pathway to 5-hmC-mediated suppression of melanoma progression, suggesting a new strategy for epigenetic cancer therapy.

The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway[subscript 1, 2]. BRAF(V600E) mutations are also present in benign melanocytic naevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes[subscript 4, 5]. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

During vertebrate embryogenesis, hematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM) region. A zebrafish chemical genetic screen identified compounds that regulate blood flow as modulators of HSC formation. silent heart (sih) embryos that lack a heartbeat and blood circulation exhibited severely reduced HSCs. Blood flow modifiers exerted their effects after the onset of heartbeat; however, nitric oxide (NO) donors affected HSC induction even when treatment occurred prior to the initiation of circulation, and rescued HSCs in sih mutants. NO synthase (Nos) inhibitors and morpholino-knockdown of nos1 (nnos/enos) blocked HSC development. Embryonic transplantation assays demonstrated a cell-autonomous requirement for nos1. Nos3 (eNos) was expressed in HSCs in the murine AGM. Intrauterine Nos inhibition or Nos3 deficiency in mice resulted in the absence of hematopoietic clusters and reduced transplantable progenitors and HSCs. This work links blood flow to AGM hematopoiesis, and identifies NO as a conserved downstream regulator of HSC development.

Advantageous organismal and technical attributes of the zebrafish are being increasingly applied to study cancer biology. Along with other tumor models, zebrafish that develop melanomas have been generated. In both genetics and phenotype, zebrafish melanomas are strikingly similar to their human counterparts. For this reason, studies in the zebrafish are poised to make significant contributions to melanoma biology. In this review, we summarize important features of human melanoma and discuss how the zebrafish can be used to address many questions that remain unanswered about this devastating disease.

The synthetic multivulva (synMuv) genes negatively regulate Ras-mediated vulval induction in the nematode Caenorhabditis elegans. The synMuv genes define three classes, A, B, and C, such that double mutants carrying mutations in genes of any two classes are multivulva. The class B synMuv genes include lin-35, a homolog of the retinoblastoma (Rb) tumor suppressor gene, as well as homologs of genes that function with Rb in transcriptional regulation. We screened for additional synMuv mutations using a strategy different from that of previous synMuv genetic screens. Some of the mutations we recovered affect new synMuv genes. We present criteria for assigning synMuv mutations into different genetic classes. We also describe the molecular characterization of the class B synMuv gene lin-65.

The synthetic multivulva (synMuv) genes define two functionally redundant pathways that antagonize RTK/Ras signaling during Caenorhabditis elegans vulval induction. The synMuv gene lin-35 encodes a protein similar to the mammalian tumor suppressor pRB and has been proposed to act as a transcriptional repressor. Studies using mammalian cells have shown that pRB can prevent cell cycle progression by inhibiting DP/E2F-mediated transcriptional activation. We identified C. elegans genes that encode proteins similar to DP or E2F. Loss-of-function mutations in two of these genes, dpl-1 DP and efl-1 E2F, caused the same vulval abnormalities as do lin-35 Rb loss-of-function mutations. We propose that rather than being inhibited by lin-35 Rb, dpl-1 DP and efl-1 E2F act with lin-35 Rb in transcriptional repression to antagonize RTK/Ras signaling during vulval development.

Two studies suggest how mutations in the APC tumor suppressor gene can contribute to chromosomal aberrations in precancerous cells.