Zhiqiang Zeng

A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo

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 BRAFV600E to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAFV600Emitf 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.

Combined zebrafish-yeast chemical-genetic screens reveal gene–copper-nutrition interactions that modulate melanocyte pigmentation

SUMMARY Hypopigmentation is a feature of copper deficiency in humans, as caused by mutation of the copper (Cu2+) transporter ATP7A in Menkes disease, or an inability to absorb copper after gastric surgery. However, many causes of copper deficiency are unknown, and genetic polymorphisms might underlie sensitivity to suboptimal environmental copper conditions. Here, we combined phenotypic screens in zebrafish for compounds that affect copper metabolism with yeast chemical-genetic profiles to identify pathways that are sensitive to copper depletion. Yeast chemical-genetic interactions revealed that defects in intracellular trafficking pathways cause sensitivity to low-copper conditions; partial knockdown of the analogous Ap3s1 and Ap1s1 trafficking components in zebrafish sensitized developing melanocytes to hypopigmentation in low-copper environmental conditions. Because trafficking pathways are essential for copper loading into cuproproteins, our results suggest that hypomorphic alleles of trafficking components might underlie sensitivity to reduced-copper nutrient conditions. In addition, we used zebrafish-yeast screening to identify a novel target pathway in copper metabolism for the small-molecule MEK kinase inhibitor U0126. The zebrafish-yeast screening method combines the power of zebrafish as a disease model with facile genome-scale identification of chemical-genetic interactions in yeast to enable the discovery and dissection of complex multigenic interactions in disease-gene networks.

Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

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 Gα 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 GNA11R183C and GNA11Q209L 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 GNA11R183C 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.

Knockdown of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene results in retinal degeneration

PURPOSE The authors investigated the expression and function of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene. METHODS Zebrafish RP2 (ZFRP2) cDNA was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was examined by RT-PCR. The deduced peptide sequence was aligned with RP2 orthologues from different species. Translational suppression (knockdown) of zebrafish RP2 was carried out by antisense morpholino-injection. The phenotype of ZFRP2 knockdown morphants was characterized by immunohistology and histology. Human wild-type and mutant RP2 mRNAs were coinjected with ZFRP2 morpholinos to test whether human RP2 mRNA could rescue ZFRP2 knockdown phenotypes. RESULTS ZFRP2 encodes a protein of 376 amino acids containing an N-terminal tubulin folding cofactor C-like domain and a C-terminal nucleoside diphosphate kinase-like domain. It shares 63% to 65% amino acid identity with human, mouse and bovine RP2. RP2 is expressed at the earliest stages of zebrafish development and persists into adulthood. Knockdown of RP2 in zebrafish causes a curved body axis and small eye phenotype, associated with increased cell death throughout the retina. Human wild-type RP2 mRNA could rescue the body curvature phenotype of ZFRP2 morphants, and the eye size of the resultant morphants was significantly increased over that of morphants in which ZFRP2 had been depleted. CONCLUSIONS Zebrafish RP2 is widely expressed throughout development. ZFRP2 knockdown caused retinal degeneration in zebrafish. Human RP2 could partially rescue the small eye phenotype of ZFRP2 morphants.

Zebrafish have a competent p53-dependent nucleotide excision repair pathway to resolve ultraviolet B-induced DNA damage in the skin

Ultraviolet (UV) light is a primary environmental risk factor for melanoma, a deadly form of skin cancer derived from the pigmented cells called melanocytes. UVB irradiation causes DNA damage, mainly in the form of pyrimidine dimers (cis-syn cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts), and organisms have developed complex multiprotein repair processes to cope with the DNA damage. Zebrafish is becoming an important model system to study the effects of UV light in animals, in part because the embryos are easily treated with UV irradiation, and the DNA damage repair pathways appear to be conserved in zebrafish and mammals. We are interested in exploring the effects of UV irradiation in young adult zebrafish, so that we can apply them to the study of gene-environment interactions in models of skin cancer. Using the Xiphophorus UV melanoma model as a starting point, we have developed a UV irradiation treatment chamber, and established UV treatment conditions at different ages of development. By translating the Xiphophorus UV treatment methodology to the zebrafish system, we show that the adult zebrafish skin is competent for nucleotide excision DNA damage repair, and that like in mammalian cells, UV treatment promotes phosphorylation of H2AX and a p53-dependent response. These studies provide the groundwork for exploring the role of UV light in melanoma development in zebrafish.

ALDH2 Mediates 5-Nitrofuran Activity in Multiple Species

Summary 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.

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf.

Coordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF4TΔ2B promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.

A zebrafish model for nevus regeneration.

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.

MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

Summary Altered phosphodiesterase (PDE)-cyclic AMP (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 mechanism underlying 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.

Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy

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).