Search

Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.

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.

Dermal invasion is a hallmark of malignant melanoma. Although the molecular alterations that drive the progression of primary melanoma to metastatic disease have been studied extensively, the early progression of noninvasive primary melanoma to an invasive state is poorly understood. To elucidate the mechanisms underlying the transition from radial to vertical growth, the first step in melanoma invasion, we developed a zebrafish melanoma model in which constitutive activation of ribosomal protein S6 kinase A1 drives tumor invasion. Transcriptomic analysis of ribosomal protein S6 kinase A1–activated tumors identified metabolic changes, including up-regulation of genes associated with oxidative phosphorylation. Vertical growth phase human melanoma cells show higher oxygen consumption and preferential utilization of glutamine compared to radial growth phase melanoma cells. Peroxisome proliferator activated receptor γ coactivator (PGC)-1α, has been proposed as a master regulator of tumor oxidative phosphorylation. In human primary melanoma specimens, PGC1α protein expression was found to be positively associated with increased tumor thickness and expression of the proliferative marker Ki-67 and the reactive oxygen species scavenger receptor class A member 3. PGC1α depletion modulated cellular processes associated with primary melanoma growth and invasion, including oxidative stress. These results support a role for PGC1α in mediating glutamine-driven oxidative phosphorylation to facilitate the invasive growth of primary melanoma.

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap−/− mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap−/− mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis., National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant P30DK034854), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK090311), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant R24OD017870), National Institutes of Health (U.S.) (Grant P30DK034854), National Institutes of Health (U.S.) (Grant 1R01DK090311), National Institutes of Health (U.S.) (Grant 1R01DK105198), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK105198), National Institutes of Health (U.S.) (Grant R24OD017870), National Institute of General Medical Sciences (NIGMS) (Grant T32GM007753), NHMRC (Grant 1146558), National Cancer Institute (U.S.) (Grant 5P01CA120964), National Cancer Institute (U.S.) (Grant 5P30CA006516)

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, differ in their speed of dermal invasion but converge biologically once they invade and metastasize. Herein, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) was significantly hyperactivated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and did not directly correlate with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using siRNA and the pharmacological inhibitor BI-D1870 compared with superficial spreading cells. Gene expression microarray analyses revealed that RSK1 orchestrated a program of gene expression that promoted cell motility and invasion. Differential overexpression of the prometastatic matrix metalloproteinase 8 and tissue inhibitor of metalloproteinases 1 in metastatic nodular compared with metastatic superficial spreading melanoma was observed. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Together, these data reveal a novel role for activated RSK1 in the progression of nodular melanoma and suggest that melanoma originating from different histologic subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize.

Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation.

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2E450fs (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAFV600E-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo., Direct visualisation of heterogeneous cell populations in live animals has been challenging. Here, the authors optimize cell transplantation into optically clear immune-deficient zebrafish, and use intravital imaging to track and to assess functional diversity of individual cancer cells in vivo.

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.

Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436 +/- 35 tilted vs. 362 +/- 34 untilted; p < 0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity.

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.

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.

Zebrafish is emerging as a unique model organism for studying cancer genetics and biology. For several decades zebrafish have been used to study vertebrate development, where they have made important contributions to understanding the specification and differentiation programs in many tissues. Recently, zebrafish studies have led to important insights into thyroid development, and have been used to model endocrine cancer. Zebrafish possess a unique set of attributes that make them amenable to forward and reverse genetic approaches. Zebrafish embryos develop rapidly and can be used to study specific cell lineages or the effects of chemicals on pathways or tissue development. In this review, we highlight the structure and function of endocrine organs in zebrafish and outline the major achievements in modeling cancer. Our goal is to familiarize readers with the zebrafish as a genetic model system and propose opportunities for endocrine cancer research in zebrafish.

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.

Medullary thyroid cancer (MTC) is a neuroendocrine cancer that arises from malignant transformation of calcitonin-producing C-cells. A majority of MTC is associated with somatically acquired mutations in the proto-oncogene Rearranged during transfection (RET), resulting in constitutive activation of downstream effectors of the ERK and AKT signaling pathways. While surgical treatment may be curative in patients with localized MTC, a significant fraction of patients harbor residual disease and ultimately relapse. Much of the research on MTC has converged on inhibition of RET, however, studies suggest that other signaling pathways may contribute to disease. As a result, there is a growing need to identify novel targets for MTC. The zebrafish is well regarded as a model to study of development due its genetic tractability, conservation of pathways and rapid organ development; characteristics that also make it a fitting model to study human cancers. In this study we take advantage of these traits to 1) perform an in vivo chemical screen to identify modulators of C-cell development in zebrafish and 2) develop an in vivo oncogenic RET model in zebrafish to test the putative ‘hits’ from our screen. We hypothesize that the nexus of development and cancer modeling in the zebrafish will lead to therapies of potential use in treating MTC. We screened compounds modulating pathways involved in development, kinase signaling and chromatin modification for their ability to influence C-cell development and found that separate inhibition of the FGF and GSKβ; signaling pathways results in marked down-regulation of markers associated with C-cell differentiation. In parallel, we have introduced human oncogenic RET into an analogous cell type of neural crest origin in zebrafish which results in their early transformation, and are testing the ability of the identified ‘hits’ to suppress transformation of these cells. Our results implicate the FGF and GSKβ; pathways in C-cell development and encourage further investigation of their potential as therapeutic targets for MTC. Moreover, our studies highlight the use of zebrafish as model to identify novel targeted therapies in cancer. This abstract is also presented as Poster B44. Citation Format: Jacques A. Villefranc, Aida Alazar, Yariv Houvras. Linking development and cancer modeling in zebrafish identifies putative therapeutic targets in medullary thyroid cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR04.

The activating BRAF kinase mutation V600E occurs in ∼60% of human nevi and melanomas. A transgenic strain of zebrafish expressing BRAF-V600E under the control of the mitfa promoter on a p53 mutant background develops melanomas with 100% penetrance. Previously, we showed that a transposon-based vector, MiniCoopR, allows us to program melanocytes to express a candidate gene in a specialized zebrafish strain Tg(mitfa:BRAFV600E); mitfa-/-; p53-/-. This strain lacks melanocytes, but the lineage can be rescued by MiniCoopR injection at the 1-cell stage. This genetic system allows us to create cohorts of transgenic animals that express candidate genes and assay the effects on tumor development. Previous work identified two histone methyltransferases (HMTs), SETDB1 and SUV39H1, which cooperate with BRAF-V600E to accelerate the onset and severity of melanoma development. A number of HMTs display alterations in expression level or copy number in human cancers compared to normal tissues, and we tested a panel of methyltransferases using the MiniCoopR system. We hypothesize that HMTs promote tumorigenesis by altering the epigenetic program of melanocytes. In order to explore the effects on melanocyte specification and differentiation, we overexpressed and depleted candidate HMTs in zebrafish embryos and performed in situ hybridization for lineage-specific markers, such as crestin, sox10, mitfa, dct, and tyr. HMTs that exert specific developmental phenotypes were further characterized by performing chromatin immunoprecipitation (ChIP). Our goal is to correlate the processes affected in the developing embryo with the epigenetic program to elucidate the mechanism by which HMTs influence tumorigenesis. Additionally, we are developing a strain of zebrafish Tg(mitfa:BRAFV600E); mitfa-/-; alb-/-; p53-/-; Tg(mitfa:GFP) in which melanocytes rescued with MiniCoopR express GFP. These rescued melanocytes can be programmed to express candidate HMTs and allow us to visualize melanocyte and melanoma development in vivo. The work presented here will help correlate cancer phenotypes with specific chromatin-modifying enzymes that may be the basis for new drug development and biomarker discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5020. doi:1538-7445.AM2012-5020