Browsing by Subject "Zebrafish"
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Item Chemical Genetic Studies of Tumor Suppressors in the Developing Zebrafish(2010-11-02) Verduzco, Daniel; Amatruda, James F.The zebrafish is a powerful disease model system. Its distinct advantages allow it to be used for high-throughput genetic and chemical screens with high translational potential. One mutant isolated from a screen for cell cycle deficiencies is the zebrafish cdc25a mutant standstill. This mutant suffers from a G2/M arrest due to activation of the checkpoint protein ATM such that ATM knockdown or inhibition rescues the cell cycle. Additional knockdown studies reveal that the unique zebrafish cdc25 gene cdc25d is responsible for cell cycle progression in the absence of functional Cdc25a or ATM. Assays to measure genomic stress reveal that ATM is activated in the absence of DNA damage. Activation of ATM in the absence of DNA damage is a unique occurrence, but further studies seeking regulators of ATM reveal that ATM is constitutively active at a basal level. This basal ATM activity is sufficient to attenuate cell cycle rate. Other studies involving the use of zebrafish to study cancer involve a screen using peptoids. The peptoid is a class of compound that is modular, easy and inexpensive to synthesize, and stable in vivo. Initial tests reveal that peptoids are able to enter the zebrafish embryo, remain stable, and exert biological functions. Screening of 3,744 compounds for peptoids that specifically affect zebrafish mutant for p53 reveal a group of 4 structurally similar peptoids. Further studies of these peptoids reveal that they are able to perform their function on mouse embryonic fibroblasts in vitro. Finally, I sought the role of the DNA damage marker H2AX in early zebrafish development. While studying the DNA damage response in cdc25a mutants, I discovered that H2AX is present and cyclic in pre-midblastula transition embryos. This phosphorylation is necessary for synchronization and relies on the ability of ATM to phosphorylate H2AX on Ser-139 such that inhibition of ATM or ablation of the phosphorylation site leads to desynchronization and improper development.Item Discovery of Novel Anesthetic Compounds Using Zebrafish Larvae(2018-01-23) Lima, Maria; Yang, Xiaoxuan; Forman, StuartINTRODUCTION: Novel general anesthetic compounds for use in medicine can provide increased treatment options for patients undergoing invasive medical procedures such as surgery. Zebrafish larvae have recently emerged as a platform for high throughput screening of neuroactive compounds. In our lab, we are using zebrafish larvae to conduct a high throughput screen of over 2,000 uncharacterized drug compounds for possible anesthetic properties. Furthermore, we are testing new combinations of etomidate and MPAB, two known anesthetics acting on the GABA-A receptor, to determine agonistic or antagonistic interactions; we aim to reduce toxic side effects of various drug combinations and to exploit the beneficial properties of these drugs to improve treatment efficaciousness. METHODS: After a 0.2 second bright white light stimulus in dark-adapted 7 day old post-fertilization Tu zebrafish larvae, the Photomotor response (PMR) was analyzed using a specialized motion tracking video system (ViewPoint Zebralab). This bright stimulus startles the zebrafish into a brief burst of movement. Anesthetic effects of new compounds were qualified by calculating the PMR inhibition, representing the decrease in movement after the stimulus and sedative effects were quantified by recording pre-stimulus basal activity inhibition, representing the normal zebrafish movement during periods of no stimulus. RESULTS: Several prospective novel anesthetic compounds have been identified and our lab is currently performing more screens to assess the reversibility and potency of each drug. Specifically, we discovered Compound 84 which shows a significantly higher potency (IC50 = 8.99 μM , 95% IC: 6.38 to 12.7 μM) and normal reversibility (representing the zebrafish ability to recover from drug PMR inhibition overnight). CONCLUSION: Zebrafish photomotor response is a promising method for high throughput identification of novel anesthetics. Additionally, co-administration of MPAB with etomidate can potentially reduce dosage requirements in anesthetics, therefore, providing beneficial drug combination options to reduce side effects in higher risk patients.Item FishATLAS: Assessment of Organotropism Determination Through Imaging Informatics of Xenografted Zebrafish(December 2021) Saucier, David Hamilton; Fiolka, Reto; Danuser, Gaudenz; Amatruda, James F.; Whitehurst, Angelique Wright; Brekken, Rolf A.; Mason, Ralph P.Ewing sarcoma patients with metastatic disease have a 5-year survival rate of approximately 28%. The hallmark of this disease is an aberrant transcription factor made by a fusion of Chromosomes 11 and 22 called EWSFLI1. EWSFLI1 expression levels have been correlated with differing responses in cell metastatic propensity, but much remains to be elucidated. Indeed, many current models fail to meet the statistical rigor that is needed for exceedingly spontaneous, rare events like metastasis. To address this need, FishATLAS utilizes zebrafish human cancer cell xenograft images after high fidelity registration using a novel diffeomorphic transformation to display metastatic hot spots of different cancer cell conditions to begin to grasp the deeper underpinnings of organotropism in vivo with individual cancers. Utilizing a suite of statistical tests, FishATLAS determines at a global whole-fish scale and the local microenvironment, if there are statistically different cell hotspots when comparing two or more different conditions. As it stands, data for EWSFLI1, its target SOX6, a non-transformed cell line NIH3T3, TC32 subclones, and melanoma cell lines have all shown unique distributions of metastatic hot spots. These findings serve as a tool for drug discovery and later environmental re-mapping in FishATLAS by allowing transgenic fish images (such as vasculature and lymphatics) to be overlayed onto any historical data set. These can then be used to determine a given microenvironment's contributions to secondary sites of metastasis. In the case of EWSFLI1 and its target SOX6, there was a marked difference upon shRNA-mediated knockdown that removed a population of hotspots in the upper somitic veins while some were persistent post genetic perturbation. SOX6 shRNA KD data indicates that the somite and inter-somitic arteries are more sensitive for metastatic colonization. Previous studies and our current accumulator data suggest these to be regions with higher oxidative stress, guiding insights for oxidative-mechanistic therapy. These and other conditional accumulations of sparse metastatic hotspots demonstrate the power of FishATLAS as a longitudinal assay of cellular and genetic conditions across all cancers.Item High-Throughput Chemical Screen for Inhibitors of EWS-FLI1 Using a Trangenic Zebrafish Model of Ewing Sarcoma(2015-01-26) Burton, Barbara; He, Christy F.; Neumann, Joanie C.; Zmau, Daniel P.; Amatruda, James F.Ewing Sarcoma is the second most common primary bone tumor of children and adolescents. With a lack of targeted therapies, survival remains poor despite remarkably intense chemotherapeutic regimens. 85% of Ewing cases are caused by a translocation between the EWS gene on chromosome 22 and the FLI1 gene on chromosome 11 [t(11;22)(q24;q12)]. The fusion EWS-FLI1 gene codes for a chimeric transcription factor which activates and suppresses expression of a diverse array of target genes, and is an enticing candidate for therapeutic investigation. EWS-FLI1 activity can be readily observed in a homozygous transgenic zebrafish model, in which the fusion gene is inserted downstream of the Microphthalmia-Associated Transcription Factor promoter, localizing EWS-FLI1 expression to melanocytes derived from the neural crest. These fish present with an increased melanocyte count on the dorsum of the head compared to wild type. We hypothesized that compounds which suppress EWS-FLI1 activity will prevent the formation of this phenotype in homozygous zebrafish larvae. A high-throughput chemical screen was performed using two libraries of FDA approved compounds. One compound was found to significantly reduce melanocyte count in EWS-FLI1 homozygous zebrafish compared to unexposed homozygous controls (p < 0.0001) in a dose-dependent manner. Furthermore, treated homozygous larvae displayed melanocyte phenotype similar to that of untreated wild type controls. From these results, we conclude that this compound interferes with EWS-FLI1 activity in the mitf:EWS-FLI1 homozygous zebrafish model. Further testing on Ewing Sarcoma cell lines will determine the potential significance of this compound as a specific inhibitor of tumor growth.Item Identification of Developmental Signaling Pathways with a Novel Role in Regulating Zebrafish Primordial Germ Cell Migration(2013-07-01) Boldt, Clayton Ryan; Lum, Lawrence; Amatruda, James F.; Cobb, Melanie H.; Buszczak, MichaelNormal cell migration is critical for embryonic patterning, organ development and immune response. Primordial germ cell (PGC) migration in zebrafish has proven a valuable model for the study of cell motility. In zebrafish, PGC migration is guided principally by the chemokine Sdf-1a and its receptors Cxcr4b in germ cells, and Cxcr7b in somatic cells. While a role has also been suggested for PI3K signaling, the possible contribution of other signaling pathways in PGC migration is not fully understood. In this study, I used inhibitors of early developmental signaling pathways to identify those with a novel role in PGC migration. Among these compounds, the most significant effects were from the Tankyrase inhibitor IWR-1, which blocks β-catenin-dependent Wnt signaling. IWR-1 treatment during periods of active PGC migration results in fewer germ cells reaching the gonad. Treatment with the Porcupine inhibitor IWP-L6, which blocks Wnt production, did not result in substantial effects on PGC migration, suggesting that the effects of IWR-1 may not be dependent on β-catenin activity, but on the direct IWR-1 target, Tankyrase. Treatment with XAV939, an additional Tankyrase inhibitor, phenocopied the effects of IWR-1. Germ cell-targeted inhibition of Wnt signaling did not phenocopy the effects of IWR-1 and XAV939, arguing against a germ cell-autonomous role for Wnt signaling in PGC migration. We observed significant changes in the expression and patterning of sdf-1a, cxcr4b and cxcr7b following IWR-1 treatment, but not Wnt inhibition. Thus, I conclude that Tankyrase has an important role in patterning the PGC migratory environment during early development. I also sought to determine the eventual fate of ectopic PGCs in zebrafish. I created a transgenic line with GFP-labeled PGCs that has enabled us to follow ectopic germ cells during the first two weeks of development. Daily monitoring of ectopic PGCs revealed that they are not cleared, as in mice, but persist in their ectopic locations. In several instances, I observed ectopic germ cells undergoing morphological changes, followed by loss of GFP expression. The results of these observations lend credibility to the hypothesis that extragonadal germ cell tumors could arise from PGCs that do not migrate properly during development.Item A Mutation in Alk6b Causes Impaired Germ Cell Differentation and Testicular Germ Cell Tumors in Zebrafish(2010-11-02) Neumann, Joanie; Amatruda, James F.Germ cell tumors (GCTs) affect infants, children and young adults and are increasing in incidence worldwide. GCTs arise from pluripotent germ cells and can exhibit differentiated and undifferentiated histologies, which vary in their malignant potential and response to treatment. The pathways that determine tumor cell differentiation are not known, impeding the development of new therapies. Thus, the treatment of GCTs has remained static since the introduction 30 years ago of cisplatin which, while effective, causes severe side effects including hearing loss, infertility and kidney damage. We identified a zebrafish mutant line with a high incidence of GCT during a forward genetic screen to identify cancer susceptibility loci. Homozygous adult males develop tumors consisting of undifferentiated spermatogonia by 4 months of age while heterozygous males develop tumors around 7 to 9 months of age. We used interval haplotype analysis and high-resolution recombinational mapping to localize the mutation to a 0.82 cM interval on zebrafish chromosome 10. We identified a premature termination codon in Alk6b (Activin Receptor-like Kinase 6b) in the mutant animals. Alk6b is a member of the TGF-beta/BMP superfamily of receptors. BMP signaling has diverse roles including regulation of cell proliferation, differentiation, embryonic development, germ cell specification and gonadogenesis. Misregulation of the BMP signaling pathway has been implicated in various human cancers. In agreement with a critical role for Alk6b in controlling germ cell differentiation, we find evidence of impaired BMP signal transduction in the zebrafish GCTs, as well as evidence of alterations in the expression level of BMP target genes. We have also examined BMP signaling in a series of 40 clinically-annotated human GCTs of diverse histologic subtypes. In agreement with the predictions made from our zebrafish model, we find that undifferentiated GCTs such as dysgerminomas lack BMP signaling activity, whereas signaling is maintained in the differentiated subtype of Yolk Sac Tumors. These results confirm the relevance of the zebrafish model for understanding germ cell tumorigenesis, and will foster the development of improved, targeted therapy of human GCTs.Item Pumilio Regulates the Epithelial-Mesenchymal Transition and Gastrulation During Zebrafish Development(2013-05-13) Damoulis, Vanessa Ann; Castrillon, Diego H.; Amatruda, James F.; Johnson, Jane E.; Buszczak, MichaelThe pumilio family of RNA binding proteins act as translational repressors to control developmental patterning events of invertebrates, germline stem cell maintenance, and neuronal growth through deadenylation, inhibition of translational elongation, and allow access of microRNAs to their targets. pumilio proteins have yet to be identified and functionally characterized in the zebrafish, Danio rerio. Here, three putative pumilio homologs in zebrafish are identified by their characteristic PUF (pumilio and FBF) binding domains and found to be expressed during early development and enriched in the immature oocytes of the adult ovary. Loss of each homolog results in developmental defects, arising from improper formation of the shield and dorsal organizer structures during the first steps of gastrulation. Gastrulation events necessitate both proper patterns of gene signaling and cell motility, as gained by epithelial-to-mesenchymal transitions (EMT), to occur. In pum1 morphants, both the presentation of wild-type signaling gradients and EMT events are inhibited. Through in silico and in vivo analysis, acvr1b, an Activin receptor, was identified as a target for pum1. acvr1b assists in patterning the gastrulating embryo and, through control of miR-200a expression, inhibits EMT events. Thus, pum1 through its direct translational repression of acvr1b is able to modulate levels of miR-200a and control both patterning and EMT events in the early vertebrate embryo.Item The Role of Autophagy in Early Development and Tumor Suppression Using a Zebrafish Model System(2013-06-25) Lee, Eunmyong; Brugarolas, James B.; Abrams, John M.; Cleaver, Ondine; Levine, Beth; Amatruda, James F.Autophagy is an evolutionarily conserved lysosomal degradation pathway which involves the sequestration of cytoplasmic components into a double membraned structure called the autophagosome. By using genetically manipulated autophagy-deficient models, important roles for autophagy in development and tumorigenesis have been suggested. Genetic analyses indicate that autophagy is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryonic development. To address this question, the role of autophagy in early development was examined using zebrafish, a model system for studying vertebrate tissue and organ morphogenesis. Active autophagy was observed in multiple tissues during early embryonic development, as evidenced by the presence of autophagosomes in electron microscope images or GFP-LC3 puncta in autophagy reporter fish line Tg(cmv