Modeling pediatric brain and central nervous system cancer in zebrafish

Brain and central nervous system (CNS) cancers are the leading cause of cancer-related death in children (Ostrom QT et al., 2015). Low-grade brain and CNS tumors that require minimal surgical resection due to their location in critical regions are associated with long-term morbidity throughout the life of the child (Armstrong GT et al., 2011). Children diagnosed with high-grade, aggressive brain and CNS tumors generally have a poor outcome and suffer significant deficits in neurological and neuroendocrine function as a result of intensive therapy (Fangusaro J et al., 2012). Effective, targeted therapeutics for the treatment of pediatric brain and CNS cancer are needed. Gaining a better understanding of the molecular mechanisms underlying pediatric brain and CNS cancer initiation and progression will benefit the development of therapeutics. Animal models are an important component for the improvement of our understanding of the biology of these cancers (Huszthy PC et al., 2012). The zebrafish has recently emerged as a system for modeling human diseases including brain and CNS cancers. We have characterized two distinct zebrafish brain and CNS tumor models, a low-grade, glial-like tumor model and a high-grade CNS primitive neuroectodermal-like tumor model. The transgenic Tg(flk1:RFP)is18 zebrafish line develops low-grade glial-like tumors in the optic pathway including the retina, optic nerve and optic tract. These tumors exhibited histological features similar to those observed in human pediatric pilocytic astrocytoma. Differential gene expression analysis revealed a neuroglial progenitor signature in the tumors of the retina. The second brain tumor model helped establish the use of nuclease-mediated somatic mutagenesis in zebrafish for the study of tumor suppressor function in cancer. In this model we generated genetic mosaic adults using TALENs targeting the rb1 (retinoblastoma1) tumor suppressor. These mosaic adults developed predominantly undifferentiated, primitive neuroectodermal tumors (67% of tumors) as well as differentiated, glial-like tumors (33% of tumors). This was the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish and highlighted the utility of site-specific nucleases as a rapid, simple, and cost efficient method to screen potentially hundreds of candidate tumor suppressor genes that impact tumorigenesis. These models will be useful for the study of pediatric brain and CNS tumorigenesis

Rapid Tumor Induction in Zebrafish by TALEN-Mediated Somatic Inactivation of the Retinoblastoma1 Tumor Suppressor rb1

Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems

Modeling pediatric brain and central nervous system cancer in zebrafish

Brain and central nervous system (CNS) cancers are the leading cause of cancer-related death in children (Ostrom QT et al., 2015). Low-grade brain and CNS tumors that require minimal surgical resection due to their location in critical regions are associated with long-term morbidity throughout the life of the child (Armstrong GT et al., 2011). Children diagnosed with high-grade, aggressive brain and CNS tumors generally have a poor outcome and suffer significant deficits in neurological and neuroendocrine function as a result of intensive therapy (Fangusaro J et al., 2012). Effective, targeted therapeutics for the treatment of pediatric brain and CNS cancer are needed. Gaining a better understanding of the molecular mechanisms underlying pediatric brain and CNS cancer initiation and progression will benefit the development of therapeutics. Animal models are an important component for the improvement of our understanding of the biology of these cancers (Huszthy PC et al., 2012). The zebrafish has recently emerged as a system for modeling human diseases including brain and CNS cancers. We have characterized two distinct zebrafish brain and CNS tumor models, a low-grade, glial-like tumor model and a high-grade CNS primitive neuroectodermal-like tumor model. The transgenic Tg(flk1:RFP)is18 zebrafish line develops low-grade glial-like tumors in the optic pathway including the retina, optic nerve and optic tract. These tumors exhibited histological features similar to those observed in human pediatric pilocytic astrocytoma. Differential gene expression analysis revealed a neuroglial progenitor signature in the tumors of the retina. The second brain tumor model helped establish the use of nuclease-mediated somatic mutagenesis in zebrafish for the study of tumor suppressor function in cancer. In this model we generated genetic mosaic adults using TALENs targeting the rb1 (retinoblastoma1) tumor suppressor. These mosaic adults developed predominantly undifferentiated, primitive neuroectodermal tumors (67% of tumors) as well as differentiated, glial-like tumors (33% of tumors). This was the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish and highlighted the utility of site-specific nucleases as a rapid, simple, and cost efficient method to screen potentially hundreds of candidate tumor suppressor genes that impact tumorigenesis. These models will be useful for the study of pediatric brain and CNS tumorigenesis.</p

MBP1 Null Mutant Strains of Candida albicans Do Not Show Defects in Responding to Oxidative Stress.

Color poster with text and images describing research conducted by Staci Solin, advised by Daniel P. Herman.Candida albicans is the most frequently isolated fungal pathogen in humans. Morphogenesis, the transition from a yeast to filamentous morphology, has been demonstrated to play an important role in the organism's ability to cause systemic disease. To further characterize the role of the Mbp1 protein in C. albicans, we tested the survival of wild-type, heterozygous, and null mutant strains in the prescence of various peroxides.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Defective Morphogenesis in MBP1 Null Mutant Strains of Candida albicans is Specific to Solid Media.

Color poster with text, table, and images (Spring 2009)Candida albicansis a pathogenic yeast capable of undergoing morphogenesis, which is the transition from yeast to hyphalmorphologies. We have previously cloned and partially characterized the MBP1gene of C. albicansand shown that null mutant strains that cannot synthesize the Mbp1 protein are defective in morphogenesis under nitrogen limiting conditions on solid media. To further characterize the function of the Mbp1 gene, the ability of wild-type and MBP1null mutant strains to undergo morphogenesis was assessed when grown in liquid media under conditions that should stimulate morphogenesis.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Rapid Tumor Induction in Zebrafish by TALEN-Mediated Somatic Inactivation of the Retinoblastoma1 Tumor Suppressor rb1

Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer.This article is from Scientific Reports 5 (2015): 13745, doi:10.1038/srep13745. Posted with permission.</p