Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish

The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish have immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations, however, co-expressing a constitutively active human AKT1 resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myoxma. All tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and that the binary transgenic approach is a useful tool for studying interaction of oncogenes and oncogenic pathways in zebrafish

Spectral analysis of Gene co-expression network of Zebrafish

We analyze the gene expression data of Zebrafish under the combined framework of complex networks and random matrix theory. The nearest neighbor spacing distribution of the corresponding matrix spectra follows random matrix predictions of Gaussian orthogonal statistics. Based on the eigenvector analysis we can divide the spectra into two parts, first part for which the eigenvector localization properties match with the random matrix theory predictions, and the second part for which they show deviation from the theory and hence are useful to understand the system dependent properties. Spectra with the localized eigenvectors can be characterized into three groups based on the eigenvalues. We explore the position of localized nodes from these different categories. Using an overlap measure, we find that the top contributing nodes in the different groups carry distinguished structural features. Furthermore, the top contributing nodes of the different localized eigenvectors corresponding to the lower eigenvalue regime form different densely connected structure well separated from each other. Preliminary biological interpretation of the genes, associated with the top contributing nodes in the localized eigenvectors, suggests that the genes corresponding to same vector share common features.Comment: 6 pages, four figures (accepted in EPL

Histopathologic Alterations Associated with Global Gene Expression Due to Chronic Dietary TCDD Exposure in Juvenile Zebrafish

The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption

Beroepsmatige blootstelling aan ozon in een drinkwaterproductiebedrijf

Gemeten blootstellingsniveaus aan ozon laten dusdanig lage waarden zien, dat onder normale omstandigheden een kans op normoverschrijding niet aanwezig i

Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish

The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish have immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations, however, co-expressing a constitutively active human AKT1 resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myoxma. All tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and that the binary transgenic approach is a useful tool for studying interaction of oncogenes and oncogenic pathways in zebrafish

Roles of brca2 (fancd1) in Oocyte Nuclear Architecture, Gametogenesis, Gonad Tumors, and Genome Stability in Zebrafish

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture

Novel Multi-Linear Quantitative Brain Volume Formula For Manual Radiological Evaluation Of Brain Atrophy

This research article published by Research Square, 2020The brain is a dynamic organ that develops and involutes in volume. The process of volume loss known as brain atrophy commonly occurs in elderly. However, some conditions have been implicated to provoke this paradoxical process in childhood and making it important to have methods and techniques of quantifying brain volume. Automated quantitative methods are very important in brain atrophy assessment but these tools have limited availability in developing countries. The simplified linear radiological methods are poorly reproducible and hence there is a need to develop an alternative formula that is reproducible and applicable at all healthcare levels

Histopathologic Alterations Associated with Global Gene Expression Due to Chronic Dietary TCDD Exposure in Juvenile Zebrafish

Correction 25 Aug 2014: The PLOS ONE Staff (2014) Correction: Histopathologic Alterations Associated with Global Gene Expression Due to Chronic Dietary TCDD Exposure in Juvenile Zebrafish. PLOS ONE 9(8): e106605. https://doi.org/10.1371/journal.pone.0106605The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption

Oncogenic KRAS promotes malignant brain tumors in zebrafish

BACKGROUND: Zebrafish have been used as a vertebrate model to study human cancers such as melanoma, rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a human version of oncogenic KRAS (KRAS[superscript G12V]). METHODS: Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression. Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated. RESULTS: We demonstrated that transient transgenic expression of KRAS[superscript G12V] in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS[superscript G12V] induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS[superscript G12V] induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord. CONCLUSIONS: Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and for discovering oncogenic RAS inhibitors.Keywords: gfap, krt5, Oncogenic KRAS (KRAS[superscript G12V]), Zebrafish, Drug screening, Brain tumor

A Digenean Metacercaria (Apophallus sp.) and a Myxozoan (Myxobolus sp.) Associated with Vertebral Deformities in Cyprinid Fishes from the Willamette River, Oregon

A high prevalence of vertebral deformities has been observed in various fishes, especially cyprinids, from certain regions of the Willamette River for many years. One proposed source of these deformities is exposure to toxicants. Histological evaluation of affected chiselmouth Acrocheilus alutaceus revealed that all lesions associated with vertebrae were associated with metacercariae of digenean trematodes. Approximately half of the northern pikeminnow Ptychocheilus oregonensis had infections in which metacercariae were associated with these lesions. Metacercariae were also associated with vertebral lesions in three of four affected peamouth Mylocheilus caurinus. Many metacercariae that were present within the vertebral bodies were associated with bony dysplasia and bony proliferation in all three species. We also evaluated the association of the metacercariae with the vertebral deformities, using intact fish that had been cleared with trypsin. Fish from the affected regions had a much higher prevalence of metacercariae and deformities and a greater abundance of metacercariae than those in the reference site. Chiselmouths had more deformities and metacercariae than northern pikeminnow. In all fish species, 77% of deformities were directly associated with metacercariae; in chiselmouths, about 95% of the deformities exhibited this relationship. Two types of metacercariae were identified in affected fish: Apophallus sp. (Heterophyidae) and a neascus type (Strigeidida). The Apophallus sp. appeared to be more closely associated with the skeleton deformities. A Myxobolus sp. morphologically similar to M. cyprini was also associated with the vertebral lesions in about 50% of the northern pikeminnow and 5% of the chiselmouths. Intact plasmodia were found in somatic muscle, and lesions containing free spores were often located at bone surfaces. This survey demonstrates that metacercariae (probably Apophallus sp.) and a Myxobolus sp. are major causes of the vertebral deformities seen in cyprinid fishes from certain regions of the Willamette River.This is the publisher’s final pdf. The published article is copyrighted by the American Fisheries Society and can be found at: http://www.tandfonline.com/toc/uahh20/current#.UgvTRHfAF8F