No abstract

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Deltalike ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets. (Blood. 2012; 119(11):2679-2687) IntroductionAngiogenic balance and endothelial cell fate are determined by the extracellular signals generated by angiogenic growth factors (stimuli) and inhibitors. 1,2 Molecular mechanisms that determine angiogenic balance have been extensively studied; however, our understanding of the key intracellular events remains incomplete. Recent studies have shown that growing vasculature follows the gradients of VEGF, which are sensed by the nonproliferative endothelial tip cells that direct further expansion of the vascular sprout. The density and morphology of the growing vasculature is dictated by the frequency of tip cells. Following behind tip cells, proliferating stalk cells ensure sprout lengthening and lumen formation. Their fate is maintained by Delta like ligand 4 (Dll4), which is produced by the tip cells. Dll4 binds Notch on adjacent stalk cells, and the resulting signal represses tip fate and ensures proliferation and sprout lengthening toward the VEGF source. 3 Stalk cell proliferation and neovessel integrity depend on VEGF and other pro-angiogenic cytokines, such as basic fibroblast growth factor, which through cognate receptors activate mitogenic kinases converging on Erk1/2. 4 In normal tissues, VEGF release from the extracellular matrix is tightly controlled and improper VEGF gradients cause abnormally high numbers of tip cells and aberrant vascular patterns. 5,6 A large family of Sprouty (Spry) genes regulates secondary branching of the tubular structures in the kidney, lung, and ear. 7 This family encodes proteins Spry1 through 4 and sprouty-related domain 1 (SPRED1) and SPRED2. In...

No abstract

Transplantable tumors are an accepted gold standard in cancer studies in rodents. The progress of this model in zebrafish has long been constrained by the lack of true inbred lines in zebrafish. We have generated several lines of homozygous diploid clonal zebrafish lines, which allow serial transplantations of tumor cells from one fish to another without sublethal gamma-irradiation. The spectrum of transplantable tumors that were initially induced and maintained in inbred clonal zebrafish lines was limited to different types of spontaneous and diethylnitrosamine-induced hepatic tumors. However, this model can readily be extended to a broad range of extrahepatic tumors, transgenic tumors with defined mechanisms of induction and fluorescence-tagged tumor lines. These models will further facilitate in-depth analysis of invasive tumor growth, angiogenesis, metastasis and tumor-initiating cells by in vivo imaging and provide a cost-effective system for high-throughput (HTP) screening of anticancer therapeutics, including biological response modifiers. In addition, homozygous zebrafish lines are an indispensable tool for immunogenetics, mapping of quantitative trait loci and other genetic applications. The whole procedure, from generation of a gynogenetic female homozygous fish (a founder) to obtaining 3-4 consecutive passages of a syngeneic tumor, takes approximately 12-18 months. This time-frame largely depends on methods of tumor induction, tumor type and tumor growth rate.

No abstract

Earlier we suggested a new hypothesis of the possible evolutionary role of hereditary tumors (Kozlov, Evolution by tumor Neofunctionalization, 2014), and described a new class of genes – tumor specifically expressed, evolutionarily novel (TSEEN) genes - that are predicted by this hypothesis (Kozlov, Infect Agents Cancer 11:34, 2016). In this paper we studied evolutionarily novel genes expressed in fish tumors after regression, as a model of evolving organs. As evolutionarily novel genes may not yet have organismal functions, we studied the acquisition of new gene functions by comparing fish evolutionarily novel genes with their human orthologs. We found that many genes involved in development of progressive traits in humans (lung, mammary gland, placenta, ventricular septum, etc.) originated in fish and are expressed in fish tumors and tumors after regression. These findings support a possible evolutionary role of hereditary tumors, and in particular the hypothesis of evolution by tumor neofunctionalization.Research highlightsEarlier we described a new class of genes that are tumor-specifically expressed and evolutionarily novel (TSEEN). As the functions of TSEEN genes are often uncertain, we decided to study TSEEN genes of fishes so that we could trace the appearance of their new functions in higher vertebrates. We found that many human genes which are involved in development of progressive traits (placenta development, mammary gland and lung development etc.,) originated in fishes and are expressed in fish tumors.

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

The activities of 1,2-dibromopropane (DBP) and 1,1,3-tribromopropane (TBP) were studied in seven genotoxicity assays, (i) SOS-induction in E. coli, (ii) DNA repair in primary rat hepatocyte culture, (iii) the Salmonella/microsome assay, (iv) a host-mediated assay using Salmonella, (v) the somatic mutation and recombination assay in Drosophila melanogaster, (vi) HGPRT-mutagenesis assay in ARL 18 cells, and (vii) micronucleus formation assay in mouse polychromatophylic erythrocytes (PCE), forestomach (FS), glandular stomach (GS), duodenum (D), jejunum (J), cecum (C) and liver (L). The halopropanes were also tested for tumor formation in the fish Danio rerio. DBP was active in assays (ii), (v), (vii FS) and (vii L). TBP was positive in assays (ii) and (iii), strongly positive in (vii L) and borderline positive in (iv). However, neither DBP nor TBP induced tumors in fish, in contrast to the carcinogenic 1,2-dibromo-3-chloropropane. The genotoxicity and potential carcinogenicity of DBP and TBP in mammals is discussed.