Activated N-Ras signaling regulates arterial-venous specification in zebrafish

Abstract

BackgroundThe aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown.MethodsA conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection.ResultsIn the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality.ConclusionsOur studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and tumorigenic angiogenesis.

Chemical biology reveals CARF as a positive regulator of canonical Wnt signaling by promoting TCF/β-catenin transcriptional activity

“…For the ectopic expression, Tol2 transposon-mediated transient transgenesis was applied and performed as previously described [ 36 ]. flk1-△N β-catenin-p2a-mcherry transgene constructs within Tol2 vectors (~45 ng/μl) were mixed with transposase mRNA (~50 ng/μl ) and 0.2 m KCl, and then injected into 1-cell stage embryos [ 37 ]. wnt8 morpholinos (MOs, wnt8 -ORF1 MO+ wnt8 -ORF2 MO) (0.6 ng, the sequences have been described previously [ 24 ]), CARF morpholino (MO) (5′-CCTCTTCTTGCCGCCATCACTCTAA-3′, 2 ng), and p53 morpholino (MO) (5′-TCTTGGCTGTCGTTTTGCGCCATTG-3′, 4 ng) were injected into 1-cell stage embryos.…”

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis

“…For the ectopic-expression, Tol2 transposon-mediated transient transgenesis was applied and performed as previously described [ 84 ]. A series of topbp1 transgene constructs within Tol2 vectors (~40 ng/μl) were mixed with transposase mRNA (~60 ng/μl) and 0.2 M KCl, and then injected into 1-cell stage embryos, respectively [ 85 ]. The volume of the mixture injected was about 0.5nL.…”

“…Cytoplasmic and nuclear extracts were prepared from the 3dpf embryos with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s instruction. The immunoblotting was carried out as previously described [ 85 ], with rabbit anti-phospho-Chk1 (Ser345) (133D) antibody (Cell Signaling Technology), rabbit anti-γH2AX antibody, rabbit anti-zebrafish TopBP1 antibody (generated by 840–940 amino acid of zebrafish TopBP1 protein as antigen), mouse anti-GAPDH antibody (1D4) (Santa Cruz), mouse anti-alpha-tubulin antibody (Sigma) or rabbit anti-Histon3 (H3) antibody (Abcam).…”

The Gridlock transcriptional repressor impedes vertebrate heart regeneration by restricting expression of lysine methyltransferase

“…WT zebrafish of the AB strain were used for generating grl mutants and various transgenic lines. Published transgenic lines used in this study included Tg(cmlc2:mCherry) ( Palencia-Desai et al, 2011 ), Tg(flk1:EGFP) ( Jin et al, 2005 ), Tg(flk1:mCherry) ( Ren et al, 2013 ), Tg(tcf21:nucEGFP) ( Wang et al, 2011 ) and Tg(cmlc2:CreER) ( Kikuchi et al, 2010 ). The Institutional Animal Care and Use Committee at East China Normal University advises animal care and research.…”