Activated N-Ras signaling regulates arterial-venous specification in zebrafish
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JOURNAL OF HEMATOLOGY & ONCOLOGY
RESEARCH
Open Access
Activated N-Ras signaling regulates arterial-venous specification in zebrafish Chun-Guang Ren1*, Lei Wang1, Xiao-E Jia1, Yi-Jie Liu2, Zhi-Wei Dong1, Yi Jin2, Yi Chen2, Min Deng1, Yong Zhou1, Yi Zhou3, Rui-Bao Ren2*, Wei-Jun Pan1* and Ting-Xi Liu1,2
Abstract Background: The 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. Methods: A 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. Results: In 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. Conclusions: Our 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. Keywords: Vasculogenesis, Arteriogenesis, N-Ras
Background RAS proteins are small GTPases that have been proved to be essential for the control of cell proliferation, survival, and differentiation [1,2]. Ras genes are evolutionally and functionally conserved in all eukaryotic organisms. In mammals, the 3 Ras genes encode 4 highly * Correspondence: [email protected]; [email protected]; [email protected] 1 Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China 2 Shanghai Institute of Hematology, RuiJin Hospital, Shanghai
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