Project description:Vascular endothelial growth factor (VEGF) regulates vasculogenesis by using its tyrosine kinase receptors. However, little is known about whether Sec14-like phosphatidylinositol transfer proteins (PTP) are involved in this process. Here, we show that zebrafish sec14l3, one of the family members, specifically participates in artery and vein formation via regulating angioblasts and subsequent venous progenitors' migration during vasculogenesis. Vascular defects caused by sec14l3 depletion are partially rescued by restoration of VEGFR2 signaling at the receptor or downstream effector level. Biochemical analyses show that Sec14l3/SEC14L2 physically bind to VEGFR2 and prevent it from dephosphorylation specifically at the Y1175 site by peri-membrane tyrosine phosphatase PTP1B, therefore potentiating VEGFR2 signaling activation. Meanwhile, Sec14l3 and SEC14L2 interact with RAB5A/4A and facilitate the formation of their GTP-bound states, which might be critical for VEGFR2 endocytic trafficking. Thus, we conclude that Sec14l3 controls vasculogenesis in zebrafish via the regulation of VEGFR2 activation.
Project description:We isolated GFP-positive and negative cell populations, by fluorescence-activated cell sorting (FACS), from Tg(fli1a:EGFP)y1 embryos at 22-24 hpf, then extracted their RNAs for high-through put sequencing based on Illumina HiSeq2000 to find out enriched genes in vascular, hematopoietic and pharyngeal arch cells
Project description:Hypoxia controls reparative angiogenesis. MiRNAs are master regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs modulate vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony forming cells (ECFCs), a well-characterized subtype of endothelial progenitor. Under hypoxic conditions, miR-130a overexpression enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3-dependent transcription, and accumulation of HIF1α via translational inhibition of DDX6. These findings unveil a new role for miR-130a in hypoxia, whereby it modulates the VEGFR2/STAT3/HIF1α axis to increase the vasoregenerative capacity of ECFCs.
Project description:Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis.
Project description:Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis. Approximately 50 pg of etsrp RNA was microinjected into zebrafish embryos at the 1-4 cell stages. Three separate batches of approximately 150 microinjected embryos and uninjected controls were frozen on dry ice at the tailbud stage. Total RNA was purified using the RNAquous-4PCR kit (Ambion). Approximately 10-20 µg RNA from each batch was sent to the NimbleGen facility, were it was processed, labeled and hybridized to standard NimbleGen 385K gene expression array. Briefly, RNA was converted into cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA was random-prime labeled with Cy3-nonamers and hybridized to the microarrays for 16 hours at 42°C. The zebrafish microarray was based on Zv6 genome assembly and contained approximately 32,000 genes with approximately 12 probes of 60mer oligonucleotides per each gene. The arrays were washed, dried, and scanned at 5μm resolution using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA). Data were extracted from scanned images using NimbleScan software (Roche NimbleGen). Quantile normalization was performed across replicate arrays, and RMA (Robust Multichip Average) analysis was performed to generate gene expression values. Average ratios of expression values of etsrp-injected vs. control were calculated from three experiments. Results were sorted by the fold change from the highest to the lowest. Genes that displayed values of p>0.05 or showed expression levels less than 2-fold above the background level in both control and experimental samples were eliminated from further analysis.
Project description:RNA sequencing of lung tissue from transgenic mice in order to investigate the effect of a single tyrosine to phenylalanine exchange in the endothelial receptor VEGFR2 at position Y949. This exchange creates a mouse with unleaky blood vessels which is an advantage in several diseases such as cancer and cardiovascular disease.