Figure 4
Figure 4. miR-30b and 30c regulate dll4 expression and angiogenic sprouting in vivo. (A) Expression of miR-30 in whole zebrafish embryos after microinjection of miR-30 mimics at the 1- to 4-cell stage measured by qRT-PCR. Expression is relative to uninjected control embryos. Values indicate the amount of injected mimic. Error bars indicate SD of qRT-PCR experiment which was performed in triplicate. n = 1 but RNA was collected from 20 to 30 embryos per condition. (B) Representative in situ hybridization showing expression of dll4 mRNA levels in the developing vasculature of WT zebrafish embryos and embryos injected with 0.05 ng (miR-30b) and 0.1 ng (miR-30c) miRNA mimics. Values indicate the number of embryos with the predominant, displayed phenotype versus the total number of embryos assayed. Images were taken using a Nikon 1200F camera on a Nikon E1500 dissecting microscope and acquired using ACT-1 software (Nikon). Images were cropped using Adobe Photoshop CS4. (C) Expression of dll4 mRNA in whole zebrafish embryos after microinjection with miR-30 mimics measured by qRT-PCR. Expression is relative to uninjected control embryos. Values indicate amount of injected mimic. Error bars indicate SD of qRT-PCR which was performed in triplicate. n = 1 but RNA was collected from 20 to 30 embryos per condition. (D) DA diameter in embryos injected with dll4 MO or miR-30 mimic relative to uninjected embryos (means + SEM, n = 3). Six DA measurements were made per embryo using Adobe Photoshop CS4. Differences between uninjected embryos and embryos injected with dll4 MO or miR-30 mimics were significant (***P < .001). (E) Trunk vasculature in uninjected Tg(kdrl:EGFP) embryos or embryos injected with the indicated miRNA mimic and/or TP. Left panels: Advanced sprouting and aberrant endothelial cell migration (yellow arrowheads) at 25 hpf. Right panels: Increased branching of the ISVs (red arrowheads) at 72 hpf. Fluorescent images were taken using an Axiocam (Zeiss) on an Axiovision Lumar V12 dissecting microscope (Zeiss) and acquired using AxioVision v4.8 software. Images were cropped and arrowheads were added using Adobe Photoshop CS4.

miR-30b and 30c regulate dll4 expression and angiogenic sprouting in vivo. (A) Expression of miR-30 in whole zebrafish embryos after microinjection of miR-30 mimics at the 1- to 4-cell stage measured by qRT-PCR. Expression is relative to uninjected control embryos. Values indicate the amount of injected mimic. Error bars indicate SD of qRT-PCR experiment which was performed in triplicate. n = 1 but RNA was collected from 20 to 30 embryos per condition. (B) Representative in situ hybridization showing expression of dll4 mRNA levels in the developing vasculature of WT zebrafish embryos and embryos injected with 0.05 ng (miR-30b) and 0.1 ng (miR-30c) miRNA mimics. Values indicate the number of embryos with the predominant, displayed phenotype versus the total number of embryos assayed. Images were taken using a Nikon 1200F camera on a Nikon E1500 dissecting microscope and acquired using ACT-1 software (Nikon). Images were cropped using Adobe Photoshop CS4. (C) Expression of dll4 mRNA in whole zebrafish embryos after microinjection with miR-30 mimics measured by qRT-PCR. Expression is relative to uninjected control embryos. Values indicate amount of injected mimic. Error bars indicate SD of qRT-PCR which was performed in triplicate. n = 1 but RNA was collected from 20 to 30 embryos per condition. (D) DA diameter in embryos injected with dll4 MO or miR-30 mimic relative to uninjected embryos (means + SEM, n = 3). Six DA measurements were made per embryo using Adobe Photoshop CS4. Differences between uninjected embryos and embryos injected with dll4 MO or miR-30 mimics were significant (***P < .001). (E) Trunk vasculature in uninjected Tg(kdrl:EGFP) embryos or embryos injected with the indicated miRNA mimic and/or TP. Left panels: Advanced sprouting and aberrant endothelial cell migration (yellow arrowheads) at 25 hpf. Right panels: Increased branching of the ISVs (red arrowheads) at 72 hpf. Fluorescent images were taken using an Axiocam (Zeiss) on an Axiovision Lumar V12 dissecting microscope (Zeiss) and acquired using AxioVision v4.8 software. Images were cropped and arrowheads were added using Adobe Photoshop CS4.

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