Loss of dll4 regulation by miR-30 synergizes with partial Vegfa signaling knockdown to block angiogenesis. (A) Representative in situ hybridization showing expression of dll4 mRNA in the developing vasculature of zebrafish embryos injected with 10 ng of the indicated TP. Values indicated 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. (B) Expression of dll4 mRNA in whole zebrafish embryos after microinjection of dll4-TP^miR-30 measured by qRT-PCR (means + SEM, n = 5). Expression is relative to embryos injected with dll4-TP^control. Values indicate the amount of injected TP as ng/embryo. Differences between dll4-TP^control and dll4-TP^miR-30 injected embryos were significant (*P < .05, ***P < .001). (C) Trunk vasculature in uninjected Tg(fli1a:EGFP) embryos or embryos injected with the indicated MO and/or TP at 28 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 using Adobe Photoshop CS4. (D) Quantification of ISV sprouting defects after injection of the MOs and/or TPs indicated. Columns are the average of 2 independent experiments with 15 to 30 embryos counted per sample per experiment. Moderate phenotype: embryos lacking 1 ISV but with a minimum of 6 ISVs. Severe phenotype: embryos with 5 or less ISVs. To determine statistical significance, each observation of no phenotype, moderate phenotype and severe phenotype was given the value 0, 1, and 2, respectively, and then a Wilcoxon rank-sum test was performed. The difference between embryos injected with dll4-TP^miR-30 and embryos injected with dll4-TP^miR-30 + kdr MO was significant (***P < .001). Embryos were examined using an Axiovision Lumar V2 dissecting microscope (Zeiss) for quantification to be performed. (E) Representative in situ hybridization showing expression of kdrl mRNA in the developing vasculature of a WT embryo and embryos injected with 10 ng of the indicated TP. Values indicated 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.