(A) Schematic representation of the zebrafish embryonic vasculature, with red square marking the subintestinal vessels (SIVs). (B?G) Confocal images at 3 dpf showing ectopic sprouts (arrowheads) arising in the SIVs of apoba?/? apoBb.1?/? double mutants (E), and apoba?/? apoBb.1MO (F) and stalactite (stl) mutants (G), but not in WT (B), apoBa?/? (C), or apoBb.1?/? (D) animals. (H and I) Quantification of the number (H) and length (I) of ectopic sprouts at 3 dpf. n = 3, nWT = 45, napoBa?/? = 55, napoBb.1?/? = 23, napoBa?/? apoBb.1?/? = 31, napoBa?/? apoBb.1MO = 40, napoBb.1?/? apoBaMO = 21. The data are shown as the mean ± SEM, calculated using ANOVA followed by Tukey?s multiple-comparison test. ****P < 0.0001, **P < 0.01. (J and K) Confocal images at 5 dpf of apoBa?/? and apoba?/? apoBb.1MO embryos in the Tg(12xNRE:Egfp) reporter background. White arrowheads in apoBa?/? apoBb.1MO embryos point to endothelial cells (ECs) with active Notch signaling, in ectopic sprouts that failed to retract. n = 2, napoBa?/? = 10, napoBa?/? apoBb.1MO = 12. (L and M) Alkaline phosphatase (AP) staining of the SIVs at 3 dpf, showing inhibition of ectopic sprouting following intravascular injection of DiI-LDL into apoBa?/? apoBb.1MO. (N) Quantification of number and length of ectopic sprouts following intravascular injection of DiI-LDL (n = 3, napoBa?/? = 57, napoBa?/?+LDL = 43, napoBa?/? apoBb.1MO = 67, napoBa?/? apoBb.1MO+LDL = 57). The data are shown as the mean ± SEM, calculated using ANOVA followed by Tukey?s multiple-comparison test. ****P < 0.0001, ***P < 0.001. (O) AP staining of the SIVs of 3 dpf apoBa?/? apoBb.1MO embryos treated with OA, as quantified (P) (n = 3, napoBa?/? apoBb.1MO = 20, napoBa?/? apoBb.1MO+6?g OA = 19, napoBa?/? apoBb.1MO+10?g OA = 21, napoBa?/? apoBb.1MO+20?g OA = 22). The data are shown as the mean ± SEM, calculated using ANOVA followed by Tukey?s multiple-comparison test. Scale bar: (B?G) 50 ?m, (J?O) 100 ?m. P < 0.05 (considered significant versus control group).
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