Fig. 2

miR-9 Controls Brain Vasculature Development by Limiting VEGF-A Expression

(A) Schematic representation of the miR-9 MO and miR-9 RNA mimic effects. In the presence of the miR-9 MO, miR-9 is paired with the MO, inhibiting the mRNA degradation. The miR-9 RNA mimic is ubiquitously expressed to allow the degradation of the miR-9 targets.

(B) Whole-mount in situ hybridization against vegfaa in embryos at 48 hpf. vegfaa mRNA behaves like a miR-9 target, showing an increase in the miR-9 knockdown and a decrease in the miR-9 gain of function.

(C and D) Whole-mount in situ hybridization against miR-9 in control (C) and miR-9 morphant (D) shows that miR-9 knockdown does not affect brain and eye morphogenesis at 72 hpf. In miR-9 morphant Tg(kdrl:mCherry) brain at 72 hpf, the mesencephalic central artery (MMCtA), hindbrain and retinal blood vessels are affected (asterisks in D). In the hindbrain, primordial hindbrain channels (PHBCs) are thicker, and central arteries (CtAs) are disorganized and thinner and show aberrant multidirectional sprouting.

(E) Confocal projections of EGFP labeling in Tg(kdrl:egfp) at 72 hpf showing ECs (E'; EGFP+/DAPI+) in posterior PHBCs in control or miR-9 morphant larvae.

(F) Quantification of the size, in microns, of PHBCs (the region of interest [ROI] indicated in E) (control, n = 33; or miR-9 MO, n = 28) and the total number of ECs recruited to form the posterior PHBCs (control, n = 12; or miR-9 MO, n = 10) at 72 hpf.

(G) Quantification of the number of CtAs found on each hemi-hindbrain in the control (n = 20) or the miR-9 MO dilution series (ns = 34, 28, and 56 for croissant dilutions) at 72 hpf.

(H and I) Tg(kdrl:mCherry) brain at 72 hpf showing blood vessel formation in the hindbrain (H), and quantification of the number of CtAs found on each hemi-hindbrain (I), of control larvae (n = 40), SU5416-treated larvae (0.06 μM; n = 48), miR-9-morphant larvae (n = 48), and miR-9-MO larvae treated with 0.06 μM SU5416 (n = 30). Dorsal view of the brain with anterior up. Lateral view of the retina.

Scale bars: 100 μm for in situ hybridization (ISH) in (B)–(D) and 10 μm for immunolabeling in (C)–(E') and (H). Error bars represent SD. ∗∗∗p < 0.0005, determined by t test, two-tailed; n.s, not significant.