Fig. 4

Fig. 4 The sema3fa-deficient embryos present with leaky vessels within the outer retina. (A–C) Vessels (white) in ventral views of the eyes of live imaged Tg(kdrl:mCherry) 7 dpf larvae. In WT (A: N = 6, n = 15/17) larvae the extraocular choroid plexus (yellow arrowhead) has begun to branch over the back of the retina. Heterozygous (B: N = 4, n = 9/13) and homozygous (C: N = 6, n = 12/17) semafa^ca304 eyes exhibit a dramatic increase in the choroid vasculature as compared to WT. (D–O) Sema3fa deficiency permits the entry of leaky vessels into the neural retina. Thick (40 µm) cryosections through the eyes of embryos injected at 8 dpf (E–I) or 10 dpf (J–O) with 2,000,000 MW rhodamine dextran and fixed 24 hours later. Sections are counterstained with nuclear label (Hoechst). Blood vessels (yellow arrowheads) infiltrate aberrantly into the outer retina of homozygous embryos at 8 dpf (I: N = 2, n = 4/4) and 10 dpf (O: N = 3, n = 10/10), but are not present in WT embryos (F: N = 2, n = 4/4 for 8 dpf; L: N = 2, n = 10/10 for 10 dpf). Dextran is present in the neural retina at 8 and 10 dpf in homozygous mutant eyes (H, I: 8 dpf, N = 2, n = 4/4; N, O: 10 dpf, N = 3, n = 8/10), but is essentially absent from WT (E, F: 8 dpf, N = 2, n = 4/4; K, L: 10 dpf, N = 3, n = 9/10) eyes. Dashed line represents the boundary between the RPE and neural retina. Boxed areas in E, H, K, and N are shown magnified in F, I, L, and O. (P) Average numbers of TUNEL+ apoptotic cells in whole mount WT, heterozygous, and homozygote eyes at 7 dpf (P > 0.99; N = 2, n = 10 for each genotype). (Q, R) An example of the peripheral retina of a WT (Q) and semafa^ca304 (R) 10 dpf larvae, where penetrating pathologic vessels in the mutant appear to disrupt retinal cellular architecture (arrow). (S,T) vegfaa expression in plastic sections of WT (S) and sema3fa mutant (T) 7 dpf retinas. (U) RT-qPCR for vegfaa and vegfab performed on mRNA isolated from WT (n = 5) and semafa^ca304 (n = 5) 7 dpf eyes. br, brain; INL, inner nuclear layer; L, lens; ONL, outer nuclear layer; RGCL, retinal ganglion cell layer. Error bars are SD. Scale bar: D: 100 µm; F, I, L, O: 20 µm.