Aggf1 is involved in Bmp-mediated CV formation. (A) Expression patterns of aggf1 mRNA in zebrafish embryos at 24, 30 and 36hpf, as detected by whole-mount in situ hybridization. Sense probe was used to confirm the specificity of the hybridization reaction. Regions from the yolk tube to the tail are enlarged beneath. (B) Expression of aggf1 mRNA in 36hpf embryos injected without (control) or with hsp70l:noggin3-FLAG or hsp70l:bmp2b-FLAG plasmid and heat shocked at 24hpf. The caudal regions are enlarged to the right. (C) Confocal stack GFP images of the caudal region of 48hpf Tg(fli1:GFP) embryos injected with control morpholino oligonucleotide (MO) or two independent MOs against aggf1 (MO1 and MO2). Single-scan confocal images of these embryos are shown to the right. (D) The CV phenotypes observed in C were quantified as in Fig. 2D. (E) Confocal stack images of the caudal region of 48hpf Tg(fli1:GFP) embryos injected with hsp70l:bmp2b-FLAG plasmid together with either control MO or aggf1 MO1. Arrowheads indicate ectopic venous vessels originating from the CVP. (F) Quantification of ectopic venous vessel formation observed in E, showing the area covered by ectopic venous vessels relative to that in control MO-injected embryos. Data are mean±s.e.m. Control MO, n=10; aggf1, MO1 n=10. (G) Confocal stack fluorescence images of 32hpf Tg(fli1:Gal4FF);(UAS:NLS-GFP) embryos injected with control MO or aggf1 MO1 and subjected to TUNEL staining, as in Fig. 2G. (H) Percentage of TUNEL-positive ECs among the NLS-GFP-expressing ECs in the CVP of embryos injected with either control MO or aggf1 MO1 as observed in G. Data are mean±s.e.m. Control MO, n=16; aggf1 MO1, n=16. (F,H) *P<0.05, ***P<0.001; n.s., not significant. Scale bars: 50µm.
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