LOXL2 is required for proper formation of intersegmental vessels in zebrafish. (A) Semiquantitative RT-PCR of loxl2a and actin mRNA from zebrafish embryos was performed at different developmental stages (20-72 hpf). (B) Whole-mount in situ hybridization of loxl2a in zebrafish embryos at 24 and 48 hpf using a sense probe as a control. Bar: 50 μm. (C) LOXL2 and actin were immunodetected in protein extracts of 24 and 48 hpf embryos. Proteins were sequentially extracted in an NP40/Deoxycholate-soluble fraction (S) and in an SDS-soluble fraction (IS). (D) Zebrafish embryos (48 hpf) injected with mismatch (ms-MO) or loxl2a (loxl2a-MO1) morpholinos were sorted according to the presence (+) or the absence (−) of ISV blood circulation. SDS-soluble proteins were subjected to immunoblotting for actin and LOXL2 detection. (E) Confocal images of Tg(fli1:egfp)y1 zebrafish embryos at 24 and 48 hpf, injected with either mismatch (ms-MO) or loxl2a morpholinos (loxl2a-MO1). Bar: 100 μm. (F) Brightfield images of 48 hpf embryos untreated (control) or treated with β-APN at 3 hpf and 23 hpf. Bar: 250 μm. (G) Quantification of the number of perfused ISV in 48 hpf embryos untreated or treated with β-APN at 3 hpf or 23 hpf (n = 30-32 embryos per condition). (H) Confocal images of Tg(fli1:egfp)y1 zebrafish embryos at 48 hpf, untreated or treated with β-APN at 3 hpf or 23 hpf. Bar: 100 μm. See also supplemental Figure 5 and supplemental Videos 1-3.