Vash1 regulates formation of trunk lymphatic vasculature. (A,B) Tg[fli1a:EGFP]y1 labels parachordal lymphangioblasts (PL, indicated by asterisks) of control (A) and vash1 KD embryos (B) at 52 hpf. (C) Quantification of the percentage of somites with PLs in embryos injected with control and vash1 MO, as well as the rescue with 150 pg vash1 mRNA. Each point corresponds to one embryo. (D) Quantification of percentage of PLs connected to a venous ISV in embryos injected with control and vash1 MO; 6-8 somites. In C, n=62 for controls, n=62 for vash1 morphants and n=59 for vash1 morpholino and RNA-injected embryos. In D, n=25 for controls and n=23 for vash1 morphants. Three biological replicates were carried out. *P<0.0332, **P<0.02, ****P<0.0001 (Kruskal–Wallis in C, Mann–Whitney test in D). (E-G) Zebrafish trunk of 4 dpf Tg[kdr-l:ras-Cherrys916,fli1a:EGFPy1] zebrafish embryos. Arrowheads indicate GFP- and mCherry-positive putative ISV-to-ISV connections in vash1 KD embryos (F, ISV-to-ISV connection in F′). The main axial lymphatic in the zebrafish trunk – the thoracic duct (TD) – is GFP-positive, mCherry-negative (E,E′, arrow), and absent in the vash1 KD embryo (F, ISV-to-ISV connection in F″). The percentage of somites with TD was quantified (G). n=60 control and n=63 morphants analysed. ****P<0.0001 (Mann–Whitney test). (H) Strategy for CRISPR mutation of exon 4 of vash1 includes design of a duplex guide RNA (dgRNA) to target the codons that translate into lysine and cystein (in bold) of positions 174 and 175 of the zebrafish Vash1 protein, crucial for the carboxypeptidase function. (I,J) Trunk region of Crispr/Cas injected vash1 knockout (KO) Tg[fli1a:EGFP]y1 embryos (J) in comparison with the control (I). Embryos injected with control gRNA and Cas9 exhibit regular PL coverage (I, asterisks) and no mutation. vash1 CRISPants – embryos with confirmed CRISPR mutations – lack PLs (J). n=30 control embryos, n=14 CRISPants.