Sox18/Vegfd double knockout mouse embryos display defects of the major vessels. (A) Immunofluorescence staining on transverse section of 11.5 dpc embryo using NEUROPILIN-2 (green), CAVEOLIN (red), and CD34 (blue) revealed the dilation of the DA and CV in the double knockout embryo compared with the control. (B) Frontal view of 11.5 dpc embryos whole mount stained with PECAM (red) and VEGFR3 (green) (i,iii). Rendering of the major vasculature using IMARIS software (i-iv), showing the dilated left, right, and midline DA in the double knockout embryo (iv) compared with control embryos Sox18^+/−, Vegfd^+/+ (ii, white arrowhead). Asterisk indicates the missing interarterial branching in double knockout embryo. (C) In situ hybridization shows both the expression profile of Sox18 and Vegfd on transverse sections of mouse embryo at 11.5 dpc. Sox18 mRNA was detected in the endothelium of both DA and CV at 11.5 dpc (red). Vegfd mRNA was detected in the vicinity of the DA and CV (purple). (D) In situ hybridization shows the expression profile of vegfd in zebrafish at 24 hpf. (i,iii) vegfd mRNA was detected at the tailbud (i, black arrow), neurons (iii, black arrows), and trunk (iii, bracket). (ii,iv) Sense probe was used as a negative control for the specificity of vegfd signal in in situ. Dashed lines (i,ii) indicate the magnified area in (iii-iv). A, anterior; CSG, cervical sympathetic ganglia; D, dorsal; H, heart; L, lateral; M, medial. Scale bar represents 200 μm (A), 100 μm (C), 200 μm (Dii), and 100 μm (Div).