The sox18sa12315 mutant behaves as expected for a null allele. (A) On the left is a schematic representation of the Sox18 protein, with the HMG-box domain in blue. The G > A transition and the premature stop codon introduced in the sa12315 mutant are indicated. Fragments of the electropherograms derived using Sanger sequencing of the region surrounding the mutation in wt (sox18+/+), heterozygous (sox18+/−) or homozygous mutants (sox18−/−) are reported on the right. The restriction site for the BstNI/MvaI enzymes (boxed sequence) is disrupted by the mutation. (B) Embryos derived from sa12315 heterozygote matings and injected with subcritical doses of sox7-MO were collected in several independent experiments and analyzed in vivo at 2 dpf and 3 dpf or fixed at around 30 hpf for ISH, as shown in (C). The histogram on the right shows the trunk–tail circulatory phenotypes observed at 3 dpf. In control embryos, i.e., uninjected or injected with a standard control MO (first and second bars, respectively), trunk–tail circulatory defects are present in a small percentage of embryos. On the contrary, the partial knockdown of sox7 causes a blockage in trunk–tail circulation in a dose-dependent manner (third and fourth bars). Circulatory defects are genotype-dependent (see Table S1). (C) ISHs were performed on embryos derived from sa12315 heterozygote matings and injected with subcritical doses of sox7-MO, as shown in (B), fixed at around 30 hpf. Upper panels show control ISH performed with the endothelial marker cdh5, showing no gross alteration in embryos of the three different genotypes. Lower panels show ISHs performed with a probe for vsg1/plvapb, whose expression was particularly downregulated in double partial sox7/sox18 morphants [29]. Higher magnification images of the trunk–tail regions of the embryos are also shown. Experiments were repeated twice; all plavpb stained embryos and a subset of cdh5 stained embryos were genotyped; numbers in each image refer to a single experiment. Lateral views, anterior to the left. Pictures were taken at 40× and 63× magnification, for lower and higher magnification images respectively.
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