Fig. S2

Generation and verification of an esama mutant allele (A) Schematic representation of the wild-type and esama^ubs19 mutant allele. Top: the amino acid sequence of wild-type Esama (total of 428aa) flanking the region of mutagenesis and schematic drawing of wild-type Esama. Esama harbors two N-terminal extracellular Ig-like domains (one Vand one C2-type), a single transmembrane domain and a long cytoplasmic tail harboring a MAGI-1 binding domain. Bottom: the 10bp deletion leads to a frame shift, which alters Esama's sequence after the first 37aa and leads to a premature stop after a total of 70aa (altered amino acid sequence highlighted in red). This premature stop leaves only a very short peptide. (B and C) Genotyping PCR strategy (B) and examples of amplicons produced by wild-type, esama^ubs19 heterozygous or homozygous mutants. (A) The external primers (P30 and P55) flank exon 2 and amplify both, mutant and wild-type alleles (482bp). This amplicon is outcompeted by the smaller wild-type and ubs19 products. P99 specifically anneals to the wild-type sequence and produces a band of 356bp with P55. P101 is specific for the mutation and together with P30 generates a 142bp product. (D and E) Confocal images of wild-type (D) and esama^ubs19 (E) Tg(fli1a:EGFP)y1 (blue) embryos stained for Esama (green) and Zo-1 (red), single channels are shown in inversed contrast, anterior to the left. The architecture of SeAs looks similar between wild-type and esama^ubs19 (compare the EGFP channels D and E); there are no obvious angiogenic defects in the mutant. The staining for Esama confirms the loss of protein in the mutant background (E'). Importantly, the Esama antibody recognizes epitopes in the cytosolic C-terminal portion of the protein (Sauteur et al., 2014), excluding the possibility of splice variants in the esama^ubs19 mutant background. The Esama antibody shows cross-reactivity with the myotome, which remains visible in the mutant (demarcated by red asterisks in D' and E'). aa, amino acid; TM, transmembrane domain, scale bars, 20μm.