Ppa-egl-4 genome location, EGL-4 protein sequence alignment, and protein expression. (A) The position of Ppa-egl-4 on integrated genetic linkage and physical maps. Ppa-egl-4 is located at 167 cM on Chromosome IV on Supercontig 85. Nearby subcontigs 85.x flanking the contig85.28 containing egl-4 are shown (≈30 kb, not to scale) with the genotyped strain background indicated above. The only region containing the Washington donor sequence is between contigs 85.25 and 85.32, spanning ≈110 kb. The 780-bp deletion containing the second exon (tu374), which results in frame shift and a premature stop codon, is shown below the diagram of the egl-4 gene structure. Genetic distances are indicated in parentheses. (B) Amino acid sequence alignment of egl-4 orthologs among P. pacificus strains California and Washington, C. elegans N2, and Pristionchus sp. 11. (C) Western blot of whole adult hermaphrodite protein extracts, using an anti-Cel-EGL-4 antibody. Additional loading and antibody controls are shown in Fig. S3 A and B. (D) P. pacificus California adult hermaphrodites immunostained with anti-Cel-EGL-4 antibody show staining in head neurons. (Scale bars, 20 μm.)