Fig. S3

A CRISPR/Cas9 generated five base deletion of zebrafish pitpnc1a leads to a truncated protein lacking key functional residues, related to Figure 3.

A) The pitpnc1a Δ5 allele leads to a truncated 336 bp open reading frame. B) Alignment of the full length Pitpnc1a and predicted truncated protein of the pitpnc1a Δ5 allele. Critical amino acids for binding of the inositol ring of phosphatidylinositol (T59, K61, E86, and N90, mouse numbering) are highlighted, demonstrating the truncated protein lacks two of these critical residues. C) DNA extracted from whole larvae and subjected to high resolution melt curve analysis (HRMA) is able to distinguish the wild type (+/+, red), heterozygous (+/-, yellow), and homozygous mutant (-/-, green) pitpnc1a genotypes. D) In situ hybridization revealed the expression of the dorsal forebrain markers egr3, eomesa, and tbr1a are unaffected in pitpnc1a-/- animals. Dorsal views; anterior to the top. E) In situ hybridization for an anterior hypothalamic marker, npvf, is unaffected in pitpnc1a-/- animals. Ventral views; anterior to the top. F) Representative optical pERK slices (plane 88 of the Z-brain) from wild type and mutant brains stained with pERK/tERK and linearly registered to the Z-Brain reference using the tERK channel. White arrowheads point to examples of areas with upregulated pERK in mutant brains. Images were normalized for intensity using the Stack Normalizer plugin https://imagej.nih.gov/ij/plugins/normalizer.html in Fiji. G) Single channels for the unthresholded maximum projections for the mutant upregulated (green, left) and downregulated (magenta, right) pERK signals.