- Title
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Pitpnc1a Regulates Zebrafish Sleep and Wake Behavior through Modulation of Insulin-like Growth Factor Signaling
- Authors
- Ashlin, T.G., Blunsom, N.J., Ghosh, M., Cockcroft, S., Rihel, J.
- Source
- Full text @ Cell Rep.
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pitpnc1a -/- Larvae Are Behaviorally Hyperactive (A) CRISPR/Cas9 generated a 5-base pair deletion in exon 2 of pitpnc1a. (B) Pitpnc1a protein is detected in pitpnc1a+/+ brain lysates, weaker in pitpnc1a+/-, and undetectable in pitpnc1a-/-. (C) An activity plot across a 14:10 hr day-night cycle (zoomed in C′) reveals hyperactivity in 6 dpf pitpnc1a-/-. The shaded ribbons represent ± SEM. (D and E) Bar plots (±SEM) of the mean day (D) and night (E) activity. pitpnc1a-/- animals are significantly more active during both day and night (one-way ANOVA, Tukey’s post hoc test). (F) At 36 hpf, pitpnc1a-/- embryos twitch significantly more than pitpnc1a+/+ (mean ± SEM, one-way ANOVA). (G) At both 36 and 52 hpf, pitpnc1a-/- embryos are significantly more touch sensitive (pooled data from 5 and 4 independent experiments, respectively, and repeated in a blinded pitpnc1a+/- in-cross; chi-square test). |
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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. |
IGFBP2 dampens IGF signaling in zebrafish larvae, related to Figure 4. (A-C’) Confocal images of 48 hpf tailfins labeled with anti-pIGFR antibody (green) reveals numerous puncta in both pitpnc1a -/- (B, B’) and pitpnc1a +/+ (C, C’) embryos, which are nearly eliminated after soaking in 1 ng/μL IGFBP2 (C, C’). D) IGFBP2 dose-dependently reduces brain c-fos levels and reduces growth in pitpnc1a -/- embryos. E-G) pitpnc1a -/- animals have slightly larger brains than pitpnc1a+/+ larvae. E) Inter-ocular distance (red line) and brain length (green) were measured relative to total body length (not shown). F) Mutant brains are significantly (4.2%) larger than wild type (p=0.03, one-tailed t-test) as measured by interocular distance G) and trending (1.3%) larger by length (p=0.13, one-tailed t-test). Scale bars, A-C’, 20 μm. |