Extended Data Fig. 8: Characterization of MCH:epNTR, H6408 and mchr1a mchr1b double mutants.
a, Schematic of the location of MCH-positive cells in the lateral hypothalamus of the zebrafish larvae. b, Confocal maximum projection shows MCH cells from the Tg(−5kbMCH:epNTR-tagRFPT) transgenic fish before treatment with the ablation agent metronidazole (MTZ). Faint signal of projections to the pituitary are seen (dashed region and asterisk). Scale bar, 50 μm. c, MCH cells are ablated in Tg(−5kbMCH:epNTR-tagRFPT) larva treated with metronidazole. d–f, Tg(−5kbMCH:epNTR-tagRFPT) larva stained with MCH antibody show lateral hypothalamic location and projection to the pituitary gland (d) (green) co-localizing to tagRFP-T expression (e, magenta; f, white). g, Alignment of wild-type MCHR1a protein sequence and predicted truncated mutant protein resulting from CRISPR deletion. h, Sequencing confirmation of loss of TATG sequence from exon 2 of mchr1a gene from genotyping of homozygous MCHR1a mutant. i, Alignment of wild-type MCHR1b protein sequence and predicted truncated mutant protein resulting from TILLING mutant with early stop codon. j, Sequencing confirmation of TGG to TGA substitution from exon 1 of mchr1b gene from genotyping of homozygous MCHR1b mutant. k–m, MCHR-antagonist (H6408)-treated fish display increased activity and disrupted onset of ependymal cell and PMT-wave dynamics. k, Top, timeline of experiment and addition of pharmacological agents during the 2-h Ca2+ imaging experiment (red triangle indicates the addition of carbachol). Bottom, proportion of activation of ependymal cells (open circle) and/or PMT wave (filled circle), witnessed in the imaging session between carrier control (n = 8 fish) and H6408 (n = 7 fish) is plotted against the mean latency to activation (in min) ± s.e.m. *P < 0.05 χ2 test. l, Top, actimetry of wild-type zebrafish (n = 20) treated with carrier versus MCHR antagonist (H6408; n = 20) in 10-min bins over a 24-h period, showing mean seconds of activity per bin (bold) ± s.e.m. (shaded). Bottom, sleep depicted as minutes of sleep per 10-min bin (bold) ± s.e.m. across a 24-h period. Black triangle depicts the day-to-night transition, white triangle depicts the night-to-day transition and the orange triangle shows the time of feeding. m, Box plots showing spread of total activities and sleep between between carrier (E3)-stimulated versus H6408-treated groups, separated further by day and night. Blue lines denote median and black circles indicate the mean value. *P < 0.05, two-sided Student’s t-test. See Supplementary Information for reproducibility information.
