Phylogenetic trees of the P2YR family and the generation of a P2RY11^−/− mutation in zebrafish using CRISPR-Cas9 editing. A. Phylogenetic analysis was conducted on the predicted protein sequences of 8 families of P2Y receptors across 7 species using the neighbor-joining method. B. Structure of the zebrafish P2RY11 gene and protein. A guide RNA was designed to specifically target exon 2. The P2RY11^−/− allele was created by deleting 8 bases in exon 2. The "-" indicates the deleted nucleotides. The mutation results in a frameshift and premature truncation of the protein. The blue boxes represent the transmembrane domains of P2RY11 protein. C. Western blot and statistical analysis (n = 3) of the P2RY11 protein in the tail of the P2RY11^−/− and P2RY11^+/+ adult (3 mpf) zebrafish. D. The expression level of P2RY11 mRNA in the 3-dpf P2RY11^−/− and P2RY11^+/+ larvae was analyzed by qRT- PCR (n = 20). T-test, ***p < 0.001, **** p < 0.0001

Morphological analysis of P2RY11^−/− zebrafish larvae. A, Morphology of P2RY11^−/− and P2RY11^+/+ larvae at 48, 72, and 96 hpf. B-D, Statistical analysis for the head (B), eye (C), and pericardium (D) of P2RY11^−/− and P2RY11^+/+ larvae at 48 hpf (n = 28), 72 hpf (n = 23), and 96 hpf (n = 28). Scale bar: 500 μm. T-test for analysis, *p < 0.05, ** p < 0.01 ***p < 0.001, ****p < 0.0001

Deficiency of P2RY11 reduces the HCRT expression. A, Reduced expression of hcrt mRNA in P2RY11^−/− mutants at 3 dpf detected by RT-qPCR (n = 20). B, Western blot (n = 10) and statistical analysis (n = 3) of the HCRT protein in P2RY11^−/− mutants at 3 dpf detected by western blot. C, Decreased HCRT neurons in P2RY11 MO injected larvae at 3 dpf (n = 20). D, Reduced expression of hcrt mRNA in P2RY11 MO morphants at 3dpf (n = 20). E, Western blot (n = 20) and statistical analysis (n = 3) of the P2RY11 protein in P2RY11 MO morphants at 3dpf. T-test (A, B, E) and one way ANOVA (D) were conducted. *p < 0.05, **p < 0.01 ***p < 0.001

Abnormal sleep/wake pattern in P2RY11^−/− mutants. A, B, showing time sequence photos of rest total (A) and waking activity (B). The red line represents the mutant group, while the blue line indicates the WT group. C-F, Parameter analysis showed that rest time (C) and rest bout length (D) were prolonged in P2RY11^−/− mutants, with increased sleep frequency (E) during the daytime. Meanwhile, the waking activity (F) were decreased in the daytime recordings. The bars in black and white blow the x-axis indicate the tested nighttime and daytime periods, respectively. (t-test, n = 12, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Effect of P2RY11 mutation on the expression of genes related to the immune system process. A, Quantitative analysis of the expression of il6, tnfa, il1b, il4, il10 and tgfb. (n = 20). B, Gene Ontology analysis of the DE genes related to the immune system process. C, The relationship of the enriched GO terms. D, Heatmap demonstrating downregulation of genes in P2RY11^−/− mutants involved in the main enriched terms (n = 3). E, qRT-PCR validation of the RNA-seq data. Downregulation of cxcl20, cora1a, ccl39.3, ccl34b.1, cyba, mpeg1, mmp13a, lyz (n = 20). T-test was performed, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Deficiency of P2RY11 reduced macrophages and neutrophils and their accumulation at the injury site following the fin amputation. A, The Sudan black B (SB) signal in siblings (upper) and P2RY11 mutants (lower) at 3 dpf (t-test, n = 10). B, The neutral red signal in siblings (upper) and P2RY11 mutants (lower) at 3 dpf in CHT (t-test, n = 10). C, recruited neutrophils to wounds following tail fin amputation. SB staining (upper) showed the recruitment of neutrophils at 2 hpi in 3dpf-siblings and P2RY11 mutants (t-test, n = 10). SB⁺ cells were significantly reduced in P2RY11^−/− mutants in the quantified region (the black rectangle). The recruitment of neutrophils (RPF⁺ cells in the white rectangle) was also abolished in P2RY11 MO morphants at 3 dpf in the transgenic lyz:DsRed compared with the control morpholino-injected group (cm) and wild type group (WT) (one-way ANOVA, n = 11). D, recruited macrophages to wounds following tail fin amputation. Neutral red staining revealed the recruitment of macrophages at 6 hpi in 3dpf-siblings and P2RY11 mutants (t-test, n = 12). Neutral red⁺ cells were significantly reduced in P2RY11 mutants within the quantified region (the black rectangle). The recruitment of macrophages (GFP⁺ cells in the white rectangle) was also abolished in P2RY11 MO morphants at 3 dpf in the transgenic mpeg1: GFP compared with the control morpholino-injected group (CM) and wild- type group (WT) (one-way ANOVA, n = 12). E, Migration speed of neutrophils and macrophages in P2RY11 knock-down larvae during the process toward the wound edge (one-way ANOVA, n = 5). F, Quantitative analysis of il6, tnfa, il1b, il4, il10, and tgfb from tails of WT and mutants at 6 hpi (t-test, n = 20). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Scale bar: 200 μm

Diagram illustrating the effect of P2RY11 mutation on the immune response and the relationship of P2RY11 mutation with NT1