Establishment of P2RY11 mutant

To elucidate the relationship between zebrafish P2Y11 and its corresponding orthologues in different taxa, a phylogenetic analysis were performed using the protein sequences of 8 families of P2Y receptors across 7 species (shrew, zebrafish, human, macaques, rat, mouse, and frog) using the neighbor-joining method (Fig. 1A). The P2Y receptors can be categorized into two clades. The P2Y11 cluster forms one clade with clusters of P2Y1, P2Y2, P2Y4, and P2Y6. Clusters of P2Y8, P2Y12, P2Y13, and P2Y14 forms another clade. As expected, zebrafish P2Y11 is located in the cluster of P2Y11.

Fig. 1

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

To investigate the role of P2RY11 in sleep and inflammation, a P2RY11mutated zebrafish line was generated by targeting the exon 2 of P2RY11 (Fig. 1B). A P2RY11 mutation with an 8- bp deletion was identified, causing a frameshift mutation in the protein-coding region, which consequently triggered premature termination of translation. To verify the mutation, the expression of P2RY11 mRNA and protein were measured in WT and P2RY11 mutants. The results showed that the expression of P2RY11 was decreased in mRNA level and undetected at the protein level in P2RY11 mutants (Fig. 1C, D). Therefore, the generated P2RY11 (-8 bp) mutant is a loss-of-function mutation.

Morphological analysis of P2RY11-deficient zebrafish larvae

To examine whether P2RY11 deficiency would affect early development, we observed morphologic characteristics from 4-cell stage. No difference was found between the P2RY11−8 bp mutant and its siblings from 4-cell stage to 24 hpf. With development progressing, the eyes and head of P2RY11−8 bp larvae became significantly smaller than those of their siblings at 48, 72 and 96 hpf (Fig. 2A-C). Pericardial edema was also evident in P2RY11−8 bp larvae (Fig. 2A). The size of the pericardium was significantly larger when compared with the control siblings at 48, 72, and 96 hpf (Fig. 2D). In summary, the deficiency of P2RY11 could not affect the early embryonic development but impact later growth of the eye, head, and heart regions.

Fig. 2

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 decreases the expression of HCRT

Given the association of P2RY11 with NT1, the expression of HCRT was examined in P2RY11-deficient larvae at 3 dpf. Both hcrt mRNA and HCRT protein were reduced in P2RY11−8 bp larvae compared to their WT controls (Fig. 3A, B). In order to investigate the impact of P2RY11 deficiency on HCRT neurons, we knocked down the expression of P2RY11 in Tg(hcrt:GFP) and observed a decrease in number of HCRT neurons and mRNA levels of hcrt in the P2RY11 deficient larvae (Fig. 3C-E).

Fig. 3

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

Deficiency in P2RY11 increases sleepiness and reduced waking activity during the daytime

To examine whether the deficiency of P2RY11 could affect the sleep patterns of zebrafish larvae, we conducted a sleep/wake analysis from 96 to 144 hpf. The results showed that P2RY11−8 bp larvae exhibited reduced activity and increased rest during both night and daytime compared to their WT controls (Fig. 4A, B). The parameters regarding sleep/wake behavior were analyzed. P2RY11 deficiency could significantly increase duration of rest, the number of rest bouts, and length of rest bouts only during the daytime period (Fig. 4C-E). Accordingly, both total activity and waking activity of P2RY11−8 bp larvae were considerably lower than those of their WT controls in either daytime recorded (Fig. 4F; Fig. S1). Thus, P2RY11−8 bp larvae exhibited a changed sleep/wake pattern similar to the excessive daytime sleepiness observed in narcolepsy patients.

Fig. 4

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)

The lack of P2RY11 affects the expression of genes involved in the immune system process

Since P2RY11 is expressed in different immune cell types, and its activation exerts both pro- and anti-inflammatory effect (Gruenbacher et al. 2021), here we conducted a qRT-PCR analysis to measure the mRNA levels cytokines to detect the impact of P2RY11 deficiency on inflammatory status. Comparing with WT controls, the mRNA levels of il6, tnfa, and il1b were up-regulated, while the mRNA levels of il4, il10 and tgfb were down-regulated in the P2RY11−8 bp larvae (Fig. 5A).

Fig. 5

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

To analyze the function of P2RY11, RNA sequencing analysis was performed and 1693 differential expressed (DE) genes were identified. GO term analysis were further conducted on DE genes using the Metascape database. The results showed that the plasma membrane signaling receptor complex (GO:0098802), calcium ion binding (GO:0005509), and protein refolding (GO:0042026) were the most enriched terms in the up-regulated DE genes (Fig. S2A, B). While metabolic processes (GO:008152), cellular locations (GO:0051179) and cellular processes (GO:009987) were the most enriched terms in the down-regulated DE genes (Fig. S2C). In addition, the immune system process (GO:0002376) was also enriched in down-regulated DE genes of P2RY11−8 bp compared to WT controls (Fig. S2C). Based on the genes related to the immune system process, we further conducted GO term enrichment analysis and found that the defense response, leukocyte chemotaxis, and inflammatory response were the main enriched terms (Fig. 5B, C). Clustering analysis of the enriched GO term of the immune system process was conducted and showed that genes involved in neutrophil chemotaxis (cxcl8a, cxcl20, ccl25b, ccl39.3, wasb, lta4h, ccl44), inflammatory response (pycard, caspb, lta4h, anxa1c), defense response (tfa, mpeg1, hamp, lygl1, npsn, tnfrsf14l, wfdc2), and macrophage chemotaxis (mmp13a, cyba, cxcr3.2) were downregulated in P2RY11−8 bp compared to their WT controls (Fig. 5D). The expression of ccl34.b, coro1a, ccl39.3, cxcl20, cyba, mmp13a, lyz, and mpeg1 were detected to valid the RNA- seq data. It was found that mRNA levels of the selected genes were significantly reduced in P2RY11−8 bp larvae, consistent with the RNA-seq results (Fig. 5E). Therefore, the above results show that the absence of P2RY11 reduces the mRNA expression of several genes related to the chemotaxis of macrophages and neutrophils, inflammatory response, and defense response.

The lack of P2RY11 decreases the number of macrophages and neutrophils, as well as their accumulation at the injury site following fin amputation

Since P2RY11−8 bp larvae showed decreased expression of mpeg1 and lyz, which are specifically expressed in macrophages and neutrophils, respectively, we used Sudan Black staining and Neural red staining to label macrophages and neutrophils in P2RY11−8 bp larvae. We found that mutation of P2RY11 obviously reduced the numbers of neutrophils and macrophages in the caudal hematopoietic tissue (CHT) located at the ventral side of the tail (Fig. 6A, B).

Fig. 6

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

Given the reduction of genes related to the chemotaxis of macrophage and neutrophil in P2RY11−8 bp larvae, a caudal fin injury was induced to stimulate non-infectious inflammatory responses and access the chemotaxis of macrophages and neutrophils. The numbers of neutrophils and macrophages at the damaged site reached their peaks at 2 hpi and 6 hpi, respectively. Thus, larvae were collected at 2 hpi to detect the number of neutrophils in the cut region. SB staining revealed that depletion of P2RY11 could significantly reduce the accumulation of neutrophils in the wound at 2 hpi, consistent with the findings in the P2RY11MO-injected transgenic line Tg (lyz: Dsred) (Fig. 6C). The macrophages were also examined in the cut region at 6 hpi in P2RY11−8 bp larvae and P2RY11MO-injected transgenic line Tg (mpeg1: GFP). Neutral red staining revealed that the number of macrophages at the wounded site of P2RY11−8 bp larvae was conspicuously lower than the control group (Fig. 6D). Similarly, larvae injected with P2RY11MO displayed a reduced accumulation of macrophages in the cut region at 6 hpi compared with the WT groups (Fig. 6D). Additionally, the migration of macrophages and neutrophils was detected and found to be slower in P2RY11 knocked-down larvae compared to WT larvae.

The cytokines in the tail at 6 hpi was also examined using qRT-PCR. It was found that tnfa and il1b was much higher in P2RY11−8 bp larvae than in the WT group, whereas il6 showed no difference between WT and P2RY11−8 bp larvae. Il4, il10, and tgfb showed decreased expression in P2RY11−8 bp larvae, especially conspicuously for tgfb expression. This suggests that the deficiency of P2RY11 exaggerated inflammatory responses and affect the balance of the inflammatory response during tissue damage (Fig. 6E).

Taken together, the above results show that the depletion of P2RY11 could affect the production of cytokines and the accumulation of the two types of leucocytes in the damaged tissue.