Loss of cnr1 leads to reduction in both mature and progenitor MCC populations: (A) live imaging between different treatment groups between 24 and 72 hpf with the percentage of embryos with edema between 48–72 hpf (arrows indicate pericardial edema); scale bar = 200 μm; (B) 28 ss WT, cnr1 MO stained via WISH using the mature MCC marker odf3b and cetn4; scale bar = 50 μm; (C,D) number of odf3b⁺ and cetn4⁺ cells per nephron at 28 ss; (E) 24 ss WT, cnr1 MO stained via WISH using the progenitor MCC marker pax2a and jag2b; scale bar = 50 μm; (F,G) number of pax2a⁺ and jag2b⁺ cells per nephron at 24 ss. Data presented on graphs are represented as mean ± SD; **** p < 0.0001 (t-test).

cnr1 agonism leads to reduction in mature MCC population: (A) 28 ss WT, 0.01 mM MA and 0.01 mM AN-treated embryos stained via WISH using the mature MCC marker odf3b and cetn4; scale bar = 50 μm; (B,C) number of odf3b⁺ and cetn4⁺ cells per nephron at 28 ss; (D) 24 ss WT, 0.01 mM MA and 0.01 mM AN-treated embryos stained via WISH using the progenitor MCC marker pax2a and jag2b; scale bar = 50 μm; (E,F) number of pax2a⁺ and jag2b⁺ cells per nephron at 24 ss. Data presented on graphs are represented as mean ± SD; * p < 0.05 and **** p < 0.0001 (ANOVA).

cnr1 antagonism leads to reduction in mature MCC population: (A) 28 ss WT, 0.02 mM AM-251 and 0.02 mM AM-281-treated embryos stained via WISH using the mature MCC marker odf3b and cetn4; scale bar = 50 μm; (B,C) number of odf3b⁺ and cetn4⁺ cells per nephron at 28 ss; (D) 24 ss WT, 0.02 mM AM-251 and 0.02 mM AM-281-treated embryos stained via WISH using the progenitor MCC marker pax2a and jag2b; scale bar = 50 μm; (E,F) number of pax2a⁺ and jag2b⁺ cells per nephron at 24 ss. Data presented on graphs are represented as mean ± SD;** p < 0.01 (ANOVA).

Loss of cnr1 leads to cilia defects across embryonic tissues: (A) 28 hpf whole-mount IF for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red) and DAPI (nucleus, blue) in the proximal segment of WT, cnr1 MO and embryos treated with 0.02 mM AM-251; scale bar = 50 μm; (B) proximal cilia length at 28 hpf; (C) fluorescence intensity plot of α-tubulin intensity within the proximal segment at 28 hpf; (D) number of basal bodies in the proximal segment at 28 hpf; (E) percentage of ciliated basal bodies/total basal bodies in the proximal segment at 28 hpf; (F) 10 ss whole-mount IF for acetylated α-tubulin (cilia, green), anti-PKC (membrane boundary, red) and DAPI (nucleus, blue) in the KV of WT and cnr1 MO embryos; scale bar = 50 μm; (G) 4 dpf whole-mount IF for acetylated α-tubulin (cilia, green) and DAPI (nucleus, blue) in the neuromast of WT and cnr1 MO embryos; scale bar = 25 μm; (H) KV cilia length at 10 ss; (I) neuromast cilia length at 4 dpf; (J) 4 dpf whole-mount IF for acetylated α-tubulin (cilia, green) and DAPI (nucleus, blue) in the anterior macula and crista of WT and cnr1 MO embryos; scale bar = 25 μm; (K) 4 dpf whole-mount IF for acetylated α-tubulin (cilia, green), γ-tubulin (basal bodies, red) and DAPI (nucleus, blue) in the nasal placode of WT and cnr1 MO embryos; scale bar = 25 μm; (L,M) nasal MCC area and nasal placode area at 4 dpf; (N) number of nasal MCCs at 4 dpf; (O) 55 hpf WT and cnr1 MO embryos stained via WISH using heart marker myl7; scale bar = 50 μm; (P) phenotype percentage of heart looping phenotypes at 55 hpf. Data presented on graphs are represented as mean ± SD; ** p < 0.01 *** p < 0.001 and **** p < 0.0001 (t-test (H,I,L–N), ANOVA (B–E) and Fisher’s exact test (P)).

cnr1 regulates renal MCC development via cAMP signaling and independently: (A,D) 28 ss embryos between experimental groups stained via WISH using the mature MCC marker odf3b; scale bar = 50 μm; (B,E) number of odf3b⁺ cells per nephron at 28 ss; (C,F) phenotype percentage of MCC phenotypes at 28 ss. Data presented on graphs are represented as mean ± SD; ** p < 0.01 *** p < 0.001 and **** p < 0.0001 (ANOVA (Figure 4B,E) and Fisher’s exact test with Holm’s correction (Figure 4C,F)).

Roadmap delineating the relationship between Cnr1 and players in renal MCC development.