Figure 4—source data 1.

Figure 4—source data 1.

(A) Predicted domain structure of zebrafish Ccbe1. Ccbe1 consists of a signal peptide (SP), an EGF domain, a calcium-binding EGF domain (Ca-EGF), and two collagen repeat domains (ColA and ColB). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed CRISPR RNA (crRNA). (B) Experimental flows of the microinjection experiments for panels (C and D). Injection cocktails containing Cas9 protein with or without the three ccbe1 crRNA:tracrRNA duplex complexes were injected into one-cell stage embryos produced from Tg(kdrl:EGFP);Tg(lyve1:DsRed) fish crossed with wild-type AB counterparts. Injected progeny were analyzed at 120 hpf for the presence or absence of Tg(lyve1:DsRed)⁺ FGPs in the dorsal meningeal surfaces of the optic tectum. (C) Brightfield and confocal images of 120 hpf Tg(kdrl:EGFP);Tg(lyve1:DsRed) larvae after no injection or Cas9 protein injection with and without the three ccbe1 crRNA:tracrRNA complexes. Although the larvae injected with the ccbe1 crRNA ribonucleoprotein (RNP) complexes exhibited no apparent differences in their gross morphology compared to uninjected and Cas9 injected controls, they completely lacked Tg(lyve1:DsRed)⁺ FGPs (white arrows) in the dorsal meningeal surfaces of the optic tectum. (D) Percentage of 120 hpf larvae of indicated treatment with and without Tg(lyve1:DsRed)⁺ FGPs in the dorsal meningeal surfaces of the optic tectum (n = 20 animals examined for each group). (E) Experimental flows of the microinjection experiments for panel (F and G). (F) Percentage of 54 hpf larvae of indicated treatment with and without the DLV (the number of the animals examined per treatment is listed in the panel). A significant fraction of the embryos injected with the ccbe1 crRNA RNP complexes lacked the DLV at this stage. (G) Quantification of DLV lengths of the fish of indicated treatment that formed the DLV at 54 hpf. (H) Experimental flows of the microinjection experiments for panels (I–K). (I) Dorsal views of 96 hpf vegfab^+/+ and vegfab^-/- larvae that carried Tg(kdrl:EGFP) and Tg(lyve1:DsRed) transgenes after Cas9 protein injection with and without the three ccbe1 crRNA:tracrRNA complexes. While control solution injected vegfab^+/+ and vegfab^-/- larvae formed Tg(lyve1:DsRed)⁺ FGPs in the dorsal surfaces of the brain meninges, those injected with the ccbe1 crRNA RNP complexes completely lacked FGPs regardless of vegfab genotypes. A significant fraction of vegfab^-/- larvae in both injection groups exhibited the ‘No DLV’ phenotype. (J) Percentage of 96 hpf larvae of indicated genotype and treatment with and without the DLV (the number of the animals examined per genotype is listed in the panel). vegfab^-/- larvae injected with the ccbe1 crRNA RNP complexes displayed a markedly increased penetrance of the ‘No DLV’ phenotype. (K) Quantification of PCeV, MCeV, and MsV formation at 96 hpf. vegfab^-/- larvae injected with the Cas9 control solution as well as vegfab^+/+ larvae injected with the ccbe1 crRNA RNP complexes did not exhibit a defect in PCeV formation. However, vegfab^-/- larvae injected with the ccbe1 crRNA RNP complexes exhibited severe defects in PCeV formation. Scale bars: 50 μm in (C) (fluorescence image), (J); 1 mm in (C) (brightfield image).

Figure 4—source data 1.