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. 2021 Oct 22;9:735598. doi: 10.3389/fcell.2021.735598

FIGURE 2.

FIGURE 2

Larvae injected with three unique flt4 dgRNPs phenocopied the complete loss of brain meningeal perivascular FGPs observed in flt4–/– fish. (A) Schematic representation of the dorsal view of the zebrafish larval head. The boxed area indicates the approximate region where the confocal images of panels (B–D) were captured. (B–D) Dorsal head views of 6 dpf flt4+/+, flt4+/–, and flt4–/– larvae carrying Tg(fli1:nEGFP) and Tg(lyve1:DsRed) transgenes. While flt4+/+ and flt4+/– larvae formed Tg(fli1:nEGFP);Tg(lyve1:DsRed)-double positive FGPs in the dorsal meningeal surfaces over the optic tectum (arrows, B,C), flt4–/– larvae completely lacked FGPs (D). (E) Quantification of FGPs over the optic tectum for each genotype at 6 dpf (n = 17 for flt4+/+, n = 39 for flt4+/–, and n = 21 for flt4–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 3 (E3) and E6 on the flt4 genomic locus. (G) Predicted domain structure of zebrafish Flt4. Flt4 consists of a signal peptide (SP), six immunoglobulin-like domains (Ig), a transmembrane domain (TM), and two tyrosine kinase domains (TyrKc). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing flt4 crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three flt4 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 and 10 dpf for FGP formation over the optic tectum. (L,M) Brightfield images of the 6 dpf larvae injected with Cas9 protein with (M) or without (L) the three flt4 crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Dorsal head views of the 6 (N,O) and 10 (P,Q) dpf Tg(fli1:nEGFP);Tg(lyve1:DsRed) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three flt4 crRNA1-3. While larvae injected with Cas9 alone formed FGPs over the optic tectum at 6 and 10 dpf (arrows, N,P), those injected with the three flt4 dgRNPs displayed a complete lack of the FGPs at both stages (O,Q). (R) Quantification of FGPs over the optic tectum at 6 dpf (n = 28 for uninjected; n = 24 for Cas9 controls; n = 32 for crRNA1, crRNA2, and crRNA3; and n = 35 for crRNA1-3). Larvae injected with all three flt4 dgRNPs failed to form FGPs. Fish injected with the individual flt4 dgRNPs displayed varying degrees of FGP formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).