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

FIGURE 8.

FIGURE 8

Simultaneous disruptions of the Vegfr2 zebrafish paralog genes using a triple dgRNP injection method. (A) Experimental workflow of the phenotypic analysis presented in panels (B–I). Adult kdr+/–;kdrl+/– fish carrying the Tg(kdrl:EGFP) transgene were incrossed, and progeny generated from the crosses were imaged to quantify the formation of the dorsal aorta and aISVs at 32 hpf. (B–G) Lateral views of kdr+/+ (B), kdr+/– (C), kdr–/– (D), kdr+/+;kdrl–/– (E), kdr+/–;kdrl–/– (F), and kdr–/–;kdrl–/– (G) trunk vasculature visualized by Tg(kdrl:EGFP) expression at 32 hpf. No obvious trunk vascular defects were observed in kdr+/+, kdr+/–, and kdr–/– embryos (B–D). kdr+/+;kdrl–/– embryos exhibited stalled aISV growth (E), and this aISV growth defect was exacerbated in kdr+/–;kdrl–/– embryos (F). In all kdr–/–;kdrl–/– embryos examined at 32 hpf, the dorsal aorta failed to form (G). DA: dorsal aorta; PCV: posterior cardinal vein. (H) Percentage of 32 hpf embryos of indicated genotype exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per genotype is listed in the panel). (I) Quantification of aISV lengths of 32 hpf embryos of the indicated genotypes (n = 10 for kdr+/+, n = 23 for kdr+/–, n = 16 for kdr–/–, n = 17 for kdr+/+;kdrl–/–, and n = 30 for kdr+/–;kdrl–/–; 5 aISV lengths measured per embryo). kdr+/–;kdrl–/– embryos displayed significantly shorter average aISV lengths than those of kdr+/+;kdrl–/– embryos. (J) Experimental workflow of the microinjection experiments for panels (K–R). Injection cocktails containing Cas9 protein with and without the three kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage embryos generated from incrosses of kdr+/– fish carrying the Tg(kdrl:EGFP) transgene. Injected progeny were imaged to quantify the formation of the dorsal aorta and aISVs at 32 hpf. (K–P) Lateral trunk views of the 32 hpf kdr+/+ (K,N), kdr+/– (L,O), and kdr–/– (M,P) embryos injected with Cas9 protein with (N–P) or without (K–M) the three kdrl crRNA:tracrRNA duplexes (kdrl dgRNPs). Trunk vasculature was visualized by Tg(kdrl:EGFP) expression. No obvious trunk vascular defects were observed in kdr+/+, kdr+/–, and kdr–/– embryos injected with Cas9 (K–M). In contrast, kdr+/+ embryos injected with the triple kdrl dgRNPs displayed the stalled aISV phenotype (N). Exacerbated aISV defects were observed in the kdrl dgRNPs-injected kdr+/– embryos (O). A nearly half of the kdrl dgRNPs-injected kdr–/– embryos exhibited a complete absence of the dorsal aorta (P). (Q) Percentage of 32 hpf embryos of the indicated genotype and treatment exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per group is listed in the panel). (R) Quantification of aISV lengths of 32 hpf embryos of the indicated genotype and treatment (n = 11 for Cas9-injected kdr+/+, n = 27 for Cas9-injected kdr+/–, n = 17 for Cas9-injected kdr–/–, n = 31 for kdrl dgRNPs-injected kdr+/+, and n = 47 for kdrl dgRNPs-injected kdr+/–; 5 aISV lengths per embryo measured). kdr+/–embryos injected with the kdrl dgRNPs exhibited significantly shorter average aISV lengths than those of the same dgRNPs-injected kdr+/+ fish. (S) Experimental workflow of the microinjection experiments for panels (T–W). Injection cocktails containing Cas9 protein with and without the three kdr/kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage embryos generated from incrosses of fish carrying the Tg(kdrl:EGFP) transgene. Injected progeny were imaged to observe the formation of the dorsal aorta and aISVs at 32 hpf. (T–Y) Lateral trunk views of the 32 hpf Tg(kdrl:EGFP) embryos injected with Cas9 protein (T) or with the three kdr (U) or kdrl (X) dgRNPs, or the combined six kdr and kdrl dgRNPs (Y). No obvious trunk vascular defects were observed in embryos injected with Cas9 (T) and the three kdr dgRNPs (U). In contrast, kdrl dgRNPs-injected embryos displayed the stalled aISV phenotype (X). The combined injections of the kdr and kdrl dgRNPs led to a complete absence of the dorsal aorta (Y) as observed in 32 hpf kdr–/–;kdrl–/– embryos. (Z) Percentage of 32 hpf embryos of the indicated treatment exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per group is listed in the panel). (V) Percentage of 32 hpf embryos exhibiting an absence of the dorsal aorta following the cytoplasmic injections of kdr or kdrl triple dgRNPs, or these combined six dgRNPs (the number of the animals examined per group is listed in the panel). (W) Percentage of 24 hpf dead embryos following the cytoplasmic injections of kdr or kdrl triple dgRNPs, or these combined six dgRNPs (the number of the animals examined per group is listed in the panel). A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (I,R). Fisher’s exact test was used to calculate P values for panels (H,Q,Z). Scale bars: 50 μm.