Real-time imaging of blood coagulation and angiogenesis during development in a zebrafish model of type I antithrombin deficiency

Abstract

Severe type I antithrombin (AT) deficiency is considered to cause embryonic lethality. Although several pathological analyses using mice or zebrafish have been attempted, the previous studies did not unveil the detailed mechanism leading to lethality in the early developmental stage. In order to solve this problem, we established type I AT deficient zebrafish by the CRISPR/Cas9 system into Tg(gata1:dsRed) and Tg(fli1a:GFP) lines, so that we could conduct real-time imaging of thrombosis and angiogenesis using fluorescence stereo zoom microscopy. The established zebrafish AT (zAT) mutants harbored frameshift mutations which resulted to be type I AT deficient, unable to secrete zAT protein into blood. Both heterozygous (zAT^+/-) and homozygous (zAT^-/-) mutants showed reduced survival rate and diverse thrombosis up to 9 days post fertilization. In addition, blood vessel formation was delayed at 30 hpf in zAT^-/-, which was recovered normally by 5 dpf and had little effect on survival. Notably, we analyzed the differences in gene expression profiles under AT-depleted conditions by real-time quantitative PCR, and zAT^-/- juvenile zebrafish showed increased PLG gene expression and decreased F2 gene expression. Our in vivo study revealed the effects of AT deficiency on embryos during development from the aspects of coagulation and vascular formation.

Keywords: Angiogenesis; Antithrombin deficiency; Genome editing; Thrombosis; Zebrafish.

Fig. 1

Characterization of zAT mutant zebrafish demonstrating hereditary type I AT deficiency. (A) Sequencing analysis of exon 1 of the SERPINC1 gene in wild-type and zAT mutant zebrafish revealed the deletion of five bases (ACTTA; shown by the red dotted line), and the insertion of a single base (C; shown by the blue dotted line). Ribbon models illustrate the structural differences between the wild-type (blue ribbon) and mutant (red ribbon) zAT proteins as predicted by ColabFold. (B) Genotyping of zAT^+/+, zAT^+/−, and zAT^−/− was performed by detecting a four-base deletion by Native-PAGE. (C) SERPINC1 mRNA levels showed no significant difference between zAT^+/+ and zAT^+/− (N.S., not significant; p = 0.37; n = 3), but were significantly lower in zAT^−/− compared to zAT^+/+ (p < 0.05; n = 3). (D, E) Detection and band quantification of zAT protein in zebrafish plasma by western blot analysis using an anti-zAT antibody.

Fig. 2

Survival curves and thrombosis sites of zAT mutants. (A) Illustration of a juvenile zebrafish. Arteries are shown in red and veins in blue. The dotted rectangular area and the alphabet correspond to the multipanel figures in Fig. 2. The locations of the heart, pectoral fin, and PCV are indicated by arrows. The positioning of the blood vessels and heart was based on previous studies by Lee et al.. (B) Comparative survival rates between zAT^+/+ zebrafish and zAT^+/− or zAT^−/− zebrafish, illustrating survival differences across genotypes. The sample sizes are N = 84, 68, and 76 for zAT^+/+, zAT^+/−, and zAT^−/− zebrafish, respectively. N.S., not significant; *p < 0.05. The survival rates for zAT^+/+, zAT^+/−, and zAT^−/− zebrafish on the 9dpf were 100%, 88.24%, 88.16%, respectively. (C, D) Fluorescent images of erythrocytes in zAT^+/+ zebrafish, focusing on the heart, pectoral fin vessel (PFV), and posterior cardinal vein (PCV). (E–H) Fluorescence imaging depicting thrombosis in zAT mutant zebrafish. Red fluorescence represents erythrocytes. White arrows or areas circled by white dotted lines indicate thrombus locations in the heart (E), pectoral fin (F), and PCV (G). An instance of a beating heart with restricted systemic blood flow is shown in (H). (I) Immunofluorescence staining of juvenile zebrafish displaying the same symptoms as in (H), using an anti-fibrin antibody, with blue fluorescence indicating fibrin. The zebrafish is outlined with a white dotted line. (J) Incidence of thrombosis in the heart, PFV, PCV, and systemic circulation corresponding to (E–H), respectively. Sample sizes for heart, PFV, and systemic are N = 45 for zAT^+/+ zebrafish, N = 12 for zAT^+/− zebrafish, and N = 24 for zAT^−/− zebrafish. The number of samples for PCV is N = 45 for zAT^+/+ zebrafish, N = 35 for zAT^+/− zebrafish, and N = 40 for zAT^−/− zebrafish.

Fig. 3

Impact of severe zAT deficiency on vascular development. (A) Representative images of intersegmental vessel (ISV) development in zebrafish of different genotypes at 30 h post-fertilization (hpf). Red arrows indicate ISVs that have completed formation, while blue arrows indicate ISVs that have not yet completed formation. (B) Percentage of ISVs that have successfully completed formation across different genotypes. (C) Measurements of the length of ISVs in different genotypes. (D) Angle between each ISV and the dorsal aorta in different genotypes. (E) Distance between two adjacent ISVs in different genotypes. (F) Representative images at 5 days post-fertilization (dpf) showing overall vessel formation in different genotypes. Statistical significance is noted where applicable (N.S., not significant; **p < 0.01; *p < 0.05).

Fig. 4

Gene expression analysis in juvenile zebrafish at 7 days post-fertilization (dpf) by quantitative real-time PCR. Gene expression levels of protein S (A), protein C (B), plasminogen (C), prothrombin (D), coagulation factor V (E), and coagulation factor X (F) were compared between wild-type and mutant zebrafish. N.S., not significant; **p < 0.01; *p < 0.05.