Abstract
Purpose :
Rodent-based studies have shown that ANGPT1 is indispensable for the development and maintenance of the ocular lymphatic network, which is essential for normal aqueous humor drainage. In humans, ANGPT1 loss-of-function mutations cause primary congenital glaucoma with variable expressivity. In this work, we characterized an angpt1 knock-out zebrafish model allowing for high throughput characterization of potential disease-causing variants as well as investigation of novel gene-based therapeutics targeting the ANGPT/TIE2 signaling pathway.
Methods :
Using ABTL zebrafish embryos, angpt1 was disrupted by injection of a splice-site morpholino before 4 cell stage. Microangiography, ocular histology, and visual motor responses (VMR) were compared between morphants and controls at day 5 post-fertilization (5dpf). Phenotype recovery using wild type and mutant human mRNA was also evaluated in the morphants. An adult fish knock-out line was created using CRISPR/Cas9, generating an 11 bp deletion in exon 5 of angpt1. The phenotypic features of the crispant embryos with different genotypes (WT/WT, WT/del, and del/del) were compared.
Results :
Morpholino-induced skipping of exon 3 and the exon 5 11bp deletion in crispants was confirmed by next-generation sequencing. Phenotypic characterization of morphants showed abnormal ocular and trunk vasculature compared with controls at 5dpf (P<0.05). Additionally, the ventral limbal structures, which anatomically correspond to the aqueous humor outflow pathway in adult zebrafish, the ventral canalicular network, were abnormal in morphants (P<0.05). The VMR test showed reduced sensitivity to light in morphants compared to controls (Pt-test<0.001). Compared with wild type crispant embryos, homozygous 11bp deletion carriers (crispants) also showed malformation of the vasculature (P<0.05), ventral limbal structures (P=0.019), and VMR reduction (P=0.005). Furthermore, human mRNAs containing c.A62G (p.N21S) and c.C239T (p.S80F), two rare ANGPT1 variants found in human glaucoma cases, failed to rescue the VMR phenotype compared with wild type mRNA (P<0.05), suggesting deleterious variant effects.
Conclusions :
Loss of angpt1 in zebrafish embryos recapitulates several essential ocular and functional phenotypes from existing animal models. Further work offers opportunities for high throughput evaluation of human ANGPT1 variants as well as exploring novel gene-based therapeutic approaches.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.