FishCOLler: Pilot In Vivo Models of COL4A1/A2 Syndrome in Tractable Fish Embryos Recapitulate Neurovascular and Ocular Pathology and Demonstrate Utility for Whole-Organism Variant Testing and Mechanistic Investigation ^†

Abstract

Collagen IV α1/α2 heterotrimers are the major constituents of all basement membranes (BM). Consistently, COL4A1/A2 mutations cause a complex multisystem disorder. While mouse models are invaluable, they alone cannot support the rapid functional validation needed for clinical translation. The FishCOLler project establishes zebrafish as a scalable in vivo platform to model COL4A1/A2 disease, employ rapid assays to monitor key disease traits, and enable mechanistic studies. Our first fish disease faithfully models patient symptoms, i.e., brain hemorrhage and ocular dysgenesis. The work supports FishCOLler as a platform for rapid variant interpretation, therapeutics testing, and highlights potential consequences of gene dosage modulation strategies.

Background and objectives: Collagen IV α1 and α2 chains, assembled into a 2α1:1α2 heterotrimer, are the most abundant components of basement membranes (BMs) across all tissues and animal phyla. Consistently, mutations in COL4A1 and COL4A2, particularly within glycine-rich domains critical for heterotrimer formation, cause a multisystem disorder characterized by cerebrovascular disease (including microbleeds and intracerebral hemorrhage), porencephaly, epilepsy, ocular dysgenesis, and myopathy [1,2,3]. Yet, despite their fundamental role, the molecular mechanisms through which collagen IV α1/α2 networks maintain tissue integrity and provide signaling platforms during tissue development and homeostasis remain poorly understood.

This gap in knowledge, coupled with the growing number of uncharacterized patients COL4A1/A2 variants and the overlooked allelic heterogeneity, significantly hampers patient stratification, the development of preventive strategies against life-threatening events (i.e., brain hemorrhage in children), and the advancement of mechanism-based therapies. Mouse COL4A1/A2 mutants originally described by Gould et al. [1] have been instrumental in identifying the syndrome and remain central to understanding its biology. However, rodent models alone cannot fulfill the need for a speedy functional validation and mechanisms exploration at a whole-organism level, required for improving diagnostics and to fast-track translational approaches.

Here, we present rationale, experimental approaches and first results of the FishCOLler project, founded via the “Seed Grant initiative” by “Fondazione Telethon” in collaboration with “Associazione famiglie COL4A1/A2” and launched in November 2024.

This pilot project aims at 1. establishing proof-of-concept in vivo models of COL4A1/A2 syndrome in the convenient zebrafish model, suited for rapid genetic and whole-organism screens, 2. developing rapid quantitative assays to score key disease features at the whole-organism level, and 3. beginning to benchmark the models to provide a scalable platform for functional variant testing and therapeutic hypothesis evaluation.

Approaches and results: Focusing initially on the ortholog of COL4A1, we employed an antisense oligonucleotide-based approach to model graded loss-of-function states in fish embryos. We obtained and validated the col4a1 loss of function models, which show phenotypes mirroring human disease traits. To ensure phenotypic stratification with respect to multi-organ pathology, we parallelly developed in vivo assays for 1. quantifying collagen IVα1 secretion in BM on whole-mount samples; 2. assessing susceptibility to spontaneous and trauma-induced hemorrhage in the entire brain without sectioning nor surgery, normally required in rodent models; and 3. ranking anterior eye segment dysgenesis (ASD) defects. After reduced collagen IVa1 secretion in BMs, observed for several severe patients COL4A1 mutations, could be recapitulated and traced in col4a1 knockdown (KD) fish, we measured phenotypes occurrence. We discuss hallmark disease features in col4a1 KD fish, including developmental delay, brain ventriculomegaly, impaired brain growth, generalized vascular fragility, and ocular malformations, phenocopying patients’ clinical manifestations. We also measured hemorrhagic susceptibility and anemia, providing an in vivo severity marker. Last, we scored and report signs of ASD often observed in children, including visible lens fiber defects resulting in cataract of variable severity.

Translational relevance: The data support our in vivo FishCOLler platform for rapid, cost-effective validation of COL4A1 and COL4A2 VUS, to accelerate the transition from discovery to clinical interpretation and targeted management. Our approach and results begin to demonstrate how zebrafish COL4A1/A2 disease models can help optimize the design of large, resource-intensive rodent animal studies, significantly reducing animal distress, while still providing the essential foundational knowledge required to enable preclinical studies and, ultimately, clinical trials. Of note, given the dominant nature of most COL4A1 and A2 pathogenic variants, gene dosage modulation is emerging as a potential therapeutic avenue. Our zebrafish COL4A1/A2 disease modeling offers indeed a unique opportunity to rapidly test the consequences of collagen IVα1/a2 manipulation at the organismal level and to evaluate the suitability of such emerging strategies.

We also discuss the latest CRISPR/Cas9-based strategies that we began to use to generate stable zebrafish KO and KI mutants, laying the groundwork for future long-term mechanistic and therapeutic studies.

Conclusions: Our initial data demonstrate that zebrafish is a powerful, complementary model system for COL4A1/A2 syndrome, supporting rapid disease modeling, mechanistic dissection, and therapeutic exploration. Disease traits across cerebral, vascular, and ocular systems can be robustly scored within three days after transient model generation obtained via microinjection. This dramatically shortens experimental timelines compared to traditional models. The findings also underscore the necessity of careful evaluation when considering therapeutic modulation of collagen IVα1 deposition in BMs. Altogether, the data presented show that the in vivo “FishCOLler platform” we generated has the potential to advance and support the translational pipeline for COL4A1/A2 syndrome. Last, the results also point to a conserved role of collagen IV a1/a2 in neurovascular and eye development.