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Generation and characterization of a novel gne Knockout Model in Zebrafish
Hagay Livne1,2, Tom Avital1, Shmuel Ruppo3
1Faculty of Marine Sciences, Ruppin Academic Center, Michmoret, Israel.
Frontiers in Cell and Developmental Biology
|November 10, 2022
Summary
We developed a zebrafish model for GNE Myopathy, revealing that GNE gene mutations cause severe muscle defects and impact cell cycle and DNA repair, offering a new avenue for therapeutic target discovery.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
- Zebrafish Models
Background:
- GNE Myopathy is a rare, inherited neuromuscular disorder caused by mutations in the GNE gene, which encodes a key enzyme in sialic acid biosynthesis.
- The exact mechanism by which GNE mutations lead to muscle pathology remains unclear, and existing cellular and mouse models have limitations.
- Additional functions of the GNE gene in muscle tissue have been hypothesized, necessitating further investigation.
Purpose of the Study:
- To investigate the functions of the GNE gene in muscle using zebrafish genetic and transgenic models.
- To characterize the phenotype of GNE deficiency in zebrafish using macroscopic, microscopic, and molecular analyses.
- To establish a relevant animal model for GNE Myopathy.
Main Methods:
- Generation of transgenic zebrafish expressing a mutated human GNE gene.
- Creation of GNE-knockout (KO) zebrafish using CRISPR/Cas9 technology.
- Phenotypic characterization including macroscopic observation, muscle histology, and RNA sequencing at multiple developmental stages (3, 5, and 7 days post-fertilization).
Main Results:
- Transgenic fish with a mutated human GNE gene showed normal development.
- GNE-KO zebrafish exhibited lethality around 8-10 days post-fertilization, with phenotypes including body axis curvature, swim bladder deflation, and reduced movement and heart rate.
- Muscle histology revealed severe fiber disorganization in GNE-KO larvae by 5 days post-fertilization, and RNA sequencing indicated involvement of cell cycle and DNA damage/repair pathways.
- Sialic acid supplementation did not rescue the GNE-KO phenotype or improve lifespan.
Conclusions:
- The study successfully established a GNE-knockout zebrafish model, which is the first animal model demonstrating clear muscle defects relevant to GNE Myopathy.
- The findings suggest that GNE deficiency impacts muscle integrity through mechanisms involving cell cycle regulation and DNA repair processes.
- This zebrafish model provides a valuable platform for further research into GNE Myopathy pathogenesis and the identification of novel therapeutic targets.
