Abstract: PO1341
Stable Genetic Deletion of Gapvd1 in Drosophila Results in a Nephrocyte-Restricted Phenotype
Session Information
- Genetic Diseases of the Kidneys: Non-Cystic - II
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Lang, Konrad, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Milosavljevic, Julian, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Heinkele, Helena, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Gerstner, Lea, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Hermle, Tobias F., Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
Group or Team Name
- Hermle Lab
Background
Mutations in the gene GAPVD1 cause of nephrotic syndrome in humans. GAPVD1 interacts with RAB5 and but the subcellular localization of GAPVD1 is unclear. Silencing of Gapvd1 the podocyte-like Drosophila nephrocytes by RNA-interference resulted in mislocalization of fly nephrin.
Methods
We generated conditional knockdowns and a stable genetic deletion of Drosophila Gapvd1 by CRISPR/Cas9 and used microhomology-mediated end joing to introduce a genomic HA-tag into the Gapvd1 c-terminus. We performed a functional analysis of the novel fly models.
Results
We generated twin frameshift mutations at the second and third exons of the Drosophila Gavpd1 gene. Animals carrying these mutantion were homozygous viable without any overt phenotype. However, the podocyte-like nephrocytes revealed a severly altered slit diaphragm morphology with mislocalization of fly nephrin and the orthologue of NEPH1 and partial loss of both proteins from the surface. This phenotype was similar but considerably stronger than the phenotype observed by RNAi-mediated silencing. This suggests that the homozygous frameshift mutations result in a null allele. The phenotype was further confirmed by conditional CRISPR/Cas-mediated silencing using two different gRNAs. Deletion of Gapvd1 in the Drosophila model thus results in a phenotype that manifests exclusively in disturbed slit diaphragms formation. This recapitulates the phenotype observed in human patients that was limited to nephrotic syndrome, supporting the use of Drosophila model for this genetic disease. To study the subcellular localization of Drosophila Gapvd1, we introduced an HA-tag into the c-terminus of the Gapvd1 locus. Immunofluorescence of nephrocytes derived from the knock-in lines showed co-localization of the protein with Drosphila Rab5, supporting that Gapvd1 primarily resides in early endosomes. We overexpressed human GAPVD1 in nephrocytes, that equally localized in early endosomes. Gain-of-function of the human gene entailed reduced tracer endocytosis in nephrocytes, suggesting a dominant negative effect.
Conclusion
We established suitable new Drosophila models to study the function of Gapvd1 in nephrocytes as an invertebrate podocyte model.
Funding
- Government Support – Non-U.S.