Characterization of zebrafish lines with mutations in the nephronophthisis-associated genes nphp1, nphp2, nphp4 and nphp8

Abstract: Nephronophthisis (NPH) is a rare autosomal recessive genetic kidney disease. It is the most common genetic cause of end-stage renal failure in children and young adults and is characterized by its phenotypic and genotypic heterogeneity. To this date, mutations in more than 25 different NPHP genes have been associated with NPH. Most of these genes encode proteins that localize to the cilium and mutations in these genes affect ciliary function. Thus, NPH is considered a ciliopathy.
I used zebrafish, a well-established model system in biomedical research, to characterize chemically induced point mutations in the nphp1, nphp2, nphp4, and nphp8 genes. Two successive generations of mutant fish were assessed for typical ciliopathy-associated phenotypes, including glomerular cysts formation, cloaca malformation, aberrant heart-looping, heart edema, hydrocephalus and abnormal body curvature. Recently, discrepancies between phenotypes caused by morpholino oligonucleotides (MO)-mediated target gene depletion, and mutants with defined mutations have uncovered the ability of zebrafish to compensate defective gene functions. Although still poorly understood, several concepts including transcriptional adaptation triggered by mRNA decay have been implicated to explain this phenomenon of “genetic robustness”. We questioned whether this acquired compensation is passed down to the progeny of mutant zebrafish. We observed that typical ciliopathy phenotypes (i.e. glomerular cysts and cloaca malformation) were further reduced between F1 and F2 generations, suggesting evolutionary compensation. The second (F2) generation of maternal zygotic mutant fish allowed the assessment of the lack of maternal RNA contribution. Zebrafish lines carrying more severe mutations and displaying stronger phenotypes in F1 tended to compensate the loss of protein function more successfully in F2. Mutations in the nphp8 gene, which are associated with severe human disease, seem less capable to compensate than zebrafish with mutations in the nphp4 gene. Genetic compensation could be confirmed in nphp4-deficient mutants via MO-induced knockdown of the nphp4 gene, since these F2 mutants became largely refractory to additional gene depletion.
Not only do zebrafish, carrying defined point mutations in nphp1 and nphp4, display reduction of ciliopathy-related phenotypes between F1 and F2, but so do mutant lines with larger CRISPR/Cas9-mediated deletions, further revealing partial compensation in the maternal zygotic F2 zebrafish. Finally, CRISPR/Cas9 was used in this work to generate a new loss-of-function nphp2 mutation. Simultaneous use of two sgRNAs, targeting the first and the last exon respectively, successfully deleted most of the genes‘ open-reading frame, creating a new model to study nphp2 function in zebrafish in further experiments

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2023

Klassifikation und Themen

• Schlagwort: Embryonalentwicklung ; Zebrabärbling ; Niere ; Zebrabärbling ; Zystenniere ; Glomerulopathie

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2023
• Urheber: Kayser, Nicolas
• Beteiligte Personen und Organisationen: Yakulov, Toma ; Walz, Gerd ; Moser, Martin ; Klinik für Innere Medizin IV. Freiburg im Breisgau ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Weitere Informationen

• DOI: 10.6094/UNIFR/241914
• URN: urn:nbn:de:bsz:25-freidok-2419143
• Rechteinformation: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:46 MEZ