Inversin (NPHP2) and Vangl2 are required for normal zebrafish cloaca formation

Area of Science:

• Genetics and Developmental Biology
• Cell Biology
• Molecular Medicine

Background:

• Nephronophthisis (NPH) is an autosomal recessive ciliopathy caused by mutations in over 20 NPHPs genes.
• NPHP gene products regulate ciliary trafficking and have extraciliary functions, including planar cell polarity (PCP) signaling.
• NPHP2/Inversin is implicated in both primary cilia structure and PCP signaling pathways.

Purpose of the Study:

• To characterize the tissue-specific functions of zebrafish Nphp2 using the invs^sa36157 mutant line.
• To investigate the interplay between NPHP genes and PCP pathway members in zebrafish development.
• To elucidate the molecular mechanisms underlying NPH-associated phenotypes, particularly cloaca malformations.

Main Methods:

• Utilized the zebrafish mutant line invs^sa36157 with a premature stop codon in NPHP2.
• Employed morpholino-mediated knockdown to deplete nphp1 and vangl2 in zebrafish embryos.
• Performed time-lapse imaging and in situ hybridization to analyze cell migration, tissue development, and apoptosis.

Main Results:

• Zebrafish invs^sa36157 mutants displayed mild ciliopathy, increased glomerular and cloaca cyst formation, and susceptibility to nphp1/nphp2/nphp8 module depletion.
• Simultaneous depletion of nphp1 and vangl2 in invs^sa36157 mutants significantly increased cloaca malformations, with cells failing to form the cloaca opening despite correct migration.
• Reduced apoptotic activity was observed in double knockdown embryos, indicating a role for apoptosis in cloacal morphogenesis.

Conclusions:

• NPHP1, NPHP2/Inversin, and VANGL2 are critical for normal cloaca formation in zebrafish.
• The study highlights the complex interplay of ciliopathy genes and PCP signaling in epithelial development.
• Findings provide insights into the molecular mechanisms underlying ciliopathy phenotypes and associated developmental defects.