Xenopus rsph6a.L: A Conserved Model for Human RSPH4A associated Ciliopathy

Abstract

Mutations in the radial spoke head protein RSPH4A are a primary cause of Primary Ciliary Dyskinesia (PCD). While mouse models serve as the gold standard for confirming pathogenicity, high costs and invasive procedures limit their utility for initial high-throughput screening. Conversely, while foundational studies in Chlamydomonas reinhardtii have provided structural insights, their evolutionary distance from mammals limits direct translational application. To address these limitations, we evaluated Xenopus laevis as a cost-effective vertebrate model that offers distinct experimental advantages. Although Xenopus possesses a single rsph6a.L gene, our phylogenetic and structural analyses reveal that it shares high conservation with mammalian RSPH4A and RSPH6A, retaining functional domains commonly affected in human patients. We depleted rsph6a.L using CRISPR-Cas12a and Morpholino oligonucleotides. Immunofluorescence analysis revealed that ciliary axoneme assembly remained intact; however, bead flow assays demonstrated a significant reduction in fluid flow velocity. This defect was attributed to a loss of planar beating directionality and a transition to aberrant rotational motion. These findings demonstrate that rsph6a.L is essential for the coordination of mucociliary clearance and validate the Xenopus system as a conserved, rapid screening tool for assessing RSPH4A-associated PCD pathogenicity.