Iroquois Transcription Factor irx5b Regulates Multiciliated Cell Development During Zebrafish Nephrogenesis

Background: Chronic Kidney Disease (CKD) is a major global health burden, affecting nearly 10% of the population and leading to millions of deaths annually. Understanding the genetic factors underlying nephron development is key to addressing renal dysfunction. In zebrafish, the transcription factor irx2a is essential for kidney development, acting downstream of retinoic acid signalling to promote multiciliated cell (MCC) fate and proper nephron segmentation. Loss of irx2a leads to reduced MCCs and nephron dysfunction, linking it to renal pathogenesis. We propose that irx5b may also contribute to MCC formation, given its expression in the distal early nephron segment, which corresponds to MCC localization. Methods: Here, we used loss-of-function approaches to investigate the role of irx5b during zebrafish embryogenesis. Whole-mount in situ hybridization was used to assess nephron segmentation, immunohistochemistry was used to visualize cilia morphology. Nephron physiology and renal function were evaluated via FITC-dextran clearance assays. Double loss of function with irx2a was used to test genetic interactions influencing MCC fate. Results: Loss of irx5b led to a significant reduction in MCCs within the zebrafish pronephros, accompanied by shortened distal nephron segments and impaired renal fluid clearance. irx5b-deficient embryos exhibited pericardial edema and hydrocephaly, consistent with MCC dysfunction. Immunohistochemistry revealed disrupted cilia structure with fewer ciliated basal bodies. FITC-dextran assays confirmed delayed excretion. Notably, irx5b and irx2a double deficiency resulted in enhanced MCC loss, suggesting functional redundancy in regulating MCC development. Conclusion: Our findings suggest that irx5b acts as a key regulator of multiciliated cell development and nephron patterning in zebrafish. Its loss impairs cilia formation and renal function, while co-regulation with irx2a suggests functional redundancy. These insights advance our understanding of MCC biology and may inform future studies on CKD and related ciliopathies. Funding: Private Foundation Support