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Worldwide incidence of kidney diseases has been rising. Thus, recent research has focused on zebrafish, whose fast development and innate regeneration capacity allow identifying factors influencing renal processes. Among these poorly studied factors are extracellular matrix (ECM) proteins like Fibronectin (Fn) essential in various tissues but not yet evaluated in a renal context. We utilized early nat and han zebrafish mutant embryos and carrier adults to investigate Fn's role during kidney development and regeneration. The locus natter (nat) encodes Fn and the locus han encodes Hand2, which results in increased Fn deposition. Our results show that Fn impacts identity maintenance and morphogenesis during development and influences conditions for neonephrogenic cluster formation during regeneration. Histological analysis revealed disrupted pronephric structures and increased blood cell accumulation in Fn mutants. Despite normal expression of specification markers (pax2, ATPα1a.1), structural abnormalities were evident. Differences between wild-type and mutation-carriers suggest a haploinsufficiency scenario. These findings reveal a novel function for ECM in renal development and regeneration, with potential implications for understanding and treating kidney diseases., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Uribe-Montes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)