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Fig. 2 Rfc1 loss-of-function leads to premature death, head morphological anomalies and motility defects in zebrafish.A–C Generation of an rfc1 mutant zebrafish line carrying a 20-bp deletion in exon 5, confirmed by Sanger sequencing (B). This deletion induces a frameshift resulting in a premature stop codon at position 182 (p.S182X), upstream of all three conserved RFC1 functional domains: BRCT, ATPase+ AAA-type, and DNA replication domains (C). D RT-qPCR quantification of rfc1 transcript levels showing a significant reduction in heterozygous (rfc1+/−, n = 3) and homozygous mutant (rfc1−/−, n = 3) larvae compared to wild-type (rfc1+/+, n = 2). E Survival curve of zebrafish larvae showing premature lethality of rfc1−/− individuals by 10 days post-fertilization (dpf). F Representative images of larvae at 3 and 5 dpf showing reduced head and eye size in rfc1−/− larvae, but not in heterozygous siblings. G, H Quantification of craniofacial parameters in rfc1 mutant larvae. G Head length at 3 dpf (n = 22 rfc1+/+, 26 rfc1+/–, 26 rfc1–/–) and 5 dpf (n = 16 rfc1+/+, 41 rfc1+/–, 19 rfc1–/–). H Eye diameter at 3 dpf (n = 22 rfc1+/+, 28 rfc1+/–, 26 rfc1–/–) and 5 dpf (n = 16 rfc1+/+, 22 rfc1+/–, 20 rfc1–/–). I Quantification of total distance swum over 1 h (lights off), demonstrating significant hypomotility in rfc1−/− larvae at 5 dpf compared to siblings (N = 3, n = 101 rfc1+/+, 160 rfc1+/−, 107 rfc1−/−). J Representative swimming tracks recorded over 10 min at 5 dpf, illustrating reduced locomotor activity in rfc1−/− larvae. Data in (D, G–I) are presented as mean ± SEM; individual dots represent biological replicates. Statistical analysis: one-way ANOVA with multiple comparisons. ns not significant (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001); **** (p ≤ 0.0001).