Loss of fignl2 leads to smaller eyes, pericardial edema, and reduced swimming velocity. (A) Mutated alleles generated using CRISPR/Cas9 lead to complete loss of the AAA-type ATPase domain. Underlined sequences are the CRISPR targets, wherein the bases in bold are the proto-spacer adjacent motifs (sequences are shown only on the sense strand). (B–D) Representative phenotype observation and edema index measurement in Fign mutants (B), Fignl1 mutants (C), and Fignl2 mutants (D) at 4 dpf. (E) Percentage of the filial embryos generated by crossing of fign^+/ntu702 × fignl2^+/ntu702, fignl1^+/ntu704 × fignl2^+/ntu704 and fignl2^+/ntu705 × fignl2^+/ntu705, showing partial embryonic lethality of fignl2 loss-of-function. Significance of differences compared to the wild type group is shown as p values on top of each dataset, and those between different mutant genotypes are shown on a horizontal line indicating groups used for comparison. (F) Statistics of pericardial edema index (PEI), n = (6,6,6) and (6,6,6) and (5,8,6); *p < 0.05, showing that the severity of edema was negatively correlated with functional fignl2 alleles. (G) Heart rate of the filial embryos generated by crossing of fignl2^+/ntu705 × fignl2^+/ntu705 showing abnormalities in the cardiovascular system in the fignl2 mutants, n = 6,10,8. Eyes of the filial embryos generated by crossing of fignl2^+/ntu705 × fignl2^+/ntu705 showing abnormalities in the cardiovascular system in the fignl2 mutants, n = 5,8,6. (H) Statistics of swimming velocity with 10-min light-dark cycle of fign, fignl1, and fignl2 mutants at 5 dpf, n = (12,25,9) and (14,22,11), and (20,19,9), showing weakened swimming ability of fignl2 mutants. Circles indicate a p < 0.1, asterisks (*) indicate a p < 0.05, and (**) indicates a p < 0.01, as indicated by the Student's t-test between the wild type and homozygous mutant groups.

Expression of fign, fignl1, and fignl2 in zebrafish embryos. (A) Expression analysis of fignl2 using in situ hybridization. (a1) fignl2 expression is observed in the nervous system and somites at 24 hpf, and weak staining is also observed in caudal blood vessels (arrowhead) (Enlarged in a1′). (a2)in situ hybridization using a fignl2 sense probe as control. (B) Single-cell expression analysis of fign, fignl1, and fignl2 during zebrafish development (a re-analysis of GEO GSE112294). (b1) Single-cells in the dataset (4–24 hpf) are mapped to different tissues. (b2–b4) Expression of fign, fignl1, and fignl2 in the single-cell dataset. (b5) Violin plot of fign, fignl1, and fignl2 expression in the single-cell expression dataset of zebrafish embryos (4–24 hpf). (b2′-b4′) Expression of fign, fignl1, and fignl2 in endothelial cells (18 and 24 hpf). (b5′) Violin plot of fign, fignl1, and fignl2 expression in endothelial cells (18 and 24 hpf). (b1′′) Neuronal cells were isolated by elavl3 expression. (b2′′-b4′′) Expression of fign, fignl1, and fignl2 in elavl3-positive cells. (b5′′) Violin plot of fign, fignl1, and fignl2 expression in elavl3-positive cells. (b5′′′) Violin plot of fign, fignl1, and fignl2 expression in mnx1 positive cells. Expression data for violin plots are normalized and presented as counts per 10⁴.

Depletion of fignl2 causes morphological changes in intersegmental vessels (ISVs) and caudal primary neurons. (A) Schematic diagram of morpholino oligonucleotide (MO) induced fignl2 knockdown causing aberrant splicing. (B) MO treatment results in mRNA size change in fignl2 RT-PCR amplicon. The pound sign (#) shows some amplicons of the original size detected after treatment with 0.3 mM MO. (C) MO treatment results in a 34-bp deletion in fignl2 mRNA. (D)fignl2 knockdown causes pericardial edema in developing zebrafish. Graphical representation of edema index (D′), eye size (D′′), heart rate (D′′′) in Ctrl and fignl2 knockdown zebrafish. Ctrl MO, n = 10; fignl2 MO, n = 11. (E)fignl2 knockdown causes morphological changes in ISVs, arrows showing malformations. Graphical representation (E′) of the percentage of different form of ISV malformations caused by fignl2 knockdown. Ctrl MO, n = 10; fignl2 MO, n = 11. (F)fignl2 knockdown causes morphological changes in caudal primary neurons, including greater axon length and increased branching. Representative axons were shown enlarged in (Fa) and (Fb). Graphical representation of axon length (F′), primary branch numbers (F′′) in Ctrl and fignl2 knockdown zebrafish. Ctrl MO, n = 10; fignl2 MO, n = 11. (G)fignl2 mutants shows more frequent twitching. Twitching was counted in 10 s. Ctrl MO, n = 10; fignl2 MO, n = 11.