Genomic analysis of zebrafish UNG orthologs. (a) The genomic region encoding unga on chromosome 5. CAGE-Seq data clearly indicate active transcription from the annotated transcriptional start site of the gene. (b) A detailed look at the genomic region with the annotated ungb gene reveals multiple other genes that overlap the annotation. We also note that while many of these other genes are actively transcribed, as suggested by the mapping of CAGE-Seq datasets, an active transcriptional start site is not visible for ungb. (Figure was generated with GDV, for links and details see Materials and Methods section).

In silico analysis of the developmental expression of zebrafish UNG paralogs. (a) Expression dynamics of unga during the earliest stages of development show a gradual decrease in maternal transcript with no significant zygotic contribution after ZGA (Data from [46]). (b) The analysis of single-cell datasets also demonstrates the gradual clearance of maternal products, following a burst of unga expression during later stages of somatogenesis. During the same timeframe, ungb expression cannot be observed (Data from [48]). (c) GO analysis of genes that show significant co-expression with unga during early embryogenesis shows that the highest level of unga expression can be observed in actively dividing cell populations.

Creation and initial characterization of unga mutant zebrafish. (a) Targeted mutagenesis of unga results in a novel allele (elu24) carrying a 7 bp deletion in the second exon of the gene. (b) Wild-type and homozygous unga^elu24/elu24 adult zebrafish (scale bar: 0.33 cm). (c) The expression of unga is significantly reduced in unga^elu24/elu24 animals as shown by qPCR measurements (The Mann–Whitney test was used to calculate the p-values).

Sensitivity to DNA damage is increased in unga mutant zebrafish retinas. (a,b) AO staining of control and DEB-treated 2 dpf wild-type embryos shows an increased level of cell death upon the administration of the genotoxic agent. (c,d) DEB-treated unga mutant embryos also show elevated levels of apoptosis. However, unlike in wild-type controls, large clusters of AO-positive cells appear in these animals. Scale bar: 100 μm. (e) Quantification of apoptosis as measured by the number of AO foci in the control and DEB-treated embryos of the relevant genotypes (The Mann–Whitney test was used to calculate the p-values). (f–i) HE staining of the retinal sections of the wild-type and unga mutant embryos reveal that upon DEB treatment, a few dying (or missing) cells can be seen in the wild-type animals (black arrowheads). In contrast, large clusters of cells with apoptotic character are apparent in the mutant retinas (black arrows). Scale bar: 100 μm.

Homozygous unga mutant zebrafish males are subfertile. (a) Ratio of fertilized eggs in the indicated crosses. (b) Expression of unga in the differentiating germ cells of adult zebrafish. ScRNAseq data used in this reanalysis were derived from [52]. (c,d) Toluidine blue staining of testis sections from wild-type (c) and mutant (d) testes. Scale bar: 50 μm. (e–h) Comparative statistics of various sperm concentration (e) and motility (f–h) parameters between wild-type and unga mutant males (The Mann–Whitney test was used to calculate the p-values).

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