A–Dcmyb WISH identified HSPCs from 2 to 4 dpf in smu471 mutants (right) compared with their siblings (left). Acmyb⁺ signals were not altered at 2 dpf in the VDA region (blue arrowheads indicate signals in the region). B In the CHT region, cmyb⁺ signals were decreased slightly at 2.5 dpf in smu471 mutants (red arrowheads indicate signals in the region). C, D Severely decreased cmyb⁺ signals in the CHT region at 3 dpf (C) with a further reduction at 4 dpf (D) in smu471 mutants. The boxed regions in the lower right corner are magnifications. Scale bars: 200 µm.

A Genetic map of the candidate region on chromosome 5 (Chr 5) located between two SSLP markers, z20360 (5 recombinants in 1200 smu471 mutants) and z21082 (4 recombinants in 1200 smu471 mutants). There are twenty candidate genes in this mapped region. B, C Sequencing of the candidate gene cDNAs showed a transition of T to A at sart3 exon 6, which predicted a stop codon (B) and truncated Sart3 protein (C). D Rescue assay. Zebrafish sart3 mRNA (zf sart3 mRNA) overexpression restored defective cmyb expression in smu471 mutants. E A 14-bp deletion and 4-bp addition in sart3 exon 1 were identified in CRISPR/Cas9 generated mutants. Sequence underlined in red indicates the CRISPR/Cas9 target in sart3. F Premature Sart3 protein generated by the sart3^(Δ14,+4) mutant. Gcmyb⁺ signals decreased in 4 dpf sart3^(Δ14,+4) mutants. Hcmyb + signals decreased in the 4 dpf bi-allelic sart3^{(Δ14,+4)/smu471} mutants. The red arrowheads indicate the cmyb signals. Scale bars: 200 µm.

A, B Decreased HSPC proliferation in sart3^smu471 mutants. A The BrdU incorporation assay. The white arrowheads indicate proliferating HSPCs (cd41:eGFP^low; BrdU double positive cells). Green indicates eGFP, red indicates BrdU. B Statistical analysis of the percentage of CHT localized cd41^low HSPCs that incorporate BrdU (Student’s t-test, means ± SD; siblings, n = 28; sart3^smu471 mutants, n = 16; ****P < 0.0001). C, D Increased HSPC death in sart3^smu471 mutants. C The TUNEL assay. The white arrowheads indicate HSPCs undergoing cell death (cd41:eGFP^low; TUNEL double positive cells). Green indicates eGFP, red indicates TUNEL. D Statistical analysis of the percentage of CHT localized cd41^low HSPCs labeled by TUNEL (Mann–Whitney U test, means ± SD; siblings, n = 24; sart3^smu471 mutants, n = 27; *P < 0.05). Scale bars: 50 µm.

A Distance heatmap analysis of WT siblings and sart3^smu471 mutants. The color scale is shown on the left. The deeper the blue block the less different the samples are. B Spliceosome and p53 signaling pathway enrichment in sart3^smu471 mutants by KEGG analysis of the RNA-seq data. C Heatmap demonstrating upregulation of p53 pathway genes in sart3^smu471 mutants (data was normalized by Z-score, Z-score was shown on the right). D, E RT-qPCR validation of the RNA-seq data. Upregulation of p53 and its downstream target genes p21, ccng1, baxa, mdm2, gadd45, and casp8 in sart3^smu471 whole larvae (D) and HSPCs (E) at 4 dpf (Student’s t-test, means ± SD, n ≥ 15 per group for whole larvae, n ≥ 2 × 10⁴ per group for HSPCs, ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.0001). F Increased p53 protein in 4 dpf sart3^smu471 mutants by western blot analysis. GAPDH was used as the loading control. G, H Phenotype restoration in p53 knockdown sart3^smu471 mutants. Red arrowheads indicate WISH signals of cmyb at 4 dpf in the CHT region (G) and red circles indicate rag1 signals at 5 dpf in the thymus region (H). Scale bars: 200 µm.

A Overlap of gene expression change (padj < 0.05) with alternative splicing (padj < 0.05) in sart3^smu471 mutants from RNA-seq data. The overlapping number is 607. B p53 signaling pathway enrichment by GO term enrichment and KEGG pathway analyses from the 607 overlapped genes. The horizontal axis represents the significance, −log10(p). C Heatmap of p53 signaling pathway-related gene changes exhibited in (B) (data was normalized by Z-score, Z-score was shown on the right). The p53 negative regulator, mdm4, is boxed. Dmdm4 alternative splicing and primers designed for PCR detection of exon 6 skipping. The mdm4 full-length transcript (mdm4-FL) decreases and truncated mdm4 transcript (mdm4-S) increase in sart3^smu471 mutants. E Decreased expression of mdm4-FL by RT-qPCR at 2.5–4 dpf sart3^smu471 mutants (Student’s t-test, means ± SD, n ≥ 15 per group, ****P < 0.0001). F The procedure for the mdm4 overexpression rescue of reduced HSPCs in sart3^smu471 mutants. hs: heat shock. G Partial restoration of cmyb signals in sart3^smu471 mutants by mdm4 overexpression (OE). Red arrowheads indicate cmyb signals by WISH in the CHT region of 4 dpf larvae. Scale bars: 200 µm.

Schematic diagram of HSPC development regulated by Sart3-Mdm4-p53 pathway in zebrafish. Sart3, which could recycle U4 and U6 snRNPs into spliceosome assembly to affect pre-mRNA splicing [43], promotes HSPC development (current study). Mutation of Sart3 (highlighted in burst) reduces efficient recycling of U4 and U6 snRNPs (broken lines) so that normal splicing of mdm4 is disrupted. Mdm4 exon 6 (E6) skipping by sart3 mutation results in increased truncated mdm4 transcript (mdm4-S) and decreased mdm4 full-length transcript (mdm4-FL). The insufficiency of Mdm4 (orange rectangle) triggers activation of p53 pathway and promotes HSPCs to undergo proliferation arrest and apoptosis in zebrafish early hematopoiesis.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.