Figure 5

Figure 5 mybl2b is a functionally essential target of Mettl16 in HSPC development.

(A) Venn diagram showing the overlap between hypomethylated transcripts and differentially expressed genes in mettl16^−/− mutants compared with siblings. (B) Integrative Genomics Viewer (IGV) tracks displaying MeRIP-seq (upper panels) and RNA-seq (lower panels) read distribution in mybl2b mRNA of siblings and mettl16^−/− mutants. The red arrow at the bottom of the tracks indicates the position of the m⁶A peak. (C, D) qPCR analysis showing that the mRNA expression level of mybl2b, trpa1b, and ntrk2b in mettl16^−/− embryos at 3 dpf and 4 dpf. n ≥ 15 per group, performed with three biological replicates. (E, F) qPCR analysis showed the mRNA expression level of mybl2b in mettl16 morphants at 3 dpf and 4 dpf. n ≥ 15 per group, performed with three biological replicates. (G) m⁶A enrichment in mybl2b mRNA in mettl16 deficient zebrafish at 3 dpf by meRIP-qPCR. n ≥ 200 per group, performed with three biological replicates. (H, I) WISH (H) and qPCR (I) analysis showed that the decreased expression of HSPC marker cmyb in mettl16 morphants at 4 dpf was restored by mybl2b mRNA co-injection. Numbers at the bottom right indicate the number of embryos with similar staining patterns among all embryos examined, n = 3 independent experiments (H). Scale bars, 100 μm (H). n ≥ 10 per group, performed with three biological replicates (I). (J, K) Flow analysis of the cell cycle showing the increase of HSPCs in the G0/G1 phase in mettl16 morphants was restored by mybl2b mRNA co-injection. n ≥ 200 per group, performed with three biological replicates. Data information: In (C–I, K), data were represented as mean ± SEM, *adjusted P < 0.05, ** adjusted P < 0.01, *** adjusted P < 0.001, **** adjusted P < 0.0001, n.s. non-significant, Student’s t-test (C–G), one-way ANOVA analysis with post hoc test of Tukey’s multiple comparison correction (H, I, K). Source data are available online for this figure.