Fig. 3: Mettl14 knockout decreases RGC proliferation in vivo.

a–c, Coronal sections of E17.5 brains stained with antibodies recognizing BrdU, PH3 and Pax6. Pregnant mothers received a BrdU pulse 30 min before embryo collection. d, Quantification of immunostaining from E17.5 sections. Numbers of Pax6⁺, BrdU⁺ and PH3⁺ cells were determined and normalized to those in comparable sections from nondeleted mice; one-way ANOVA (n = 3 brain sections for all experimental groups; Pax6⁺, P = 0.0005, F(2, 6) = 34.41; BrdU⁺, P = 0.0231, F(2, 6) = 7.531; PH3⁺, P = 0.0002, F(2, 6) = 47.73) followed by Bonferroni’s post hoc test (Pax6⁺, WT versus KO, P = 0.0004, 95% C.I. = 0.2814–0.6025, WT versus Het, P = 0.0584, 95% C.I. = –0.006443–0.3146; BrdU⁺, WT versus KO, P = 0.0194, 95% C.I. = 0.08378–0.7348, WT versus Het, P = 0.7612, 95% C.I. = –0.2218–0.4292; PH3⁺, WT versus KO, P = 0.0002, 95% C.I. = 0.2976–0.5796, WT versus Het, P = 0.2287, 95% C.I. = –0.05332–0.2288). e,f, Coronal sections of E15.5 (e) and E17.5 (f) brains stained with both anti-BrdU that recognizes BrdU only and anti-IdU that also recognizes BrdU. Pregnant mothers received one IdU injection, followed by one BrdU injection 1.5 h later. After another 0.5 h, the embryonic brains were collected for analysis. g, Quantification of the percentage of IdU⁺BrdU^– cells, representing cells that left S-phase during the 1.5-h chase, among total IdU⁺ cells. One-way ANOVA (n = 3 brain sections for all experimental groups; E15.5, P = 0.0025, F(2, 6) = 19.21; E17.5, P = 0.0075, F(2, 6) = 12.35) followed by Bonferroni’s post hoc test (E15.5, WT versus KO, P = 0.0067, 95% C.I. = 2.835–12.6, WT versus Het, P = 0.5802, 95% C.I. = –6.787–2.973; E17.5, WT versus KO, P = 0.0107, 95% C.I. = 2.347–13.19, WT versus Het, P = 0.9999, 95% C.I. = –5.598–5.244). h,i, Coronal sections of E15.5 (h) and E17.5 (i) brains stained with antibodies recognizing Ki67 and BrdU. Pregnant mothers received one BrdU injection 24 h before embryo collection. j, Quantification of the percentage of BrdU⁺Ki67^– cells, representing cells that exited the cell cycle during 24 h, among total BrdU⁺ cells. One-way ANOVA (n = 3 brain sections for all experimental groups; E15.5, P = 0.0173, F(2, 6) = 8.589; E17.5, P = 0.0016, F(2, 6) = 22.51) followed by Bonferroni’s post hoc test (E15.5, WT versus KO, P = 0.014, 95% C.I. = 4.493–29.92, WT versus Het, P = 0.6051, 95% C.I. = –7.885–17.54; E17.5, WT versus KO, P = 0.01, 95% C.I. = 3.932–21.2, WT versus Het, P = 0.1249, 95% C.I. = –15.28–1.99). k,l, Immunostaining of coronal sections of E17.5 brain with antibodies to the intermediate progenitor marker Tbr2 and the proneural marker Neurod2. Dashed white lines indicate border of VZ/SVZ area. Similar results were obtained from three independent experiments. Scale bars represent 100 μm. Graphs represent the mean ± s.d. Dots represent data from individual data points. ns, non-significant. *P < 0.05, **P < 0.01, ***P < 0.001.