Fig 1: WTAP-mediated m6A depletion increases GRN levels, and influences cell–cell communication essential for planarian regeneration. (A) Integrative genomics viewer tracks displaying the distributions of methylated RNA immunoprecipitation sequencing (MeRIP-seq) data that normalized by RNA-seq data along grn in both control and wtap knockdown planarians at 7 and 11 dpa (top). Barplot showing the enrichment of m6A modification on grn. The p values were determined using Wilcoxon-test. (B) Whole-mount fluorescent in situ hybridization showing expressions of grn (red) and dph1 (green) in control, wtap knockdown (wtap RNA interference [RNAi]), double knockdown of wtap and grn (wtap + grn RNAi), double knockdown of wtap and cdk7 (wtap + cdk7 RNAi), double knockdown of wtap and cdk9 (wtap + cdk9 RNAi) planarians at 7 dpa. Hoechst indicates DNA staining. Scale bar, 200 µm (left panels), 10 µm (magnified panels). (C) Barplot showing the number of grn +/dph1 + cells of control (control), wtap knockdown (wtap RNAi), double knockdown of wtap and grn (wtap + grn RNAi), double knockdown of wtap and cdk7 (wtap + cdk7 RNAi), double knockdown of wtap and cdk9 (wtap + cdk9 RNAi) planarians in (B). Data were analysed by the two-tailed unpaired Student's t-test (bottom). (D) Immunofluorescence showing the expression and localization of GRN protein in control (control), wtap knockdown (wtap RNAi), double knockdown of wtap and grn (wtap + grn RNAi) planarians at 7 dpa. Hoechst indicates DNA staining. Scale bar, 100 µm. (E) Bright-field images showing the phenotypes of control (control), wtap knockdown (wtap RNAi), double knockdown of wtap and grn (wtap + grn RNAi) planarians at 7 dpa. Scale bar, 300 µm. Bottom left number, number of planarians with phenotype versus total number tested. (F) Barplot showing percentage of regenerated worms in control (control), wtap knockdown (wtap RNAi), double knockdown of wtap and grn (wtap + grn RNAi) planarians at 7 dpa. Error bars represent standard deviation. Data are the mean ± SD (n = 3 independent experiments). The p values were determined using a two-sided unpaired Student's t-test. (G) Whole-mount fluorescent in situ hybridization showing expressions and localizations of pc2 (red) and smedwi-1 (green) of control (control), wtap knockdown (wtap RNAi), double knockdown of wtap and grn (wtap + grn RNAi), double knockdown of wtap and cdk7 (wtap + cdk7 RNAi) and double knockdown of wtap and cdk9 (wtap + cdk9 RNAi) planarians at 7 dpa. Dotted line, amputation plane (n = 3). Scale bar, 200 µm. (H) Quantification of percentage of pc2 positive regenerated neurons in each group of (G). Error bars represent standard deviation. Data are the mean ± SD (n = 3 independent experiments). The p values were determined using a two-sided unpaired Student's t-test. See also Figures S5–S7.