Characterization of Mettl3 conditional knockout mice

Consistent with a previous report [35], we detected ubiquitous occupation of METTL3 in various brain regions by immunostaining (S1A Fig), indicating that METTL3 might be involved in the development of mammalian brains. To investigate the functions of METTL3-mediated m⁶A in brain development, we introduced two loxP sites into the Mettl3 genomic region flanking exons 2–4, a region encoding the methyltransferase catalytic domain in B6D2F1 mice by CRISPR/Cas9 system-assisted homologous recombination (S1B Fig). The resulting Mettl3^flox/flox mice were crossed with Nestin-Cre transgenic mice to generate Mettl3 conditional knockout mice (genotype Mettl3^flox/flox;Nestin-Cre, named as cKO) (S1B and S1C Fig). Genotyping results confirmed the specific deletion of Mettl3 exons 2–4 in the brain tissue of newborn cKO mice, whereas the Mettl3 coding sequence in their tail tissue remains intact (S1C–S1G Fig). Immunohistochemical staining (S1A Fig) and western blot (S1H Fig) further confirmed that the cKO mice failed to produce METTL3 protein and its isoforms in the whole brain, whereas in other nonneural tissues, such as liver, METTL3 protein remained unperturbed. Littermates of the cKO mice with the Mettl3^flox/flox or Mettl3^flox/+ genotypes were used as the controls (Ctrl).

The cKO pups had a significantly decreased body weight growth rate as compared to that of the Ctrl (Fig 1A) and were only approximately half the size of their Ctrl littermates by P14 (S2A Fig). The majority of cKO pups died before P20 without artificial feeding (Fig 1B). In addition, the cKO mice demonstrated balance disorders and altered gaits after P7 that became severe at around P14 (S1 Movie). When lifted by the tail, the cKO mice displayed dramatic tremors with tightly curled legs and forward bended head (Fig 1C and S2 and S3 Movies), demonstrating typical features of cerebellar ataxia [36]. We then analyzed the behavior of the mice in an open field test; the cKO mice only generated intermittent and slow movements with much reduced speed and moving distance, and were seldom capable of moving out of the central region (Fig 1D and 1E and S2B–S2D Fig). The smaller body size, premature death, and ataxia-like movement disorders indicated severe developmental defects in the cKO mouse brain. We then performed magnetic resonance imaging (MRI) analysis and identified smaller brain sizes and enlarged ventricles in the brains of Mettl3 cKO mice (S2E Fig), which is consistent with the results in another m⁶A depletion model via Nestin-Cre mediated conditional knockout of Mettl14 [28]. Furthermore, in addition to the cortex, we also observed shrunk and unstructured cerebellums in the Mettl3 cKO mice (Fig 1F–1H). It is tempting to speculate that the developmental defects in the cerebellums caused by depletion of METTL3 might contribute to the movement disorders of the cKO pups. Ultra high-pressure liquid chromatography tandem-mass spectrometry (UHPLC-MS/MS) analysis showed that the m⁶A modification on mRNAs of cKO mouse brain tissues were nearly wiped out (S2F Fig), which indicated that the brain developmental defects might be caused by m⁶A depletion.

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Fig 1. Characterization of the Mettl3 conditional knockout mice.

(A) Body weight changes of the Ctrl and cKO mice during the first 2 wk after birth. (B) Kaplan–Meier survival curves showing the survival rates of both Ctrl and cKO mice. (C) Tail suspension test of both Ctrl and cKO mice. (D, E) Open field test of Ctrl and cKO mice for their speed (D) and total traveled distance (E). (F) MRI of mice sagittal brain section. Scale bar, 1 mm. (G, H) Surface areas of the whole brain (G) and cerebellum (H) of Ctrl and cKO mice. Sagittal section. The data underlying this figure can be found in S1 Data. Data shown are means ± SEM, n = 4. **p-value < 0.01, ***p-value < 0.001, Student t test. cKO, Mettl3 conditional knockout; Ctrl, control; Mettl3, methyltransferase-like 3; MRI, magnetic resonance imaging; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.2004880.g001

Mettl3 conditional knockout causes cerebellar hypoplasia in mice

A previous report has demonstrated that the m⁶A RNA methylation level in mouse cerebellum is generally higher than that in other brain regions [35]. Consistently, our western blot showed relatively higher expressions of METTL3 and METTL14 in the cerebellum than those in the cerebral cortex of wild-type mouse (S3A Fig). UHPLC-MS/MS analysis also detected twice the amount of m⁶A on mRNA from the cerebellum in comparison to that from other brain regions of the wild-type mouse (S3B Fig). We then focused on the effect of METTL3 depletion on the development of the cerebellum. We first systematically investigated the expression pattern of METTL3 in developing mouse cerebellum by immunohistochemical staining, and we found evident and ubiquitous immunostaining signals in the EGL, PCL, ML, and IGL of the cerebellum from embryonic day 16.5 (E16.5) to P14 (S4A–S4C Fig). Interestingly, the METTL3 expression level in the proliferating GCPs within the outer EGL was shown to be relatively lower than that of the newborn CGCs within the inner EGL at P7 (S4B Fig). We confirmed that METTL3 was depleted in the cKO cerebellums as early as E16.5, when no substantial structural defect was seen in the cerebellar region of the cKO mouse brains (S4A–S4C Fig). Macroscopic histological examination showed shrunken size, loss of weight, and remarkable failure in foliation of the cKO cerebellums (Fig 2A–2C). Further immunohistological analysis revealed drastic loss of CGCs in the IGL layer of the cKO cerebellums (Fig 2D and 2E and S4D Fig). In spite of the fact that the number of Purkinje cells in the cKO cerebellums remained almost the same as that in the Ctrl mice, they failed to maintain the well-organized single-cell layer (Fig 2D and 2F and S4D Fig). In addition, Purkinje cells in cKO cerebellums had much shorter dendrites and exhibited reduced calbindin 1 (CABL1, also known as D-28K) staining signal (Fig 2D, 2G and 2H and S4D Fig), indicating defective Purkinje cell maturation in the cKO cerebellums. Glial fibrillary acid protein (GFAP) staining also showed that the Bergmann glia lost their scaffold organization pattern in the cKO cerebellums (Fig 2D and S4D Fig). Based on the above, we concluded that depletion of METTL3 caused severe cerebellar hypoplasia in mice, indicating that m⁶A played important roles in regulating not only the development of the cortex [28] but also the cerebellar region of the brain.

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Fig 2. Mettl3 conditional knockout causes cerebellar hypoplasia in mice.

(A) Macromorphological comparison between brains of the Ctrl and cKO mice. (B, C) Weight of the whole brain (B) and cerebellum (C) of the Ctrl and cKO mice. (D) Histological abnormalities of cerebellum in cKO mice shown by HE staining, NeuN, D-28K, and GFAP immunohistochemical staining. Scale bar, 200 μm. (E, F) Density difference of granule cells (E) and Purkinje cells (F) in the Ctrl and cKO mouse cerebellums. (G, H) Dendrite length (G) and mean calbindin D-28K staining intensity. (H) Difference of Purkinje cells in the Ctrl and cKO cerebellums. P14 mice were used in all studies. Data related to this figure are shown in S1 Data. Data shown are means ± SEM. For cell number count in (E) and (F), n = 3; for dendrite length and D-28K staining intensity in (G) and (H), n = 10. ***p-value < 0.001, Student t test. cKO, Mettl3 conditional knockout; Ctrl, control; D-28K, calbindin 1; EGL, external granular layer; GFAP, glial fibrillary acid protein; HE, hematoxylin and eosin; IGL, internal granular layer; Mettl3, methyltransferase-like 3; ML, molecular layer; NeuN, neuronal nuclei; PCL, Purkinje cell layer; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.2004880.g002

Depletion of METTL3 induces apoptosis of newborn granule cells

We further assessed the proliferation potential of GCPs using an in vivo bromodeoxyuridine (BrdU) labeling assay. The cerebellar tissues were dissected from P7 mice 2 h post–BrdU injection and costained with BrdU/antigen identified by monoclonal antibody Ki 67 (Ki67)/DAPI. The numbers of Ki67⁺ cells exhibited no difference between the Ctrl and cKO samples, indicating that GCPs maintain the ability to proliferate in cKO EGLs (Fig 3A and 3E), although with a slightly reduced rate (S5A and S5B Fig). However, both TUNEL and Cleaved Caspase-3 immunostaining revealed a significantly increased cell apoptosis rate in cKO cerebellum, especially in the EGL region (Fig 3B, 3C, 3F and 3G). Histological examination also detected a large number of karyorrhexis and karyopyknosis, the characteristic nuclear forms of apoptotic cells, in the EGL of cKO cerebellums (S5C Fig). Consistent with the enhanced apoptotic signals, the BrdU⁺/Ki67⁻ cells, representing postmitotic newborn granule neurons, were drastically reduced in cKO cerebellums at 48 h post–BrdU injection (Fig 3D and 3H). These data demonstrated that the severe depletion of CGCs in cKO cerebellums mainly resulted from the abnormal postmitotic apoptosis of newborn granule cells.

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Fig 3. Mettl3 conditional knockout induces apoptosis of newborn granule cells.

(A) BrdU (green), Ki67 (red), and DAPI (blue) immunofluorescent staining of Ctrl and cKO mouse cerebellums 2 h after BrdU injection. (B) TUNEL (green) and PI (red) staining of P7 Ctrl and cKO mouse cerebellums. (C) Cleaved Caspase-3 (green), Ki67 (red), and DAPI (blue) immunofluorescent staining of P7 Ctrl and cKO mouse cerebellums. (D) BrdU (green), Ki67 (red), and DAPI (blue) immunofluorescent staining of Ctrl and cKO mouse cerebellums 48 h after BrdU injection. (E) Proportion of Ki67⁺/DAPI⁺ cells in the EGL of P7 Ctrl and cKO mice. (F, G) Density of TUNEL⁺ (F) and Cleaved Caspase-3⁺ (G) cells in P7 Ctrl and cKO mouse cerebellums. (H) Density of BrdU⁺/Ki67⁻ cells in the EGL of Ctrl and cKO mice 48 h post–BrdU injection. Scale bars in (A–D), 200 μm for left panels; 25 μm for right panels. Data related to this figure are shown in S1 Data. Data shown are means ± SEM. Three sections of each sample were analyzed. ***p-value < 0.001, Student t test. BrdU, bromodeoxyuridine; cKO, Mettl3 conditional knockout; Ctrl, control; EGL, external granular layer; Ki67, antigen identified by monoclonal antibody Ki 67; Mettl3, methyltransferase-like 3; PI, Propidium iodide; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.2004880.g003

m⁶A depletion induces dramatic gene expression changes in the cerebellum

To further investigate the underlying mechanisms for the cerebellar developmental defects in Mettl3 cKO mice, we performed RNA sequencing (RNA-seq) and m⁶A sequencing (m⁶A-seq) using mRNAs extracted from the cerebellums of Ctrl and cKO mice at both P7 and P14. Over 10,000 and 15,000 differential m⁶A peaks were identified in the cerebellar mRNAs of P7 and P14 Ctrl mice, respectively, whereas in Mettl3 cKO mice, only a few hundred (background level) differential m⁶A peaks were detected (S1, S2 and S3 Tables). Consistent with previous reports [16, 37], the m⁶A peaks in cerebellar RNAs were also enriched in the regions with RRACH motif (Fig 4A and S6A Fig) and tended to occur near stop codons and within 3′UTRs of mRNAs (Fig 4B and S6B Fig). Among the 15,614 expressed genes in the cerebellum of the P7 Ctrl mice, transcripts of 6,838 genes contain m⁶A peaks (Fig 4C). Because the presence of m⁶A peaks could facilitate mRNA degradation [16], we focused on the 696 genes with both m⁶A peak loss and elevated expression in the cKO cerebellums (Fig 4C), as this group of transcripts was likely stabilized after m⁶A depletion. Gene ontology analysis revealed that most of these genes were enriched in neural development-associated biological processes and the apoptotic signaling pathway (Fig 4D and S7A and S7B Fig). Similar results were also obtained from the m⁶A-seq and RNA-seq data at P14 (S6A–S6D and S7C and S7D Figs).

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Fig 4. m⁶A depletion induces dramatic gene expression changes in the cerebellum.

(A) The most enriched sequence motif of m⁶A peaks in mRNAs from P7 mouse cerebellums. (B) Distribution of m⁶A peaks along the 5′UTR, CDS, and 3′UTR regions of total cerebellar mRNAs from P7 Ctrl mice after normalized with length. (C) Venn diagram representing the relationships between cerebellar expressed genes and m⁶A modification. Green circle represents all expressed genes in P7 Ctrl mouse cerebellums, brown circle represents genes with m⁶A modifications in P7 Ctrl mouse cerebellums, red circle represents genes with up-regulated expression in P7 Mettl3 cKO mouse cerebellums as compared to the Ctrl, and blue circle represents genes with down-regulated expression in P7 Mettl3 cKO mouse cerebellums as compared to the Ctrl. Numbers represent the counts of genes in each group. (D) Significantly enriched (p-value ≤ 0.01, Benjamini–Hochberg multiple testing correction) GO terms of genes with up-regulated expression in P7 cKO cerebellums and with m⁶A peaks. CDS, coding sequence; cKO, Mettl3 conditional knockout; GO, gene ontology; JAK-STAT, Janus kinase (JAK), Signal Transducer and Activator of Transcription protein (STAT); m⁶A, N⁶-methyladenosine.

https://doi.org/10.1371/journal.pbio.2004880.g004

m⁶A depletion enhances the stability of development- and apoptosis-associated gene transcripts

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis confirmed the up-regulation of genes with m⁶A loss (S8A and S8C Fig) in cKO cerebellums, including essential factors for cerebellar development such as Atoh1, Cxcr4, Notch2 and its ligand Jag1 [32, 38–41], as well as neuronal apoptosis such as Dapk1, Fadd, and Ngfr [42–44] (Fig 5A and S6E Fig). We further measured the degradation rates of these mRNAs in neural stem cell (NSC) lines established from the Ctrl and cKO neonatal mice (Fig 5B) using the actinomycin-D mediated transcription inhibition assay. We detected prolonged mRNA half-lives of these development- and apoptosis-associated genes in the cKO NSCs (Fig 5C). Because there were no reported apoptosis phenotypes in the developing cortex [28], we compared the expression levels of the apoptosis-promoting genes that showed extended half-lives with the depletion of m⁶A in both the cerebellar region and the cortex of wild-type mice by quantitative polymerase chain reaction (qPCR), and the results displayed higher expression levels of these genes in the cerebellar region than in the cortex (Fig 5D). Furthermore, in vitro cultured CGCs isolated from P7 mice showed elevated expression levels of these genes with the depletion of METTL3 (Fig 5E). All lines of evidence indicated that these apoptosis-promoting genes functioned predominantly in the cerebellum CGCs rather than in the cortex. Our data demonstrated that the depletion of m⁶A resulted in a dysregulated gene expression profile during the development of the cerebellum, especially for genes related to cerebellar development and cell apoptosis.

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Fig 5. m⁶A depletion enhances the stability of development- and apoptosis-associated gene transcripts.

(A) qRT-PCR results confirmed the up-regulated expression in P7 cKO cerebellums of selected genes. The major functions of detected genes are shown under the lines. (B) Immunostaining for Sox2 (red), Nestin (green), and Mettl3 (purple) in neural stem cell lines established from Ctrl and Mettl3 cKO neonatal mice. Scale bar, 100 μm. (C) The RNA half-lives of genes detected in (A). Values and error bars in (A) and (C) represent the mean and SEM of three independent experiments. (D) qRT-PCR results confirmed higher expression levels of apoptosis-promoting genes (Dapk1, Fadd, Ngfr) in cerebellums of P7 wild-type mice than those in the cortex. (E) qRT-PCR results confirmed the up-regulated expression of the apoptosis-promoting genes in the cKO CGCs. Data related to this figure are shown in S1 Data. Data shown are means ± SEM. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001, Student t test. CGC, cerebellar granule cell; cKO, Mettl3 conditional knockout; Ctrl, control; Mettl3, methyltransferase-like 3; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.2004880.g005

m⁶A depletion induces exon exclusion in synapse-associated genes

Consistent with a previous report that m⁶A depletion tends to produce exon-excluded transcripts [17], we also found that the majority of exon-excluded transcripts in cKO cerebellums (as compared with the Ctrl) were derived from m⁶A-containing transcripts in the Ctrl at both P7 and P14 (Fig 6A and S9A Fig). Interestingly, genes with exon-excluded transcripts in cKO were enriched in synapse-associated pathways, including transmission across chemical synapses and protein–protein interactions at synapses (Fig 6B and S9B Fig). More specifically, we found that genes associated with the two major glutamate receptors, the N-methyl-D-aspartic acid (NMDA) receptors and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, were highly enriched in the pathway analysis (Fig 6B). NMDA receptors were reported to be essential for the maturation and survival of CGCs [44, 45]. We confirmed that the m⁶A-modified exons (S8B and S8C Fig) in these synapse-associated genes, such as Grin1, Atp2b3, Grm1, and Lrp8, were excluded in cKO cerebellums by RT-PCR (Fig 6C and S9C Fig). The MiniGene assay confirmed the exclusion of exon 21 (C1 region) of Grin1 (S10A Fig) and exon 19 of Lrp8 (S10B Fig) with the depletion of m⁶A by either Mettl3 knockdown or mutation of the m⁶A modification sites (S10C and S10D Fig), indicating the regulatory role of m⁶A modification in RNA splicing and function of the ion channel associated genes.

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Fig 6. m⁶A depletion induces exon exclusion in synapse-associated genes.

(A) Genes producing exon-excluded transcripts in P7 cKO cerebellums as compared with the Ctrl. (B) Enriched pathways of genes producing exon-excluded transcripts in P7 cKO cerebellums as compared with the Ctrl. Enriched pathways under the “Neuronal system” are expanded on the right. (C) Production of exon-excluded transcripts of major synapse-associated genes in P7 cKO cerebellums. Left, IGV tracks displaying the RNA-seq reads coverage in P7 Ctrl (blue) and cKO (red) cerebellum, the excluded exons are shaded with pink columns. Middle, calculated exon-exclusion percentage in P7 cKO cerebellums by the PSI value. Right, semiquantitative PCR detection of exon-excluded transcripts in P7 cKO cerebellums. (D) Images of the CGCs in the calcium detection assay. Grin1₀₀₁ or Grin1₀₁₁ was introduced into the cKO CGCs by electroporation and was co-expressed with mCherry (red). Fluo-4 (rainbow) indicated the cellular calcium concentration in the CGCs. Scale bar, 5 μm. (E) Statistical analysis of the cellular calcium concentration in the calcium detection assay. (F) CCK8 assay shows the survival rate of the Ctrl CGCs with overexpression of mCherry and cKO CGCs with overexpression of Grin1₀₀₁ or Grin1₀₁₁ in 3 DIV and 5 DIV. (G) Schematic diagram illustrating the mechanisms underlying the regulatory functions of METTL3-mediated m⁶A modification in cerebellar development. Red dots represent m⁶A modification. The box and line symbols in the right part of the diagram represent pre-spliced RNAs, with exons shown as boxes and introns shown as black lines. Exons in post-spliced RNAs are connected by red lines. Further information about this figure can be found in S1 Data. The data were represented as means ± SEM. For calcium concentration detection, n = 10; for CCK8 assay, n = 3. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, Student t test. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BF, bright field; CCK8, Cell Counting Kit-8; CGC, cerebellar granule cell; cKO, Mettl3 conditional knockout; CREB, cAMP responsive element binding protein; Ctrl, control; DIV, days in vitro; Grin1₀₀₁, C1 region excluded variant of Grin1; Grin1₀₁₁, C1 region included variant of Grin1; IGV, integrative genomics viewer; METTL3, methyltransferase-like 3; m⁶A, N⁶-methyladenosine; NMDA, N-methyl-D-aspartic acid; PKMT, Lysine methyltransferase; PPAR, peroxisome proliferator-activated receptor; PSI, percent spliced in index; RNA-seq, RNA sequencing; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.2004880.g006

The NMDA receptor is a heterodimer of glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) and glutamate ionotropic receptor NMDA type subunit 2 (GRIN2) and is permeable to Ca²⁺ when activated by glutamate. The Grin1 gene contains three alternatively spliced exons (ASEs), namely N1, C1, and C2, and generates at least eight splicing variant isoforms [46]. The C1 region has been shown to be a regulatory domain for calcium influx and is reported to be affinitive to calmodulin (CaM) [47] and inhibit the NMDA receptor in a Ca²⁺-dependent way [48]. To further investigate the effect of the altered splicing of Grin1 induced by depletion of m⁶A on the survival of CGCs, we isolated and cultured CGCs from both Ctrl and cKO mouse cerebellums. We overexpressed Grin1₀₀₁ (C1 excluded variant) or Grin1₀₁₁ (C1 included variant) in the cKO CGCs by electroporation. We found that cKO CGCs had a much higher intracellular calcium concentration than that of the Ctrl ones, whereas overexpression of Grin1₀₁₁, but not mCherry or Grin1₀₀₁, reduced the cellular calcium concentration in the cKO CGCs (Fig 6D and 6E). Consistently, Cell Counting Kit-8 (CCK8) assay detected an increased survival rate of the cKO CGCs with Grin1₀₁₁ overexpression (Fig 6F). Our findings established that the altered Grin1 splicing induced by m⁶A depletion resulted in a substantial increase in intracellular calcium concentration and consequently the apoptosis of the CGCs.

Ethics statement

The study was approved by the Research Ethic Committee in the Institute of Zoology, Chinese Academy of Sciences in Beijing, China (Approval number: IOZ20150076).

Animals

Mouse strains used in this study were B6D2F1 (C57BL/6×DBA2), C57BL/6, and DBA2. B6D2F1 and DBA2 mice were purchased from Beijing Vital River Laboratory Animal Center. C57BL/6 Nestin-Cre mice were purchased from the Jackson Laboratory. All mice were housed under specific pathogen-free (SPF)-grade conditions in the animal facilities of the Institute of Zoology, Chinese Academy of Sciences.

Generation of Mettl3^flox/flox; Nestin-Cre mice

The T7-Cas9 plasmid and the T7-sgRNA backbone used in this study were from Qi Zhou’s lab [66]. T7-sgRNA backbone harbored a T7 promoter followed by two reverse orientated Bbsl restriction sites, which was followed by a small guide RNA scaffold. The T7-sgRNA plasmid construction was performed as previously described [66]. The annealed sgRNA oligonucleotides synthesized from Beijing Genomics Institute (BGI) have two different overhangs that are complementary with the corresponding sticky ends of Bbsl-digested T7-gRNA vector to ensure directional cloning. Oligonucleotides used for CRISPR-Cas9 system-assisted homologous recombination through embryo injection were synthesized in Integrated DNA Technologies. All oligonucleotides were listed in S4 Table.

The sgRNAs and Cas9 mRNA were transcribed by the HiScribe T7 In Vitro Transcription Kit (New England Biolabs) using endonuclease linearized DNA templates according to the manufacturer’s instructions. Cas9 mRNA was capped with the m7G (5′) ppp (5′) G RNA Cap Structure Analog (New England Biolabs) by T7 RNA transcriptase. RNAs were dissolved in DEPC-H₂O (New England Biolabs) and used for intracytoplasmic RNA microinjection following previously reported procedures [66]. Briefly, one-cell-stage embryos were collected from female B6D2F1 mice at 0.5 day post-coitum (dpc). Each embryo was microinjected with 25 ng/μL L-sgRNA, 25 ng/μL R-sgRNA, 100 ng/μL Cas9 mRNA, 50 ng/μL L-oligo DNA, and 50 ng/μL R-oligo DNA into the cytoplasm. Injected embryos were subsequently implanted into the oviducts of pseudopregnant B6D2F1 female mice. Full-term pups were obtained by natural labor at 19.5 dpc.

All pups were genotyped and two Mettl3^flox/+ founder mice were obtained. The viripotent Mettl3^flox/+ founder mice were self-bred or mated with Nestin-Cre mice to generate Mettl3^flox/flox and Mettl3^flox/+; Nestin-Cre mice, respectively. The Mettl3^flox/flox and Mettl3^flox/+; Nestin-Cre mice were interbred to generate Mettl3^flox/+; Nestin-Cre, Mettl3^flox/flox, Mettl3^flox/+, and Mettl3^flox/flox; Nestin-Cre mice.

Genotyping

Genomic DNA was extracted using the Mouse Direct PCR Kit (Bimake). Briefly, mouse tissues were mixed with 100 μL Buffer L and 2 μL Protease Plus, and incubated at 55 °C for 30 min, then 100 °C for 5 min according to the manufacturer’s instructions.

First, all mouse pups were genotyped to identify the insertion of loxP sites in Mettl3 using tail DNA extract. The length of the PCR product was 222 bp/182 bp (loxP/wt) in Mettl3 intron 1 with primers F1/R1 and 335 bp/295 bp (loxP/wt) in Mettl3 intron 4 with primers F2/R2. Pups with loxP inserted both in Mettl3 intron 1 and intron 4 in the same allele of Mettl3 were identified as Mettl3^flox/+. Pups with loxP inserted both in Mettl3 intron 1 and intron 4 in both alleles of Mettl3 were identified as Mettl3^flox/flox. The PCR product for Nestin-Cre was 410 bp with primers Nestin-Cre-F/Nestin-Cre-R. Primers F1/R2 were used to further confirm Mettl3 deletion in the brain, producing PCR product with 318 bp/2,554 bp (cKO/wt) in length. All sequences of primers for genotyping are listed in S5 Table.

Western blot

Mouse brain tissues were harvested after cervical dislocation and were homogenized either by grinding into homogenous powder using mortar and pestle cooled in liquid-nitrogen bath or by mechanical homogenizer using beating beads. Pulverized tissue samples were lysed in RIPA buffer (0.5% NP-40, 50 mM Tris-Cl, pH 8, 150 mM NaCl, 1 mM EDTA) supplemented with proteinase and phosphatase inhibitors. The lysate was centrifuged at 12,000 g at 4 °C for 20 min to remove cell debris. Pierce Coomassie Plus (Bradford) assay kit (Thermo) was used to determine the protein concentration. The fraction (50–100 μg) was separated by 10% SDS-PAGE and analyzed by immunoblotting with corresponding antibodies, anti-Mettl3 (1:1,000, Abcam, ab195352), anti-Mettl14 (1:1,000, Atlas Antibodies, HPA038002), and anti-γ-ACTIN (1:3,000, Santa Cruz, SC65638).

Tail suspension test

Tail suspension test was performed as described by Pierre-Olivier Frappart [36]. Mice at P14 were hung by the tail. Postures and movements of the mice were observed.

Open field test

The assay was performed as previously described [67] using P14 Ctrl and cKO mice. The apparatus was a square-shaped arena (750 × 750 mm², length × width), which was divided into two concentric squares, the central field (325 × 325 mm² arena) and the peripheral field, and was illuminated evenly at 15 lux. Animals were placed in the center of the arena, and their locomotion was recorded by a camera hung over the arena. A 10-min-long video was analyzed by the TopScan software. Locations of the animals in the area were recorded at the speed of one image per s. The frequency crossing the border, latency in the central field, total moving distance, and velocity of mice recorded in the apparatus were analyzed.

MRI and stereological measurements

All mice for MRI were anesthetized with 10% chloralic hydras (Sigma) by intraperitoneal injection. Mice were fixed on the scanning coil with a self-made scaffold. Body temperature was maintained by circulating water through warming pads. MRI was conducted using a Bruker 7.0 T MRI equipment (Bruker Medical Systems, CLinScan) and was performed at P14 for both Ctrl and cKO mice. Both the sagittal section and transverse section were scanned. For image acquisition, rapid acquisition relaxation enhanced (RARE) sequence was used. The MRI parameters included T2W repetition time = 2,500 ms, T2W echo time = 60 ms, field of view = 12 × 12 mm, matrix = 240 × 240 pixels, resolution = 50 × 50 μm, and slice thickness = 0.6 mm. MRI images acquired were processed by ImageJ (https://imagej.nih.gov/ij/) to calculate the area of the whole brain section or the cerebellar region. To be brief, the outline of the whole brain or the cerebellum in the image was drawn out as region of interest (ROI); the number of pixels contained within the ROI was calculated by the software. As the resolution is 50 × 50 μm, each pixel represents an area of 2.5 × 10⁻³ mm². Three repeats were conducted for each calculation.

BrdU labeling

In order to examine the proliferation activity of GCPs in the cerebellar EGL, we injected P7 mice with BrdU (Sigma) intraperitoneally at a dose of 50 μg BrdU/g body weight [36]. Mice were humanely killed 2 h later for immunofluorescence staining. To investigate the generation and migration of CGCs, BrdU-injected mice were kept for another 2 d until they were humanely killed at P9 for staining.

Macromorphological observation

To compare the gross appearance and histological organization of the cerebellums of both Ctrl and cKO mice, mice at P14 were humanely killed by breaking the neck. The whole brain or cerebellum was dissected, weighed, or fixed with 4% paraformaldehyde and photographed.

Hematoxylin and eosin analysis

Dissected brains were cut into sagittal blocks and fixed with 4% paraformaldehyde followed by dehydration (70%, 80%, 90%, 100% ethanol) and paraffin embedding. Sections 3 μm in thickness were cut from the paraffin-embedded tissue blocks with a Leica slicing machine (Leica Biosystems) and mounted on poly-D-lysine coated glass slices (Zhong Shan Golding Bridge Biotechnology). Slices were heated at 65 °C for 2 h and then immersed in xylene to remove paraffin. After a series of rehydration processes (100%, 100%, 90%, 80%, 70% ethanol), slices were stained with hematoxylin and eosin (HE) using standard methods and imaged with a Leica Aperio VERSA 8 microscope (Leica Biosystems).

Immunohistochemical analysis

For immunohistochemical staining, rehydrated sections or coverslips were incubated with primary antibodies overnight. Primary antibodies used in this study were for Mettl3 (ab195352, 1:250, Abcam), neuronal nuclei (NeuN, MAB377, 1:500, Millipore), calbindin D-28K (C9848, 1:2,000, Sigma), and glial fibrillary acid protein (GFAP, Z033429, 1:1,000, Dako). Appropriate Horseradish peroxidase conjugated secondary antibodies (anti-Mouse IgG and anti-Rabbit IgG, Vector laboratories) were used according to manufacturer’s recommendations. A 3, 3′-diaminobenzidine kit (DAB, Vector laboratories) was used for color developing. Images were obtained using standard methods and imaged with a Leica Aperio VERSA 8 microscope (Leica Biosystems). Immunohistochemical images were analyzed with Image-Pro Plus (Media Cybernetics). At least three repeats were conducted for each calculation.

Immunofluorescence analysis

Immunofluorescence staining was performed as previously described, with a few modifications [68]. Briefly, rehydrated sections or coverslips were incubated with primary antibodies overnight. Primary antibodies used in this study were for BrdU (C8434, 1:500, Sigma), Ki67 (ab15580, 1:500, Abcam), Cleaved Caspase-3 (9661s, 1:200, Cell Signaling Technology), Phospho-Histone H3 (3377s, 1:500, Ser10, Cell Signaling Technology), Nestin (MAB353, 1:1,000, Millipore,), Sox2 (SC-17320, 1:1,000, Santa Cruz), and Mettl3 (ab195352, 1:1,000, Abcam). Appropriate fluorophore (FITC, Cy3, and Cy5) conjugated secondary antibodies (Jackson ImmunoResearch, 1:1,000) were used according to the manufacturer’s recommendations. Sections were counterstained with DAPI (Invitrogen) and mounted on slices with Dako Fluorescence Mounting Medium (Dako). Fluorescent images were obtained using a Carl Zeiss LSM 780 confocal system. For quantification of Ki67 and Phospho-Histone H3 positive cell ratio, we used three pairs of mice at P7 and quantified three sections of the cerebellum vermis for each Ctrl and cKO mouse, respectively. We lined out the EGL region manually, and the total numbers of Ki67⁺-, Phospho-Histone H3- and DAPI-positive cells in the EGL region were analyzed with Columbus Image Analyzing System (Perkin Elmer). For the quantification in the assays of long-term BrdU labelling, TUNEL, and Cleaved-Caspase 3 staining, the positive cells in the whole section of the cerebellum vermis were quantified. Three pairs of mice and three sections for each of Ctrl and cKO mouse were used for quantification.

TUNEL analysis

The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s instruction. Briefly, sections were permeabilized by proteinase K and labeled with rTdT reaction mix for 1 h at 37 °C; the reaction was stopped by 2 × SSC. Sections were counterstained by PI (Invitrogen) and mounted on slices with Dako Fluorescence Mounting Medium (Dako). Images were obtained using a Carl Zeiss LSM 780 confocal system. Fluorescent images were analyzed with Columbus Image Analyzing System (Perkin Elmer). At least three repeats were conducted for each calculation.

RNA isolation

Total RNA from mouse cerebellums was extracted using TRIzol reagent (Invitrogen). mRNAs were purified with Dynabeads mRNA Purification Kit (Thermo) according to the manufacturer’s manual, followed by DNase I (M6101, Promega) treatment to remove genomic DNA. RNA concentration was measured using Nanodrop 1000 (Thermo). For quality control, only samples with OD 260/280 nm ratio more than 2 and 260/230 nm values in the range of 2.0–2.2 were used for subsequent experiments. The integrity of RNAs was checked by RNA gel electrophoresis, and only total RNAs having a 28S band twice as bright as the 18S band were used for further study. Two sets of samples were collected as biological replicates.

UHPLC-MS/MS analysis of mononucleotides

A total of 400 ng RNA was mixed with 0.1 U Nuclease P1 (Sigma) and 1.0 U calf intestinal phosphatase (New England Biolabs) in the final reaction volume of 50 μL, adjusted with water and incubated at 37 °C overnight. The digested RNA solutions were filtered by ultrafiltration tubes (MW cutoff: 3 kDa, Pall, Port Washington), then subjected to UHPLC-MS/MS analysis for detection of m⁶A. The UHPLC-MS/MS analysis was performed with an Agilent 1290 UHPLC system coupled with a G6410B triple quadrupole mass spectrometer (Agilent Technologies). A CAPCELL PAK C18 column (100 × 2.1 mm I.D., 3 μm particle size, SHISEIDO) was used for UHPLC separation of mononucleotides. UHPLC separation parameters were used as follows: 0–6.5 min, 5.0% B; 6.6–11.0 min, 20.0% B; 11.1–16.0 min, 100% B; 16.1–24 min, 5.0% B. Solvent A was an aqueous solution of 0.1% formic acid, and solvent B was 100% methanol. The mass spectrometer was operated in the positive ion mode. A multiple reaction monitoring (MRM) mode was adopted, using m/z 282→150 for m⁶A (collision energy, 15 eV) and m/z 268→136 for A (10 eV). The injection volume for each sample was 5 μL, and the amount of m⁶A and A was calibrated by standard curves. Nitrogen was used for nebulizing and desolvation gas of MS detection. The nebulization gas was set at 40 psi, the flow rate of desolvation gas was 9 L/min, and the source temperature was set at 300 °C. Capillary voltage was set at 3,500 V. High purity nitrogen (99.999%) was used as collision gas. Each sample was analyzed at least three times.

Methylated RNA immunoprecipitation

Methylated RNA immunoprecipitation (MeRIP) was performed as previously described [69]. Briefly, purified mRNA was randomly fragmented to size around 100 nucleotides using Ambion RNA fragmentation reagents and then subjected to immunoprecipitation (IP) with anti-m⁶A antibody (202003, Synaptic Systems) and protein-A magnetic beads (88845, Pierce) in MeRIP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40) supplemented with RNase inhibitor. m⁶A-containing mRNA fragments were eluted with m⁶A in MeRIP buffer and were purified using TRIzol reagent. For MeRIP-seq, two sets of samples were collected for duplicated biological repeats. For MeRIP-qRT-PCR, the same procedures were carried out except that the purified mRNA was fragmented into about 200 nucleotides; three biological repeats were conducted.

RNA-seq and m⁶A-seq

Poly (A) RNA from 1 mg total RNA or purified mRNA and purified m⁶A-containing fragments were used to generate the cDNA libraries, respectively, according to TruSeq RNA Sample Prep Kit protocol. All samples were sequenced by Illumina HiSeq-3000 with paired-end 101-bp read length. About 100 million and 150 million 2*101 paired-end reads in cKO and Ctrl mice were obtained, respectively.

Bioinformatic analysis

Raw RNA-seq reads were mapped to the mouse reference genome (mm10) using HISAT2 (Version 2.0.4) with parameters “—novel-splicesite-infile” [70]. StringTie (Version 1.3.1c) was used to construct full-length transcript assembly and estimate transcript abundance with default parameters [71]. Transcripts were filtered by TACO (Version 0.5.1) [72] with default parameters. Differentially expressed genes between the Ctrl and cKO mice at P7 and P14 were identified using edgeR (Version 3.10.0) [73] with fold change ≥ 1.5 and adjusted p-value ≤ 0.1 (Benjamini–Hochberg multiple testing correction) as the thresholds.

To identify decreased or increased m⁶A peaks in Ctrl and cKO m⁶A-seq against RNA-seq, two replicates for each group were used for differential peak analysis. The sequences were normalized according to total library size prior to peak calling (see S6 Table for normalization factors). A peak calling prioritization pipeline (PePr, version 1.1.18) was used to identify differential binding sites of m⁶A caused by Mettl3 conditional knockout [74]. A 200-bp sliding window approach and intergroup normalization were performed for m⁶A peak calling after removing PCR duplication, and a p-value threshold of 1e-5 (Wald test) was reported. The m⁶A peaks were annotated by bedtools (version 2.25.0) [75]. The enriched sequence motifs among m⁶A peaks were identified by HOMER (version 4.7) [76] with motif lengths of 5 nt, 6 nt, 7 nt, and 8 nt, respectively.

Gene Ontology biological processes and Reactome gene sets enrichment analysis were carried out using Metascape with p-value < 0.01 (Banjamini–Hochberg multiple testing correction), with the expressed genes (FPKM > 0.1) in Ctrl mouse cerebellums as the background [77]. For the enriched terms, Kappa scores were used as the similarity metric when performing hierarchical clustering, and then subtrees with similarity > 0.3 were considered as a cluster [77]. Enriched terms were selected according to the Metascape cluster results.

To analyze alternative splicing, the PSI method provided by Schafer and colleagues was used [78]. Briefly, annotations on an exon sequence were created through DEXseq (Version 1.20.2) [79], based on assembled transcripts. The PSI ratios for all exon parts were estimated by modifying the code provided by Schafer and colleagues [77]. PSI comparison supported by at least 10 inclusion and exclusion reads was considered for further analysis. Exons with ΔPSI ≥ 10% in Ctrl mice compared to cKO mice were considered as ASEs. ASEs overlapped with m⁶A-modified genes in Ctrl were further analyzed to uncover the target exons and their splicing pattern affected by METTL3.

Cell cultures

For establishment of the neural stem cell (NSC) lines, neonatal mouse brains were cut into small pieces by ophthalmic scissors and digested with 0.25% Typsin-EDTA (Gibco) for 15 min in 37 °C, 5% CO₂. The dissociated cell suspension was plated on Matrigel (BD Biosciences) coated 6-well plates. The basal culture medium contained Neurobasal Medium (Gibco) and DMEM/F12 Medium (Gibco) at a ratio of 1:1, plus 0.5% N-2 Supplement (Gibco), 1% B-27 Serum-Free Supplement (Gibco), 2 mM GlutaMAX-I (Gibco), bFGF (20 ng/mL, R & D systems), and EGF (20 ng/mL, R & D systems). For immunofluorescent staining, NSCs were plated on coverslips coated with poly-L-ornithine (Sigma) and Matrigel (BD Biosciences), successively. HeLa cells were cultured in 37 °C, 5% CO₂ in DMEM Medium (Gibco) plus 10% FBS (Gibco), 2 mM GlutaMAX-I (Gibco), and 0.01 mM beta-mercapto-thanol (beta-ME, Gibco).

CGCs were isolated from P7–P8 mouse pups as previously described [80]. To be brief, cerebellums were dissected and washed by HBSS (Gibco) and dissociated with 0.25% Typsin-EDTA (Gibco). Cells were suspended and subjected to Percoll (GE Healthcare) gradient (30% and 65% Percoll) separation. The astroglia and other heavier cells were separated by pre-plating on a poly-D-lysine (Sigma) coated dish for 20 min in a 5% CO₂, 37 °C incubator. Purified CGCs were plated on poly-D-lysine coated plates and cultured in Neurobasal Medium (Gibco) containing 2% B-27 Serum-Free Supplement (Gibco), 2 mM GlutaMAX-I (Gibco), and 15 μM glutamate (Gibco) in a 5% CO₂, 37 °C incubator. After 20 h, 10 μM Cytosine arabinoside (Ara-C, Sigma) was added to arrest the proliferation of nonneuronal cells.

Mouse CGC electroporation

The plasmids Pmax-mCherry, Pmax-Grin1₀₀₁-T2A-mCherry, or Pmax-Grin1₀₁₁-T2A-mCherry were constructed using the Pmax-GFP backbone (Lonza). Approximately 5 × 10⁶ mouse CGCs were resuspended in 100 μL P3 Solution from the Lonza Nucleofector P3 Primary Cell 4D X Kit (Lonza). Additionally, 10 μg of the appropriate plasmid was added to the cell suspension. The cell suspension was then placed in the cuvette and electroporated using a Lonza Nucleofector 4D device (Lonza) under the presupposed program CD150. Cells were then resuspended and cultured in CGC culture medium.

Calcium imaging and measurement

Electroporated CGCs were plated on a 20 mm confocal dish (Nest) precoated with poly-D-lysine. Culture medium of 3 DIV neurons was replaced by 2 mL of Locke’s solution containing 154 mM NaCl, 5 mM KCl, 3.6 mM NaHCO₃, 5 mM HEPES, 1.5 mM CaCl₂, 1.2 mM MgCl₂, and 5.6 mM glucose, pH 7.4, and incubated for 10 min at room temperature. Cells were subsequently incubated for 20 min in 2 mL of Locke’s solution containing 10 pM Fluo-4 (Thermo) and then washed with 2 mL of Locke’s solution for 10 min before starting the experiment. All measurements were made at room temperature (25 °C). Fluo-4 (green) and mCherry (indicating the expression of appropriate plasmid, red) fluorescence were imaged with an inverted Perkin-Elmer microscope. Images were digitized in an image processor connected to a computer equipped with VOLOCITY (version 6.0) software. The formula [Ca²⁺]_i = Kd_Fluo-4 × [F − F_min/ F_max − F] [81] was used to calculate the intracellular calcium concentration of mouse CGCs. F_max and F_min were obtained by perfusing cells with a salt solution containing 10 mM CaCl₂, and subsequently with a Ca²⁺-free salt solution containing 10 mM EGTA (Calcium Calibration Buffer Kit, Thermo).

CCK8 assay

Electroporated CGCs were seeded on 48 multi-wells (Corning) coated with poly-D-lysine. At DIV 1, DIV 3, and DIV 5, cells were incubated with CGC culture medium containing 1 × CCK8 (Solarbio) for 4 h in a 5% CO₂, 37 °C incubator. The absorbance was measured using a scanning multi-well spectrophotometer (ELISA reader) at a wavelength of 450 nm. The survival rates of CGCs were calculated as OD450_DIV3/OD450_DIV1 × 100% or OD450_DIV5/OD450_DIV1 × 100%.

RNA half-life detection

NSCs were plated on 6-well plates, 5 × 10⁵ per well, for 2 d, then cells were treated with actinomycin-D (10 μg/mL, Sigma) and collected for qRT-PCR analysis 3 h, 6 h, 9 h, or 12 h later. The half-life was calculated according to the following equation: ln (C_i/C₀) = −kt_i, where k is degradation rate, C_i is the mRNA value at time i, and t_i is the time interval in hours. Three repeats were conducted for each calculation.

qRT-PCR and semiquantitative RT-PCR

In the qRT-PCR experiments, 1 μg of total RNA treated with DNase I was reversely transcribed into cDNA by the Reverse Transcription System (Promega). SYBR Green PCR Master Mix (Toyobo) was used in all qRT-PCR experiments. The relative fold expression changes of genes were calculated using the 2^-ΔΔCt method, with Actb as internal control. Semiquantitative RT-PCR products were amplified for 30 cycles. Primers used in these experiments are listed in S7 Table.

MiniGene splicing assay

For miniGene-based splicing analysis of exon 19 in Lrp8 and exon 21 in Grin1, a fragment containing either wild-type (miniGene) or mutated m⁶A motif (miniGene-M) was inserted into the pSpliceExpress reporter vector (Addgene). For Lrp8, two m⁶A motifs (GAACC) were mutated to GATCC. For Grin1, one m⁶A motif (AGACA) was mutated to AGTCA. MiniGene or miniGene-M was transfected into HeLa cells with control siRNA (siControl) or siRNAs for knockdown of Mettl3 (siMettl3) using Lipofectamine 3000 (Invitrogen), respectively. Forty-eight hours after transfection, total RNA was isolated and reverse transcription was carried out to produce the cDNA. Then, cDNA was used as template for alternative splicing change detection through semiquantitative RT-PCR. AlphaView was used to analyze the density of bands on agar gels. Ratios of exon exclusion were quantified based on the density of bands. All the primers and siRNAs used for this assay are listed in S8 Table.

Statistical analysis

All statistical analyses (unless stated otherwise) were performed using the R package for Statistical Computing. For experimental data quantification, Student t test was applied using GraphPad Prism 6 software, and the error bar was shown based on SEM (unless stated otherwise). P value < 0.05 was considered statistically significant.