Ythdf2-mediated m6A mRNA clearance modulates neural development in mice

Abstract

Background: N ⁶ -methyladenosine (m⁶A) modification in mRNAs was recently shown to be dynamically regulated, indicating a pivotal role in multiple developmental processes. Most recently, it was shown that the Mettl3-Mettl14 writer complex of this mark is required for the temporal control of cortical neurogenesis. The m⁶A reader protein Ythdf2 promotes mRNA degradation by recognizing m⁶A and recruiting the mRNA decay machinery.

Results: We show that the conditional depletion of the m⁶A reader protein Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development. We demonstrate that neural stem/progenitor cell (NSPC) self-renewal and spatiotemporal generation of neurons and other cell types are severely impacted by the loss of Ythdf2 in embryonic neocortex. Combining in vivo and in vitro assays, we show that the proliferation and differentiation capabilities of NSPCs decrease significantly in Ythdf2 ^-/- embryos. The Ythdf2 ^-/- neurons are unable to produce normally functioning neurites, leading to failure in recovery upon reactive oxygen species stimulation. Consistently, expression of genes enriched in neural development pathways is significantly disturbed. Detailed analysis of the m⁶A-methylomes of Ythdf2 ^-/- NSPCs identifies that the JAK-STAT cascade inhibitory genes contribute to neuroprotection and neurite outgrowths show increased expression and m⁶A enrichment. In agreement with the function of Ythdf2, delayed degradation of neuron differentiation-related m⁶A-containing mRNAs is seen in Ythdf2 ^-/- NSPCs.

Conclusions: We show that the m⁶A reader protein Ythdf2 modulates neural development by promoting m⁶A-dependent degradation of neural development-related mRNA targets.

Keywords: N 6 -methyladenosine (m6A); Neural development; Neurogenesis; Ythdf2; mRNA clearance.

Fig. 1

Ythdf2^−/− mice are embryonic lethal. a The gene-targeting strategy to disrupt the Ythdf2 gene in mouse. Conditional Ythdf2 gene-targeted mouse contains LoxP sites flanking the 5′ UTR and exon 1 of the endogenous Ythdf2 locus. WT_F wild-type forward primer, WT_R wild-type reverse primer, KO_F Ythdf2^−/− forward primer, WT_R Ythdf2^−/− reverse primer, Ex exon. b Numbers of offspring from heterozygous Ythdf2^+/− intercrosses. The number and genotype of embryos at E12.5/E14.5 and postnatal are indicated. c PCR analysis of embryo tail DNA showing a 271-bp wild-type band (WT) and a 550-bp targeted band (KO) with primers displayed in a. d Western blot analysis of the Ythdf2 expression in wild-type and Ythdf2^−/− embryos. Two samples for each genotype. Actin was used as loading control. e Numbers of embryos per litter at E12.5/E14.5 and E18.5 from wild-type or heterozygous intercrosses. Error bars represent mean ± standard deviation, n = 7 litters. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test

Fig. 2

Ythdf2 is required for normal embryonic cortical development. a Sagittal brain sections of E12.5 and E14.5 were stained with H&E. An enlarged view of the forebrain cortex is shown. Scale bar indicates 20 μm. b Thickness of the cortical layer in Ythdf2^+/−, Ythdf2^−/−, and their wild-type littermates at E12.5 and E14.5. Error bars represent mean ± standard deviation, n = 3 embryos and 3 technical replicates. c Immunostaining of E12.5 and E14.5 brain sagittal sections for Dcx in wild-type, Ythdf2^+/−, and Ythdf2^−/− littermates. Nuclei were counterstained with DAPI. VZ ventricular zone, SVZ subventricular zone, IZ intermediate zone, CP cortical plate. d Ratio of the thickness of Dcx-immunolabeled neuronal layers over cortical layers in Ythdf2^+/− and Ythdf2^−/− compared with wild type. Error bars represent mean ± standard deviation, n = 3 embryos and 3 technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test

Fig. 3

The number of basal progenitors and mitotic capability of apical progenitors depends on Ythdf2. a Immunostaining of E12.5 and E14.5 sagittal sections with Tbr2 (green) and Sox2 (red) antibodies in wild type, Ythdf2^+/−, and Ythdf2^−/− embryos. VZ ventricular zone, SVZ subventricular zone, IZ intermediate zone, CP cortical plate. Nuclei were counterstained with DAPI. b Percentage of Tbr2⁺ cells over Tbr2⁺/Sox2⁺ at E12.5 and E14.5. Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. Scale bars, 20 μm. c Immunostaining of E12.5 and E14.5 sagittal sections with Phh3 (green) and Sox2 (red) antibodies in wild type, Ythdf2^+/−, and Ythdf2^−/−embryos. Nuclei were counterstained with DAPI. d Number of Phh3⁺ cells per 400 μm of the cortical wall at E12.5/E14.5 from c. Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. Scale bars, 20 μm

Fig. 4

Ythdf2^−/− NSPCs exhibit decreased proliferation and defects in natural differentiation in vitro. a Number of viable NSPCs at 0, 24, 72, and 120 h monitored by signal intensity of Presto Blue reagent. Proliferation rate was calculated by normalizing to wild type at 0 h. b mRNA expression levels of Ythdf2 during NSPC differentiation. Cells were collected at differentiation Day 0 (D0), 3, and 5. Isolated total RNAs were applied for RT-qPCR analysis. Actin was used as normalization control. c Immunostaining of Map2⁺ and Gfap⁺ cells differentiated from E14.5 neurospheres at D5 and D7. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. d Percentage of Map2 or Gfap positive cells. Error bars represent mean ± standard deviation, n = 3 biological repeats and 3 technical replicates. e Mean number of primary neurites per neuron (Map2⁺). n = 20 neurons for each biological repeat. f Mean length of the longest neurite of neurons (Map2⁺). n = 20 neurons for each biological repeat. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test

Fig. 5

Overview of m⁶A methylomes in wild-type and Ythdf2^−/− neurospheres. a The m⁶A contents of mRNAs isolated from wild type and Ythdf2^−/− were quantified by LC-MS/MS. b Sequencing motif in m⁶A peaks verified in wild type and Ythdf2^−/− with HOMER database. c Distribution of m⁶A peaks along transcripts in wild type and Ythdf2^−/−. d Scatter plot showing m⁶A peaks with increased (red) or decreased (green) levels. e Representative m⁶A distribution along Nrp2 transcript. Enrichment coverage of m⁶A and input are displayed as red and blue, respectively. Grey lines define coding sequence (CDS) borders

Fig. 6

Ythdf2 is required for regulating mRNA decay of m⁶A-modified neuron-related gene targets. a m⁶A enrichment of target sites in gene candidates, verified by m⁶A IP combined with RT-qPCR. Non-m⁶A-modified gene Actin was used as negative control. b Gene expression of gene candidates, verified by RT-qPCR with input RNA. Non-changed gene Actin was used as negative control. c Ythdf2 binding levels of gene candidates, verified by Ythdf2 RIP combined with RT-qPCR. Non-m⁶A-modified gene Actin was used as negative control. d Representative mRNA profile of Nrp2 at 0-, 2-, and 4-h time points after actinomycin D (5 μg/ml) treatment (h.p.t.) in wild type and Ythdf2^−/−. Error bars represent mean ± standard deviation, n = 2 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test