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Proper development of the mammalian cerebral cortex relies on precise gene expression regulation, which is controlled by genetic, epigenetic, and epitranscriptomic factors. Here we generate RNA demethylase Fto and methyltransferase Mettl3 cortical-specific conditional knockout mice, and detect severe brain defects caused by Mettl3 deletion but not Fto knockout. Transcriptomic profiles using RNA sequencing indicate that knockout of Mettl3 causes a more dramatic alteration on gene transcription than that of Fto. Interestingly, we conduct ribosome profiling sequencing, and find that knockout of Mettl3 leads to a more severe disruption of translational regulation of mRNAs than deletion of Fto and results in altered translation of crucial genes in cortical radial glial cells and intermediate progenitors. Moreover, Mettl3 deletion causes elevated translation of a significant number of mRNAs, in particular major components in m6A methylation. Our findings indicate distinct functions of Mettl3 and Fto in brain development, and uncover a profound role of Mettl3 in regulating translation of major mRNAs that control proper cortical development.

In this study, we take advantage of human induced pluripotent stem (iPS) cell-derived neural stem cells and brain organoids to study the role of p53 during human brain development. We knocked down (KD) p53 in human neuroepithelial stem (NES) cells derived from iPS cells. Upon p53KD, NES cells rapidly show centrosome amplification and genomic instability. Furthermore, a reduced proliferation rate, downregulation of genes involved in oxidative phosphorylation (OXPHOS), and an upregulation of glycolytic capacity was apparent upon loss of p53. In addition, p53KD neural stem cells display an increased pace of differentiating into neurons and exhibit a phenotype corresponding to more mature neurons compared to control neurons. Using brain organoids, we modeled more specifically cortical neurogenesis. Here we found that p53 loss resulted in brain organoids with disorganized stem cell layer and reduced cortical progenitor cells and neurons. Similar to NES cells, neural progenitors isolated from brain organoids also show a downregulation in several OXPHOS genes. Taken together, this demonstrates an important role for p53 in controlling genomic stability of neural stem cells and regulation of neuronal differentiation, as well as maintaining structural organization and proper metabolic gene profile of neural progenitors in human brain organoids.

Retinoic acid (RA), an active derivative of vitamin A, is critical for the neural system development. During the neural development, the RA/RA receptor (RAR) pathway suppresses BMP signaling-mediated proliferation and differentiation of neural progenitor cells. However, how the stability of RAR is regulated during neural system development and how BMP pathway genes expression in neural tissue from human fetuses affected with neural tube defects (NTDs) remain elusive. Here, we report that FBXO30 acts as an E3 ubiquitin ligase and targets RARγ for ubiquitination and proteasomal degradation. In this way, FBXO30 positively regulates BMP signaling in mammalian cells. Moreover, RA treatment leads to suppression of BMP signaling by reducing the level of FBXO30 in mammalian cells and in mouse embryos with NTDs. In samples from human NTDs with high levels of retinol, downregulation of BMP target genes was observed, along with aberrant FBXO30 levels. Collectively, our results demonstrate that RARγ levels are controlled by FBXO30-mediated ubiquitination and that FBXO30 is a key regulator of BMP signaling. Furthermore, we suggest a novel mechanism by which high-retinol levels affect the level of FBXO30, which antagonizes BMP signaling during early stage development.