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While it is now appreciated that certain long noncoding RNAs (lncRNAs) have important functions in cell biology, relatively few have been shown to regulate development invivo, particularly with genetic strategies that establish cis versus trans mechanisms. Pnky is a nuclear-enriched lncRNA that is transcribed divergently from the neighboring proneural transcription factor Pou3f2. Here, we show that conditional deletion of Pnky from the developing cortex regulates the production of projection neurons from neural stem cells (NSCs) in a cell-autonomous manner, altering postnatal cortical lamination. Surprisingly, Pou3f2 expression is not disrupted by deletion of the entire Pnky gene. Moreover, expression of Pnky from a BAC transgene rescues the differential gene expression and increased neurogenesis of Pnky-knockout NSCs, as well as the developmental phenotypes of Pnky-deletion invivo. Thus, despite being transcribed divergently from a key developmental transcription factor, the lncRNA Pnky regulates development in trans.

Systematic analyses of spatiotemporal gene expression trajectories during organogenesis have been challenging because diverse cell types at different stages of maturation and differentiation coexist in the emerging tissues. We identified discrete cell types as well as temporally and spatially restricted trajectories of radial glia maturation and neurogenesis in developing human telencephalon. These lineage-specific trajectories reveal the expression of neurogenic transcription factors in early radial glia and enriched activation of mammalian target of rapamycin signaling in outer radial glia. Across cortical areas, modest transcriptional differences among radial glia cascade into robust typological distinctions among maturing neurons. Together, our results support a mixed model of topographical, typological, and temporal hierarchies governing cell-type diversity in the developing human telencephalon, including distinct excitatory lineages emerging in rostral and caudal cerebral cortex.

The human genome produces tens of thousands of long non-coding RNA (lncRNAs), transcripts larger than 200 nt that do not make proteins. Select lncRNAs are established regulators of gene expression in developmental processes and in the etiology of diseases. However, the functions of the vast majority of lncRNAs are unknown. Here we utilize high throughput descriptive and functional genomics to characterize and understand the roles of lncRNAs in development of the human brain and in cancer biology. Starting with deep RNA sequencing (RNA-seq) of developing brain tissues, we comprehensively annotated lncRNA transcript structures in the cerebral cortex, revealing thousands of novel lncRNAs, many of which were antisense or non-polyadenylated. We then applied single cell RNA-seq to quantitatively measure the transcriptomes of individual cells, revealing cell type-specific expression of lncRNAs and surprisingly high abundance of certain lncRNAs (e.g. DLX6-AS1) that appear to be low abundance at the population level, due to cellular heterogeneity in the brain. Combination of single cell RNA-seq and exome-seq were then applied to not only measure RNA expression heterogeneity, but also to map the mutational phylogenies of glioblastoma tumors, revealing a novel deletion in PDGFRA that confers survival advantage. We then developed a CRISPR interference (CRISPRi) platform to systematically repress 16,401 lncRNA loci across seven cancer and non-cancer cell lines, identifying 499 lncRNAs necessary for robust growth and proliferation. The functions of these lncRNAs were exquisitely cell type-specific and correlated with complex transcriptional networks and chromatin architecture. Finally, we applied CRISPRi screening of lncRNAs in combination with ionizing radiation therapy and identified over 200 lncRNAs whose knockdown sensitizes glioblastoma cells to radiation induced cell death. As much as these efforts advance our knowledge of lncRNA biology in development and in disease, they also raise exciting new questions for future inquiry.

The epigenetic mechanisms that enable lifelong neurogenesis from neural stem cells (NSCs) in the adult mammalian brain are poorly understood. Here we show that JMJD3, a histone H3-lysine 27 (H3K27) demethylase, acts as a critical activator of neurogenesis from adult subventricular zone (SVZ) NSCs. JMJD3 is upregulated in neuroblasts, and Jmjd3-deletion targeted to SVZ NSCs in both developing and adult mice impairs neuronal differentiation. JMJD3 regulates neurogenic gene expression via interaction at not only promoter regions, but also neurogenic enhancer elements. JMJD3 localizes at neural enhancers genome-wide in embryonic brain, and in SVZ NSCs, JMJD3 regulates the I12b enhancer of Dlx2. In Jmjd3-deleted SVZ cells, I12b remains enriched with H3K27me3, and Dlx2-dependent neurogenesis fails. These findings support a model in which JMJD3 and the poised state of key transcriptional regulatory elements comprise an epigenetic mechanism that enables the activation of neurogenic gene expression in adult NSCs throughout life.