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Glioblastoma is a devastating form of brain cancer. To identify aspects of tumor heterogeneity that may illuminate drivers of tumor invasion, we created a glioblastoma tumor cell atlas with single-cell transcriptomics of cancer cells mapped onto a reference framework of the developing and adult human brain. We find that multiple GSC subtypes exist within a single tumor. Within these GSCs, we identify an invasive cell population similar to outer radial glia (oRG), a fetal cell type that expands the stem cell niche in normal human cortex. Using live time-lapse imaging of primary resected tumors, we discover that tumor-derived oRG-like cells undergo characteristic mitotic somal translocation behavior previously only observed in human development, suggesting a reactivation of developmental programs. In addition, we show that PTPRZ1 mediates both mitotic somal translocation and glioblastoma tumor invasion. These data suggest that the presence of heterogeneous GSCs may underlie glioblastoma's rapid progression and invasion.

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

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

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 in vivo, 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 in vivo. 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.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

While thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known. Here, we identify Pinky (Pnky) as a neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain. In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Pnky interacts with the splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates neurogenesis from embryonic and postnatal NSC populations.

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.