Molecular identity of human outer radial glia during cortical development.

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.

Pluripotency factors in embryonic stem cells regulate differentiation into germ layers.

Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells.

miRNA-independent function of long noncoding pri-miRNA loci.

Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri-miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (i.e., GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (also known as MIR29HG), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster (LOC646329-miR29Δ) did not decrease cell growth, while knockdown of LOC646329-miR29Δ transcripts reduced cell proliferation. The miR-29a/b1-independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene (MKLN1), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer-like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms.

Single-cell sequencing maps gene expression to mutational phylogenies in PDGF- and EGF-driven gliomas.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However, clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy, in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type, expressing pro-angiogenic factors. In contrast, phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data.

Logic integration of mRNA signals by an RNAi-based molecular computer.

Synthetic in vivo molecular 'computers' could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that 'transduce' mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi 'computational' module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting.

Outer Radial Glia-like Cancer Stem Cells Contribute to Heterogeneity of Glioblastoma.

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 Long Noncoding RNA Pnky Is a Trans-acting Regulator of Cortical Development In Vivo.

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.

Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex.

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.

Single-cell analysis of long non-coding RNAs in the developing human neocortex.

BACKGROUND: Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that can regulate molecular and cellular processes in brain development and disease. LncRNAs exhibit cell type- and tissue-specific expression, but little is known about the expression and function of lncRNAs in the developing human brain. Furthermore, it has been unclear whether lncRNAs are highly expressed in subsets of cells within tissues, despite appearing lowly expressed in bulk populations. RESULTS: We use strand-specific RNA-seq to deeply profile lncRNAs from polyadenylated and total RNA obtained from human neocortex at different stages of development, and we apply this reference to analyze the transcriptomes of single cells. While lncRNAs are generally detected at low levels in bulk tissues, single-cell transcriptomics of hundreds of neocortex cells reveal that many lncRNAs are abundantly expressed in individual cells and are cell type-specific. Notably, LOC646329 is a lncRNA enriched in single radial glia cells but is detected at low abundance in tissues. CRISPRi knockdown of LOC646329 indicates that this lncRNA regulates cell proliferation. CONCLUSION: The discrete and abundant expression of lncRNAs among individual cells has important implications for both their biological function and utility for distinguishing neural cell types.

The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells.

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.

Activation of neuronal gene expression by the JMJD3 demethylase is required for postnatal and adult brain neurogenesis.

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.