Yuping Luo

Single-Cell Transcriptome Analyses Reveal Signals to Activate Dormant Neural Stem Cells

The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

Identification and functional analysis of long non-coding RNAs in mouse cleavage stage embryonic development based on single cell transcriptome data

BackgroundLong non-coding RNAs (lncRNAs) regulate embryonic development and cell fate decision in various ways, such as modulation of chromatin modification and post-transcription regulation of gene expression. However, the profiles and roles of lncRNAs in early mammalian development have not yet been demonstrated. Here, we reported a comprehensive analysis of mouse cleavage stage embryonic lncRNA profiles based on public single-cell RNA-seq data.ResultsWe reconstructed 50,006 high-confidence transcripts in 22,827 loci, and identified 5563 novel lncRNAs from 3492 loci expressed in mouse cleavage stage embryos. These lncRNAs share similar characteristics with previously reported vertebrate lncRNAs, such as relatively short length, low exon number, low expression level and low sequence conservation. Expression profile analysis revealed that the profiles of lncRNA vary considerably at different stages of cleavage stage embryos, suggesting that many lncRNAs in cleavage stage embryos are stage-specifically expressed. Co-expression network analysis suggested many lncRNAs in cleavage stage embryos are associated with cell cycle regulation, transcription, translation and oxidative phosphorylation to regulate the process of cleavage stage embryonic development.ConclusionsThis study provides the first catalog of lncRNAs expressed in mouse cleavage stage embryos and gives a revealing insight into the molecular mechanism responsible for early embryonic development.

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Identification of miRNA Signatures during the Differentiation of hESCs into Retinal Pigment Epithelial Cells

Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.

Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling.

Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis. We have previously reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in ischemic stroke. However, little is known about long non-coding RNAs and theirs role in angiogenesis after stroke. In this study, we identified a rat lncRNAs, Meg3, and found that Meg3 was significantly decreased after ischemic stroke. Overexpression of Meg3 suppressed functional recovery and decreased capillary density after ischemic stroke. Downregulation of Meg3 ameliorated brain lesion and increased angiogenesis after ischemic stroke. Silencing of Meg3 resulted in a proangiogenic effect evidenced by increased endothelial cell migration, proliferation, sprouting, and tube formation. Mechanistically, we showed that Meg3 negatively regulated notch pathway both in vivo and in vitro. Inhibition of notch signaling in endothelial cells reversed the proangiogenic effect induced by Meg3 downregulation. This study revealed the function of Meg3 in ischemic stroke and elucidated its mechanism in angiogenesis after ischemic stroke.

Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

ABSTRACTThe mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.

Splicing-Related Features of Introns Serve to Propel Evolution

The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to introns ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.

Generation of Human Embryonic Stem Cell Line Expressing zsGreen in Cholinergic Neurons Using CRISPR/Cas9 System.

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation, physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation, cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons.

Systematic Analysis of RNA Regulatory Network in Rat Brain after Ischemic Stroke

Although extensive studies have identified large number of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in ischemic stroke, the RNA regulation network response to focal ischemia remains poorly understood. In this study, we simultaneously interrogate the expression profiles of lncRNAs, miRNAs, and mRNAs changes during focal ischemia induced by transient middle cerebral artery occlusion. A set of 1924 novel lncRNAs were identified and may involve brain injury and DNA repair as revealed by coexpression network analysis. Furthermore, many short interspersed elements (SINE) mediated lncRNA:mRNA duplexes were identified, implying that lncRNAs mediate Staufen1-mediated mRNA decay (SMD) which may play a role during focal ischemia. Moreover, based on the competitive endogenous RNA (ceRNA) hypothesis, a stroke regulatory ceRNA network which reveals functional lncRNA:miRNA:mRNA interactions was revealed in ischemic stroke. In brief, this work reports a large number of novel lncRNAs responding to focal ischemia and constructs a systematic RNA regulation network which highlighted the role of ncRNAs in ischemic stroke.

The Nonlinear Stability of Semi-Thin Spherical Shells Under Uniformly Symmetric Circular Line Loads

Publisher Summary This chapter examines the nonlinear stability of semithin spherical shells under uniformly symmetric circular line loads. The loading capacity and buckling behavior of thin spherical joints under uniformly symmetric circular line loads are analyzed theoretically by means of nonlinear buckling theory and nonlinear numerical analyzing methods. A numerical method for calculating the loading behavior of the thin spherical joints is developed. The numerical method is practical for the joints design, especially for thin spherical joints with large diameters for which the present design code is not suitable. According to nonlinear theory of thin shells, buckling differential equations of the shell with large deflections can be expressed in polar coordinates. The uniformly symmetrical circular line load is a concentrated line load uniformly distributed on the shell surface. The discontinuity of the shear will occur in the shell at the loading line because of the concentrated line load. It makes the resolution of the buckling equations much difficult, even impossible sometimes. In modem space frame and tress structures, the welded thin spherical joints with larger diameters are often stiffened with inner ribs or rings. The loads and the shell joint structures are both symmetric for a single-direction loaded model.