ABSTRACT
Autism spectrum disorder (ASD) is an umbrella term that encompasses several disabling neurodevelopmental conditions. These conditions are characterized by impaired manifestation in social and communication skills with repetitive and restrictive behaviors or interests. Thus far, there are no approved biomarkers for ASD screening and diagnosis; also, the current diagnosis depends heavily on a physician's assessment and family's awareness of ASD symptoms. Identifying blood proteomic biomarkers and performing deep blood proteome profiling could highlight common underlying dysfunctions between cases of ASD, given its heterogeneous nature, thus laying the foundation for large-scale blood-based biomarker discovery studies. This study measured the expression of 1196 serum proteins using proximity extension assay (PEA) technology. The screened serum samples included ASD cases (n = 91) and healthy controls (n = 30) between 6 and 15 years of age. Our findings revealed 251 differentially expressed proteins between ASD and healthy controls, of which 237 proteins were significantly upregulated and 14 proteins were significantly downregulated. Machine learning analysis identified 15 proteins that could be biomarkers for ASD with an area under the curve (AUC) = 0.876 using support vector machine (SVM). Gene Ontology (GO) analysis of the top differentially expressed proteins (TopDE) and weighted gene co-expression analysis (WGCNA) revealed dysregulation of SNARE vesicular transport and ErbB pathways in ASD cases. Furthermore, correlation analysis showed that proteins from those pathways correlate with ASD severity. Further validation and verification of the identified biomarkers and pathways are warranted.
Subject(s)
Autism Spectrum Disorder , Humans , Autism Spectrum Disorder/genetics , Pilot Projects , Proteomics , Biomarkers/metabolism , Proteome/metabolismABSTRACT
Although the application of human mesenchymal stem cells (hMSCs) to repair damaged or diseased tissues has proven relatively effective, both the donor-to-donor variability in ex vivo expansion rates and the maintenance of stemness remain a bottleneck to widespread translation. Previous work from this laboratory stratified donors into those yielding hMSCs with high- or low-growth capacity; global transcriptomic analysis revealed that high-growth-capacity hMSCs were characterized by a loss of the gene encoding glutathione S-transferase theta 1 (GSTT1). These GSTT1-null hMSCs demonstrated increased proliferative rates, clonogenic potential, and longer telomeres compared with low-growth capacity hMSCs that were GSTT1-positive. Thus, this study identifies GSTT1 as a novel genomic DNA biomarker for hMSC scalability.
Subject(s)
Biomarkers/metabolism , Bone Marrow Cells/cytology , Genome, Human , Mesenchymal Stem Cells/cytology , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Proliferation/genetics , Clone Cells , Genotype , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Homozygote , Humans , Mesenchymal Stem Cells/metabolism , Transcriptome/geneticsABSTRACT
Long noncoding RNAs (lncRNAs) are abundant in the mammalian transcriptome, and many are specifically expressed in the brain. We have identified a group of lncRNAs, including rhabdomyosarcoma 2-associated transcript (RMST), which are indispensable for neurogenesis. Here, we provide mechanistic insight into the role of human RMST in modulating neurogenesis. RMST expression is specific to the brain, regulated by the transcriptional repressor REST, and increases during neuronal differentiation, indicating a role in neurogenesis. RMST physically interacts with SOX2, a transcription factor known to regulate neural fate. RMST and SOX2 coregulate a large pool of downstream genes implicated in neurogenesis. Through RNA interference and genome-wide SOX2 binding studies, we found that RMST is required for the binding of SOX2 to promoter regions of neurogenic transcription factors. These results establish the role of RMST as a transcriptional coregulator of SOX2 and a key player in the regulation of neural stem cell fate.
Subject(s)
Nerve Tissue Proteins/metabolism , RNA, Long Noncoding/metabolism , Repressor Proteins/metabolism , SOXB1 Transcription Factors/metabolism , Alternative Splicing , Binding Sites , Cell Differentiation , Cell Line , Co-Repressor Proteins , DNA-Binding Proteins , Gene Expression Regulation, Developmental , Humans , Neural Stem Cells , Neurogenesis , Promoter Regions, Genetic , Protein Binding , RNA Interference , RNA, Long Noncoding/genetics , RNA, Small InterferingABSTRACT
The stochastic dynamics and regulatory mechanisms that govern differentiation of individual human neural precursor cells (NPC) into mature neurons are currently not fully understood. Here, we used single-cell RNA-sequencing (scRNA-seq) of developing neurons to dissect/identify NPC subtypes and critical developmental stages of alternative lineage specifications. This study comprises an unsupervised, high-resolution strategy for identifying cell developmental bifurcations, tracking the stochastic transcript kinetics of the subpopulations, elucidating regulatory networks, and finding key regulators. Our data revealed the bifurcation and developmental tracks of the two NPC subpopulations, and we captured an early (24 h) transition phase that leads to alternative neuronal specifications. The consequent up-regulation and down-regulation of stage- and subpopulation-specific gene groups during the course of maturation revealed biological insights with regard to key regulatory transcription factors and lincRNAs that control cellular programs in the identified neuronal subpopulations.
Subject(s)
Gene Expression Profiling/methods , Gene Regulatory Networks , Neural Stem Cells/cytology , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Cell Differentiation , Cell Lineage , Cells, Cultured , Gene Expression Regulation, Developmental , Humans , Neurogenesis , RNA, Long Noncoding/genetics , Transcription Factors/geneticsABSTRACT
Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA.
Subject(s)
Bulbo-Spinal Atrophy, X-Linked/metabolism , Bulbo-Spinal Atrophy, X-Linked/pathology , Intracellular Signaling Peptides and Proteins/physiology , Motor Neurons/metabolism , Motor Neurons/pathology , Neurites/metabolism , Neurites/pathology , Bulbo-Spinal Atrophy, X-Linked/genetics , Cell Differentiation , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Intracellular Signaling Peptides and Proteins/genetics , Phenotype , Signal TransductionABSTRACT
The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine.
Subject(s)
Autism Spectrum Disorder/genetics , Glycosyltransferases/genetics , Induced Pluripotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Animals , Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/pathology , Cell Differentiation/genetics , Central Nervous System/growth & development , Central Nervous System/pathology , Disease Models, Animal , Gene Expression Regulation, Developmental , Genome, Human , Glycosyltransferases/biosynthesis , High-Throughput Nucleotide Sequencing , Humans , Induced Pluripotent Stem Cells/pathology , Neural Stem Cells/pathology , Neurons/metabolism , Neurons/pathology , Precision Medicine , Zebrafish/genetics , Zebrafish/growth & developmentABSTRACT
Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs, including hsa-miR-135b. MiR-135b was activated during NE development, and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-ß and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Eye Proteins/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , MicroRNAs/genetics , Paired Box Transcription Factors/metabolism , Repressor Proteins/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism , Binding Sites , Bone Morphogenetic Proteins/genetics , Cell Differentiation , Cell Line , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Eye Proteins/genetics , Gene Expression Profiling , Homeodomain Proteins/genetics , Humans , MicroRNAs/metabolism , Models, Molecular , Mutation , Neural Plate , PAX6 Transcription Factor , Paired Box Transcription Factors/genetics , Repressor Proteins/genetics , Sequence Analysis, DNA , Transcription Factors/genetics , Transcription Factors/metabolism , Transforming Growth Factor beta/geneticsABSTRACT
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/metabolism , Gene Editing , Protein Engineering , RNA, Guide, Kinetoplastida , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/genetics , Endonucleases/metabolism , Genetic Variation , Humans , Models, Biological , Mutation , Pluripotent Stem Cells/metabolism , Protein Binding , Protein Multimerization , Regulatory Factor X Transcription Factors/chemistry , Regulatory Factor X Transcription Factors/geneticsABSTRACT
BACKGROUND: Development of primary open-angle glaucoma (POAG) is associated with the malfunctioning trabecular meshwork (TM). Cell therapy offers great potential for the treatment of POAG, but requires the generation of functional TM cells in vitro to replace the lost/dysfunctional cells. TM differentiation in vitro from various stem cell types must be monitored by the expression of specific markers. However, no single definitive marker of the TM has been identified. RESULTS: To identify robust markers of TM differentiation, we performed global transcriptome profiling using high-density oligonucleotide microarray on ex vivo TM tissue and cultured TM progenitors. Corneal and scleral tissues were also used in the analysis. After removal of genes expressed in the cornea and sclera, 18 genes were identified that were differentially expressed in the TM relative to the other samples. CDH23, F5, KCNAB1, FGF9, SPP1, and HEY1 were selected among the genes highly expressed in the TM, together with BDNF which was repressed, compared to progenitors for further investigation. Expression analysis by qPCR verified the differential expression and immunofluorescence of the anterior segment confirmed strong expression in the TM. CONCLUSIONS: Three independent cohort of expression studies have identified novel markers, fitting in identifying TM cells and in evaluating directed TM differentiation in vitro.
Subject(s)
Cell Differentiation/genetics , Gene Expression Profiling , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolism , Aged , Biomarkers/metabolism , Cornea/metabolism , Humans , Middle Aged , Sclera/metabolism , Stem Cells/cytologyABSTRACT
How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.
Subject(s)
Embryo, Mammalian/embryology , Endoderm/embryology , Enhancer Elements, Genetic/physiology , Gene Expression Regulation, Developmental/physiology , HMGB Proteins/metabolism , Octamer Transcription Factor-3/metabolism , SOXB1 Transcription Factors/metabolism , SOXF Transcription Factors/metabolism , Amino Acid Motifs , Animals , Binding Sites , Cell Differentiation/physiology , Cell Line , Embryo, Mammalian/cytology , Endoderm/cytology , HMGB Proteins/genetics , Mice , Octamer Transcription Factor-3/genetics , SOXB1 Transcription Factors/genetics , SOXF Transcription Factors/genetics , Transcription, Genetic/physiologyABSTRACT
The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.
Subject(s)
MicroRNAs/genetics , Motor Neurons/metabolism , Motor Neurons/pathology , Nerve Degeneration/pathology , Animals , Apoptosis/genetics , Base Sequence , Humans , Mice , MicroRNAs/metabolism , Molecular Sequence Data , Muscular Atrophy, Spinal/genetics , Nerve Degeneration/genetics , Neurogenesis/genetics , Signal Transduction/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE-1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we found that REST knockout cells lost the ability to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective downregulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. REST regulates cardiac differentiation of ESCs by negatively regulating the Wnt/ß-catenin signaling pathway and positively regulating the cardiogenic TF Gata4. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation.
Subject(s)
Cell Differentiation/genetics , GATA4 Transcription Factor/biosynthesis , Mouse Embryonic Stem Cells/metabolism , Repressor Proteins/genetics , Animals , Cell Lineage/genetics , GATA4 Transcription Factor/genetics , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Myocytes, Cardiac/metabolism , Wnt Signaling PathwayABSTRACT
The central nervous system (CNS) is a complex biological system composed of numerous cell types working in concert. The intricate development and functioning of this highly ordered structure depends upon exquisite spatial and temporal control of gene expression in the cells comprising the CNS. Thus, gene regulatory networks that control cell fates and functions play critical roles in the CNS. Failure to develop and maintain intricate regulatory networks properly leads to impaired development or neural dysfunction, which might manifest as neurological disorders. Long noncoding RNAs (lncRNAs) are emerging as important components of gene regulatory networks, working in concert with transcription factors and epigenetic regulators of gene expression. Interestingly, many lncRNAs are highly expressed in the adult and developing brain, often showing precise temporal and spatial patterns of expression. This specificity of expression and growing awareness of the importance of lncRNAs suggest that they play key roles in CNS development and function. In this review, we highlight the growing evidence for the importance of lncRNAs in the CNS and the indications that their dysregulation underlies some neurological disorders.
Subject(s)
Central Nervous System/physiopathology , RNA, Long Noncoding/genetics , Brain/growth & development , Brain/physiopathology , Central Nervous System/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Humans , Nervous System Diseases/genetics , Nervous System Diseases/physiopathology , Transcription, GeneticABSTRACT
Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome, which have been proposed to be key regulators of biological processes, including stem cell pluripotency and neurogenesis. However, at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study, we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method, hESCs were robustly and efficiently differentiated into neurons, and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified, and shown to physically interact with SOX2, and PRC2 complex component, SUZ12. Using a similar approach, we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis, and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis, and represents important evidence for an indispensable role of lncRNAs in human brain development.
Subject(s)
Cell Differentiation/genetics , Chromatin/metabolism , Neurons/cytology , RNA, Untranslated/physiology , Transcription Factors/metabolism , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Oligonucleotide Array Sequence Analysis , RNA, Untranslated/genetics , RNA, Untranslated/metabolismABSTRACT
The variant histone protein H2A.Z plays a critical role in early development. Likewise, Nanog, a master regulator of embryonic stem cells (ESCs), is essential for proper development in early embryogenesis. In this study, we establish that these two factors work together to maintain pluripotency. It is shown that H2A.Z influences the protein level of Nanog through the ubiquitin-proteasome pathway. Knockdown of H2A.Z causes differentiation of mouse ESCs and disrupts the reprogramming of somatic cells, which can be partially rescued by overexpression of Nanog. We conclude that the H2A.Z-Nanog partnership is involved in ESC pluripotency and reprogramming of somatic cells. Stem Cells 2015;33:2126-2134.
Subject(s)
Histones/metabolism , Homeodomain Proteins/metabolism , Animals , Cell Differentiation , Humans , Mice , Nanog Homeobox Protein , Pluripotent Stem Cells/metabolismABSTRACT
This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application.
Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation/physiology , Cell Proliferation/physiology , Mesenchymal Stem Cells/cytology , Animals , Cells, Cultured , Cytokines/metabolism , Humans , Mice , Wound Healing/physiologyABSTRACT
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.
Subject(s)
Body Patterning/genetics , Homeodomain Proteins/genetics , Regulatory Elements, Transcriptional/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Zebrafish Proteins/genetics , Animals , Binding Sites , Gene Expression Regulation, Developmental , Genomics , Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Wnt Signaling Pathway/genetics , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/metabolismABSTRACT
Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate.
Subject(s)
Adult Stem Cells/physiology , Gene Silencing/physiology , Lateral Ventricles/cytology , Lateral Ventricles/physiology , Neural Stem Cells/physiology , Repressor Proteins/physiology , Animals , Bone Morphogenetic Protein 6/physiology , Cell Differentiation/physiology , Cell Proliferation/physiology , Cells, Cultured , Humans , Male , Mice , Transcription Factors/physiologyABSTRACT
An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type.