Central Nervous System Progenitor Research Articles

Pax3/7 regulates neural tube closure and patterning in a non-vertebrate chordate.

Pax3/7 factors play numerous roles in the development of the dorsal nervous system of vertebrates. From specifying neural crest at the neural plate borders, to regulating neural tube closure and patterning of the resulting neural tube. However, it is unclear which of these roles are conserved in non-vertebrate chordates. Here we investigate the expression and function of Pax3/7 in the model tunicate Ciona. Pax3/7 is expressed in neural plate border cells during neurulation, and in central nervous system progenitors shortly after neural tube closure. We find that separate cis-regulatory elements control the expression in these two distinct lineages. Using CRISPR/Cas9-mediated mutagenesis, we knocked out Pax3/7 in F0 embryos specifically in these two separate territories. Pax3/7 knockout in the neural plate borders resulted in neural tube closure defects, suggesting an ancient role for Pax3/7 in this chordate-specific process. Furthermore, knocking out Pax3/7 in the neural impaired Motor Ganglion neuron specification, confirming a conserved role for this gene in patterning the neural tube as well. Taken together, these results suggests that key functions of Pax3/7 in neural tube development are evolutionarily ancient, dating back at least to the last common ancestor of vertebrates and tunicates.

Genetic Regulation of Vertebrate Forebrain Development by Homeobox Genes.

Forebrain development in vertebrates is regulated by transcription factors encoded by homeobox, bHLH and forkhead gene families throughout the progressive and overlapping stages of neural induction and patterning, regional specification and generation of neurons and glia from central nervous system (CNS) progenitor cells. Moreover, cell fate decisions, differentiation and migration of these committed CNS progenitors are controlled by the gene regulatory networks that are regulated by various homeodomain-containing transcription factors, including but not limited to those of the Pax (paired), Nkx, Otx (orthodenticle), Gsx/Gsh (genetic screened), and Dlx (distal-less) homeobox gene families. This comprehensive review outlines the integral role of key homeobox transcription factors and their target genes on forebrain development, focused primarily on the telencephalon. Furthermore, links of these transcription factors to human diseases, such as neurodevelopmental disorders and brain tumors are provided.

The endogenous progenitor response following traumatic brain injury: a target for cell therapy paradigms.

Although there is ample evidence that central nervous system progenitor pools respond to traumatic brain injury, the reported effects are variable and likely contribute to both recovery as well as pathophysiology. Through a better understanding of the diverse progenitor populations in the adult brain and their niche-specific reactions to traumatic insult, treatments can be tailored to enhance the benefits and dampen the deleterious effects of this response. This review provides an overview of endogenous precursors, the associated effects on cognitive recovery, and the potential of exogenous cell therapeutics to modulate these endogenous repair mechanisms. Beyond the hippocampal dentate gyrus and subventricular zone of the lateral ventricles, more recently identified sites of adult neurogenesis, the meninges, as well as circumventricular organs, are also discussed as targets for endogenous repair. Importantly, this review highlights that progenitor proliferation alone is no longer a meaningful outcome and studies must strive to better characterize precursor spatial localization, transcriptional profile, morphology, and functional synaptic integration. With improved insight and a more targeted approach, the stimulation of endogenous neurogenesis remains a promising strategy for recovery following traumatic brain injury.

Mitochondria in Early Forebrain Development: From Neurulation to Mid-Corticogenesis.

Function of the mature central nervous system (CNS) requires a substantial proportion of the body’s energy consumption. During development, the CNS anlage must maintain its structure and perform stage-specific functions as it proceeds through discrete developmental stages. While key extrinsic signals and internal transcriptional controls over these processes are well appreciated, metabolic and mitochondrial states are also critical to appropriate forebrain development. Specifically, metabolic state, mitochondrial function, and mitochondrial dynamics/localization play critical roles in neurulation and CNS progenitor specification, progenitor proliferation and survival, neurogenesis, neural migration, and neurite outgrowth and synaptogenesis. With the goal of integrating neurodevelopmental biologists and mitochondrial specialists, this review synthesizes data from disparate models and processes to compile and highlight key roles of mitochondria in the early development of the CNS with specific focus on forebrain development and corticogenesis.

Fate restrictions in embryonic neural progenitors.

The vertebrate central nervous system (CNS) is a fantastically complex organ composed of dozens of cell types within the neural and glial lineages. Its organization is laid down during development, through the localized and sequential production of subsets of neurons with specific identities. The principles and mechanisms that underlie the timely production of adequate classes of cells are only partially understood. Recent advances in molecular profiling describe the developmental trajectories leading to this amazing cellular diversity and provide us with cell atlases of an unprecedented level of precision. Yet, some long-standing questions pertaining to lineage relationships between neural progenitor cells and their differentiated progeny remain unanswered. Here, we discuss questions related to proliferation potential, timing of fate choices and restriction of neuronal output potential of individual CNS progenitors through the lens of lineage relationship. Unlocking methodological barriers will be essential to accurately describe CNS development at a cellular resolution.

Niche stiffness underlies the ageing of central nervous system progenitor cells.

Aging causes a decline in tissue regeneration due to a loss of function in adult stem and progenitor cell populations1. An important example is the deterioration of the regenerative capacity of the widespread and abundant population of central nervous system (CNS) multipotent stem cells known as oligodendrocyte progenitor cells (OPCs)2. A relatively overlooked potential source for this loss of function is the stem cell niche, a source of cell-extrinsic cues including chemical and mechanical signalling3,4. In this study, we show that the OPC microenvironment stiffens with age, and that this stiffening is sufficient to cause age-related OPC loss of function. Using biological and novel synthetic scaffolds to mimic the stiffness of young brain we find that isolated aged OPCs (aOPCs) cultured on these scaffolds are molecularly and functionally rejuvenated. When we disrupt mechanical signalling, OPC proliferation and differentiation rates are increased. We identify the mechanoresponsive ion channel Piezo1 as a key mediator of OPC mechanical signalling. Inhibition of Piezo1 overrides mechanical signals in vivo and allows OPCs to maintain activity in the aging CNS. We also show that Piezo1 plays an important role in regulating cell number during CNS development. Thus, we show that tissue stiffness is an important regulator of aging in OPCs, and provide new insights into how adult stem and progenitor cell function changes with age. These findings are of fundamental importance not only for the development of regenerative therapies but also for understanding the aging process itself.

A concerted metabolic shift in early forebrain alters the CSF proteome and depends on MYC downregulation for mitochondrial maturation.

Massive, coordinated cellular changes accompany the transition of central nervous system (CNS) progenitors from forebrain neurectodermal cells to specified neuroepithelial cells. We have previously found that MYC regulates the changing ribosomal and proteostatic landscapes in mouse forebrain precursors at embryonic days E8.5 and E10.5 (before and after neural tube closure; NTC) (Chau et al., 2018). Here, we demonstrate parallel coordinated transcriptional changes in metabolic machinery during this same stage of forebrain specification. Progenitors showed striking mitochondrial structural changes transitioning from glycolytic cristae at E8.5, to more traditional mitochondria at E10.5. Accordingly, glucose use shifted in progenitors such that E8.5 progenitors relied on glycolysis, and after NTC increasingly used oxidative phosphorylation. This metabolic shift was matched by changes in surrounding amniotic and cerebrospinal fluid proteomes. Importantly, these mitochondrial morphological shifts depend on MYC downregulation. Together, our findings demonstrate that metabolic shifting accompanies dynamic organelle and proteostatic remodeling of progenitor cells during the earliest stages of forebrain development.

60 Are gangliogliomas in children and adults disorders of nervous system development?

INTRODUCTION: Gangliogliomas (GGs) are neuroepithelial tumours of the central nervous system (CNS) composed of mature ganglion cells or a mixed population of ganglion and glial cells. Microarray data of low grade gliomas (LGG) including GGs revealed overexpression of the Dlx2 gene, a homeobox gene essential for interneuron migration and differentiation. We hypothesized that GGs are arrested in development, and began to explore the role of the Dlx2 gene. BRAF rearrangements and BRAF V600E point mutations have been reported in pediatric LGG. METHODS: DLX2 expression was examined in GGs using immunofluorescence (IF) and immunohistochemistry (IHC) labelling of formalin fixed paraffin embedded (FFPE) tissue sections, along with staining of glial and neuronal markers. BRAF mutations were detected using a commercial antibody and/or sequence verification of the DNA extracted from the FFPE blocks. RESULTS: In the Discovery cohort 10/30 were DLX2+ (33.3%) and in the Validation cohort 15/40 were DLX2+ (37.5%). Of these 15 cases, 15 were GFAP+ (100%), 15 were synaptophysin and/or NeuN+ (100%), and 13 were OLIG2+ (86.7%); 6 had a BRAF V600E mutation (40.0%). For the Validation cohort of 40 GGs, 28 were OLIG2+ (70.0%); 13/28 co-expressed DLX2 (46.4%). 18/40 cases had a BRAF V600 mutation(17 V600E, 1 V600G; 45.0%) and 6/18 were DLX2+ (33.3%). CONCLUSIONS: DLX2 is expressed in GGs in both neuronal and glial marker expressing tumour cells. Our results support that GGs arise from CNS progenitors arrested at the neuronal-glial cell fate “decision” point.

Central and Peripheral Nervous System Progenitors Derived from Human Pluripotent Stem Cells Reveal a Unique Temporal and Cell-Type Specific Expression of PMCAs.

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca2+ regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca2+ changes, which can ultimately correspond to changes in specific stem cell transcription states.

Conserved gene regulatory module specifies lateral neural borders across bilaterians

The lateral neural plate border (NPB), the neural part of the vertebrate neural border, is composed of central nervous system (CNS) progenitors and peripheral nervous system (PNS) progenitors. In invertebrates, PNS progenitors are also juxtaposed to the lateral boundary of the CNS. Whether there are conserved molecular mechanisms determining vertebrate and invertebrate lateral neural borders remains unclear. Using single-cell-resolution gene-expression profiling and genetic analysis, we present evidence that orthologs of the NPB specification module specify the invertebrate lateral neural border, which is composed of CNS and PNS progenitors. First, like in vertebrates, the conserved neuroectoderm lateral border specifier Msx/vab-15 specifies lateral neuroblasts in Caenorhabditis elegans Second, orthologs of the vertebrate NPB specification module (Msx/vab-15, Pax3/7/pax-3, and Zic/ref-2) are significantly enriched in worm lateral neuroblasts. In addition, like in other bilaterians, the expression domain of Msx/vab-15 is more lateral than those of Pax3/7/pax-3 and Zic/ref-2 in C. elegans Third, we show that Msx/vab-15 regulates the development of mechanosensory neurons derived from lateral neural progenitors in multiple invertebrate species, including C. elegans, Drosophila melanogaster, and Ciona intestinalis We also identify a novel lateral neural border specifier, ZNF703/tlp-1, which functions synergistically with Msx/vab-15 in both C. elegans and Xenopus laevis These data suggest a common origin of the molecular mechanism specifying lateral neural borders across bilaterians.

Spatial distribution and characterization of non-apical progenitors in the zebrafish embryo central nervous system.

Studies of non-apical progenitors (NAPs) have been largely limited to the developing mammalian cortex. They are postulated to generate the increase in neuron numbers that underlie mammalian brain expansion. Recently, NAPs have also been reported in the retina and central nervous system of non-mammalian species; in the latter, however, they remain poorly characterized. Here, we characterize NAP location along the zebrafish central nervous system during embryonic development, and determine their cellular and molecular characteristics and renewal capacity. We identified a small population of NAPs in the spinal cord, hindbrain and telencephalon of zebrafish embryos. Live-imaging analysis revealed at least two types of mitotic behaviour in the telencephalon: one NAP subtype retains the apical attachment during division, while another divides in a subapical position disconnected from the apical surface. All NAPs observed in spinal cord lost apical contact prior to mitoses. These NAPs express HuC and produce two neurons from a single division. Manipulation of Notch activity reveals that neurons and NAPs in the spinal cord use similar regulatory mechanisms. This work suggests that the majority of spinal NAPs in zebrafish share characteristics with basal progenitors in mammalian brains.

PS1 - 218 Characterization of Ganglioglioma as a Neurodevelopmental Disorder: A Cast of Arrested Development?

Gangliogliomas (GG) are low grade neuroepithelial tumours of the central nervous system (CNS) comprised of neoplastic glial and neuronal cells. There are no animal models or cell lines to study. Microarray data of a panel of low grade gliomas including GG revealed overexpression of the DLX2 homeobox gene required for tangential interneuronal migration and differentiation in the embryonic forebrain. We hypothesized that DLX2 regulates neural versus glial cell fate decisions in CNS progenitors and that GG are arrested in development. METHODS: DLX2 expression was examined along with glial fibrillary acidic protein (GFAP; glial marker) and synaptophysin and/or NeuN (neuronal markers) expression in a cohort of GG tumours using immunohistochemistry and dual immunofluorescence labelling of formalin fixed paraffin embedded (FFPE) tissue sections. BRAF mutational status was also assessed. RESULTS: In our discovery cohort (Genoa), 10/30 samples (33%) expressed DLX2. In our validation cohort (Edmonton), 22/36 (61%) expressed nuclear DLX2 and 12/22 DLX2+cases had BRAF mutations (55%; 11 V600E, 1 V600G). 12/18 cases with BRAF mutations were DLX2+(67%). One heavily pre-treated child with progressive cervicomedullary GG has had a very good partial response to BRAF inhibitor therapy. All 22 DLX2+tumours co-expressed GFAP (100%) and 21/22 (95%) also expressed synaptophysin or NeuN. CONCLUSIONS: Our results support GG as neurodevelopmental tumours arising from bipotential CNS progenitors that are arrested at the neural-glial cell fate "decision" point. Biological or differentiation-based treatments could be considered+/- BRAF inhibitors for those GG with/without the V600E mutation, respectively.

Cell of Origin and Cancer Stem Cells in Tumor Suppressor Mouse Models of Glioblastoma.

The cellular origins and the mechanisms of progression, maintenance of tumorigenicity, and therapeutic resistance are central questions in the glioblastoma multiforme (GBM) field. Using tumor suppressor mouse models, our group recently reported two independent populations of adult GBM-initiating central nervous system progenitors. We found different functional and molecular subtypes depending on the tumor-initiating cell lineage, indicating that the cell of origin is a driver of GBM subtype diversity. Using an in vivo model, we also showed that GBM cancer stem cells (CSCs) or glioma stem cells (GSCs) contribute to resistance to chemotherapeutic agents and that genetic ablation of GSCs leads to a delay in tumor progression. These studies are consistent with the cell of origin and CSCs as critical regulators of the pathogenesis of GBM.

Heterogeneous generation of new cells in the adult echinoderm nervous system.

Adult neurogenesis, generation of new functional cells in the mature central nervous system (CNS), has been documented in a number of diverse organisms, ranging from humans to invertebrates. However, the origin and evolution of this phenomenon is still poorly understood for many of the key phylogenetic groups. Echinoderms are one such phylum, positioned as a sister group to chordates within the monophyletic clade Deuterostomia. They are well known for the ability of their adult organs, including the CNS, to completely regenerate after injury. Nothing is known, however, about production of new cells in the nervous tissue under normal physiological conditions in these animals. In this study, we show that new cells are continuously generated in the mature radial nerve cord (RNC) of the sea cucumber Holothuria glaberrima. Importantly, this neurogenic activity is not evenly distributed, but is significantly more extensive in the lateral regions of the RNC than along the midline. Some of the new cells generated in the apical region of the ectoneural neuroepithelium leave their place of origin and migrate basally to populate the neural parenchyma. Gene expression analysis showed that generation of new cells in the adult sea cucumber CNS is associated with transcriptional activity of genes known to be involved in regulation of various aspects of neurogenesis in other animals. Further analysis of one of those genes, the transcription factor Myc, showed that it is expressed, in some, but not all radial glial cells, suggesting heterogeneity of this CNS progenitor cell population in echinoderms.

Neuromesodermal progenitors and the making of the spinal cord.

Neuromesodermal progenitors (NMps) contribute to both the elongating spinal cord and the adjacent paraxial mesoderm. It has been assumed that these cells arise as a result of patterning of the anterior neural plate. However, as the molecular mechanisms that specify NMps in vivo are uncovered, and as protocols for generating these bipotent cells from mouse and human pluripotent stem cells in vitro are established, the emerging data suggest that this view needs to be revised. Here, we review the characteristics, regulation, in vitro derivation and in vivo induction of NMps. We propose that these cells arise within primitive streak-associated epiblast via a mechanism that is separable from that which establishes neural fate in the anterior epiblast. We thus argue for the existence of two distinct routes for making central nervous system progenitors.

Abstract LB-132: Adult lineage-restricted CNS progenitors specify distinct glioblastoma subtypes

Abstract A central question in glioblastoma multiforme (GBM) research is the hierarchy of tumor-initiating cells, and its contribution to the malignant phenotype and genomic make-up of GBM. We examine the potential of adult lineage restricted central nervous system (CNS) progenitors to form malignant gliomas. We show that targeting of Nf1,p53 and Pten mutations in adult CNS progenitors but not stem cells gives rise to fully penetrant GBM. We identify two phenotypically and molecularly distinct GBM subtypes that arise from different adult progenitor lineages. Using multiple inducible cre transgenic lines, we demonstrate that murine GBMs are molecularly separable based on the cell of origin. The oligodendrocyte progenitor cell-derived murine tumors have parallels with a subset of human high-grade gliomas with oligodendrocytic component, tumors that have been ascribed better prognosis compared to classic malignant astrocytomas. These studies indicate that two independent sources of GBM-initiating adult progenitors are susceptible to identical mutations and point to the cell of origin as a major determinant of GBM subtype. Citation Format: Sheila R. Alcantara Llaguno, Zilai Wang, Daochun Sun, Jian Chen, Jing Xu, Euiseok Kim, Kimmo Hatanpaa, Jack Raisanen, Dennis Burns, Jane Johnson, Luis F. Parada. Adult lineage-restricted CNS progenitors specify distinct glioblastoma subtypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-132. doi:10.1158/1538-7445.AM2015-LB-132

Radial glia and neural progenitors in the adult zebrafish central nervous system.

The adult central nervous system (CNS) of the zebrafish, owing to its enrichment in constitutive neurogenic niches, is becoming an increasingly used model to address fundamental questions pertaining to adult neural stem cell (NSC) biology, adult neurogenesis and neuronal repair. Studies conducted in several CNS territories (notably the telencephalon, retina, midbrain, cerebellum and spinal cord) highlighted the presence, in these niches, of progenitor cells displaying NSC-like characters. While pointing to radial glial cells (RG) as major long-lasting, constitutively active and/or activatable progenitors in most domains, these studies also revealed a high heterogeneity in the progenitor subtypes used at the top of neurogenic hierarchies, including the persistence of neuroepithelial (NE) progenitors in some areas. Likewise, dissecting the molecular pathways underlying RG maintenance and recruitment under physiological conditions and upon repair in the zebrafish model revealed shared processes but also specific cascades triggering or sustaining reparative NSC recruitment. Together, the zebrafish adult brain reveals an extensive complexity of adult NSC niches, properties and control pathways, which extends existing understanding of adult NSC biology and gives access to novel mechanisms of efficient NSC maintenance and recruitment in an adult vertebrate brain.

Purinergic signalling in neuroregeneration.

Purinergic signalling in neuroregeneration.

HDAC inhibitors dysregulate neural stem cell activity in the postnatal mouse brain

The mammalian central nervous system (CNS) undergoes significant expansion postnatally, producing astrocytes, oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy, a condition commonly treated with valproate/valproic acid (VPA), a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs, however, may be essential for the differentiation of distinct subpopulations of neurons and glia. Here, we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7–P14, combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG, rostral migratory stream (RMS), and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed, however, that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA, OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity, we found that short term treatment with VPA in vivo reduced neurosphere numbers and size, a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor, Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively, these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered, especially in individuals whose brains are actively undergoing key postnatal time windows of development.

Differentiation of Human Fetal Multipotential Neural Progenitor Cells to Astrocytes Reveals Susceptibility Factors for JC Virus

Viral infections of the central nervous system (CNS) are of increasing concern, especially among immunocompromised populations. Rodent models are often inappropriate for studies of CNS infection, as many viruses, including JC virus (JCV) and HIV, cannot replicate in rodent cells. Consequently, human fetal brain-derived multipotential CNS progenitor cells (NPCs) that can be differentiated into neurons, oligodendrocytes, or astrocytes have served as a model in CNS studies. NPCs can be nonproductively infected by JCV, while infection of progenitor-derived astrocytes (PDAs) is robust. We profiled cellular gene expression at multiple times during differentiation of NPCs to PDAs. Several activated transcription factors show commonality between cells of the brain, in which JCV replicates, and lymphocytes, in which JCV is likely latent. Bioinformatic analysis determined transcription factors that may influence the favorable transcriptional environment for JCV in PDAs. This study attempts to provide a framework for understanding the functional transcriptional profile necessary for productive JCV infection.