miR379–410 cluster miRNAs regulate neurogenesis and neuronal migration by fine‐tuning N‐cadherin

N‐cadherin‐mediated adhesion is essential for maintaining the tissue architecture and stem cell niche in the developing neocortex. N‐cadherin expression level is precisely and dynamically controlled throughout development; however, the underlying regulatory mechanisms remain largely unknown. MicroRNAs (miRNAs) play an important role in the regulation of protein expression and subcellular localisation. In this study, we show that three miRNAs belonging to the miR379–410 cluster regulate N‐cadherin expression levels in neural stem cells and migrating neurons. The overexpression of these three miRNAs in radial glial cells repressed N‐cadherin expression and increased neural stem cell differentiation and neuronal migration. This phenotype was rescued when N‐cadherin was expressed from a miRNA‐insensitive construct. Transient abrogation of the miRNAs reduced stem cell differentiation and increased cell proliferation. The overexpression of these miRNAs specifically in newborn neurons delayed migration into the cortical plate, whereas the knockdown increased migration. Collectively, our results indicate a novel role for miRNAs of the miR379–410 cluster in the fine‐tuning of N‐cadherin expression level and in the regulation of neurogenesis and neuronal migration in the developing neocortex.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Strange bedfellows: Reelin and Notch signaling interact to regulate cell migration in the developing neocortex

Neuronal cell migration in the developing neocortex relies on the Reelin signaling cascade to establish the characteristic "inside out" lamination pattern. In this issue of Neuron, Rakic and colleagues report that Notch signaling, long known to regulate neural stem cells, also plays a critical role in mediating the effects of Reelin on neuronal cell migration.     

The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development

The switch of neural stem and progenitor cells from proliferation to differentiation during development is a crucial determinant of brain size. This switch is intimately linked to the architecture of the two principal classes of neural stem and progenitor cells, the apical (neuroepithelial, radial glial) and basal (intermediate) progenitors, which in turn is crucial for their symmetric versus asymmetric divisions. Focusing on the developing rodent neocortex, we discuss here recent advances in understanding the cell biology of apical and basal progenitors, place key regulatory molecules into subcellular context, and highlight their roles in the control of proliferation versus differentiation.     

Neural Precursor Cells from Adult Mouse Cerebral Cortex Differentiate into Both Neurons and Oligodendrocytes

Recent findings concerning adult neurogenesis in two selected structures of the mammalian brain, the olfactory bulb and dentate gyrus of the hippocampus, present the possibility that this mechanism of neurogenesis applies for all brain regions, including the cerebral neocortex. In this way, a small number of potential neural precursor cells may exist in the cerebral neocortex, but they do not normally differentiate into cortical neurons in vivo. It has, however, been reported recently that cycling cells isolated from non-neurogenic areas of adult rat cerebral cortex could generate neurons in vitro. In this study, we analyzed the lineage potential of cycling cells from the adult mouse neocortex. For the dissection of the cerebral cortex from the adult mouse, which is significantly smaller than that of the adult rat, we have modified the previous dissection protocol developed for the rat neocortex. As a result, cycling cells from adult mouse neocortex gave rise to neurons and oligodendrocytes, but not to astrocytes, whereas when the previous dissection method was used, cycling cells gave rise to neurons, oligodendrocytes and astrocytes. This discrepancy might stem from slight contamination of the dissected mouse neocortical tissue in the previous protocol used for the dissection of rat neocortex by cells from the surrounding subependymal zone, where typical adult neural stem cells exist. The results presented here will contribute to our understanding of the nature of cycling cells in the adult mammalian neocortex, and for which future stem cell research will provide new possibilities for cell replacement therapy to be used in the treatment of neurodegenerative conditions.     

Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.     

Dynamic formation of functional networks by synchronization

Cleavage plane orientation has been thought to govern the fate of neural stem cell progeny, but supporting evidence in the neocortex has been sparse. A new study by Postiglione et?al. in this issue of Neuron shows that mouse Inscuteable-mediated control of cleavage plane orientation regulates the output of neural progenitor cells.     

Principles of neural stem cell lineage progression: Insights from developing cerebral cortex

How to generate a brain of correct size and with appropriate cell-type diversity during development is a major question in Neuroscience. In the developing neocortex, radial glial progenitor (RGP) cells are the main neural stem cells that produce cortical excitatory projection neurons, glial cells, and establish the prospective postnatal stem cell niche in the lateral ventricles. RGPs follow a tightly orchestrated developmental program that when disrupted can result in severe cortical malformations such as microcephaly and megalencephaly. The precise cellular and molecular mechanisms instructing faithful RGP lineage progression are however not well understood. This review will summarize recent conceptual advances that contribute to our understanding of the general principles of RGP lineage progression.     

Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1-enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1-bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation.     

Development and evolution of the human neocortex

The size and surface area of the mammalian brain are thought to be critical determinants of intellectual ability. Recent studies show that development of the gyrated human neocortex involves a lineage of neural stem and transit-amplifying cells that forms the outer subventricular zone (OSVZ), a proliferative region outside the ventricular epithelium. We discuss how proliferation of cells within the OSVZ expands the neocortex by increasing neuron number and modifying the trajectory of migrating neurons. Relating these features to other mammalian species and known molecular regulators of the mouse neocortex suggests how this developmental process could have emerged in evolution.     

神经球方法在神经生物学中的应用及其局限性

神经球是神经干细胞在体外扩增培养过程中的一般表现形式。目前,神经球方法(neurosphere assay,NSA)已在神经干细胞性质、神经发育模型研究和作为分子载体用于神经系统疾病治疗等方面得到了广泛应用,但因神经球固有的异质性、自发融合等特性限制了其应用。本文就神经球方法在神经生物学研究中的应用及其局限性做一综述。     

CRISPR/Cas9‐induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock‐in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near‐maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.     

Hedgehog-GLI signaling regulates the behavior of cells with stem cell properties in the developing neocortex

Stem cells are crucial for normal development and homeostasis, and their misbehavior may be related to the origin of cancer. Progress in these areas has been difficult because the mechanisms regulating stem cell lineages are not well understood. Here, we have investigated the role of the SHH-GLI pathway in the developing mouse neocortex. The results show that SHH signaling endogenously regulates the number of embryonic and postnatal mouse neocortical cells with stem cell properties, and controls precursor proliferation in a concentration-dependent manner in cooperation with EGF signaling. These findings identify a crucial mechanism for the regulation of the number of cells with stem cell properties that is unexpectedly conserved in different stem cell niches.     

Behavior and Differentiation of the Neural Stem Cells in vivo

We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.     

LeX is expressed by principle progenitor cells in the embryonic nervous system, is secreted into their environment and binds Wnt-1

LeX/SSEA1/CD15 is an extracellular matrix-associated carbohydrate expressed by ES cells and by adult neural and bone marrow stem cells. It is important for cell adhesion, compaction and FGF2 responses of early embryonic stem cells; however, its function at later stages is not clear. We now show that LeX is expressed by primary mouse neural progenitor cells, including neural stem cells, neuroblasts and glioblasts, but not by their more differentiated products. LeX distinguishes highly proliferative cells even in the primitive neuroepithelium, demonstrating heterogeneity in cell potential before radial glia arise. At later stages, LeX expressing progenitors are frequently radial in morphology. Surface LeX expression can be used to enrich neural stem and progenitor cells from different CNS regions throughout development by FACS. We found that LeX expression is particularly strong in neural regions with prolonged neurogenesis, e.g., the olfactory epithelium, hippocampus, basal forebrain and cerebellum. These regions also express high levels of the growth factors FGF8 and/or Wnt-1. We show here that LeX-containing molecules in the developing nervous system bind Wnt-1. Our findings suggest that LeX, which is present on the surface of principle neural progenitors and secreted into their extracellular niche, may bind and present growth factors important for their proliferation and self-renewal.     

Brain as a paradigm of organ growth: Hedgehog-Gli signaling in neural stem cells and brain tumors

The Hedgehog-Gli (Hh-Gli) signaling pathway is essential for numerous events during the development of many animal cell types and organs. In particular, it controls neural cell precursor proliferation in dorsal brain structures and regulates the number of neural stem cells in distinct embryonic, perinatal, and adult niches, such as the developing neocortex, the subventricular zone of the lateral ventricle of the forebrain, and the hippocampus. We have proposed that Hh-Gli signaling regulates dorsal brain growth during ontogeny and that its differential regulation underlays evolutionary change in the morphology (size and shape) of dorsal brain structures. It is also critically involved in sporadic brain tumorigenesis--as well as several other human cancer--suggesting that tumors derive from stem cells or progenitors maintaining an inappropriate active Hh-Gli pathway. Importantly, we and others have demonstrated that human sporadic tumors from the brain and other organs require sustained HH-GLI signaling for sustained growth and survival. Modulating HH-GLI signaling thus represents a novel rational avenue to treat, on one hand, brain degeneration and injury by inducing controlled HH-GLI-mediated regeneration and growth, and on the other hand, to combat cancer by blocking its abnormal activity in tumor cells.