Epigenetic cues modulating the generation of cell‐type diversity in the cerebral cortex

The cerebral cortex is composed of a large variety of distinct cell‐types including projection neurons, interneurons, and glial cells which emerge from distinct neural stem cell lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that neural stem cell and radial glial progenitor lineage progression are regulated in a profound deterministic manner. In this review we focus on recent advances based mainly on correlative phenotypic data emerging from functional genetic studies in mice. We establish hypotheses to test in future research and outline a conceptual framework how epigenetic cues modulate the generation of cell‐type diversity during cortical development.     

A unified hypothesis on the lineage of neural stem cells

For many years, it was assumed that neurons and glia in the central nervous system were produced from two distinct precursor pools that diverged early during embryonic development. This theory was partially based on the idea that neurogenesis and gliogenesis occurred during different periods of development, and that neurogenesis ceased perinatally. However, there is now abundant evidence that neural stem cells persist in the adult brain and support ongoing neurogenesis in restricted regions of the central nervous system. Surprisingly, these stem cells have the characteristics of fully differentiated glia. Neuroepithelial stem cells in the embryonic neural tube do not show glial characteristics, raising questions about the putative lineage from embryonic to adult stem cells. In the developing brain, radial glia have long been known to produce cortical astrocytes, but recent data indicate that radial glia might also divide asymmetrically to produce cortical neurons. Here we review these new developments and propose that the stem cells in the central nervous system are contained within the neuroepithelial --> radial glia --> astrocyte lineage.     

Continuous live imaging of adult neural stem cell division and lineage progression in vitro

Little is known about the intrinsic specification of adult neural stem cells (NSCs) and to what extent they depend on their local niche. To observe adult NSC division and lineage progression independent of their niche, we isolated cells from the adult mouse subependymal zone (SEZ) and cultured them at low density without growth factors. We demonstrate here that SEZ cells in this culture system are primarily neurogenic and that adult NSCs progress through stereotypic lineage trees consisting of asymmetric stem cell divisions, symmetric transit-amplifying divisions and final symmetric neurogenic divisions. Stem cells, identified by their astro/radial glial identity and their slow-dividing nature, were observed to generate asymmetrically and fast-dividing cells that maintained an astro/radial glia identity. These, in turn, gave rise to symmetrically and fast-dividing cells that lost glial hallmarks, but had not yet acquired neuronal features. The number of amplifying divisions was limited to a maximum of five in this system. Moreover, we found that cell growth correlated with the number of subsequent divisions of SEZ cells, with slow-dividing astro/radial glia exhibiting the most substantial growth prior to division. The fact that in the absence both of exogenously supplied growth factors and of signals provided by the local niche neurogenic lineage progression takes place in such stereotypic fashion, suggests that lineage progression is, to a significant degree, cell intrinsic or pre-programmed at the beginning of the lineage.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.     

Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors

We have addressed the role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells. We show that mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a premature and excessive generation of astrocytic precursors. An analysis of wild-type and mutant cortical progenitors in culture showed that a large fraction of Ngn2; Mash1 double-mutant progenitors failed to adopt a neuronal fate, instead remaining pluripotent or entering an astrocytic differentiation pathway. Together, these results demonstrate that proneural genes are involved in lineage restriction of cortical progenitors, promoting the acquisition of the neuronal fate and inhibiting the astrocytic fate.     

Stem cells in the embryonic cerebral cortex: Their role in histogenesis and patterning

The cytoarchitectural simplicity of the cerebral cortex makes it an attractive system to study central nervous system (CNS) histogenesis—the process whereby diverse cells are generated in the right numbers at the appropriate place and time. Recently, multipotent stem cells have been implicated in this process, as progenitor cells for diverse types of cortical neurons and glia. Continuous analysis of stem cell clone development reveals stereotyped division patterns within their lineage trees, highly reminiscent of neural lineage trees in arthropods and Caenorhabditis elegans. Given that these division patterns play a critical part in generating diverse neural types in invertebrates, we speculate that they play a similar role in the cortex. Because stereotyped lineage trees can be observed from cells growing at clonal density, cell-intrinsic factors are likely to have a key role in stem cell behavior. Cortical stem cells also respond to environmental signals to alter the types of cells they generate, providing the means for feedback regulation on the germinal zone. Evidence is accumulating that cortical stem cells, influenced by intrinsic programs and environmental signals, actually change with development—for example, by reducing the number and types of neurons they produce. Age-related changes in the stem cell population may have a critical role in orchestrating development; whether these cells truly self-renew is a point of discussion. In summary, we propose that cortical stem cells are the focus of regulatory mechanisms central to the development of the cortical cytoarchitecture. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 162–174, 1998     

Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body

Neurogenesis is known to occur in the specific niches of the adult mammalian brain, but whether germinal centers exist in the neural-crest-derived peripheral nervous system is unknown. We have discovered stem cells in the adult carotid body (CB), an oxygen-sensing organ of the sympathoadrenal lineage that grows in chronic hypoxemia. Production of new neuron-like CB glomus cells depends on a population of stem cells, which form multipotent and self-renewing colonies in vitro. Cell fate mapping experiments indicate that, unexpectedly, CB stem cells are the glia-like sustentacular cells and can be identified using glial markers. Remarkably, stem cell-derived glomus cells have the same complex chemosensory properties as mature in situ glomus cells. They are highly dopaminergic and produce glial cell line-derived neurotrophic factor. Thus, the mammalian CB is a neurogenic center with a recognizable physiological function in adult life. CB stem cells could be potentially useful for antiparkinsonian cell therapy.     

Rostral–caudal distribution of Emx1‐lineage stem/transit amplifying cells and lineage progression in embryonic cortex depend on hedgehog signaling

Lineage progression of neural precursors to an EGF‐responsive state can be promoted by several extrinsic signals, including fibroblast growth factor 2 (FGF2) and Hedgehog (Hh). It has been suggested that EGF‐responsive precursors in the embryonic cerebral cortex originate in the ventral telencephalon in an FGF‐dependent manner and migrate dorsally. To determine whether cortical EGF‐responsive cells originate locally from dorsal precursors, we marked these precursors using Emx1‐cre and the cre reporter Z/EG and observed a local origin for EGF‐responsive cells. We also found a rostral–caudal difference in the abundance of self‐renewing, neurogenic Emx1‐lineage precursors, with more present rostrally. Deleting the Hh receptor smoothened in Emx‐1 lineage cells impaired their progression to an EGF‐responsive state. Moreover, loss of smoothened increased the proportion of neurogenic, self‐renewing Emx1‐lineage cells in caudal regions of cortex, eliminating their asymmetric distribution. Our results support the idea that Hh signaling promotes lineage progression of stem/transit amplifying cells, particularly in caudal regions of the embryonic cortex, leading to rostral–caudal differences in the abundance of neurogenic, self‐renewing precursors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1096–1109, 2014     

Glial progenitors in the CNS and possible lineage relationships among them

Glial cells are derived from stem cells that mature through specific stages of development to generate fully differentiated astrocytes and oligodendrocytes. Several types of intermediate precursors have been described and in some cases lineage relationships identified although this remains a subject of controversy. We review recent findings and discuss some possibilities. Motoneuron-oligodendrocyte precursors (MNOPs), white matter progenitor cells (WMPCs), polydendrocytes, glial restricted precursors (GRPs), astrocyte precursor cells (APCs), and oligodendroblasts are likely all derived from earlier appearing stem cells but segregate at different stages in development. Some of these precursors persist in the adult, and it is these glial progenitors rather than stem cells that respond after injury and participate in the repair process. Although which specific glial progenitor responds remains unclear, the availability of new markers will likely resolve this issue. We believe that the development of consensus sets of markers and an improvement in our ability to define stages of glial maturation will lead to a clearer appreciation of the importance of glia in the etiopathology of disease.     

Neuronal stem cells in the central nervous system and in human diseases

The process of cortical expansion in the central nervous system is a key step of mammalian brain development to ensure its physiological function. Radial glial (RG) cells are a glial cell type contributing to this progress as intermediate neural progenitor cells responsible for an increase in the number of cortical neurons. In this review, we discuss the current understanding of RG cells during neurogenesis and provide further information on the mechanisms of neurodevelopmental diseases and stem cell-related brain tumorigenesis. Knowledge of neuronal stem cell and relative diseases will bridge benchmark research through translational studies to clinical therapeutic treatments of these diseases.     

Precursor cell types in the germinal zone of the cerebral cortex

Retroviral lineage tracing experiments suggest that the cortical ventricular zone is composed of a mixture of precursor cell types. The majority generate a single cell type (neurones, astrocytes or oligodendrocytes) and the remainder generate neurones and a single type of glial cell. Pluripotential precursor cells, that have the ability to generate all three cell types, are not observed. A recent paper, however, reports that when single ventricular zone cells are cultured in isolation, a small percentage of these cells are pluripotential⁽¹⁾. This review will discuss what this knowledge tells us about cortical development.     

Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. Our data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure.     

Neural differentiation of embryonic stem cells is induced by signalling from non-neural niche cells.

BACKGROUND/AIMS: Embryonic stem cell (ESC) transplantation offers new therapeutic strategies for neurodegenerative diseases and injury. However, the mechanisms underlying integration and differentiation of engrafted ESCs are poorly understood. This study elucidates the influence of exogenous signals on ESC differentiation using in vitro modelling of non-stem/stem cell interactions. METHODS: Murine ESCs were co-cultured with endothelial cells and astrocytes or conditioned medium obtained from endothelial or astrocyte cultures. After 7 days of co-culture isolated RNA was analysed using RT-PCR for the expression of pluripotency marker oct-4, neural progenitor marker nestin, and neurofilament (NFL), an early marker of neuronal lineage commitment. The presence of the glial cell surface marker A2B5 was determined in ESCs by flow cytometry. RESULTS: Neuronal differentiation was inhibited in ESCs when grown in close vicinity to cerebral endothelial or glial cells. Under these conditions, ESC differentiation was predominantly directed towards a glial fate. However, treatment of ESCs with endothelial cell- or astrocyte-conditioned medium promoted neuronal as well as glial differentiation. CONCLUSION: Our results indicate that ESC fate is determined by endothelial and glial cells that comprise the environmental niche of these stem cells in vivo. The direction of differentiation processes appears to be dependent on humoral factors secreted by adjacent cell lines.     

Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro

The ability of hematopoietic tissue-derived adult stem cells to transdifferentiate into neural progenitor cells offers an interesting alternative to central nervous system (CNS)- or embryonic-derived stem cells as a viable source for cellular therapies applied to brain regeneration. Umbilical cord blood (CB) due to its primitive nature and it unproblematic collection appears as a promising candidate for multipotent stem cell harvest. We developed a negative immunomagnetic selection method that depletes CB from hematopoietic lineage marker-expressing cells, hence isolating a discrete lineage negative (LinNeg) stem cell population (0.1% of CB mononucleated cell [MCN] population). In liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK), CB LinNeg stem cells could expand primitive nonadherent hematopoietic progenitors (up to 47-fold) and simultaneously produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Laser scanning confocal microscopy analysis identified these adherent cells to express glial fibrillary acidic protein (GFAP). Gene expression analysis showed upregulation of primitive neuroglial progenitor cell markers including, GFAP, nestin, musashi-1, and necdin. ELISA quantification of liquid culture supernatant revealed the in vitro release of transforming growth factor beta-1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF) suggesting their contribution to CB LinNeg stem cell transdifferentiation into neuroglial progenitors. Our study supports that a single CB specimen can be pre-expanded in TPOFLK to produce both primitive hematopoietic and neuropoietic progenitors, hence widening CB clinical potential for cellular therapies.