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.     

Regulation of glial development by cystatin C

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.     

Dye coupling and connexin expression by cortical radial glia in the early postnatal subventricular zone

In this study, we have analyzed the specific contribution of the cortical radial glia (RG) for gap junctional communication (GJC) within the postnatal subventricular zone (SVZ). To specifically target RG as source of dye‐coupling in situ, we have developed a new technique that involves direct cell loading through the processes that reach the pial surface, with a mix of gap junction permeant (Lucifer yellow, LY) and nonpermeant (rhodamine‐conjugated dextran 3 KDa, RD) fluorochromes, the latter used as a marker for direct loaded cells. Tissue sections were analyzed for identification of directly loaded (LY+RD+) and coupled cells (LY+RD–) in the SVZ. Directly loaded cells were restricted to the region underlying the pial loading surface area. Coupled cells were distributed in a bistratified manner, along the outer dorsal surface of the SVZ and aligning the ventricle, leaving the SVZ core relatively free. Blocking GJC prior to pial loading greatly reduced dye coupling. Phenotypic analysis indicated that coupling by RG excludes neuroblasts and is mostly restricted to cells of glial lineage. Notwithstanding, no corresponding restriction to specific cell phenotype was found for two connexin isotypes, Cx43 and Cx45, in the postnatal SVZ. The extensive homocellular cell coupling by RG suggests an important role in the regulation of neurogenesis and functional compartmentalization of the postnatal SVZ. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 2012     

Fibrillar β‐amyloid 1‐42 alters cytokine secretion,cholinergic signalling and neuronal differentiation

Adult neurogenesis is impaired by inflammatory processes, which are linked to altered cholinergic signalling and cognitive decline in Alzheimer's disease. In this study, we investigated how amyloid beta (Aβ)‐evoked inflammatory responses affect the generation of new neurons from human embryonic stem (hES) cells and the role of cholinergic signalling in regulating this process. The hES were cultured as neurospheres and exposed to fibrillar and oligomeric Aβ_1‐42 (Aβf, AβO) or to conditioned medium from human primary microglia activated with either Aβ_1‐42 or lipopolysaccharide. The neurospheres were differentiated for 29 days in vitro and the resulting neuronal or glial phenotypes were thereafter assessed. Secretion of cytokines and the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and choline acetyltransferase (ChAT) involved in cholinergic signalling was measured in medium throughout the differentiation. We report that differentiating neurospheres released various cytokines, and exposure to Aβf, but not AβO, increased the secretion of IL‐6, IL‐1β and IL‐2. Aβf also influenced the levels of AChE, BuChE and ChAT in favour of a low level of acetylcholine. These changes were linked to an altered secretion pattern of cytokines. A different pattern was observed in microglia activated by Aβf, demonstrating decreased secretion of TNF‐α, IL‐1β and IL‐2 relative to untreated cells. Subsequent exposure of differentiating neurospheres to Aβf or to microglia‐conditioned medium decreased neuronal differentiation and increased glial differentiation. We suggest that a basal physiological secretion of cytokines is involved in shaping the differentiation of neurospheres and that Aβf decreases neurogenesis by promoting a microenvironment favouring hypo‐cholinergic signalling and gliogenesis.     

A decrease in the addition of new cells in the nucleus accumbens and prefrontal cortex between puberty and adulthood in male rats

Adolescence involves shifts in social behaviors, behavioral flexibility, and adaptive risk‐taking that coincide with structural remodeling of the brain. We previously showed that new cells are added to brain regions associated with sexual behaviors, suggesting that cytogenesis may be a mechanism for acquiring adult‐typical behaviors during adolescence. Whether pubertal cell addition occurs in brain regions associated with behavioral flexibility or motivation and whether these patterns differ between pubertal and adult animals had not been determined. Therefore, we assessed patterns of cell proliferation or survival in the prefrontal cortex and nucleus accumbens. Pubertal and adult male rats were given injections of bromo‐deoxyuridine (BrdU). To assess cell proliferation, half of the animals from each group were sacrificed 24 h following the last injection. The remaining animals were sacrificed at Day 30 following the last injection to evaluate cell survival. Adult animals had significantly lower densities of BrdU‐immunoreactive (ir) cells in the prefrontal cortex, irrespective of post‐BrdU survival time, whereas in the nucleus accumbens, adult animals had a lower density of BrdU‐ir cells at the short survival time; however, the density of BrdU‐ir cells was equivalent in pubertal and adult animals at the longer survival time. These data provide evidence that cell addition during puberty may contribute to the remodeling of brain regions associated with behavioral flexibility and motivation, and this cell addition continues into adulthood, albeit at lower levels. Higher levels of cell proliferation or survival in younger animals may reflect a higher level of plasticity, possibly contributing to the dynamic remodeling of the pubertal brain. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 633–642, 2014     

Single-Cell Analysis of Regional Differences in Adult V-SVZ Neural Stem Cell Lineages

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Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells

To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75⁺ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development. © 1997 John Wiley & Sons, Inc. J Neuroblol 32 : 722–746, 1997