Early Postnatal Subventricular Zone Research Articles

Central Nervous System Developmental Regulation of Microglia via Cytokines and Chemokines

Microglia are immune cells resident in the central nervous system (CNS). It has been gradually clarified that microglia play various roles at the developmental stage of the CNS. From embryonic to early postnatal age, microglia remove apoptotic cells by phagocytosis and refine the neural circuits by synaptic pruning. In addition, microglia promote the proliferation and differentiation of neural stem cells by releasing physiologically active substances. Our group has focused on the physiological actions of microglia via cytokines and chemokines at the early postnatal developmental stage. We found that a large number of activated microglia accumulate in the early postnatal subventricular zone (SVZ). We demonstrated that the these SVZ microglia facilitate neurogenesis and oligodendrogenesis via inflammatory cytokines including IL-1β, TNFα, IL-6, IFNγ. We have also found that microglia regulate the functional maturation of the blood brain barrier (BBB) and identified the cytokines and chemokines involved in the effects of microglia. These findings indicate that microglia are physiologically more important than ever thought to reveal robust brain functions. Furthermore, the new mode of microglial action may lead to the discovery of drug targets of the incurable CNS diseases.

Endothelin-1 signaling maintains glial progenitor proliferation in the postnatal subventricular zone

Signaling molecules that regulate neurodevelopmental processes in the early postnatal subventricular zone (SVZ) are critical for proper brain development yet remain poorly characterized. Here, we report that Endothelin-1 (ET-1), a molecular component of the postnatal SVZ, promotes radial glial cell maintenance and proliferation in an autocrine manner via Notch signaling. Loss of ET-1 signaling increases neurogenesis and reduces oligodendrocyte progenitor cell proliferation (OPC) in the developing SVZ, thereby altering cellular output of the stem cell niche. We also show that ET-1 is required for increased neural stem cell and OPC proliferation in the adult mouse SVZ following demyelination. Lastly, high levels of ET-1 in the SVZ of patients with Cathepsin A-related arteriopathy with strokes and leukoencephalopathy correlate with an increased number of SVZ OPCs, suggesting ET-1’s role as a regulator of glial progenitor proliferation may be conserved in humans. ET-1 signaling therefore presents a potential new therapeutic target for promoting SVZ-mediated cellular repair.

Neonatal hypoxia-ischemia in rat elicits a region-specific neurotrophic response in SVZ microglia

BackgroundRecent findings describe microglia as modulators of neurogenesis in the subventricular zone (SVZ). SVZ microglia in the adult rat are thought to adopt a neurotrophic phenotype after ischemic stroke. Early postnatal microglia are endogenously activated and may therefore exhibit an increased sensitivity to neonatal hypoxia-ischemia (HI). The goal of this study was to investigate the impact of cortico-striatal HI on the microglial phenotype, function, and gene expression in the early postnatal SVZ.MethodsPostnatal day (P)7 rats underwent sham or right-hemispheric HI surgery. Microglia in the SVZ, the uninjured cortex, and corpus callosum were immunohistochemically analyzed at P10, P20, and P40. The transcriptome of microdissected SVZ and cortical microglia was analyzed at P10 and P20, and the effect of P10 SVZ microglia on neurosphere generation in vitro was studied.ResultsThe microglial response to HI was region-specific. In the SVZ, a microglial accumulation, prolonged activation and phagocytosis was noted that was not observed in the cortex and corpus callosum. The transcriptome of SVZ microglia and cortical microglia were distinct, and after HI, SVZ microglia concurrently upregulated pro- and anti-inflammatory as well as neurotrophic genes. In vitro, microglia isolated from the SVZ supported neurosphere generation in a concentration-dependent manner.ConclusionsMicroglia are an inherent cellular component of the early postnatal SVZ and undergo developmental changes that are affected on many aspects by neonatal HI injury. Our results demonstrate that early postnatal SVZ microglia are sensitive to HI injury and display a long-lasting region-specific response including neurotrophic features.

Dystroglycan Suppresses Notch to Regulate Stem Cell Niche Structure and Function in the Developing Postnatal Subventricular Zone

While the extracellular matrix (ECM) is known to regulate neural stem cell quiescence in the adult subventricular zone (SVZ), the function of ECM in the developing SVZ remains unknown. Here, we report that the ECM receptor dystroglycan regulates a unique developmental restructuring of ECM in the early postnatal SVZ. Dystroglycan is furthermore required for ependymal cell differentiation and assembly of niche pinwheel structures, at least in part by suppressing Notch activation in radial glial cells, which leads to the increased expression of MCI, Myb, and FoxJ1, transcriptional regulators necessary for acquisition of the multiciliated phenotype. Dystroglycan also regulates perinatal radial glial cell proliferation and transition into intermediate gliogenic progenitors, such that either acute or constitutive loss of function in dystroglycan results in increased oligodendrogenesis. These findings reveal a role for dystroglycan in orchestrating both the assembly and function of the SVZ neural stem cell niche.

Neurogenic microglia in the early postnatal subventricular zone found by a simple stereological imaging method.

ミクログリアは脳のマクロファージ様細胞として免疫機能を担当していることが古くから知られていたが，系統的に発生過程の早い時期にマクロファージと分かれていることが明らかにされた．胎生期ミクログリアはほとんどが突起を持たない丸いamoeboid型のミクログリアであるが，生後初期にamoeboid型のまま爆発的に増加し最大数に達し，発達がすすむにつれamoeboid型のミクログリアは減少し成体においてほとんどが静止型となる．脳室下帯（subventricular zone：SVZ）は一生を通じて神経新生やグリア新生が起こる領域であるが，生後初期は特に新生細胞の種類が激しく入れ替わる時期である．主要な投射神経は胎生期に，アストロサイトは胎生期から生後初期まで，オリゴデンドロサイトは胎生後期から生後初期にかけて，介在神経は胎生後期から成体に至るまで新生が続いている．そこで我々はステレオジーイメージングを大幅に簡便化し，生後初期から生後30日齢（postnatal day 30：P30）までSVZ周辺のミクログリアを観察したところ，生後初期に活性化型のミクログリアがSVZの特に中心部に集積していることを発見した．さらに，このミクログリアがIL-1β，IL-6，TNFα，IFNγの相補的な作用を介して神経新生，オリゴデンドロサイト新生を促進していることを明らかにした．この簡易ステレオロジーイメージング法は特定細胞の経時的脳内分布の概略をつかむためには非常に有用なツールと言える．そこで，簡易ステレオロジーイメージング法プロトコルを紹介し，生後初期SVZに集積したミクログリアの分布と役割について考察を加える．

Ontogeny of CX3CR1-EGFP expressing cells unveil microglia as an integral component of the postnatal subventricular zone.

The full spectrum of cellular interactions within CNS neurogenic niches is still poorly understood. Only recently has the monocyte counterpart of the nervous system, the microglial cells, been described as an integral cellular component of neurogenic niches. The present study sought to characterize the microglia population in the early postnatal subventricular zone (SVZ), the major site of postnatal neurogenesis, as well as in its anterior extension, the rostral migratory stream (RMS), a pathway for neuroblasts during their transit toward the olfactory bulb (OB) layers. Here we show that microglia within the SVZ/RMS pathway are not revealed by phenotypic markers that characterize microglia in other regions. Analysis of the transgenic mice strain that has one locus of the constitutively expressed fractalkine CX3CR1 receptor replaced by the gene encoding the enhanced green fluorescent protein (EGFP) circumvented the antigenic plasticity of the microglia, thus allowing us to depict microglia within the SVZ/RMS pathway during early development. Notably, microglia within the early SVZ/RMS are not proliferative and display a protracted development, retaining a more immature morphology than their counterparts outside germinal layers. Furthermore, microglia contact and phagocyte radial glia cells (RG) processes, thereby playing a role on the astroglial transformation that putative stem cells within the SVZ niche undergo during the first postnatal days.

Neural Stem Cells in the Immature, but Not the Mature, Subventricular Zone Respond Robustly to Traumatic Brain Injury

Pediatric traumatic brain injury is a significant problem that affects many children each year. Progress is being made in developing neuroprotective strategies to combat these injuries. However, investigators are a long way from therapies to fully preserve injured neurons and glia. To restore neurological function, regenerative strategies will be required. Given the importance of stem cells in repairing damaged tissues and the known persistence of neural precursors in the subventricular zone (SVZ), we evaluated regenerative responses of the SVZ to a focal brain lesion. As tissues repair more slowly with aging, injury responses of male Sprague Dawley rats at 6, 11, 17, and 60 days of age and C57Bl/6 mice at 14 days of age were compared. In the injured immature animals, cell proliferation in the dorsolateral SVZ more than doubled by 48 h. By contrast, the proliferative response was almost undetectable in the adult brain. Three approaches were used to assess the relative numbers of bona fide neural stem cells, as follows: the neurosphere assay (on rats injured at postnatal day 11, P11), flow cytometry using a novel 4-marker panel (on mice injured at P14) and staining for stem/progenitor cell markers in the niche (on rats injured at P17). Precursors from the injured immature SVZ formed almost twice as many spheres as precursors from uninjured age-matched brains. Furthermore, spheres formed from the injured brain were larger, indicating that the neural precursors that formed these spheres divided more rapidly. Flow cytometry revealed a 2-fold increase in the percentage of stem cells, a 4-fold increase in multipotential progenitor-3 cells and a 2.5-fold increase in glial-restricted progenitor-2/multipotential-3 cells. Analogously, there was a 2-fold increase in the mitotic index of nestin+/Mash1- immunoreactive cells within the immediately subependymal region. As the early postnatal SVZ is predominantly generating glial cells, an expansion of precursors might not necessarily lead to the production of many new neurons. On the contrary, many BrdU+/doublecortin+ cells were observed streaming out of the SVZ into the neocortex 2 weeks after injuries to P11 rats. However, very few new mature neurons were seen adjacent to the lesion 28 days after injury. Altogether, these data indicate that immature SVZ cells mount a more robust proliferative response to a focal brain injury than adult cells, which includes an expansion of stem cells, primitive progenitors and neuroblasts. Nonetheless, this regenerative response does not result in significant neuronal replacement, indicating that new strategies need to be implemented to retain the regenerated neurons and glia that are being produced.

Microglia Enhance Neurogenesis and Oligodendrogenesis in the Early Postnatal Subventricular Zone

Although microglia have long been considered as brain resident immune cells, increasing evidence suggests that they also have physiological roles in the development of the normal CNS. In this study, we found large numbers of activated microglia in the forebrain subventricular zone (SVZ) of the rat from P1 to P10. Pharmacological suppression of the activation, which produces a decrease in levels of a number of proinflammatory cytokines (i.e., IL-1β, IL-6, TNF-α, and IFN-γ) significantly inhibited neurogenesis and oligodendrogenesis in the SVZ. In vitro neurosphere assays reproduced the enhancement of neurogenesis and oligodendrogenesis by activated microglia and showed that the cytokines revealed the effects complementarily. These results suggest that activated microglia accumulate in the early postnatal SVZ and that they enhance neurogenesis and oligodendrogenesis via released cytokines.

Developmentally regulated collagen/integrin interactions confer adhesive properties to early postnatal neural stem cells

BackgroundIt is becoming increasingly apparent that the extracellular matrix acts as an important regulator of the neural stem niche. Previously we found that neural stem and progenitor cells (NSPCs) derived from the early postnatal subventricular zone of mice adhere to a collagen/hyaluronan hydrogel, whereas NSPCs from the adult and embryonic brain do not. MethodsTo examine the specific adhesive properties of young stem cells in more detail, NSPCs isolated from embryonic, postnatal day 6 (P6), and adult mouse brains were cultured on collagen I. ResultsEarly postnatal NSPCs formed paxillin-positive focal adhesions on collagen I, and these adhesions could be prevented by an antibody that blocked integrin β1. Furthermore, we found the corresponding integrin alpha subunits α2 and α11 levels to be highest at the postnatal stage. Gene ontology analysis of differentially expressed genes showed higher expression of transcripts involved in vasculature development and morphogenesis in P6 stem cells, compared to adult. ConclusionsThe ability to interact with the extracellular matrix differs between postnatal and adult NSPCs. General significanceOur observations that the specific adhesive properties of early postnatal NSPCs, which are lost in the adult brain, can be ascribed to the integrin subunits expressed by the former furthering our understanding of the developing neurogenic niche. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

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.

Corridors of migrating neurons in the human brain and their decline during infancy

The subventricular zone (SVZ) of many adult non-human mammals generates large numbers of new neurons destined for the olfactory bulb (OB)1–6. Along the walls of the lateral ventricles, immature neuronal progeny migrate in tangentially-oriented chains that coalesce into a rostral migratory stream (RMS) connecting the SVZ to the OB. The adult human SVZ, in contrast, contains a hypocellular gap layer separating the ependymal lining from a periventricular ribbon of astrocytes7. Some of these SVZ astrocytes can function as neural stem cells in vitro, but their function in vivo remains controversial. An initial report finds few SVZ proliferating cells and rare migrating immature neurons in the RMS of adult humans7. In contrast, a subsequent study indicates robust proliferation and migration in the human SVZ and RMS8,9. Here, we find that the infant human SVZ and RMS contain an extensive corridor of migrating immature neurons before 18 months of age, but, contrary to previous reports8, this germinal activity subsides in older children and is nearly extinct by adulthood. Surprisingly, during this limited window of neurogenesis, not all new neurons in the human SVZ are destined for the OB – we describe a major migratory pathway that targets the prefrontal cortex in humans. Together, these findings reveal robust streams of tangentially migrating immature neurons in human early postnatal SVZ and cortex. These pathways represent potential targets of neurological injuries affecting neonates.

Activated microglia control neurogenesis and gliogenesis in the early postnatal subventricular zone via production of inflammatory cytokines

3D organoids enable in vitro human brain development models, but they have not yet recapitulated some essential features of brain circuit formation. Recently, several studies appearing in Nature, Nature Methods, and Cell Stem Cell generated fused organoid models of inhibitory and excitatory neuron development, which can now achieve functional circuit integration.

Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression

Oligodendrocyte precursor cells (OPCs) are lineage-restricted progenitors generally limited in vivo to producing oligodendrocytes. Mechanisms controlling genesis of OPCs are of interest because of their importance in myelin development and their potential for regenerative therapies in multiple sclerosis and dysmyelinating syndromes. We show here that the SoxE transcription factors (comprising Sox8, 9, and 10) induce multipotent neural precursor cells (NPCs) from the early postnatal subventricular zone (SVZ) to become OPCs in an autonomous manner. We performed a chromatin immunoprecipitation-based bioinformatic screen and identified Suppressor of Fused (Sufu) as a direct target of repression by Sox10. In vitro, overexpression of Sufu blocked OPC production, whereas RNAi-mediated inhibition augmented OPC production. Furthermore, mice heterozygous for Sufu have increased numbers of OPCs in the telencephalon during development. We conclude that Sox10 acts to restrict the potential of NPCs toward the oligodendrocyte lineage in part by regulating the expression of Sufu.

Gap junctions are involved in cell migration in the early postnatal subventricular zone

The massive migration of neuroblasts and young neurons through the anterior extension of the postnatal subventricular zone (SVZ), known as the rostral migratory stream (RMS) is still poorly understood on its molecular basis. In this work, we investigated the involvement of gap junctional communication (GJC) in the robust centrifugal migration from SVZ/RMS explants obtained from early postnatal (P4) rats. Cells were dye-coupled in homocellular and heterocellular pairings and expressed at least two connexins, Cx 43 and 45. Treatment with the uncoupler agent carbenoxolone (CBX, 10-100 microM) reversibly reduced outgrowth from SVZ explants, while its inactive analog, glycyrhizinic acid (GZA), had no effect. Consistent with a direct effect on cell migration, time-lapse video microscopy show that different pharmacological uncouplers cause an abrupt and reversible arrest of cell movement in explants. Our results indicate that GJC is positively involved in the migration of neuroblasts within the SVZ/RMS.

Olig2 Directs Astrocyte and Oligodendrocyte Formation in Postnatal Subventricular Zone Cells

The subventricular zone (SVZ) in the neonatal mammalian forebrain simultaneously generates olfactory interneurons, astrocytes, and oligodendrocytes. The molecular cues that enable SVZ progenitors to generate three distinct cell lineages without a temporal switching mechanism are not known. Here, we demonstrate that the basic helix-loop-helix transcription factor Olig2 plays a central role in this process. Olig2 is specifically expressed in gliogenic progenitors in the postnatal SVZ and by all glial lineages derived from this structure. By expressing normal and dominant-interfering forms of Olig2 in vivo, we show that Olig2 repressor function is both sufficient and necessary to prevent neuronal differentiation and to direct SVZ progenitors toward astrocytic and oligodendrocytic fates. Although Olig2 activity has been associated previously with motor neuron and oligodendrocyte development, our findings establish a previously unappreciated role for Olig2 in the development of astrocytes. Furthermore, these results indicate that Olig2 serves a critical role in pan-glial versus neuronal fate decisions in SVZ progenitors, making it the first intrinsic fate determinant shown to operate in the early postnatal SVZ.

Mash1 specifies neurons and oligodendrocytes in the postnatal brain

Progenitors in the telencephalic subventricular zone (SVZ) remain mitotically active throughout life, and produce different cell types at embryonic, postnatal and adult stages. Here we show that Mash1, an important proneural gene in the embryonic telencephalon, is broadly expressed in the postnatal SVZ, in progenitors for both neuronal and oligodendrocyte lineages. Moreover, Mash1 is required at birth for the generation of a large fraction of neuronal and oligodendrocyte precursors from the olfactory bulb. Clonal analysis in culture and transplantation experiments in postnatal brain demonstrate that this phenotype reflects a cell-autonomous function of Mash1 in specification of these two lineages. The conservation of Mash1 function in the postnatal SVZ suggests that the same transcription mechanisms operate throughout life to specify cell fates in this structure, and that the profound changes in the cell types produced reflect changes in the signalling environment of the SVZ.

Initial stages of radial glia astrocytic transformation in the early postnatal anterior subventricular zone.

In the early postnatal subventricular zone (SVZ), two seemingly unrelated events occur simultaneously: a massive tangential migration of neuroblasts towards the olfactory bulb, known as the rostral migratory stream (RMS), and the outward movement of radial glia (RG) undergoing astrocytic transformation. Because of the orthogonal arrangement between these two sets of cells, little, if any, relevance has been ascribed for their possible interactions. By depositing DiI at the pial surface we have studied RG transformation within the SVZ/RMS, from birth up to the end of the first postnatal week. While still within the SVZ/RMS, RG morphology changed from simple bipolar to highly complex branched profiles, attaining their highest degree of complexity at the interface of the SVZ with the overlying white matter. At this interface cell bodies of radial glia accumulate and their processes run tangentially, surrounding the SVZ/RMS. Processes of RG surrounding the SVZ/RMS could also be observed by immunostaining for vimentin, GFAP, and nestin. In contrast, in the white matter all DiI-labeled RG presented a simple bipolar profile. These results indicate that the outward radial migration of the transforming RG does not occur uniformly. Instead, the different morphologies and cell densities that RG assume when they cross the SVZ/RMS and overlying white matter imply different migratory behaviors. Finally, our data suggest that RG provide a cellular scaffold to the early postnatal SVZ/RMS, much in the same way as astrocytes in the adult RMS.