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Late in neural development, the expression of growth/differentiation factor (GDF) 15 increases in the germinal epithelium of the murine ganglionic eminence (GE) especially in progenitors with characteristics of neural stem cells (NSCs) and expressing high levels of epidermal growth factor receptor (EGFRh). However, the function of GDF15 in this region is unknown. We here show that apical EGFR immunopositive progenitors in the embryonic GE also express the receptor for GDF15 and that ablation of the latter affects the number and cell division dynamics of apically and subapically dividing progenitors. Apical proliferation is increased also in the adult mutant ventricular-subventricular zone (V-SVZ), which displays more ependymal and apical NSCs than the WT counterpart. In addition, we observed a transient increase in the number of neuronal progenitors, which was compensated by increased apoptosis. From a mechanistic point of view, we show that active EGFR is essential to maintain proliferation in the developing GE and that GDF15 affects EGFR trafficking and signal transduction. Consistent with a direct involvement of GDF15, exposure of the GE to the growth factor normalized proliferation and EGFR expression and it decreased the number of apical progenitors. A similar decrease in the number of apical progenitors was also observed upon exposure to exogenous EGF. However, this effect was not associated with reduced proliferation, illustrating the complexity of the effect of GDF15. Taken together, our results indicate that GDF15 modulates proliferation and EGF responsiveness of apical progenitors in the developing GE, thereby regulating the number of total ependymal and NSCs.

The glycoprotein Prominin-1 and the carbohydrate Lewis X stage-specific embryonic antigen 1 (LeX-SSEA1) both have been extensively used as cell surface markers to purify neural stem cells (NSCs). While Prominin-1 labels a specialized membrane region in NSCs and ependymal cells, the specificity of LeX-SSEA1 expression and its biological significance are still unknown. To address these issues, we have here monitored the expression of the carbohydrate in neonatal and adult NSCs and in their progeny. Our results show that the percentage of immunopositive cells and the levels of LeX-SSEA1 immunoreactivity both increase with postnatal age across all stages of the neural lineage. This is associated with decreased proliferation in precursors including NSCs, which accumulate the carbohydrate at the cell surface while remaining quiescent. Exposure of precursors to bone morphogenetic protein (BMP) increases LEX-SSEA1 expression, which promotes cell cycle withdrawal by a mechanism involving LeX-SSEA1-mediated interaction at the cell surface. Conversely, interference with either BMP signaling or with LeX-SSEA1 promotes proliferation to a similar degree. Thus, in the postnatal germinal niche, the expression of LeX-SSEA1 increases with age and exposure to BMP signaling, thereby downregulating the proliferation of subependymal zone precursors including NSCs.

Summary In the adult subependymal zone (SEZ), neural stem cells (NSCs) apically contacting the lateral ventricle on activation generate progenitors proliferating at the niche basal side. We here show that Tailless (TLX) coordinates NSC activation and basal progenitor proliferation by repressing the NOTCH effector Hes1. Consistent with this, besides quiescence Hes1 expression also increases on Tlx mutation. Since HES1 levels are higher at the apical SEZ, NOTCH activation is increased in Tlx−/− NSCs, but not in surrounding basal progenitors. Underscoring the causative relationship between higher HES1/NOTCH and increased quiescence, downregulation of Hes1 only in mutant NSCs normalizes NOTCH activation and resumes proliferation and neurogenesis not only in NSCs, but especially in basal progenitors. Since pharmacological blockade of NOTCH signaling also promotes proliferation of basal progenitors, we conclude that TLX, by repressing Hes1 expression, counteracts quiescence and NOTCH activation in NSCs, thereby relieving NOTCH-mediated lateral inhibition of proliferation in basal progenitors., Graphical Abstract, Highlights • TLX autonomously controls quiescence in apical NSCs by repressing Hes1 • TLX controls basal progenitor proliferation via NOTCH-mediated lateral inhibition • Downregulation of Hes1 in apical Tlx−/− NSCs resumes proliferation and neurogenesis, Ciccolini and colleagues show that Tailless (TLX) affects quiescence and lineage progression by cell-autonomous and non-cell-autonomous mechanisms. In apical neural stem cells (NSCs), TLX downregulates Hes1, thereby decreasing NOTCH signaling. This promotes transcription of the proneural gene Mash1 and NSC activation. Moreover, it also decreases NOTCH-mediated lateral inhibition of basal progenitor proliferation.

In the adult mammalian brain, the apical surface of the subependymal zone (SEZ) is covered by many motile ependymal cilia and a few primary cilia originating from rare intermingled neural stem cells (NSCs). In NSCs the primary cilia are key for the transduction of essential extracellular signals such as Sonic hedgehog (SHH) and platelet-derived growth factor (PDGF). Despite their importance, the analysis of NSC primary cilia is greatly hampered by the fact that they are overwhelmingly outnumbered by the motile cilia. We here take advantage of flow cytometry to purify the two cilia types and allow their molecular characterization. Primary cilia were identified based on immunoreactivity to the marker adenylate cyclase type III (AC3) and differential levels of prominin-1 whereas motile cilia displayed immunoreactivity only to the latter. Consistent with the morphological differences between the two classes of cilia, enrichment of motile cilia positively correlated with size. Moreover, we observed age-dependent variations in the abundance of the two groups of ciliary organelles reflecting the changes associated with their development. The two cilia groups also differed with respect to the expression of signaling molecules, since PDGF receptor (PDGFR)α, smoothened (Smo) and CXC chemokine receptor (CXCR)4 were only detected in isolated primary but not motile cilia. Thus, our novel method of cilia isolation and characterization by flow cytometry has the potential to be extended to the study of cilia from different tissues and organs, providing a powerful tool for the investigation of primary cilia in physiological and pathological conditions.

Activation of γ-aminobutyric A receptors (GABAARs) in the subependymal zone (SEZ) induces hyperpolarization and osmotic swelling in precursors, thereby promoting surface expression of the epidermal growth factor receptor (EGFR) and cell cycle entry. However, the mechanisms underlying the GABAergic modulation of cell swelling are unclear. Here, we show that GABAARs colocalize with the water channel aquaporin (AQP) 4 in prominin-1 immunopositive (P+) precursors in the postnatal SEZ, which include neural stem cells. GABAAR signaling promotes AQP4 expression by decreasing serine phosphorylation associated with the water channel. The modulation of AQP4 expression by GABAAR signaling is key to its effect on cell swelling and EGFR expression. In addition, GABAAR function also affects the ability of neural precursors to swell in response to an osmotic challenge in vitro and in vivo. Thus, the regulation of AQP4 by GABAARs is involved in controlling activation of neural stem cells and water exchange dynamics in the SEZ.

Niche homeostasis in the postnatal subependymal zone of the lateral ventricle (lSEZ) requires coordinated proliferation and differentiation of neural progenitor cells. The mechanisms regulating this balance are scarcely known. Recent observations indicate that the orphan nuclear receptor Tlx is an intrinsic factor essential in maintaining this balance. However, the effect of Tlx on gene expression depends on age and cell-type cues. Therefore, it is essential to establish its expression pattern at different developmental ages. Here, we show for the first time that in the neonatal lSEZ activated neural stem cells (NSCs) and especially transit-amplifying progenitors (TAPs) express Tlx and that its expression may be regulated at the posttranscriptional level. We also provide evidence that in both cell types Tlx affects gene expression in a positive and negative manner. In activated NSCs, but not in TAPs, absence of Tlx leads to overexpression of negative cell cycle regulators and impairment of proliferation. Moreover, in both cell types, the homeobox transcription factor Dlx2 is downregulated in the absence of Tlx. This is paralleled by increased expression of Olig2 in activated NSCs and glial fibrillary acidic protein in TAPs, indicating that in both populations Tlx decreases gliogenesis. Consistent with this, we found a higher proportion of cells expressing glial makers in the neonatal lSEZ of mutant mice than in the wild type counterpart. Thus, Tlx playing a dual role affects the expression of distinct genes in these two lSEZ cell types.

In the adult subventricular zone (SVZ), astroglial stem cells generate transit-amplifying precursors (TAPs). Both stem cells and TAPs form clones in response to epidermal growth factor (EGF). However, in vivo, in the absence of sustained EGF receptor (EGFR) activation, TAPs divide a few times before differentiating into neuroblasts. The lack of suitable markers has hampered the analysis of stem cell lineage progression and associated functional changes in the neonatal germinal epithelium. Here we purified neuroblasts and clone-forming precursors from the neonatal SVZ using expression levels of EGFR and polysialylated neural cell adhesion molecule (PSANCAM). As in the adult SVZ, most neonatal clone-forming precursors did not express the neuroglia proteoglycan 2 (NG2) but displayed characteristics of TAPs, and only a subset exhibited antigenic characteristics of astroglial stem cells. Both precursors and neuroblasts were PSANCAM+; however, neuroblasts also expressed doublecortin and functional voltage-dependent Ca2+ channels. Neuroblasts and precursors had distinct outwardly rectifying K+ current densities and passive membrane properties, particularly in precursors contacting each other, because of the contribution of gap junction coupling. Confirming the hypothesis that most are TAPs, cell tracing in brain slices revealed that within 2 days the majority of EGFR+ cells had exited the cell cycle and differentiated into a progenitor displaying intermediate antigenic and functional properties between TAPs and neuroblasts. Thus, distinct functional and antigenic properties mark stem cell lineage progression in the neonatal SVZ. Disclosure of potential conflicts of interest is found at the end of this article.

A simple procedure to isolate neural stem cells would greatly facilitate direct studies of their properties. Here, we exploited the increase in EGF receptor (EGFR) levels, that occurs in late development stem cells or in younger precursors upon exposure to FGF-2, to isolate cells expressing high levels of EGFR (EGFR(high)) from the developing and the adult brain. Independently of age and region of isolation, EGFR(high) cells were highly enriched in multipotent precursors and displayed similar antigenic characteristics, with the exception of GFAP and Lex/SSEA-1 that were mainly expressed in adult EGFR(high) cells. EGFR levels did not correlate with neurogenic potential, indicating that the increase in EGFR expression does not directly affect differentiation. Instead, in the brain, many EGFR(high) precursors showed tangential orientation and, whether isolated from the cortex or striatum, EGFR(high) precursors displayed characteristics of cells originating from the ventral GZ such as expression Dlx and Mash-1 and the ability to generate GABAergic neurons and oligodendrocytes. Moreover, migration of EGFR(high) cells on telencephalic slices required EGFR activity. Thus, the developmentally regulated increase in EGFR levels may affect tangential migration of multipotent precursors. In addition, it can be used as a marker to effectively isolate telencephalic multipotent precursors from embryonic and adult tissue.

The transcription factor CREB (cAMP-response element binding protein) regulates differentiation, migration, survival and activity-dependent gene expression in the developing and mature nervous system. However, its specific role in the proliferation of embryonic neural progenitors is still not completely understood. Here we investigated how CREB regulates proliferation of mouse embryonic neural progenitors by a conditional mutant lacking Creb gene in neural progenitors. In parallel, we explored possible compensatory effects by the genetic ablation of another member of the same gene family, the cAMP-responsive element modulator (Crem). We show that CREB loss differentially impaired the proliferation, clonogenic potential and self-renewal of precursors derived from the ganglionic eminence (GE), in comparison to those derived from the cortex. This phenotype was associated with a specific reduction of histone acetylation in the GE of CREB mutant mice, and this reduction was rescued in vivo by inhibition of histone deacetylation. These observations indicate that the impaired proliferation could be caused by a reduced acetyltransferase activity in Creb conditional knock-out mice. These findings support a crucial role of CREB in controlling embryonic neurogenesis and propose a novel mechanism by which CREB regulates embryonic neural development.

The activation of epidermal growth factor receptor (EGFR) affects multiple aspects of neural precursor behaviour, including proliferation and migration. Telencephalic precursors acquire EGF responsiveness and upregulate EGFR expression at late stages of development. The events regulating this process and its significance are still unclear. We here show that in the developing and postnatal hippocampus (HP), growth/differentiation factor (GDF) 15 and EGFR are co-expressed in primitive precursors as well as in more differentiated cells. We also provide evidence that GDF15 promotes responsiveness to EGF and EGFR expression in hippocampal precursors through a mechanism that requires active CXC chemokine receptor (CXCR) 4. Besides EGFR expression, GDF15 ablation also leads to decreased proliferation and migration. In particular, lack of GDF15 impairs both processes in the cornu ammonis (CA) 1 and only proliferation in the dentate gyrus (DG). Importantly, migration and proliferation in the mutant HP were altered only perinatally, when EGFR expression was also affected. These data suggest that GDF15 regulates migration and proliferation by promoting EGFR signalling in the perinatal HP and represent a first description of a functional role for GDF15 in the developing telencephalon.

Neural stem cells (NSCs) generate new neurons in vivo and in vitro throughout adulthood and therefore are physiologically and clinically relevant. Unveiling the mechanisms regulating the lineage progression from NSCs to newborn neurons is critical for the transition from basic research to clinical application. However, the direct analysis of NSCs and their progeny is still elusive due to the problematic identification of the cells. We here describe the isolation of highly purified genetically unaltered NSCs and transit-amplifying precursors (TAPs) from the adult subependymal zone (SEZ). Using this approach we show that a primary cilium and high levels of epidermal growth factor receptor (EGFR) at the cell membrane characterize quiescent and cycling NSCs, respectively. However, we also observed non-ciliated quiescent NSCs and NSCs progressing into the cell cycle without up-regulating EGFR expression. Thus, the existence of NSCs displaying distinct molecular and structural conformations provides more flexibility to the regulation of quiescence and cell cycle progression.

Signal-regulated changes in cell size affect cell division and survival and therefore are central to tissue morphogenesis and homeostasis. In this respect, GABA receptors (GABAARs) are of particular interest because allowing anions flow across the cell membrane modulates the osmolyte flux and the cell volume. Therefore, we have here investigated the hypothesis that GABA may regulate neural stem cell proliferation by inducing cell size changes. We found that, besides neuroblasts, also neural precursors in the neonatal murine subependymal zone sense GABA via GABAARs. However, unlike in neuroblasts, where it induced depolarization-mediated [Ca2+]i increase, GABAARs activation in precursors caused hyperpolarization. This resulted in osmotic swelling and increased surface expression of epidermal growth factor receptors (EGFRs). Furthermore, activation of GABAARs signaling in vitro in the presence of EGF modified the expression of the cell cycle regulators, phosphatase and tensin homolog and cyclin D1, increasing the pool of cycling precursors without modifying cell cycle length. A similar effect was observed on treatment with diazepam. We also demonstrate that GABA and diazepam responsive precursors represent prominin+ stem cells. Finally, we show that as in in vitro also in in vivo a short administration of diazepam promotes EGFR expression in prominin+ stem cells causing activation and cell cycle entry. Thus, our data indicate that endogenous GABA is a part of a regulatory mechanism of size and cell cycle entry of neonatal stem cells. Our results also have potential implications for the therapeutic practices that involve exposure to GABAARs modulators during neurodevelopment.