Kirsten Obernier

Embryonic origin of postnatal neural stem cells

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice

Significance The time taken by neural stem cells and intermediate progenitor cells to transit through the cell cycle, and number of times they divide, is essential information to understand how new neurons are produced in the adult rodent brain. Proliferating neural stem cells and intermediate progenitors persist in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain. This extensive germinal layer in the walls of the lateral ventricles is the site of birth of different types of interneurons destined for the olfactory bulb. The cell cycle dynamics of stem cells (B1 cells), intermediate progenitors (C cells), and neuroblasts (A cells) in the V-SVZ and the number of times these cells divide remain unknown. Using whole mounts of the walls of the lateral ventricles of adult mice and three cell cycle analysis methods using thymidine analogs, we determined the proliferation dynamics of B1, C, and A cells in vivo. Achaete-scute complex homolog (Ascl)1+ C cells were heterogeneous with a cell cycle length (TC) of 18–25 h and a long S phase length (TS) of 14–17 h. After C cells, Doublecortin+ A cells were the second-most common dividing cell type in the V-SVZ and had a TC of 18 h and TS of 9 h. Human glial fibrillary acidic protein (hGFAP)::GFP+ B1 cells had a surprisingly short Tc of 17–18 h and a TS of 4 h. Progenitor population analysis suggests that following the initial division of B1 cells, C cells divide three times and A cells once, possibly twice. These data provide essential information on the dynamics of adult progenitor cell proliferation in the V-SVZ and how large numbers of new neurons continue to be produced in the adult mammalian brain.

Molecular Diversity Subdivides the Adult Forebrain Neural Stem Cell Population

Neural stem cells (NSCs) in the ventricular domain of the subventricular zone (V‐SVZ) of rodents produce neurons throughout life while those in humans become largely inactive or may be lost during infancy. Most adult NSCs are quiescent, express glial markers, and depend on Notch signaling for their self‐renewal and the generation of neurons. Using genetic markers and lineage tracing, we identified subpopulations of adult V‐SVZ NSCs (type 1, 2, and 3) indicating a striking heterogeneity including activated, brain lipid binding protein (BLBP, FABP7) expressing stem cells. BLBP+ NSCs are mitotically active components of pinwheel structures in the lateral ventricle walls and persistently generate neurons in adulthood. BLBP+ NSCs express epidermal growth factor (EGF) receptor, proliferate in response to EGF, and are a major clonogenic population in the SVZ. We also find BLBP expressed by proliferative ventricular and subventricular progenitors in the fetal and postnatal human brain. Loss of BLBP+ stem/progenitor cells correlates with reduced neurogenesis in aging rodents and postnatal humans. These findings of molecular heterogeneity and proliferative differences subdivide the NSC population and have implications for neurogenesis in the forebrain of mammals during aging. Stem Cells 2014;32:70–84

Axonal Control of the Adult Neural Stem Cell Niche

The ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brains neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C.

Primary cilia are required in a unique subpopulation of neural progenitors

Significance The primary cilium, an antenna-like extension on the surface of many cells, is considered essential for growth factor and morphogen reception and transduction. Here we asked, what is the function of primary cilia that projects from the apical surface of neural stem cells (NSCs) into the ventricle? Surprisingly, the removal of primary cilia from fetal and early postnatal NSCs had little effect on forebrain development. Primary cilia, however, were essential for the function of NSCs in a restricted region of a major postnatal germinal niche, the ventricular–subventricular zone. Our data are consistent with primary cilia being essential for the normal proliferation of a subset of Hedgehog-responsive postnatal progenitors. The apical domain of embryonic (radial glia) and adult (B1 cells) neural stem cells (NSCs) contains a primary cilium. This organelle has been suggested to function as an antenna for the detection of morphogens or growth factors. In particular, primary cilia are essential for Hedgehog (Hh) signaling, which plays key roles in brain development. Their unique location facing the ventricular lumen suggests that primary cilia in NSCs could play an important role in reception of signals within the cerebrospinal fluid. Surprisingly, ablation of primary cilia using conditional alleles for genes essential for intraflagellar transport [kinesin family member 3A (Kif3a) and intraflagellar transport 88 (Ift88)] and Cre drivers that are activated at early [Nestin; embryonic day 10.5 (E10.5)] and late [human glial fibrillary acidic protein (hGFAP); E13.5] stages of mouse neural development resulted in no apparent developmental defects. Neurogenesis in the ventricular–subventricular zone (V-SVZ) shortly after birth was also largely unaffected, except for a restricted ventral domain previously known to be regulated by Hh signaling. However, Kif3a and Ift88 genetic ablation also disrupts ependymal cilia, resulting in hydrocephalus by postnatal day 4. To directly study the role of B1 cells’ primary cilia without the confounding effects of hydrocephalus, we stereotaxically targeted elimination of Kif3a from a subpopulation of radial glia, which resulted in ablation of primary cilia in a subset of B1 cells. Again, this experiment resulted in decreased neurogenesis only in the ventral V-SVZ. Primary cilia ablation led to disruption of Hh signaling in this subdomain. We conclude that primary cilia are required in a specific Hh-regulated subregion of the postnatal V-SVZ.

Analysis of stem cell lineage progression in the neonatal subventricular zone identifies EGFR+/NG2- cells as transit-amplifying precursors.

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. STEM CELLS 2009;27:1443–1454

Interaction between DLX2 and EGFR regulates proliferation and neurogenesis of SVZ precursors.

In the postnatal subventricular zone (SVZ) neural stem cells (NSCs) give rise to transit-amplifying precursors (TAPs) expressing high levels of epidermal growth factor receptor (EGFR) that in turn generate neuroblasts. Both TAPs and neuroblasts express distal-less (DLX)2 homeobox transcription factor but the latter proliferate less. Modulation of its expression in vivo has revealed that DLX2 affects both neurogenesis and proliferation in the postnatal SVZ. However, the mechanisms underlying these effects are not clear. To investigate this issue we have here forced the expression of DLX2 in SVZ isolated NSCs growing in defined in vitro conditions. This analysis revealed that DLX2 affects the proliferation of SVZ precursors by regulating two distinct steps of neural lineage progression. Firstly, it promotes the lineage transition from NSCs to TAPs. Secondly it enhances the proliferative response of neuronal progenitors to EGF. Thus DLX2 and EGFR signalling interact at multiple levels to coordinate proliferation in the postnatal SVZ.

Restricted nature of adult neural stem cells: re-evaluation of their potential for brain repair.

Neural stem cells (NSCs) in the walls of the lateral ventricles continue to produce new neurons and oligodendrocytes throughout life. The identification of NSCs, long-range neuronal migration, and the integration of new neurons into fully formed mature neural circuits—all in the juvenile or adult brain—has dramatically changed concepts in neurodevelopment and suggests new strategies for brain repair. Yet, the latter has to be seen in perspective: NSCs in the adult are heterogeneous and highly regionally specified; young neurons derived from these primary progenitors migrate and integrate in specific brain regions. Neurogenesis appears to have a function in brain plasticity rather than brain repair. If similar processes could be induced in regions of the brain that are normally not a target of new neurons, therapeutic neuronal replacement may one day reinstate neural circuit plasticity and possibly repair broken neural circuits.

Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein–positive Acta2+ SMC population of 95% purity was isolated. Acta2+ SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca2+-activated potassium channel. Culturing of Acta2+ SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell–derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.

GABAA Receptor Signaling Induces Osmotic Swelling and Cell Cycle Activation of Neonatal Prominin+ Precursors

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. STEM CELLS 2011;29:307–319