Astrogenesis in the murine dentate gyrus is a life‐long and dynamic process
2022; Springer Nature; Volume: 41; Issue: 11 Linguagem: Inglês
10.15252/embj.2021110409
ISSN1460-2075
AutoresJúlia Schneider, Johannes Weigel, Marie‐Theres Wittmann, Pavel Švehla, Sebastian Ehrt, Fang Zheng, Tarek Elmzzahi, Julian Karpf, Lucía Paniagua‐Herranz, Onur Basak, Arif B. Ekici, André Reis, Christian Alzheimer, Felipe Ortega, Sabine Liebscher, Ruth Beckervordersandforth,
Tópico(s)Anesthesia and Neurotoxicity Research
ResumoArticle22 April 2022Open Access Source DataTransparent process Astrogenesis in the murine dentate gyrus is a life-long and dynamic process Julia Schneider Julia Schneider orcid.org/0000-0002-2128-7392 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Johannes Weigel Johannes Weigel Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Marie-Theres Wittmann Marie-Theres Wittmann orcid.org/0000-0003-3924-7572 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Pavel Svehla Pavel Svehla orcid.org/0000-0002-5680-3895 Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany Graduate School of Systemic Neurosciences, Ludwig-Maximilians University Munich, Munich, Germany Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Sebastian Ehrt Sebastian Ehrt Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany Contribution: Investigation Search for more papers by this author Fang Zheng Fang Zheng Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation Search for more papers by this author Tarek Elmzzahi Tarek Elmzzahi orcid.org/0000-0003-1168-8114 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Department of Molecular Immunology in Neurodegeneration, German Centre for Neurodegenerative Diseases Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Julian Karpf Julian Karpf Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation Search for more papers by this author Lucía Paniagua-Herranz Lucía Paniagua-Herranz Department of Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain Instituto de Investigación Sanitaria San Carlos (IdISSC), Spain Contribution: Investigation Search for more papers by this author Onur Basak Onur Basak Department of Translational Neuroscience, University Medical Centre Utrecht (UMCU), Utrecht, Netherlands Contribution: Investigation Search for more papers by this author Arif Ekici Arif Ekici orcid.org/0000-0001-6099-7066 Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation Search for more papers by this author Andre Reis Andre Reis orcid.org/0000-0002-6301-6363 Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Christian Alzheimer Christian Alzheimer Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Felipe Ortega de la O Felipe Ortega de la O orcid.org/0000-0001-6398-9520 Department of Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain Instituto de Investigación Sanitaria San Carlos (IdISSC), Spain Contribution: Resources, Supervision, Funding acquisition Search for more papers by this author Sabine Liebscher Sabine Liebscher orcid.org/0000-0001-5633-8981 Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany Graduate School of Systemic Neurosciences, Ludwig-Maximilians University Munich, Munich, Germany Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Contribution: Resources, Supervision, Funding acquisition Search for more papers by this author Ruth Beckervordersandforth Corresponding Author Ruth Beckervordersandforth [email protected] orcid.org/0000-0002-3617-7567 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Julia Schneider Julia Schneider orcid.org/0000-0002-2128-7392 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Johannes Weigel Johannes Weigel Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Marie-Theres Wittmann Marie-Theres Wittmann orcid.org/0000-0003-3924-7572 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Pavel Svehla Pavel Svehla orcid.org/0000-0002-5680-3895 Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany Graduate School of Systemic Neurosciences, Ludwig-Maximilians University Munich, Munich, Germany Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Sebastian Ehrt Sebastian Ehrt Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany Contribution: Investigation Search for more papers by this author Fang Zheng Fang Zheng Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation Search for more papers by this author Tarek Elmzzahi Tarek Elmzzahi orcid.org/0000-0003-1168-8114 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Department of Molecular Immunology in Neurodegeneration, German Centre for Neurodegenerative Diseases Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Julian Karpf Julian Karpf Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation Search for more papers by this author Lucía Paniagua-Herranz Lucía Paniagua-Herranz Department of Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain Instituto de Investigación Sanitaria San Carlos (IdISSC), Spain Contribution: Investigation Search for more papers by this author Onur Basak Onur Basak Department of Translational Neuroscience, University Medical Centre Utrecht (UMCU), Utrecht, Netherlands Contribution: Investigation Search for more papers by this author Arif Ekici Arif Ekici orcid.org/0000-0001-6099-7066 Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation Search for more papers by this author Andre Reis Andre Reis orcid.org/0000-0002-6301-6363 Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Christian Alzheimer Christian Alzheimer Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Felipe Ortega de la O Felipe Ortega de la O orcid.org/0000-0001-6398-9520 Department of Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain Instituto de Investigación Sanitaria San Carlos (IdISSC), Spain Contribution: Resources, Supervision, Funding acquisition Search for more papers by this author Sabine Liebscher Sabine Liebscher orcid.org/0000-0001-5633-8981 Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany Graduate School of Systemic Neurosciences, Ludwig-Maximilians University Munich, Munich, Germany Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Contribution: Resources, Supervision, Funding acquisition Search for more papers by this author Ruth Beckervordersandforth Corresponding Author Ruth Beckervordersandforth [email protected] orcid.org/0000-0002-3617-7567 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Julia Schneider1, Johannes Weigel1, Marie-Theres Wittmann1,2, Pavel Svehla3,4,5, Sebastian Ehrt3,5, Fang Zheng6, Tarek Elmzzahi1,7, Julian Karpf1, Lucía Paniagua-Herranz8,9,10, Onur Basak11, Arif Ekici2, Andre Reis2, Christian Alzheimer6, Felipe Ortega de la O8,9,10, Sabine Liebscher3,4,5,12 and Ruth Beckervordersandforth *,1 1Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 2Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 3Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany 4Graduate School of Systemic Neurosciences, Ludwig-Maximilians University Munich, Munich, Germany 5Medical Faculty, BioMedical Center, Ludwig-Maximilians University Munich, Munich, Germany 6Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 7Department of Molecular Immunology in Neurodegeneration, German Centre for Neurodegenerative Diseases Bonn, Bonn, Germany 8Department of Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain 9Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain 10Instituto de Investigación Sanitaria San Carlos (IdISSC), Spain 11Department of Translational Neuroscience, University Medical Centre Utrecht (UMCU), Utrecht, Netherlands 12Munich Cluster for Systems Neurology (SyNergy), Munich, Germany *Corresponding author. Tel: +49 91318526206; E-mail: [email protected] The EMBO Journal (2022)41:e110409https://doi.org/10.15252/embj.2021110409 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Astrocytes are highly abundant in the mammalian brain, and their functions are of vital importance for all aspects of development, adaption, and aging of the central nervous system (CNS). Mounting evidence indicates the important contributions of astrocytes to a wide range of neuropathies. Still, our understanding of astrocyte development significantly lags behind that of other CNS cells. We here combine immunohistochemical approaches with genetic fate-mapping, behavioural paradigms, single-cell transcriptomics, and in vivo two-photon imaging, to comprehensively assess the generation and the proliferation of astrocytes in the dentate gyrus (DG) across the life span of a mouse. Astrogenesis in the DG is initiated by radial glia-like neural stem cells giving rise to locally dividing astrocytes that enlarge the astrocyte compartment in an outside-in-pattern. Also in the adult DG, the vast majority of astrogenesis is mediated through the proliferation of local astrocytes. Interestingly, locally dividing astrocytes were able to adapt their proliferation to environmental and behavioral stimuli revealing an unexpected plasticity. Our study establishes astrocytes as enduring plastic elements in DG circuits, implicating a vital contribution of astrocyte dynamics to hippocampal plasticity. Synopsis Murine dentate gyrus astrocytes derive from neural stem cells and locally divide to establish the astrocytic niche during development. Here, astrogenesis is found to persist to old age, representing a dynamic process mostly driven by local astrocyte proliferation. During postnatal development, astrocytes in the murine dentate gyrus derive from perinatal radial glia-like neural stem cells, and locally proliferate to generate the astrocyte compartment in an outside-in-pattern. Dentate gyrus astrogenesis persists until adulthood and is predominantly driven by locally proliferating astrocytes. Adult astrogenesis is a dynamic process that contributes to a fixed neuron-to-astrocyte ratio within the adult dentate gyrus, which is preserved under pro-neurogenic stimuli. While neurogenesis rapidly declines upon aging, astrogenesis diminishes only much later in life leading to a shift in the neuron-to-astrocyte ratio towards astrogenesis. Introduction Development, function, and plasticity of neural networks require a fine-tuned orchestration of neurons and glial cells, each of which contributes about 50% to the total cell number of the brain (Herculano-Houzel, 2014). Neurons are considered the key active components in neural circuits facilitating information processing to foster complex processes, such as learning and memory, cognition, and mood through the propagation of electrical signals. Astrocytes, together with oligodendrocytes, belong to the class of macroglia and are vital players regulating the brain's metabolism, ion homeostasis, and the cross-talk to the periphery by contributing to the formation of the blood–brain barrier (Barres, 2008; Verkhratsky & Nedergaard, 2018). In recent years, it has become evident that astrocytes are in fact more than simply passive bystanders in the brain, but they actively contribute to information processing, by shaping and regulating synaptic connectivity and plasticity, as well as by releasing neurotransmitters and gliotransmitters themselves (Ballabh et al, 2004; Attwell et al, 2010; Eroglu & Barres, 2010; Araque et al, 2014; Chung et al, 2016). Several lines of evidence argue for an important contribution of astrocytes to neuropsychiatric and neurodegenerative diseases (Verkhratsky & Parpura, 2016). In sharp contrast to neurons and oligodendrocytes, and despite the astrocytes importance and abundance in the CNS, very little is known about how astrocytes are generated. Astrogenesis has almost exclusively been studied in the developing cortex and spinal cord (Ge et al, 2012; Tien et al, 2012; Shen et al, 2021), where embryonic neural stem cells (NSCs) called radial glia cells give rise first to neurons and then to glia cells. This so-called neurogenic-to-gliogenic switch occurs during late embryonic development [embryonic day 16.5 (E16.5); (Miller & Gauthier, 2007)] and relies on tightly interlaced mechanisms, which initially repress astrogenesis during the neurogenic phase and later on promote it during peri- and postnatal life (gliogenic phase). Proper neocortical glial expansion emerges postnatally, when seemingly differentiated astrocytes divide symmetrically to generate new astrocytes (Ge et al, 2012; Clavreul et al, 2019). After postnatal development, astrogenesis decays in most brain regions and predominantly persists in neurogenic niches, specialized areas of the brain in which new neurons and glial cells are generated throughout life (Alvarez-Buylla & Lim, 2004; Bond et al, 2015; Kempermann et al, 2015). The dentate gyrus (DG) of the hippocampus is one of those neurogenic regions in the adult brain and represents an excellent model system to study the process of astrogenesis from development to aging. The hippocampus, also known as the archicortex, emanates from the dentate neuroepithelium. At late gestational stages, a heterogeneous mixture of neural stem and precursor cells migrates away from the ventricular zone toward the hippocampal fissure, where neural progenitors accumulate and form the anlage of the future DG. At postnatal week two, proliferation becomes more restricted to the developing subgranular zone (SGZ), where radial glia-like NSCs (rNSCs) reside throughout life (Pleasure et al, 2000; Urbán & Guillemot, 2014). The generation and integration of new neurons into existing circuitries is a central process to hippocampal plasticity and involved in higher cognitive functions, such as learning, memory formation, and mood regulation (Ming & Song, 2011; Kempermann et al, 2015). Notably, adult neurogenesis has been reported across mammalian species (Patzke et al, 2015), but its existence in the human brain is a currently highly debated topic with conflicting results (Spalding et al, 2013; Boldrini et al, 2018; Cipriani et al, 2018; Sorrells et al, 2018; Moreno-Jiménez et al, 2019; Seki et al, 2019; Tobin et al, 2019; Franjic et al, 2022). Adult neurogenesis is highly dynamic and can be promoted by physiological stimuli, such as voluntary exercise, which significantly increases the proliferation of neural progenitor cells (van Praag et al, 1999b). During aging, neurogenesis gradually declines due to rNSC pool exhaustion and decreased proliferation of neuronal progenitors until it is fully ceased in aged mice (Kuhn et al, 1996; Seki, 2002; Bondolfi et al, 2004; Garcia et al, 2004; Heine et al, 2004; Encinas et al, 2011). Astrocytes are key components of the hippocampal neurogenic niche, and multiple lines of evidence suggest a major impact of astrocytes on DG plasticity by positively regulating rNSCs function and neuronal maturation (Lie et al, 2005; Song et al, 2012; Ehret et al, 2015; Sultan et al, 2015; Schneider et al, 2019). While adult rNSCs and neurogenesis have been in the focus of attention over the last two decades, the generation of astrocytes in the archicortex is a fundamentally understudied process. Like granule neurons, dentate astrocytes originate from developmental and adult rNSCs (Berg et al, 2019). Neurogenesis and astrogenesis in the developing DG occur simultaneously (Bond et al, 2020) in analogy to what has been recently suggested for neocortical development (Shen et al, 2021). During adult stages, astrocytes in the DG are continuously generated either by asymmetric division or trans-differentiation from rNSCs (Bonaguidi et al, 2011; Encinas et al, 2011). With the aim to gain insights into the mechanisms governing astrogenesis, we here assessed basic principles of astrocyte generation and dynamics in the DG throughout the life span of a mouse. Our data reveal that astrocytes originated from perinatal rNSCs, which give rise to proliferating astrocytes expanding the astrocyte compartment. We observed a temporal order in the generation of astrocytes located within different DG compartments, indicating an outside-in-pattern of astrogenesis, as shown already for granule neurons (Mathews et al, 2010). Importantly, our data revealed that the predominant source of newborn astrocytes in the adult DG are not rNSCs, but are locally dividing niche astrocytes, which are able to adapt their proliferation in response to pro- or anti-neurogenic stimuli (voluntary exercise and aging, respectively), thus revealing an unexpectedly high level of astrocyte plasticity. Results Astrogenesis in the developing murine DG To comprehensively understand how and when astrocytes are generated in the developing DG, we here combined genetic fate-mapping with thymidine analogue tracing and cell cycle marker analysis. To unambiguously determine the origin of DG astrocytes, we performed fate-mapping experiments using a tamoxifen-inducible mouse model [NestinCreERT2; (Yamaguchi et al, 2000)], in which the Cre recombinase is expressed in rNSCs. These mice were crossed to a reporter mouse line with a floxed STOP codon in front of a GFP [CAG CAT GFP; (Nakamura et al, 2006)] to monitor recombination events (Fig 1A). These mice will be from now on referred to as NestinCreERT2; GFP animals. Administration of tamoxifen allows to specifically define the timepoint at which the Cre recombinase can enter the nucleus, leading to the expression of the GFP reporter in rNSCs and their progeny. Since the first astrocytes have been reported to arise at the beginning of postnatal development (Bond et al, 2020), we specifically targeted perinatal rNSCs by tamoxifen administration at birth (Fig 1A and B) and analyzed their progeny at postnatal days 3 (P3), P7, P10, and P14 in distinct DG compartments, such as hilus, granular zone (GZ), and molecular layer (ML; Fig 1B). First, we specified whether the NestinCreERT2; GFP mouse line is a valid tool for rNSCs lineage tracing by determining its recombination efficiency and specificity (Fig EV1-EV4). The specificity (number of NESTIN+/GFP+ cells of all GFP+ cells) was 92.61% (Fig EV1A), showing that perinatal rNSCs were the main target of our recombination strategy. Due to their high density, it was impossible to reliably count the number of NESTIN+ cells during early postnatal DG stages. Since perinatal rNSCs were highly proliferative (50.53%; Fig EV1B), we instead estimated the recombination efficiency by assessing the number of proliferating GFP+ cells out of all proliferating cells expressing the cell cycle marker KI67 (Fig EV1C). Using this approach, we observed that recombination occurred in 63.21% of all proliferating cells, in line with previously observed recombination efficiencies in (adult) NESTIN+ rNSCs (Lagace et al, 2007). To identify astrocytes, we performed immunohistochemical stainings against glial fibrillary acidic protein (GFAP), the calcium-binding protein S100β and Acyl-CoA synthetase bubble gum family member 1 (ACSBG1), a marker for gray matter astrocytes in the developing brain (Li et al, 2012; Takeuchi et al, 2020). At P14 and P27, we observed an almost complete overlap between ACSBG1 and GFAP expression in the DG, confirming the specificity of ACSBG1 as an astrocytic marker also in the DG (Fig EV1D). To corroborate that astrocytes derive from NESTIN+ rNSCs, we searched for recombined cells, which downregulated NESTIN, but expressed astrocyte-specific marker. Indeed, we identified cells that co-expressed GFP and ACSBG1, GFAP, or S100β, indicating that NESTIN+ rNSCs give rise to astrocytes in the postnatal DG (Fig 1C). Of note, early postnatal astrocytes predominantly expressed ACSBG1, while GFAP and S100β expression was initiated later (Fig 1C). To investigate the timing of rNSC-derived astrogenesis, the total numbers of ACSBG1+/GFP+ cells were assessed at the specific timepoints (Fig 1D and E). In the first postnatal week, we observed only few ACSBG1+ astrocytes, some of which co-expressed the GFP reporter. The number of GFP+ astrocytes drastically increased at P10 and P14, revealing that rNSC-mediated astrogenesis mainly occurred in the second postnatal week (Fig 1D and E; Table EV1). We corroborated our findings using another astrocyte marker, the enzyme Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1; Fig EV1E). In the adult DG, we observed that distinct astrocyte subtypes populate different DG layers (ML, GZ, and hilus), where they exhibit a compartment-specific morphology (Beckervordersandforth et al, 2014). Furthermore, it has been reported that the developing DG is formed in an outside-in layering pattern (Mathews et al, 2010), in which embryonically derived cells contribute preferentially to the outer granule cell layer, while neurons born postnatally remain closer to the hilus (Angevine, 1965; Schlessinger et al, 1975; Bayer, 1980; Crespo et al, 1986). Thus, we next analyzed the time course of astrogenesis in distinct DG layers and observed that the ML was populated by large numbers of astrocytes already at P10, whereas in the GZ and hilus astrocytes appeared only in the subsequent days (Fig 1F; Table EV1). Hence, our data demonstrate that, in analogy to what has been reported for DG neurons (Mathews et al, 2010), rNSCs also generate astrocytes in an outside-in-pattern. Figure 1. Astrogenesis in the postnatal dentate gyrus (DG) A. Scheme illustrates developmental fate-mapping experiment using NestinCreERT2; CAG CAT GFP (from now on referred to as NestinCreERT2; GFP) mice to monitor radial glia-like neural stem cells (rNSCs) and their generated progeny. B. Experimental scheme of tamoxifen injection paradigm: perinatal mice were injected twice (12 h apart) to label early perinatal rNSCs and killed at postnatal day (P) 3, 7, 10, or 14 to analyze the rNSC-generated progeny within the DG layers hilus, granular zone (GZ), and molecular layer (ML). C. Confocal images of rNSC-derived astrocytes expressing GFP (green) and astrocyte-specific markers (ACSBG1, GFAP, and S100β; magenta) at different timepoints (P7, P10, and P14); lower pictures depict single-channel images the astrocyte markers (gray) used above. D. Representative pictures of recombined astrocytes (GFP+/ACSBG1+; arrowheads) during DG development (P3, P7, P10, and P14). E, F. Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the total DG (E) or in distinct DG compartments (F), that is, hilus, GZ, and ML indicated by different colors. G. Representative images of proliferating astrocytes (ACSBG1+, cyan; arrowheads) in combination with cell cycle markers KI67 or MCM2 (magenta; P3, P7: KI67; P10, P14: MCM2). H. Confocal image of proliferating astrocytes at P10 (ACSBG1+, cyan; MCM2+, magenta) that did not express NESTIN (white). I, J. Quantification of total proliferating astrocytes (ACSBG1+/KI67+ for P3/P7; ACSBG1+/MCM2+ for P10/P14) per area at distinct timepoints per total DG (I) or hilus, GZ and ML (J). K. Confocal images of proliferating rNSC-derived astrocytes (GFP+/ACSBG1+/MCM2+; arrowheads) at P10. Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), (E, I) indicated by red dots; all values are indicated in Table EV1; dotted lines mark GZ borders; scale bars (C, H, K) = 10 µm, (D, G) = 50 µm. Source data are available online for this figure. Source Data for Figure 1 [embj2021110409-sup-0012-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Astrogenesis and neurogenesis in the developing DG Quantification of percentage of NESTIN+ cells out of all recombined cells in the DG of P0-induced NestinCreERT2; GFP mice at P3. Quantification of percentage of proliferating NESTIN+ cells out of all proliferating cells (KI67+) in the DG of P3 NestinCreERT2; GFP mice. Quantification of percentage of recombined cells (GFP+) out of all proliferating cells at P3 of P0-induced NestinCreERT2; GFP mice. Representative images showing that ACSBG1 (magenta) overlapped with GFAP (white) expression at P14 and P27 DG astrocytes. Representative pictures of recombined astrocytes (GFP+/ALDH1L1+; green and magenta, respectively; arrowheads) at P3, P7, P10, P14 in P0-induced NestinCreERT2; GFP mice. Confocal pictures of proliferating astrocytes labeled with ALDH1L1 (cyan; arrowheads) and cell cycle markers KI67 or MCM2 (magenta; P3, P7: KI67; P10, P14: MCM2) at distinct timepoints of postnatal DG development in NestinCreERT2; GFP animals. Representative images of P28 NestinCreERT2; GFP animals, which received one BrdU injection at postnatal day 3, 7, 14, or 21. Most newly generated cells (BrdU+, magenta) were identified as granule neurons (NEUN; cyan), or astrocytes (GFAP; green). Representative images of DGs at P10 and P14 of NestinCreERT2; GFP animals, which were tamoxifen-induced at P0. Many recombined cells (GFP, green) were identified as proliferating (MCM2+; magenta) IPCs (TBR2+; cyan). Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (A, C) n = 5, (B) n = 6; dotted lines border SGZ and GZ; scale bars = 50 µm.Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2A. Adult astrogenesis in
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