Article3 February 2020free access Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone Jun Wu School of Medicine, Tsinghua University, Beijing, China IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Wen-Jia Tian IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Yang Liu Peking University-Tsinghua University-National Institute of Biological Sciences (PTN) Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing, China Search for more papers by this author Huanhuan J Wang IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Jiangli Zheng School of Medicine, Tsinghua University, Beijing, China IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Xin Wang MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Han Pan orcid.org/0000-0002-2870-8778 School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Ji Li School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Junyu Luo Peking University-Tsinghua University-National Institute of Biological Sciences (PTN) Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Xuerui Yang orcid.org/0000-0002-7731-2147 MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Lester F Lau Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author H Troy Ghashghaei WM Keck Center for Behavioral Biology, Program in Genetics, Program in Comparative Biomedical Sciences, Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA Search for more papers by this author Qin Shen Corresponding Author [email protected] orcid.org/0000-0002-9000-494X Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Tongji University Brain and Spinal Cord Clinical Research Center, Shanghai, China Search for more papers by this author Jun Wu School of Medicine, Tsinghua University, Beijing, China IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Wen-Jia Tian IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Yang Liu Peking University-Tsinghua University-National Institute of Biological Sciences (PTN) Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing, China Search for more papers by this author Huanhuan J Wang IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Jiangli Zheng School of Medicine, Tsinghua University, Beijing, China IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Xin Wang MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Han Pan orcid.org/0000-0002-2870-8778 School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Ji Li School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Junyu Luo Peking University-Tsinghua University-National Institute of Biological Sciences (PTN) Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Xuerui Yang orcid.org/0000-0002-7731-2147 MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Lester F Lau Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author H Troy Ghashghaei WM Keck Center for Behavioral Biology, Program in Genetics, Program in Comparative Biomedical Sciences, Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA Search for more papers by this author Qin Shen Corresponding Author [email protected] orcid.org/0000-0002-9000-494X Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China Tongji University Brain and Spinal Cord Clinical Research Center, Shanghai, China Search for more papers by this author Author Information Jun Wu1,2,3,4,‡, Wen-Jia Tian2,3,4,5,‡, Yang Liu6,7,8, Huanhuan J Wang2,3,4,5, Jiangli Zheng1,2,3,4, Xin Wang7,9, Han Pan9, Ji Li1, Junyu Luo6, Xuerui Yang7,9, Lester F Lau10, H Troy Ghashghaei11 and Qin Shen *,3,4,12 1School of Medicine, Tsinghua University, Beijing, China 2IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China 3Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China 4Frontier Science Center for Stem Cell Research, Ministry of Education, School of Life Sciences and Technology, Tongji University, Shanghai, China 5Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China 6Peking University-Tsinghua University-National Institute of Biological Sciences (PTN) Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China 7MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China 8China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 9School of Life Sciences, Tsinghua University, Beijing, China 10Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA 11WM Keck Center for Behavioral Biology, Program in Genetics, Program in Comparative Biomedical Sciences, Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA 12Tongji University Brain and Spinal Cord Clinical Research Center, Shanghai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 21 65981458; E-mail: [email protected] EMBO J (2020)39:e101679https://doi.org/10.15252/embj.2019101679 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 Adult neural stem cells (NSCs) reside in specialized niches, which hold a balanced number of NSCs, their progeny, and other cells. How niche capacity is regulated to contain a specific number of NSCs remains unclear. Here, we show that ependyma-derived matricellular protein CCN1 (cellular communication network factor 1) negatively regulates niche capacity and NSC number in the adult ventricular–subventricular zone (V-SVZ). Adult ependyma-specific deletion of Ccn1 transiently enhanced NSC proliferation and reduced neuronal differentiation in mice, increasing the numbers of NSCs and NSC units. Although proliferation of NSCs and neurogenesis seen in Ccn1 knockout mice eventually returned to normal, the expanded NSC pool was maintained in the V-SVZ until old age. Inhibition of EGFR signaling prevented expansion of the NSC population observed in CCN1 deficient mice. Thus, ependyma-derived CCN1 restricts NSC expansion in the adult brain to maintain the proper niche capacity of the V-SVZ. Synopsis The maintenance and activation of NSCs are regulated by niche components in the adult brain. Matricellular protein CCN1 is secreted from ependymal cells and negatively regulates the number of NSCs through restricting niche capacity in the adult V-SVZ. Matricellular protein CCN1 is specifically expressed in the ependymal cells in the adult V-SVZ. CCN1 restricts the capacity of the V-SVZ NSC niche and the number of NSCs. CCN1 suppresses the expansion of NSC pool through inhibiting EGFR signaling. Introduction In the adult rodent brain, neurogenesis occurs mainly in two regions: the ventricular–subventricular zone (V-SVZ) lining the lateral walls of the lateral ventricles and the subgranular zone (SGZ) in the hippocampal dentate gyrus (Kriegstein & Alvarez-Buylla, 2009; Suh et al, 2009; Bond et al, 2015). In both regions, neural stem cells correspond to a subpopulation of astroglial cells and continue to generate neurons and glia throughout life (Kriegstein & Alvarez-Buylla, 2009). In the adult V-SVZ, B1 NSCs are surrounded and anchored by an array of ependymal cells, forming a pinwheel structure along the planar plane of the ventricular surface (Mirzadeh et al, 2008; Shen et al, 2008; Shook et al, 2012). One or more B1 NSCs are contained in the center of the pinwheel, separated by the surrounding ependymal cells (Mirzadeh et al, 2008), establishing NSC units that are spatially dispersed in the V-SVZ. B1 cells are largely quiescent NSCs (qNSC) (Codega et al, 2014), characterized by lack of EGFR and the presence of the membrane protein vascular cell adhesion molecule 1 (VCAM1) on the apical endfeet, which directly contact the ependymal cells and protrude into the CSF (Kokovay et al, 2012; Llorens-Bobadilla et al, 2015; Basak et al, 2018). Upon activation, a small percent of active NSCs (aNSC) self-renew to maintain the stem cell pool, some of which return to quiescence, while the majority progress through a lineage from transient-amplifying cells (C cells) to neuroblasts (A cells), generating olfactory bulb (OB) neurons after several rounds of division (Doetsch et al, 1999a; Calzolari et al, 2015; Basak et al, 2018; Obernier et al, 2018). Despite long-term self-renewal, the abundance of NSCs decreases progressively during aging, which is attributed to consuming differentiation divisions and age-related changes in cell-intrinsic and niche-related factors (Conover & Shook, 2011a; Shook et al, 2012; Capilla-Gonzalez et al, 2014; Obernier et al, 2018). To compensate for the loss of NSCs, strategies to stimulate self-renewal and proliferation of NSC are often exploited. However, expanding NSC pool by over-activation of NSCs would compromise the long-term potential of neurogenesis and lead to premature exhaustion of stem cells if the activated stem cells are not allowed to return to quiescence (Kippin et al, 2005; Mira et al, 2010; Furutachi et al, 2013; Jones et al, 2015; Urban et al, 2016; Neuberger et al, 2017; Zhou et al, 2018). In addition, in a closed stem cell niche, the amount of stem cells is limited by the physical constrain of niche architecture. For example, in the embryonic epidermis, active proliferation of progenitor cells leads to crowding, which subsequently triggers delamination and differentiation to maintain a stabilized niche size (Chacon-Martinez et al, 2018). In the small intestine, slow-dividing and label-retaining stem cells are held in the specific locations at a constant amount under homeostatic condition (Barker et al, 2007; Takeda et al, 2011). Hence in a given stem cell niche, the number of quiescent stem cells and the self-renewal potential of activated stem cells determine the ability of the niche to sustain a life-long supply of newborn cells. Accumulating evidence from lineage tracing and single-cell sequencing studies indicates that in the NSC niches, there is bidirectional transition between the states of qNSCs and aNSCs (Urban et al, 2016; Basak et al, 2018; Kester & van Oudenaarden, 2018; Obernier et al, 2018). However, the cellular and molecular mechanisms controlling the capacity of the NSC niche to hold a balanced number of qNSCs and aNSCs remain unclear. Ependymal cells, occupying the majority of the ventricular surface area, are the closest neighbors of B1 cells in the V-SVZ NSC niche. Although it has been shown that ependymal cell-derived secreted factors, such as PEDF and Noggin, regulate NSC self-renewal and neurogenic potential (Lim et al, 2000; Peretto et al, 2004; Ramirez-Castillejo et al, 2006), how ependymal cells control the NSC niche and stem cell behavior remains largely unknown. Cellular communication network factor 1 (Ccn1), originally named as Cyr61, was first identified as an immediate-early gene. It belongs to the CCN family of genes encoding secreted proteins with a high degree of sequence homology and conserved tetramodular organization (Obrien et al, 1990; Perbal et al, 2018). CCN1 has been shown to promote growth factor-induced proliferative responses through interacting with various cell surface integrins (Obrien et al, 1990; Babic et al, 1998). Upon secretion, CCN1 binds to the cell membrane and extracellular matrix (ECM) to elicit diverse cellular functions, including cell adhesion, migration, proliferation, survival, apoptosis, differentiation senescence, and tumor invasion (Chen & Lau, 2009). Although it has been shown that Ccn1 is expressed in the developing and adult brain, and high level of CCN1 correlates with a poorer prognosis in glioblastoma patients (Haseley et al, 2012; Ishida et al, 2015; Otani et al, 2017), the expression pattern and function of CCN1 in the adult NSC niche have not been studied yet. In this study, we investigated the cellular and molecular relationship between NSCs and ependymal cells. We identified CCN1 as an ependyma-specific niche factor, and our results imply that ependymal cells restrict niche capacity and NSC pool expansion in the adult V-SVZ. Results CCN1 is specifically expressed in ependymal cells in the adult V-SVZ We characterized the expression pattern of CCN1 in the adult V-SVZ by immunostaining on forebrain coronal sections and V-SVZ whole-mounts from 8- to 12-week mice. Two different antibodies revealed similar patterns. In coronal sections, CCN1 was detected selectively in the apical cell layer lining the lateral ventricle (Fig 1A). From the en face view of the V-SVZ whole-mounts prepared from the lateral walls of the lateral ventricles, CCN1 was found in S100β+ cells with a large apical surface, indicating CCN1 is expressed in ependymal cells (Figs 1B and EV1A). To determine whether CCN1 was also expressed in apical B1 cells, which are embedded in the ependymal layer, we labeled B1 cells with VCAM1 and GFAP antibodies and found that CCN1 was expressed in the surrounding ependymal cells, but not in VCAM1+ B1 cells located in the center of the pinwheel organization (Fig 1C). We traced the GFAP+ fibers of VCAM1+ B1 cells from the ventricular surface deep into the V-SVZ in a series of confocal sections and found no CCN1 immunostaining signal in the cell bodies or processes of B1 NSCs (Fig 1D). We confirmed that CCN1 was only expressed in ependymal cells, but not in NSCs, oligodendrocytes, or neuroblasts based on the single-cell RNA-sequencing dataset from the adult V-SVZ (Luo et al, 2015) (Fig EV1B). Additionally, we did not detect CCN1 in blood vessels (Fig EV1C). Thus, CCN1 expression is restricted to ependymal cells in the adult V-SVZ. Figure 1. CCN1 is specifically expressed in ependymal cells in the adult V-SVZ Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle. Adult V-SVZ whole-mounts stained for CCN1 (white) and S100β (green). Scale bar: 10 μm. Adult V-SVZ whole-mounts stained for CCN1 (white), GFAP (green), and VCAM1 (magenta). Arrowhead points to a VCAM1+ B1 cell surrounded by CCN1-expressing ependymal cells. Scale bar: 10 μm. Confocal images tracing a VCAM1+ GFAP+ cell reveal CCN1 expression is absent in B1 cells. Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit. White arrowheads point to the process of the B1 cell. Scale bar: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Expression of CCN1 in the adult V-SVZ Whole-mount staining for CCN1 (white) and β-catenin (green) in the adult V-SVZ. Scale bar: 10 μm. FPKM of Ccn1 mRNA in adult V-SVZ cells (Luo et al, 2015). n = 10 (ependymal cells), 2 (neural stem cells), 9 (oligodendrocytes), and 11 (neuroblasts). Data are represented as mean ± SEM. *P = 0.0159, **P = 0.0045, ****P < 0.0001, n.s. not significant, one-way ANOVA followed by Fisher's post hoc test. V-SVZ whole-mount staining for CCN1 (white) and CD31 (red), showing no CCN1 expression in the blood vessel. Scale bar: 10 μm. Source data are available online for this figure. Download figure Download PowerPoint CCN1 negatively regulates niche capacity and B1 cell number in the adult V-SVZ In the adult V-SVZ, it has been shown that ependymal cells but not the B1 cells express the transcription factor FOXJ1 (Jacquet et al, 2009; Shah et al, 2018). To confirm the specificity of the Foxj1CreERT2 mouse line (Muthusamy et al, 2014), we treated adult Foxj1CreERT2; RosamT/mG mice with tamoxifen (TAM) for 5 consecutive days, which resulted in specific recombination in the ependymal layer, with 40% of the ependymal cells showing GFP expression (Fig EV2A). Click here to expand this figure. Figure EV2. VCAM1 expression marks quiescent NSCs and NSC units Specific labeling of ependymal cells in Foxj1CreERT2; RosamTmG mice 1 week after TAM treatment. Scale bar: 20 μm. Western blotting showing reduced CCN1 protein level in the V-SVZ of Ccn1cKO mice. n = 3, *P = 0.0123. Whole-mount staining for γ-tubulin (green), β-catenin (white), and acetylated-tubulin (magenta). Scale bar: 10 μm. Whole-mount staining for γ-tubulin (cyan), GFAP (red), and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in a NSC unit. Scale bar: 10 μm. Confocal images of adult V-SVZ whole-mounts showing scattered distribution of VCAM1+ B1 cells (arrowheads). Scale bar: 50 μm. Proportions of B1 cells that express VCAM1. n = 4, n.s. not significant. Whole-mount staining for γ-tubulin (cyan), β-catenin (red), and VCAM1 (white). Arrowheads point to VCAM1+ NSC units. Scale bar: 10 μm. Average numbers of B1 cells in a NSC units. n = 4, **P = 0.0021. Data information: In (B, F, and H), data are presented as mean ± SEM. Paired Student's t-test (B). Unpaired Student's t-test (F, H). Source data are available online for this figure. Download figure Download PowerPoint To investigate the function of ependyma-derived CCN1 in the adult V-SVZ, we crossed Foxj1CreERT2 mice with Ccn1flox/flox mice (Kim et al, 2013) to specifically delete Ccn1 in ependymal cells. We treated both Foxj1CreERT2; Ccn1flox/flox (Ccn1cKO) and Ccn1flox/flox (Ctrl) mice with TAM for 5 days at 8 weeks of age. We found that ependymal cells that had lost CCN1 expression distributed randomly in the ventricular surface, forming a mosaic pattern in the V-SVZ 1 week post-induction (Fig 2A). CCN1 protein level was notably reduced in the V-SVZ of Ccn1cKO mice 1 week after TAM treatment (Fig EV2B). Loss of CCN1 did not lead to noticeable abnormalities in the shape and surface morphology of the V-SVZ whole-mounts, as revealed by reconstructed whole view of the lateral wall of the lateral ventricle (Fig 2B). Figure 2. CCN1 restricts NSC pool size and niche capacity in the adult V-SVZ V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm. V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), and 10 μm (right). Densities (cells/mm2) of B1 cells in the adult V-SVZ. n = 4, ***P = 0.0005. V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red), and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left) and 10 μm (right). Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n = 4, **P = 0.002. Densities (units/mm2) of NSC units in the adult V-SVZ. n = 4, **P = 0.0022. Distribution of NSC units with different numbers of B1 cells. n = 4, ****P < 0.0001 (1 B1 cell/unit), **P = 0.0025 (2 B1 cells/unit), **P = 0.0063 (3 B1 cells/unit), *P = 0.0385 (4 B1 cells/unit). Data information: In (C, E, F, and G), data are presented as mean ± SEM. Unpaired Student's t-test. Source data are available online for this figure. Source Data for Figure 2 [embj2019101679-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint Remarkably, staining for GFAP in the whole-mounts revealed significantly more GFAP+ cells in the V-SVZ of Ccn1cKO mice compared with Ctrl mice (Fig 2B). We focused on the GFAP+ B1 cells, which bear a single primary cilium and are intermingled with multi-ciliated ependymal cells. From the en face view of the V-SVZ whole-mounts, we found it difficult to discern the single cilium of B1 cells among tufts of ependymal motile cilia as revealed by acetyl-tubulin staining (Fig EV2C), but γ-tubulin staining, which labels the basal bodies of cilia, facilitated the identification and quantification of the ventricle-contacting apical B1 cells in combination with GFAP staining (Fig 2B). We found that the number of GFAP+ cells with single γ-tubulin+ basal body (B1 cells) was dramatically increased in the adult Ccn1cKO V-SVZ compared with control (261.7 ± 45.5/mm2 in Ctrl; 871.7 ± 78.6/mm2 in Ccn1cKO) (Fig 2C). To assess the impact on qNSCs, we stained whole-mounts for VCAM1, which is expressed in the apical processes of qNSC (Fig EV2D). VCAM1+ qNSCs accounted for 63.69 ± 8.26% of total B1 cells in the V-SVZ of control mice (Fig EV2E and F). There was a 3.5-fold increase in the number of VCAM1+ B1 cells in CCN1-deficient mice (168.3 ± 34.8/mm2 in Ctrl; 606.7 ± 76.6/mm2 in Ccn1cKO) (Fig 2D and E), while the percentage of VCAM1+ B1 cells was similar between Ccn1cKO and Ctrl mice (Fig EV2F). An overview of the VCAM1 staining signals revealed that VCAM1+ clusters were evenly spaced in the V-SVZ whole-mounts (Fig EV2E). Together with β-catenin and γ-tubulin staining, it is evident that VCAM1+ cell-centered pinwheel structures dispersed over the ventricular surface in a unitary pattern, which we designated as NSC units (Fig EV2G). We found the density of NSC units was considerably higher in Ccn1cKO mice compared with controls (148.3 ± 30.5/mm2 in Ctrl; 396.7 ± 38.0/mm2 in Ccn1cKO) (Fig 2F). Furthermore, the average number of B1 cells within each unit was also notably increased in Ccn1cKO mice (Fig EV2H). While most of the units contained a single B1 cell in Ctrl mice, loss of CCN1 enabled NSC units to hold up to four B1 cells (Fig 2G). Thus, the level of CCN1 protein regulates the niche capacity for B1 NSCs in the adult V-SVZ. Enlarged niche capacity persists in the aging V-SVZ of Ccn1cKO mice To assess the long-term effect of Ccn1 deletion on V-SVZ niche capacity and NSC maintenance, we analyzed V-SVZ whole-mounts 16 months after TAM administration (Fig 3A). Remarkably, there were still more B1 cells and VCAM1+ B1 cells in the V-SVZ of aged Ccn1cKO mice compared with aged controls (Fig 3A–C). The density of NSC units remained higher in aged Ccn1cKO mice (33.3 ± 1.7/mm2 in Ctrl; 103.3 ± 14.8/mm2 in Ccn1cKO) (Fig 3D), but the number of B1 cells contained in each unit dropped to levels indistinguishable from controls (Fig 3E). Interestingly, the density of VCAM1+ B1 cells was 3.6-fold higher in aged Ccn1cKO mice compared with aged controls (33.3 ± 1.7/mm2 in Ctrl; 118.3 ± 14.2/mm2 in Ccn1cKO) (Fig 3B), which was similar to the fold change in young adult mice (Fig 2E). Thus, loss of CCN1 did not lead to unlimited increase in niche capacity, but the initially expanded niche and NSC pool were maintained in the V-SVZ during aging. Figure 3. The enlarged V-SVZ niche persists till aging in Ccn1cKO mice Densities (cells/mm2) of B1 cells in the aged V-SVZ. n = 3, **P = 0.0099. Densities (cells/mm2) of VCAM1+ B1 cells in the aged V-SVZ. n = 3, **P = 0.0041. V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red), and VCAM1 (white) in aged mice. Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left) and 10 μm (right). Densities (units/mm2) of NSC units in the aged V-SVZ. n = 3, **P = 0.0093. Average numbers of B1 cells in a NSC unit. n = 3, n.s. not significant. Data information: In (A, B, D, and E), data are presented as mean ± SEM. Unpaired Student's t-test. Source data are available online