@article{zhang_xiao_johnson_cai_horowitz_mennicke_coffey_haider_threadgill_eliscu_et al._2023, title={Bulk and mosaic deletions of Egfr reveal regionally defined gliogenesis in the developing mouse forebrain}, volume={26}, ISSN={["2589-0042"]}, DOI={10.1016/j.isci.2023.106242}, number={3}, journal={ISCIENCE}, author={Zhang, Xuying and Xiao, Guanxi and Johnson, Caroline and Cai, Yuheng and Horowitz, Zachary K. and Mennicke, Christine and Coffey, Robert and Haider, Mansoor and Threadgill, David and Eliscu, Rebecca and et al.}, year={2023}, month={Mar} }
 @article{cai_zhang_li_ghashghaei_greenbaum_2023, title={COMBINe enables automated detection and classification of neurons and astrocytes in tissue-cleared mouse brains}, volume={3}, ISSN={["2667-2375"]}, DOI={10.1016/j.crmeth.2023.100454}, abstractNote={Tissue clearing renders entire organs transparent to accelerate whole-tissue imaging; for example, with light-sheet fluorescence microscopy. Yet, challenges remain in analyzing the large resulting 3D datasets that consist of terabytes of images and information on millions of labeled cells. Previous work has established pipelines for automated analysis of tissue-cleared mouse brains, but the focus there was on single-color channels and/or detection of nuclear localized signals in relatively low-resolution images. Here, we present an automated workflow (COMBINe, Cell detectiOn in Mouse BraIN) to map sparsely labeled neurons and astrocytes in genetically distinct mouse forebrains using mosaic analysis with double markers (MADM). COMBINe blends modules from multiple pipelines with RetinaNet at its core. We quantitatively analyzed the regional and subregional effects of MADM-based deletion of the epidermal growth factor receptor (EGFR) on neuronal and astrocyte populations in the mouse forebrain.}, number={4}, journal={CELL REPORTS METHODS}, author={Cai, Yuheng and Zhang, Xuying and Li, Chen and Ghashghaei, H. Troy and Greenbaum, Alon}, year={2023}, month={Apr} }
 @article{li_moatti_zhang_ghashghaei_greenbaum_2022, title={Deep learning-based autofocus method enhances image quality in light-sheet fluorescence microscopy: publishers note (vol 12, pg 5214, 2021)}, volume={13}, ISSN={["2156-7085"]}, DOI={10.1364/BOE.450829}, abstractNote={[This corrects the article on p. 5214 in vol. 12, PMID: 34513252.].}, number={1}, journal={BIOMEDICAL OPTICS EXPRESS}, author={LI, Chen and Moatti, Adele and Zhang, Xuying and Ghashghaei, H. Troy and Greenbaum, Alon}, year={2022}, month={Jan}, pages={373–373} }
 @article{li_rai_ghashghaei_greenbaum_2022, title={Illumination angle correction during image acquisition in light-sheet fluorescence microscopy using deep learning}, volume={13}, ISSN={["2156-7085"]}, DOI={10.1364/BOE.447392}, abstractNote={Light-sheet fluorescence microscopy (LSFM) is a high-speed imaging technique that provides optical sectioning with reduced photodamage. LSFM is routinely used in life sciences for live cell imaging and for capturing large volumes of cleared tissues. LSFM has a unique configuration, in which the illumination and detection paths are separated and perpendicular to each other. As such, the image quality, especially at high resolution, largely depends on the degree of overlap between the detection focal plane and the illuminating beam. However, spatial heterogeneity within the sample, curved specimen boundaries, and mismatch of refractive index between tissues and immersion media can refract the well-aligned illumination beam. This refraction can cause extensive blur and non-uniform image quality over the imaged field-of-view. To address these issues, we tested a deep learning-based approach to estimate the angular error of the illumination beam relative to the detection focal plane. The illumination beam was then corrected using a pair of galvo scanners, and the correction significantly improved the image quality across the entire field-of-view. The angular estimation was based on calculating the defocus level on a pixel level within the image using two defocused images. Overall, our study provides a framework that can correct the angle of the light-sheet and improve the overall image quality in high-resolution LSFM 3D image acquisition.}, number={2}, journal={BIOMEDICAL OPTICS EXPRESS}, author={Li, Chen and Rai, Mani Ratnam and Ghashghaei, H. Troy and Greenbaum, Alon}, year={2022}, month={Feb}, pages={888–901} }
 @article{muthusamy_williams_o'toole_brudvig_adler_weimer_muddiman_ghashghaei_2022, title={Phosphorylation-dependent proteome of Marcks in ependyma during aging and behavioral homeostasis in the mouse forebrain}, volume={1}, ISSN={["2509-2723"]}, url={http://dx.doi.org/10.1007/s11357-022-00517-3}, DOI={10.1007/s11357-022-00517-3}, abstractNote={Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging.}, number={4}, journal={GEROSCIENCE}, publisher={Springer Science and Business Media LLC}, author={Muthusamy, Nagendran and Williams, Taufika I and O'Toole, Ryan and Brudvig, Jon J. and Adler, Kenneth B. and Weimer, Jill M. and Muddiman, David C. and Ghashghaei, H. Troy}, year={2022}, month={Jan} }
 @article{li_moatti_zhang_ghashghaei_greenabum_2021, title={Deep learning-based autofocus method enhances image quality in light-sheet fluorescence microscopy}, volume={12}, ISSN={["2156-7085"]}, url={http://dx.doi.org/10.1364/boe.427099}, DOI={10.1364/BOE.427099}, abstractNote={Light-sheet fluorescence microscopy (LSFM) is a minimally invasive and high throughput imaging technique ideal for capturing large volumes of tissue with sub-cellular resolution. A fundamental requirement for LSFM is a seamless overlap of the light-sheet that excites a selective plane in the specimen, with the focal plane of the objective lens. However, spatial heterogeneity in the refractive index of the specimen often results in violation of this requirement when imaging deep in the tissue. To address this issue, autofocus methods are commonly used to refocus the focal plane of the objective-lens on the light-sheet. Yet, autofocus techniques are slow since they require capturing a stack of images and tend to fail in the presence of spherical aberrations that dominate volume imaging. To address these issues, we present a deep learning-based autofocus framework that can estimate the position of the objective-lens focal plane relative to the light-sheet, based on two defocused images. This approach outperforms or provides comparable results with the best traditional autofocus method on small and large image patches respectively. When the trained network is integrated with a custom-built LSFM, a certainty measure is used to further refine the network's prediction. The network performance is demonstrated in real-time on cleared genetically labeled mouse forebrain and pig cochleae samples. Our study provides a framework that could improve light-sheet microscopy and its application toward imaging large 3D specimens with high spatial resolution.}, number={8}, journal={BIOMEDICAL OPTICS EXPRESS}, publisher={The Optical Society}, author={Li, Chen and Moatti, Adele and Zhang, Xuying and Ghashghaei, H. Troy and Greenabum, Alon}, year={2021}, month={Aug}, pages={5214–5226} }
 @article{cai_zhang_kovalsky_ghashghaei_greenbaum_2021, title={Detection and classification of neurons and glial cells in the MADM mouse brain using RetinaNet}, volume={16}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0257426}, abstractNote={The ability to automatically detect and classify populations of cells in tissue sections is paramount in a wide variety of applications ranging from developmental biology to pathology. Although deep learning algorithms are widely applied to microscopy data, they typically focus on segmentation which requires extensive training and labor-intensive annotation. Here, we utilized object detection networks (neural networks) to detect and classify targets in complex microscopy images, while simplifying data annotation. To this end, we used a RetinaNet model to classify genetically labeled neurons and glia in the brains of Mosaic Analysis with Double Markers (MADM) mice. Our initial RetinaNet-based model achieved an average precision of 0.90 across six classes of cells differentiated by MADM reporter expression and their phenotype (neuron or glia). However, we found that a single RetinaNet model often failed when encountering dense and saturated glial clusters, which show high variability in their shape and fluorophore densities compared to neurons. To overcome this, we introduced a second RetinaNet model dedicated to the detection of glia clusters. Merging the predictions of the two computational models significantly improved the automated cell counting of glial clusters. The proposed cell detection workflow will be instrumental in quantitative analysis of the spatial organization of cellular populations, which is applicable not only to preparations in neuroscience studies, but also to any tissue preparation containing labeled populations of cells.}, number={9}, journal={PLOS ONE}, author={Cai, Yuheng and Zhang, Xuying and Kovalsky, Shahar Z. and Ghashghaei, H. Troy and Greenbaum, Alon}, year={2021}, month={Sep} }
 @article{wu_tian_liu_wan_zheng_wang_pan_li_luo_yang_et al._2020, title={Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone}, volume={39}, ISSN={["1460-2075"]}, DOI={10.15252/embj.2019101679}, abstractNote={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}, number={5}, journal={EMBO JOURNAL}, author={Wu, Jun and Tian, Wen-Jia and Liu, Yang and Wan, Huanhuan J. and Zheng, Jiangli and Wang, Xin and Pan, Han and Li, Ji and Luo, Junyu and Yang, Xuerui and et al.}, year={2020}, month={Mar} }
 @article{johnson_ghashghaei_2020, title={Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex}, volume={147}, ISSN={["1477-9129"]}, DOI={10.1242/dev.186056}, abstractNote={Cellular and molecular mechanisms underlying the switch from self-amplification of cortical stem cells to neuronal and glial generation are incompletely understood, despite their importance for neural development. Here, we have investigated the role of the transcription factor specificity protein 2 (Sp2) in expansive and neurogenic divisions of the developing cerebral cortex by combining conditional genetic deletion with the mosaic analysis with double markers (MADM) system in mice. We find that loss of Sp2 in progenitors undergoing neurogenic divisions results in prolonged mitosis due to extension of early mitotic stages. This disruption is correlated with depletion of the populations of upper layer neurons in the cortex. In contrast, early cortical neural stem cells proliferate and expand normally in the absence of Sp2. These results indicate a stage-specific requirement for Sp2 in neural stem and progenitor cells, and reveal mechanistic differences between the early expansive and later neurogenic periods of cortical development.This article has an associated 'The people behind the papers' interview.}, number={4}, journal={DEVELOPMENT}, author={Johnson, Caroline A. and Ghashghaei, H. Troy}, year={2020}, month={Feb} }
 @article{johnson_ghashghaei_2020, title={The people behind the papers - Caroline Johnson and Troy Ghashghaei}, volume={147}, ISSN={["1477-9129"]}, DOI={10.1242/dev.188904}, abstractNote={ABSTRACT Cortical development involves a switch from the self-amplification of stem cells to the generation of neuron and glia by progenitors. A new paper in Development investigates the molecular control of mitosis in these two stages, using simultaneous labelling and gene knockout in clones in the developing mouse brain. We caught up the paper's two authors Caroline Johnson and her supervisor Troy Ghashghaei, Professor of Neurobiology at the College of Veterinary Medicine at North Carolina State University, to find out more.}, number={4}, journal={DEVELOPMENT}, author={Johnson, Caroline and Ghashghaei, Troy}, year={2020}, month={Feb} }
 @article{muthusamy_brumm_zhang_carmichael_ghashghaei_2018, title={Foxj1 expressing ependymal cells do not contribute new cells to sites of injury or stroke in the mouse forebrain}, volume={8}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-018-19913-x}, abstractNote={Abstract The stem cell source of neural and glial progenitors in the periventricular regions of the adult forebrain has remained uncertain and controversial. Using a cell specific genetic approach we rule out Foxj1+ ependymal cells as stem cells participating in neurogenesis and gliogenesis in response to acute injury or stroke in the mouse forebrain. Non stem- and progenitor-like responses of Foxj1+ ependymal cells to injury and stroke remain to be defined and investigated.}, journal={SCIENTIFIC REPORTS}, author={Muthusamy, Nagendran and Brumm, Andrew and Zhang, Xuying and Carmichael, S. Thomas and Ghashghaei, H. Troy}, year={2018}, month={Jan} }
 @article{brudvig_cain_sears_schmidt-grimminger_wittchen_adler_ghashghaei_weimer_2018, title={MARCKS regulates neuritogenesis and interacts with a CDC42 signaling network}, volume={8}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-018-31578-0}, abstractNote={Abstract Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks −/− cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.}, journal={SCIENTIFIC REPORTS}, author={Brudvig, J. J. and Cain, J. T. and Sears, R. M. and Schmidt-Grimminger, G. G. and Wittchen, E. S. and Adler, K. B. and Ghashghaei, H. T. and Weimer, J. M.}, year={2018}, month={Sep} }
 @article{ehling_butler_thi_ghashghaei_bäumer_2018, title={To scratch an itch: Establishing a mouse model to determine active brain areas involved in acute histaminergic itch}, volume={5}, ISSN={2451-8301}, url={http://dx.doi.org/10.1016/J.IBROR.2018.10.002}, DOI={10.1016/J.IBROR.2018.10.002}, abstractNote={Strategies to efficiently control itch require a deep understanding of the underlying mechanisms. Several areas in the brain involved in itch and scratching responses have been postulated, but the central mechanisms that drive pruritic responses are still unknown. Histamine is recognized as a major mediator of itch in humans, and has been the most frequently used stimulus as an experimental pruritogen for brain imaging of itch.Histaminergic itch via histamine and the selective histamine H4 receptor (H4R) agonist, ST-1006, recruit brain nuclei through c-fos activation and activate specific areas in the brain.An acute itch model was established in c-fos-EGFP transgenic mice using ST-1006 and histamine. Coronal brain sections were stained for c-fos immunoreactivity and the forebrain was mapped for density of c-fos + nuclei.Histamine and ST-1006 significantly increased scratching response in c-fos-EGFP mice compared to vehicle controls. Mapping c-fos immunostained brain sections revealed neuronal activity in the cortex, striatum, hypothalamus, thalamus, amygdala, and the midbrain.Histaminergic itch and selective H4R activation significantly increased the density of c-fos + nuclei in the medial habenula (MHb). Thus, the MHb may be a new target to investigate and subsequently develop novel mechanism-based strategies to treat itch and possibly provide a locus for pharmacological control of pruritus.}, journal={IBRO Reports}, publisher={Elsevier BV}, author={Ehling, Sarah and Butler, Ashley and Thi, Stephanie and Ghashghaei, H. Troy and Bäumer, Wolfgang}, year={2018}, month={Dec}, pages={67–73} }
 @article{muthusamy_zhang_johnson_yadav_ghashghaei_2017, title={Developmentally defined forebrain circuits regulate appetitive and aversive olfactory learning}, volume={20}, ISSN={["1546-1726"]}, DOI={10.1038/nn.4452}, abstractNote={In this study, the authors reveal distinct developmental programs underlying innate and learned olfactory behaviors by demonstrating that chemogenetic inactivation of neurons generated in neonatal mice impairs the behavioral response to aversive odorants, whereas inactivation of adult-born neurons impairs learning of novel food-related odors. Postnatal and adult neurogenesis are region- and modality-specific, but the significance of developmentally distinct neuronal populations remains unclear. We demonstrate that chemogenetic inactivation of a subset of forebrain and olfactory neurons generated at birth disrupts responses to an aversive odor. In contrast, novel appetitive odor learning is sensitive to inactivation of adult-born neurons, revealing that developmentally defined sets of neurons may differentially participate in hedonic aspects of sensory learning.}, number={1}, journal={NATURE NEUROSCIENCE}, author={Muthusamy, Nagendran and Zhang, Xuying and Johnson, Caroline A. and Yadav, Prem N. and Ghashghaei, H. Troy}, year={2017}, month={Jan}, pages={20–23} }
 @article{ren_ao_timothy m. o'shea_burda_bernstein_brumm_muthusamy_ghashghaei_carmichael_cheng_et al._2017, title={Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury}, volume={7}, ISSN={["2045-2322"]}, DOI={10.1038/srep41122}, abstractNote={Abstract Ependyma have been proposed as adult neural stem cells that provide the majority of newly proliferated scar-forming astrocytes that protect tissue and function after spinal cord injury (SCI). This proposal was based on small, midline stab SCI. Here, we tested the generality of this proposal by using a genetic knock-in cell fate mapping strategy in different murine SCI models. After large crush injuries across the entire spinal cord, ependyma-derived progeny remained local, did not migrate and contributed few cells of any kind and less than 2%, if any, of the total newly proliferated and molecularly confirmed scar-forming astrocytes. Stab injuries that were near to but did not directly damage ependyma, contained no ependyma-derived cells. Our findings show that ependymal contribution of progeny after SCI is minimal, local and dependent on direct ependymal injury, indicating that ependyma are not a major source of endogenous neural stem cells or neuroprotective astrocytes after SCI.}, journal={SCIENTIFIC REPORTS}, author={Ren, Yilong and Ao, Yan and Timothy M. O'Shea and Burda, Joshua E. and Bernstein, Alexander M. and Brumm, Andrew J. and Muthusamy, Nagendran and Ghashghaei, H. Troy and Carmichael, S. Thomas and Cheng, Liming and et al.}, year={2017}, month={Jan} }
 @article{beattie_postiglione_burnett_laukoter_streicher_pauler_xiao_klezovitch_vasioukhin_ghashghaei_et al._2017, title={Mosaic analysis with double markers reveals distinct sequential functions of Lgl1 in neural stem cells}, volume={94}, DOI={10.1016/j.neuron.2017.04.012}, abstractNote={The concerted production of neurons and glia by neural stem cells (NSCs) is essential for neural circuit assembly. In the developing cerebral cortex, radial glia progenitors (RGPs) generate nearly all neocortical neurons and certain glia lineages. RGP proliferation behavior shows a high degree of non-stochasticity, thus a deterministic characteristic of neuron and glia production. However, the cellular and molecular mechanisms controlling RGP behavior and proliferation dynamics in neurogenesis and glia generation remain unknown. By using mosaic analysis with double markers (MADM)-based genetic paradigms enabling the sparse and global knockout with unprecedented single-cell resolution, we identified Lgl1 as a critical regulatory component. We uncover Lgl1-dependent tissue-wide community effects required for embryonic cortical neurogenesis and novel cell-autonomous Lgl1 functions controlling RGP-mediated glia genesis and postnatal NSC behavior. These results suggest that NSC-mediated neuron and glia production is tightly regulated through the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.}, number={3}, journal={Neuron (Cambridge, Mass.)}, author={Beattie, R. and Postiglione, M. P. and Burnett, L. E. and Laukoter, S. and Streicher, C. and Pauler, F. M. and Xiao, G. X. and Klezovitch, O. and Vasioukhin, V. and Ghashghaei, T. H. and et al.}, year={2017}, pages={517-} }
 @article{johnson_wright_ghashghaei_2017, title={Regulation of cytokinesis during corticogenesis: focus on the midbody}, volume={591}, ISSN={0014-5793}, url={http://dx.doi.org/10.1002/1873-3468.12676}, DOI={10.1002/1873-3468.12676}, abstractNote={Development of the cerebral cortices depends on tight regulation of cell divisions. In this system, stem and progenitor cells undergo symmetric and asymmetric divisions to ultimately produce neurons that establish the layers of the cortex. Cell division culminates with the formation of the midbody, a transient organelle that establishes the site of abscission between nascent daughter cells. During cytokinetic abscission, the final stage of cell division, one daughter cell will inherit the midbody remnant, which can then maintain or expel the remnant, but mechanisms and circumstances influencing this decision are unclear. This review describes the midbody and its constituent proteins, as well as the known consequences of their manipulation during cortical development. The potential functional relevance of midbody mechanisms is discussed.}, number={24}, journal={FEBS Letters}, publisher={Wiley}, author={Johnson, Caroline A. and Wright, Catherine E. and Ghashghaei, H. Troy}, year={2017}, month={Aug}, pages={4009–4026} }
 @article{davis_amin_johnson_bagley_ghashghaei_nascone-yoder_2017, title={Stomach curvature is generated by left-right asymmetric gut morphogenesis}, volume={144}, ISSN={["1477-9129"]}, DOI={10.1242/dev.143701}, abstractNote={Left-right (LR) asymmetry is a fundamental feature of internal anatomy, yet the emergence of morphological asymmetry remains one of the least understood phases of organogenesis. Asymmetric rotation of the intestine is directed by forces outside of the gut, but the morphogenetic events that generate anatomical asymmetry in other regions of the digestive tract remain unknown. Here we show that the mechanisms that drive the curvature of the stomach are intrinsic to the gut tube itself. The left wall of the primitive stomach expands more than the right wall, as the left epithelium becomes more polarized and undergoes radial rearrangement. These asymmetries exist across species, and are dependent on LR patterning genes, including FoxJ1, Nodal and Pitx2. Our findings have implications for how LR patterning manifests distinct types of morphological asymmetries in different contexts.}, number={8}, journal={DEVELOPMENT}, author={Davis, Adam and Amin, Nirav M. and Johnson, Caroline and Bagley, Kristen and Ghashghaei, H. Troy and Nascone-Yoder, Nanette}, year={2017}, month={Apr}, pages={1477–1483} }
 @article{dwyer_chen_chou_hippenmeyer_nguyen_ghashghaei_2016, title={Neural Stem Cells to Cerebral Cortex: Emerging Mechanisms Regulating Progenitor Behavior and Productivity}, volume={36}, ISSN={["0270-6474"]}, DOI={10.1523/jneurosci.2359-16.2016}, abstractNote={This review accompanies a 2016 SFN mini-symposium presenting examples of current studies that address a central question: How do neural stem cells (NSCs) divide in different ways to produce heterogeneous daughter types at the right time and in proper numbers to build a cerebral cortex with the appropriate size and structure? We will focus on four aspects of corticogenesis: cytokinesis events that follow apical mitoses of NSCs; coordinating abscission with delamination from the apical membrane; timing of neurogenesis and its indirect regulation through emergence of intermediate progenitors; and capacity of single NSCs to generate the correct number and laminar fate of cortical neurons. Defects in these mechanisms can cause microcephaly and other brain malformations, and understanding them is critical to designing diagnostic tools and preventive and corrective therapies.}, number={45}, journal={JOURNAL OF NEUROSCIENCE}, author={Dwyer, Noelle D. and Chen, Bin and Chou, Shen-Ju and Hippenmeyer, Simon and Nguyen, Laurent and Ghashghaei, H. Troy}, year={2016}, month={Nov}, pages={11394–11401} }
 @article{bain_kirste_johnson_ghashghaei_collazo_ivanisevic_2016, title={Neurotypic cell attachment and growth on III-nitride lateral polarity structures}, volume={58}, ISSN={["1873-0191"]}, DOI={10.1016/j.msec.2015.09.084}, abstractNote={III-nitride materials have recently received increasing levels of attention for their potential to successfully interface with, and sense biochemical interactions in biological systems. Expanding on available sensing schemes (including transistor-based devices,) a III-N lateral polarity structure capable of introducing quasi-phase matching through a periodic polarity grating presents a novel platform for second harmonic generation. This platform constitutes a non-linear optical phenomenon with exquisite sensitivity to the chemical state of a surface or interface. To characterize the response of a biological system to the nanostructured lateral polarity structures, we cultured neurotypic PC12 cells on AlGaN with varying ratios of Al:Ga - 0, 0.4, 0.6, and 1 - and on surfaces of varying pitch to the III-polar vs. N-polar grating - 5, 10, 20 and 50 μm. While some toxicity associated with increasing Al is observed, we documented and quantified trends in cell responses to the local material polarity and nanoscale roughness. The nitrogen-polar material has a significantly higher nanoscale roughness than III-polar regions, and a 80-200 nm step height difference between the III-polar and N-polar materials in the lateral polarity configuration generates adequate changes in topography to influence cell growth, improves cell adhesion and promotes cell migration along the direction of the features. As the designed material configuration is further explored for biochemical sensing, the lateral polarity scheme may provide a route in assessing the non-specific protein adsorption to this varying nano-topography that drives the subsequent cell response.}, journal={MATERIALS SCIENCE & ENGINEERING C-MATERIALS FOR BIOLOGICAL APPLICATIONS}, author={Bain, L. E. and Kirste, R. and Johnson, C. A. and Ghashghaei, H. T. and Collazo, R. and Ivanisevic, A.}, year={2016}, month={Jan}, pages={1194–1198} }
 @article{sai_morioka_takaesu_muthusamy_ghashghaei_hanafusa_matsumoto_ninomiya-tsuji_2016, title={TAK1 determines susceptibility to endoplasmic reticulum stress and leptin resistance in the hypothalamus}, volume={129}, ISSN={0021-9533 1477-9137}, url={http://dx.doi.org/10.1242/jcs.180505}, DOI={10.1242/jcs.180505}, abstractNote={Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases including metabolic disorders. TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, and is activated by a diverse set of inflammatory stimuli. Here we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory element binding proteins (SREBPs)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER stress-induced hypothalamic leptin resistance and hyperphagic obesity under high fat diet (HFD). Thus, TAK1 is a critical regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.}, number={9}, journal={Journal of Cell Science}, publisher={The Company of Biologists}, author={Sai, Kazuhito and Morioka, Sho and Takaesu, Giichi and Muthusamy, Nagendran and Ghashghaei, H. Troy and Hanafusa, Hiroshi and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2016}, month={Mar}, pages={1855–1865} }
 @article{loziuk_meier_johnson_ghashghaei_muddiman_2016, title={TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR-MALDESI imaging of lipids and LC-MS/MS label-free proteomics}, volume={408}, ISSN={1618-2642 1618-2650}, url={http://dx.doi.org/10.1007/s00216-016-9421-3}, DOI={10.1007/s00216-016-9421-3}, abstractNote={Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of "omics" and "big data." Here, we provide an integrated approach for systematic analysis of the "lipidome" in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos.}, number={13}, journal={Analytical and Bioanalytical Chemistry}, publisher={Springer Science and Business Media LLC}, author={Loziuk, Philip and Meier, Florian and Johnson, Caroline and Ghashghaei, H. Troy and Muddiman, David C.}, year={2016}, month={Mar}, pages={3453–3474} }
 @article{rosen_bokhart_ghashghaei_muddiman_2015, title={Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI}, volume={26}, ISSN={1044-0305 1879-1123}, url={http://dx.doi.org/10.1007/s13361-015-1114-1}, DOI={10.1007/s13361-015-1114-1}, abstractNote={Analyte signal in a laser desorption/postionization scheme such as infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is strongly coupled to the degree of overlap between the desorbed plume of neutral material from a sample and an orthogonal electrospray. In this work, we systematically examine the effect of desorption conditions on IR-MALDESI response to pharmaceutical drugs and endogenous lipids in biological tissue using a design of experiments approach. Optimized desorption conditions have then been used to conduct an untargeted lipidomic analysis of whole body sagittal sections of neonate mouse. IR-MALDESI response to a wide range of lipid classes has been demonstrated, with enhanced lipid coverage received by varying the laser wavelength used for mass spectrometry imaging (MSI). Targeted MS2 imaging (MS2I) of an analyte, cocaine, deposited beneath whole body sections allowed determination of tissue-specific ion response factors, and CID fragments of cocaine were monitored to comment on wavelength-dependent internal energy deposition based on the “survival yield” method.}, number={6}, journal={Journal of The American Society for Mass Spectrometry}, publisher={Springer Science and Business Media LLC}, author={Rosen, Elias P. and Bokhart, Mark T. and Ghashghaei, H. Troy and Muddiman, David C.}, year={2015}, month={Apr}, pages={899–910} }
 @article{muthusamy_sommerville_moeser_stumpo_sannes_adler_blackshear_weimer_ghashghaei_2015, title={MARCKS-dependent mucin clearance and lipid metabolism in ependymal cells are required for maintenance of forebrain homeostasis during aging}, volume={14}, ISSN={["1474-9726"]}, DOI={10.1111/acel.12354}, abstractNote={Ependymal cells (ECs) form a barrier responsible for selective movement of fluids and molecules between the cerebrospinal fluid and the central nervous system. Here, we demonstrate that metabolic and barrier functions in ECs decline significantly during aging in mice. The longevity of these functions in part requires the expression of the myristoylated alanine-rich protein kinase C substrate (MARCKS). Both the expression levels and subcellular localization of MARCKS in ECs are markedly transformed during aging. Conditional deletion of MARCKS in ECs induces intracellular accumulation of mucins, elevated oxidative stress, and lipid droplet buildup. These alterations are concomitant with precocious disruption of ependymal barrier function, which results in the elevation of reactive astrocytes, microglia, and macrophages in the interstitial brain tissue of young mutant mice. Interestingly, similar alterations are observed during normal aging in ECs and the forebrain interstitium. Our findings constitute a conceptually new paradigm in the potential role of ECs in the initiation of various conditions and diseases in the aging brain.}, number={5}, journal={AGING CELL}, author={Muthusamy, Nagendran and Sommerville, Laura J. and Moeser, Adam J. and Stumpo, Deborah J. and Sannes, Philip and Adler, Kenneth and Blackshear, Perry J. and Weimer, Jill M. and Ghashghaei, H. Troy}, year={2015}, month={Oct}, pages={764–773} }
 @article{hammad_schmidt_zhang_bray_frohlich_ghashghaei_2015, title={Transplantation of GABAergic Interneurons into the Neonatal Primary Visual Cortex Reduces Absence Seizures in Stargazer Mice}, volume={25}, ISSN={["1460-2199"]}, DOI={10.1093/cercor/bhu094}, abstractNote={Epilepsies are debilitating neurological disorders characterized by repeated episodes of pathological seizure activity. Absence epilepsy (AE) is a poorly understood type of seizure with an estimated 30% of affected patients failing to respond to antiepileptic drugs. Thus, novel therapies are needed for the treatment of AE. A promising cell-based therapeutic strategy is centered on transplantation of embryonic neural stem cells from the medial ganglionic eminence (MGE), which give rise to gamma-aminobutyric acidergic (GABAergic) interneurons during embyronic development. Here, we used the Stargazer (Stg) mouse model of AE to map affected loci using c-Fos immunohistochemistry, which revealed intense seizure-induce activity in visual and somatosensory cortices. We report that transplantation of MGE cells into the primary visual cortex (V1) of Stg mice significantly reduces AE episodes and lowers mortality. Electrophysiological analysis in acute cortical slices of visual cortex demonstrated that Stg V1 neurons exhibit more pronounced increases in activity in response to a potassium-mediated excitability challenge than wildtypes (WT). The defective network activity in V1 was significantly altered following WT MGE transplantation, associating it with behavioral rescue of seizures in Stgs. Taken together, these findings present MGE grafting in the V1 as a possible clinical approach in the treatment of AE.}, number={9}, journal={CEREBRAL CORTEX}, author={Hammad, Mohamed and Schmidt, Stephen L. and Zhang, Xuying and Bray, Ryan and Frohlich, Flavio and Ghashghaei, H. Troy}, year={2015}, month={Sep}, pages={2970–2979} }
 @article{murlidharan_corriher_ghashghaei_asokan_2015, title={Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain}, volume={89}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.02951-14}, abstractNote={ABSTRACT Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA + ependymal lining to 2,8-linked PSA + migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA- and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain. IMPORTANCE Viruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain.}, number={7}, journal={JOURNAL OF VIROLOGY}, author={Murlidharan, Giridhar and Corriher, Travis and Ghashghaei, H. Troy and Asokan, Aravind}, year={2015}, month={Apr}, pages={3976–3987} }
 @article{muthusamy_vijayakumar_cheng_ghashghaei_2014, title={A Knock-in Foxj1(CreERT2:: GFP)Mouse for Recombination in Epithelial Cells with Motile Cilia}, volume={52}, ISSN={["1526-968X"]}, DOI={10.1002/dvg.22753}, abstractNote={The transcription factor Foxj1 is expressed by cells destined to differentiate into epithelial cells projecting motile cilia into fluid- or air-filled cavities. Here, we report the generation of an inducible knock-in Foxj1CreERT2::GFP mouse, which we show reliably induces Cre-mediated recombination for genetic studies in epithelial cells with motile cilia throughout embryonic and postnatal development. Induction during embryonic stages revealed efficient recombination in the epithelial component of the choroid plexus in the developing brain as early as E12.5. Induction during late embryonic stages showed confined recombination not only in the choroid plexus but also in the ventricular walls of the brain. Recombination induced during postnatal periods expanded to include epithelia of the lungs, testis, oviduct, and brain. Using these mice, we confirmed our recent discovery of a perinatally derived neuronal population in the mouse olfactory bulbs, which is derived from the Foxj1 lineage. Our Foxj1CreERT2::GFP knock-in mouse will be a powerful tool for studying molecular mechanisms associated with the continuum of cells that form the Foxj1 lineage, and for assessing their physiological significance during development and aging. genesis 52:350–358, 2014. © 2014 Wiley Periodicals, Inc.}, number={4}, journal={GENESIS}, author={Muthusamy, Nagendran and Vijayakumar, Akshitha and Cheng, Gang, Jr. and Ghashghaei, H. Troy}, year={2014}, month={Apr}, pages={350–358} }
 @article{nehrenberg_sheikh_ghashghaei_2013, title={Identification of neuronal loci involved with displays of affective aggression in NC900 mice}, volume={218}, ISSN={["1863-2661"]}, DOI={10.1007/s00429-012-0445-y}, number={4}, journal={BRAIN STRUCTURE & FUNCTION}, author={Nehrenberg, Derrick L. and Sheikh, Atif and Ghashghaei, H. Troy}, year={2013}, month={Jul}, pages={1033–1049} }
 @article{liang_xiao_yin_hippenmeyer_horowitz_ghashghaei_2013, title={Neural development is dependent on the function of specificity protein 2 in cell cycle progression}, volume={140}, ISSN={["0950-1991"]}, DOI={10.1242/dev.085621}, abstractNote={Faithful progression through the cell cycle is crucial to the maintenance and developmental potential of stem cells. Here, we demonstrate that neural stem cells (NSCs) and intermediate neural progenitor cells (NPCs) employ a zinc-finger transcription factor specificity protein 2 (Sp2) as a cell cycle regulator in two temporally and spatially distinct progenitor domains. Differential conditional deletion of Sp2 in early embryonic cerebral cortical progenitors, and perinatal olfactory bulb progenitors disrupted transitions through G1, G2 and M phases, whereas DNA synthesis appeared intact. Cell-autonomous function of Sp2 was identified by deletion of Sp2 using mosaic analysis with double markers, which clearly established that conditional Sp2-null NSCs and NPCs are M phase arrested in vivo. Importantly, conditional deletion of Sp2 led to a decline in the generation of NPCs and neurons in the developing and postnatal brains. Our findings implicate Sp2-dependent mechanisms as novel regulators of cell cycle progression, the absence of which disrupts neurogenesis in the embryonic and postnatal brain.}, number={3}, journal={DEVELOPMENT}, author={Liang, Huixuan and Xiao, Guanxi and Yin, Haifeng and Hippenmeyer, Simon and Horowitz, Jonathan M. and Ghashghaei, H. Troy}, year={2013}, month={Feb}, pages={552–561} }
 @article{liang_hippenmeyer_ghashghaei_2012, title={A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors}, volume={1}, ISSN={2046-6390}, url={http://dx.doi.org/10.1242/bio.20122287}, DOI={10.1242/bio.20122287}, abstractNote={Nestin-cre transgenic mice have been widely used to direct recombination to neural stem cells (NSCs) and intermediate neural progenitor cells (NPCs). Here we report that a readily utilized, and the only commercially available, Nestin-cre line is insufficient for directing recombination in early embryonic NSCs and NPCs. Analysis of recombination efficiency in multiple cre-dependent reporters and a genetic mosaic line revealed consistent temporal and spatial patterns of recombination in NSCs and NPCs. For comparison we utilized a knock-in Emx1(cre) line and found robust recombination in NSCs and NPCs in ventricular and subventricular zones of the cerebral cortices as early as embryonic day 12.5. In addition we found that the rate of Nestin-cre driven recombination only reaches sufficiently high levels in NSCs and NPCs during late embryonic and early postnatal periods. These findings are important when commercially available cre lines are considered for directing recombination to embryonic NSCs and NPCs.}, number={12}, journal={Biology Open}, publisher={The Company of Biologists}, author={Liang, H. and Hippenmeyer, S. and Ghashghaei, H. T.}, year={2012}, month={Sep}, pages={1200–1203} }
 @article{jacquet_muthusamy_sommerville_xiao_liang_zhang_holtzman_ghashghaei_2011, title={Specification of a Foxj1-Dependent Lineage in the Forebrain Is Required for Embryonic-to-Postnatal Transition of Neurogenesis in the Olfactory Bulb}, volume={31}, ISSN={["0270-6474"]}, DOI={10.1523/jneurosci.0171-11.2011}, abstractNote={Establishment of a neural stem cell niche in the postnatal subependymal zone (SEZ) and the rostral migratory stream (RMS) is required for postnatal and adult neurogenesis in the olfactory bulbs (OB). We report the discovery of a cellular lineage in the SEZ-RMS-OB continuum, the specification of which is dependent on the expression of the forkhead transcription factor Foxj1 in mice. Spatially and temporally restricted Foxj1+ neuronal progenitors emerge during embryonic periods, surge during perinatal development, and are active only for the first few postnatal weeks. We show that the development of the unique Foxj1-derived lineage is dependent on Foxj1 expression and is required for overall postnatal neurogenesis in the OB. Strikingly, the production of neurons from Foxj1+ progenitors significantly declines after the early postnatal weeks, but Foxj1-derived neurons in the OB persist during adult periods. For the first time, our study identifies the time- and region-specific activity of a perinatal progenitor domain that is required for transition and progression of OB neurogenesis from the embryonic-to-postnatal periods.}, number={25}, journal={JOURNAL OF NEUROSCIENCE}, author={Jacquet, Benoit V. and Muthusamy, Nagendran and Sommerville, Laura J. and Xiao, Guanxi and Liang, Huixuan and Zhang, Yong and Holtzman, Michael J. and Ghashghaei, H. Troy}, year={2011}, month={Jun}, pages={9368–9382} }
 @article{jacquet_ruckart_ghashghaei_2010, title={An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/2486}, DOI={10.3791/2486}, abstractNote={Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.}, number={46}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Jacquet, Benoit V. and Ruckart, Philip and Ghashghaei, H. Troy}, year={2010}, month={Dec} }
 @article{lim_piedrahita_jackson_ghashghaei_olby_2010, title={Development of a Model of Sacrocaudal Spinal Cord Injury in Cloned Yucatan MiniPigs for Cellular Transplantation Research}, volume={12}, ISSN={["2152-4998"]}, DOI={10.1089/cell.2010.0039}, abstractNote={Research into transplantation strategies to treat spinal cord injury (SCI) is frequently performed in rodents, but translation of results to clinical patients can be poor and a large mammalian model of severe SCI is needed. The pig has been considered an optimal model species in which to perform preclinical testing, and the Yucatan minipig can be cloned successfully utilizing somatic cell nuclear transfer (SCNT). However, induction of paralysis in pigs poses significant welfare and nursing challenges. The present study was conducted to determine whether Yucatan SCNT clones could be used to develop an SCI animal model for cellular transplantation research. First, we demonstrated that transection of the sacrocaudal spinal cord in Yucatan SCNT clones produces profound, quantifiable neurological deficits restricted to the tail. We then established that neurospheres could be isolated from brain tissue of green fluorescence protein (GFP) transfected SCNT clones. Finally, we confirmed survival of transplanted GFP-expressing neural stem cells in the SCI lesion and their differentiation into glial and neuronal lineages for up to 4 weeks without immunosuppression. We conclude that this model of sacrocaudal SCI in Yucatan SCNT clones represents a powerful research tool to investigate the effect of cellular transplantation on axonal regeneration and functional recovery.}, number={6}, journal={CELLULAR REPROGRAMMING}, author={Lim, Ji-Hey and Piedrahita, Jorge A. and Jackson, Lauren and Ghashghaei, Troy and Olby, Natasha J.}, year={2010}, month={Dec}, pages={689–697} }
 @article{jacquet_patel_iyengar_liang_therit_salinas-mondragon_lai_olsen_anton_ghashghaei_2009, title={Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors}, volume={16}, ISSN={["1476-5462"]}, DOI={10.1038/gt.2009.58}, abstractNote={Ongoing neurogenesis in discrete sectors of the adult central nervous system depends on the mitotic activity of an elusive population of adult stem cells. The existence of adult neural stem cells provides an alternative approach to transplantation of embryonic stem cells in cell-based therapies. Owing to the limited intrinsic fate of adult stem cells and inhibitory nature of the adult brain for neurogenesis, accommodation for circuit replacement in the brain will require genetic and epigenetic manipulation. Here, we show that a replication-incompetent Equine Infectious Anemia Virus (EIAV) is highly suitable for stable and persistent gene transfer to adult neural stem cells. The transduced regions were free of long-lasting neuroimmune responses to EIAV. Transduction in the subventricular zone was specific to the stem cell niche, but spared the progeny of adult neural stem cells that includes transit amplifying progenitors (TAPs) and migrating neuroblasts. With time, EIAV-transduced stem cells passed on the transgene to TAPs and migrating neuroblasts, which ultimately differentiated into neurons in the olfactory bulbs. We show that EIAV is highly suitable for discovery and assessment of mechanisms that regulate proliferation, migration and differentiation in the postnatal brain.}, number={8}, journal={GENE THERAPY}, author={Jacquet, B. V. and Patel, M. and Iyengar, M. and Liang, H. and Therit, B. and Salinas-Mondragon, R. and Lai, C. and Olsen, J. C. and Anton, E. S. and Ghashghaei, H. T.}, year={2009}, month={Aug}, pages={1021–1033} }
 @article{nighot_moeser_ryan_ghashghaei_blikslager_2009, title={ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum}, volume={315}, ISSN={0014-4827}, url={http://dx.doi.org/10.1016/j.yexcr.2008.10.001}, DOI={10.1016/j.yexcr.2008.10.001}, abstractNote={Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2−/− mice. Wild type, ClC-2 heterozygous and ClC-2−/− murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of 3H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation. Following ischemic injury, ClC-2−/− mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2−/− mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2−/− mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2−/− mice. These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.}, number={1}, journal={Experimental Cell Research}, publisher={Elsevier BV}, author={Nighot, Prashant K. and Moeser, Adam J. and Ryan, Kathleen A. and Ghashghaei, Troy and Blikslager, Anthony T.}, year={2009}, month={Jan}, pages={110–118} }
 @article{moy_ghashghaei_nonneman_weimer_yokota_lee_lai_threadgill_anton_2009, title={Deficient NRG1-ERBB signaling alters social approach: relevance to genetic mouse models of schizophrenia}, volume={1}, ISSN={["1866-1955"]}, DOI={10.1007/s11689-009-9017-8}, abstractNote={Abstract Growth factor Neuregulin 1 (NRG1) plays an essential role in development and organization of the cerebral cortex. NRG1 and its receptors, ERBB3 and ERBB4, have been implicated in genetic susceptibility for schizophrenia. Disease symptoms include asociality and altered social interaction. To investigate the role of NRG1-ERBB signaling in social behavior, mice heterozygous for an Nrg1 null allele ( Nrg1 +/−), and mice with conditional ablation of Erbb3 or Erbb4 in the central nervous system, were evaluated for sociability and social novelty preference in a three-chambered choice task. Results showed that deficiencies in NRG1 or ERBB3 significantly enhanced sociability. All of the mutant groups demonstrated a lack of social novelty preference, in contrast to their respective wild-type controls. Effects of NRG1, ERBB3, or ERBB4 deficiency on social behavior could not be attributed to general changes in anxiety-like behavior, activity, or loss of olfactory ability. Nrg1 +/− pups did not exhibit changes in isolation-induced ultrasonic vocalizations, a measure of emotional reactivity. Overall, these findings provide evidence that social behavior is mediated by NRG1-ERBB signaling.}, number={4}, journal={JOURNAL OF NEURODEVELOPMENTAL DISORDERS}, author={Moy, Sheryl S. and Ghashghaei, H. Troy and Nonneman, Randal J. and Weimer, Jill M. and Yokota, Yukako and Lee, Daekee and Lai, Cary and Threadgill, David W. and Anton, E. S.}, year={2009}, month={Dec}, pages={302–312} }
 @article{jacquet_salinas-mondragon_liang_therit_buie_dykstra_campbell_ostrowski_brody_ghashghaei_2009, title={FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain}, volume={136}, ISSN={["1477-9129"]}, DOI={10.1242/dev.041129}, abstractNote={Neuronal specification occurs at the periventricular surface of the embryonic central nervous system. During early postnatal periods, radial glial cells in various ventricular zones of the brain differentiate into ependymal cells and astrocytes. However, mechanisms that drive this time- and cell-specific differentiation remain largely unknown. Here, we show that expression of the forkhead transcription factor FoxJ1 in mice is required for differentiation into ependymal cells and a small subset of FoxJ1+ astrocytes in the lateral ventricles, where these cells form a postnatal neural stem cell niche. Moreover, we show that a subset of FoxJ1+ cells harvested from the stem cell niche can self-renew and possess neurogenic potential. Using a transcriptome comparison of FoxJ1-null and wild-type microdissected tissue, we identified candidate genes regulated by FoxJ1 during early postnatal development. The list includes a significant number of microtubule-associated proteins, some of which form a protein complex that could regulate the transport of basal bodies to the ventricular surface of differentiating ependymal cells during FoxJ1-dependent ciliogenesis. Our results suggest that time- and cell-specific expression of FoxJ1 in the brain acts on an array of target genes to regulate the differentiation of ependymal cells and a small subset of astrocytes in the adult stem cell niche.}, number={23}, journal={DEVELOPMENT}, author={Jacquet, Benoit V. and Salinas-Mondragon, Raul and Liang, Huixuan and Therit, Blair and Buie, Justin D. and Dykstra, Michael and Campbell, Kenneth and Ostrowski, Lawrence E. and Brody, Steven L. and Ghashghaei, H. Troy}, year={2009}, month={Dec}, pages={4021–4031} }
 @misc{ghashghaei_lai_anton_2007, title={Neuronal migration in the adult brain: are we there yet?}, volume={8}, ISSN={["1471-0048"]}, DOI={10.1038/nrn2074}, number={2}, journal={NATURE REVIEWS NEUROSCIENCE}, author={Ghashghaei, H. Troy and Lai, Cary and Anton, E. S.}, year={2007}, month={Feb}, pages={141–151} }
 @article{yokota_ghashghaei_han_watson_campbell_anton_2007, title={Radial Glial Dependent and Independent Dynamics of Interneuronal Migration in the Developing Cerebral Cortex}, volume={2}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0000794}, DOI={10.1371/journal.pone.0000794}, abstractNote={Interneurons originating from the ganglionic eminence migrate tangentially into the developing cerebral wall as they navigate to their distinct positions in the cerebral cortex. Compromised connectivity and differentiation of interneurons are thought to be an underlying cause in the emergence of neurodevelopmental disorders such as schizophrenia. Previously, it was suggested that tangential migration of interneurons occurs in a radial glia independent manner. Here, using simultaneous imaging of genetically defined populations of interneurons and radial glia, we demonstrate that dynamic interactions with radial glia can potentially influence the trajectory of interneuronal migration and thus the positioning of interneurons in cerebral cortex. Furthermore, there is extensive local interneuronal migration in tangential direction opposite to that of pallial orientation (i.e., in a medial to lateral direction from cortex to ganglionic eminence) all across the cerebral wall. This counter migration of interneurons may be essential to locally position interneurons once they invade the developing cerebral wall from the ganglionic eminence. Together, these observations suggest that interactions with radial glial scaffold and localized migration within the expanding cerebral wall may play essential roles in the guidance and placement of interneurons in the developing cerebral cortex.}, number={8}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Yokota, Yukako and Ghashghaei, H. T. and Han, Christine and Watson, Hannah and Campbell, Kenneth J. and Anton, E.S.}, editor={Chan-Ling, TailoiEditor}, year={2007}, month={Aug}, pages={e794} }
 @article{ghashghaei_weimer_schmid_yokota_mccarthy_popko_anton_2007, title={Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex}, volume={21}, DOI={10.1101/gad.1580407}, abstractNote={Radial glial cells play a critical role in the construction of mammalian brain by functioning as a source of new neurons and by providing a scaffold for radial migration of new neurons to their target locations. Radial glia transform into astrocytes at the end of embryonic development. Strategies to promote functional recovery in the injured adult brain depend on the generation of new neurons and the appropriate guidance of these neurons to where they are needed, two critical functions of radial glia. Thus, the competence to regain radial glial identity in the adult brain is of significance for the ability to promote functional repair via neurogenesis and targeted neuronal migration in the mature brain. Here we show that the in vivo induction of the tyrosine kinase receptor, ErbB2, in mature astrocytes enables a subset of them to regain radial glial identity in the mature cerebral cortex. These new radial glial progenitors are capable of giving rise to new neurons and can support neuronal migration. These studies indicate that ErbB2 signaling critically modulates the functional state of radial glia, and induction of ErbB2 in distinct adult astrocytes can promote radial glial identity in the mature cerebral cortex.}, number={24}, journal={Genes & Development}, author={Ghashghaei, H. T. and Weimer, J. M. and Schmid, R. S. and Yokota, Y. and McCarthy, K. D. and Popko, B. and Anton, E. S.}, year={2007}, pages={3258–3271} }
 @article{ghashghaei_hilgetag_barbas_2007, title={Sequence of information processing for emotions based on the anatomic dialogue between prefrontal cortex and amygdala}, volume={34}, ISSN={1053-8119}, url={http://dx.doi.org/10.1016/j.neuroimage.2006.09.046}, DOI={10.1016/j.neuroimage.2006.09.046}, abstractNote={The prefrontal cortex and the amygdala have synergistic roles in regulating purposive behavior, effected through bidirectional pathways. Here we investigated the largely unknown extent and laminar relationship of prefrontal input-output zones linked with the amygdala using neural tracers injected in the amygdala in rhesus monkeys. Prefrontal areas varied vastly in their connections with the amygdala, with the densest connections found in posterior orbitofrontal and posterior medial cortices, and the sparsest in anterior lateral prefrontal areas, especially area 10. Prefrontal projection neurons directed to the amygdala originated in layer 5, but significant numbers were also found in layers 2 and 3 in posterior medial and orbitofrontal cortices. Amygdalar axonal terminations in prefrontal cortex were most frequently distributed in bilaminar bands in the superficial and deep layers, by columns spanning the entire cortical depth, and less frequently as small patches centered in the superficial or deep layers. Heavy terminations in layers 1-2 overlapped with calbindin-positive inhibitory neurons. A comparison of the relationship of input to output projections revealed that among the most heavily connected cortices, cingulate areas 25 and 24 issued comparatively more projections to the amygdala than they received, whereas caudal orbitofrontal areas were more receivers than senders. Further, there was a significant relationship between the proportion of 'feedforward' cortical projections from layers 2-3 to 'feedback' terminations innervating the superficial layers of prefrontal cortices. These findings indicate that the connections between prefrontal cortices and the amygdala follow similar patterns as corticocortical connections, and by analogy suggest pathways underlying the sequence of information processing for emotions.}, number={3}, journal={NeuroImage}, publisher={Elsevier BV}, author={Ghashghaei, H.T. and Hilgetag, C.C. and Barbas, H.}, year={2007}, month={Feb}, pages={905–923} }
 @article{ghashghaei_weber_pevny_schmid_schwab_lloyd_eisenstat_lai_anton_2006, title={The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone}, volume={103}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0510410103}, DOI={10.1073/pnas.0510410103}, abstractNote={Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. Here, we investigate the role of neuregulins (NRGs) in this process and show that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP + astroglial cells, ependymal cells, and Dlx2 + precursors in the SVZ. Of the NRG ligands, both NRG1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP + putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2 + cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP + precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo .}, number={6}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Ghashghaei, H. T. and Weber, J. and Pevny, L. and Schmid, R. and Schwab, M. H. and Lloyd, K. C. K. and Eisenstat, D. D. and Lai, C. and Anton, E. S.}, year={2006}, month={Jan}, pages={1930–1935} }
 @article{ghashghaei_patel_olsen_anton_2005, title={752. Equine Infectious Anemia Virus Pseudotyped with the Vesicular Stomatitis Virus G-Protein, Preferentially Targets Neural Precursors in the Adult Mouse Brain}, volume={11}, ISSN={1525-0016}, url={http://dx.doi.org/10.1016/j.ymthe.2005.07.293}, DOI={10.1016/j.ymthe.2005.07.293}, abstractNote={The ongoing neurogenesis in the adult mammalian brain depends on mitotic activity of specialized stem cells in the subventricular zone. The majority of these stem cells have now been identified to have an astrocytic phenotype. Manipulation of these stem cells, especially through viral mediated gene transfer techniques, can be useful in a wide-range of experimental and potentially therapeutic settings. We used an Equine Infectious Anemia Virus (EIAV) to express Ehanced Green Fluorescent Protein (EGFP) in cells of the subventricular zone (SVZ). The virus, which was pseudotyped with the vesicular stomatitis virus G-protein (VSV-G), seemed to preferentially infect subset of glial fibrillary acidic protein expressing (GFAP+) astrocytic cells that line the ventricles. Furthermore, some of these cells may have given rise to migrating neuroblasts in the rostral migratory stream, which eventually develop into interneurons in the olfactory bulb. The preferential targeting of GFAP+ astrocytes in the SVZ by the EIAV lentivirus is potentially useful for the molecular manipulation of these stem cells.}, number={Supplement 1}, journal={Molecular Therapy}, publisher={Elsevier BV}, author={Ghashghaei, Troy and Patel, Manij and Olsen, John and Anton, Eva}, year={2005}, month={May}, pages={S292} }
 @article{anton_ghashghaei_weber_mccann_fischer_cheung_gassmann_messing_klein_schwab_et al._2004, title={Receptor tyrosine kinase ErbB4 modulates neuroblast migration and placement in the adult forebrain}, volume={7}, ISSN={1097-6256 1546-1726}, url={http://dx.doi.org/10.1038/nn1345}, DOI={10.1038/nn1345}, number={12}, journal={Nature Neuroscience}, publisher={Springer Nature}, author={Anton, E S and Ghashghaei, H T and Weber, Janet L and McCann, Corey and Fischer, Tobias M and Cheung, Isla D and Gassmann, Martin and Messing, Albee and Klein, Rudiger and Schwab, Markus H and et al.}, year={2004}, month={Nov}, pages={1319–1328} }