Identification of a sustained neurogenic zone at the dorsal surface of the adult mouse hippocampus and its regulation by the chemokine SDF-1

Abstract

We identified a previously unknown neurogenic region at the dorsal surface of the hippocampus; (the "subhippocampal zone," SHZ) in the adult brain. Using a reporter mouse in which SHZ cells and their progeny could be traced through the expression of EGFP under the control of the CXCR4 chemokine receptor promoter we observed the presence of a pool of EGFP expressing cells migrating in direction of the dentate gyrus (DG), which is maintained throughout adulthood. This population appeared to originate from the SHZ where cells entered a caudal migratory stream (aCMS) that included the fimbria, the meninges and the DG. Deletion of CXCR4 from neural stem cells (NSCs) or neuroinflammation resulted in the appearance of neurons in the DG, which were the result of migration of NSCs from the SHZ. Some of these neurons were ectopically placed. Our observations indicate that the SHZ is a neurogenic zone in the adult brain through migration of NSCs in the aCMS. Regulation of CXCR4 signaling in these cells may be involved in repair of the DG and may also give rise to ectopic granule cells in the DG in the context of neuropathology.

Keywords: CXCR4; SDF-1; adult neurogenesis; chemokines; dentate gyrus.

FIGURE 1

Expression patterns of EGFP in the adult brain of CXCR4-EGFP tg mice, (See also Supporting Information Figs. S1 and S2): The panels on the left show the sectional profiles used to generate panels on the right showing representative sagittal (A, C) and coronal (B, B’, B^″, D) sections through the brain of a 2-months old CXCR4-EGFP mouse. EGFP expression is detected in the major neurogenic niches in adult brain; in the SVZ (A, B’), the OB (A, B) and in the SGZ of the DG (A, B^″). In addition, streams of a sustained population of EGFP expressing cells with a migratory appearance are detected between the LV and the tip of the lower blade of the DG of the hippocampus as depicted in the area outlined by yellow dots, forming what is referred to as the aCMS. High magnification images of boxed areas in panel (D) are shown in (E-E^‴), illustrating streams of migratory CXCR4- EGFP cells along the aCMS pathway; at the medial wall of the LV near the FDJ (E), in the fimbria (E, E’) and along the meninges of the hippocampal fissure (E^″), including the meningeal-DG junction (E^‴), point of contact between the meninges and the lower blade of the DG. Note the continued and steady stream of CXCR4-EGFP cells between the FDJ and the SGZ of the DG and the changes of their morphology in the fimbria (E) and along the meninges in (E’, E^″). Scale bars are 250 μm in panels A, C, and D; 200 μm in panels B, B’, B^″, and 20 μm in panels E, E’, E^″, and E^‴.[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 2

CXCR4-EGFP cells along the aCMS pathway have migratory appearance and express stem/neural progenitor markers. A–H: Representative confocal images showing CXCR4-EGFP cells (green) with morphologies resembling migratory progenitors, in which a large proportion have un or bipolar morphologies, indicating their potential to migrate. A-A^″: Example of CXCR4-EGFP cells with one or two long processes (green, A) stained for the radial glia marker BLBP (A’, red). Merged images (A^″). Immuno-staining for GFAP (B, B’, B^″) and nestin (C, C’, C^″) showed the coexpression of GFAP (red) and nestin (red) in GFP cells (green), in the fimbria (B, C), and along the meninges (B’, C’), some with an apical radial glia process (arrows) with multiple endings at the meningeal-DG junction (B^″, C^″). Similarly, cells in the fimbria (D), along the meninges (D’) and at the meningeal-DG junction also stained for the neuronal stem marker SOX-2 (red), including cells at the meningeal-DG junction (D^″). Immunostaining for the neuronal progenitor marker Tbr-2, which showed it expression around the cell bodies and in the cell processes (E’), revealed that some of the cells in the SHZ and virtually all migratory cells within the fimbria (arrows, E, E^″) stained for Tbr-2 (arrows, E’, E^″).

FIGURE 3

To detect rapidly dividing cells, animals were injected with BrdU 2 h before euthanasia. Immunostaining for BrdU revealed that most of CXCR4-EGFP in the fimbria (arrows, F) incorporated BrdU (arrows, F’), indicating that these are rapidly dividing cells. Merged images (F^″). X and Y projections of the cell in inset (F^″) are shown in (F^‴). Representative images of immunostaining for BrdU (red) and SOX-2 (blue) (High magnification), showing a number of transient amplifying neural progenitors (arrows) characterized by BrdU and SOX-2 expression in the SHZ (G, G’, G^″, G^‴) and the fimbria (H, H’, H^″, H^‴) of CXCR4-EGFP tg mice. Consistent with this, using BrdU (arrows, I’, I^‴) with Tbr-2 (arrows, I^″, I^‴), we showed that these transient amplifying neural progenitors are fated to become neurons as characterized by BrdU and Tbr-2 expression in the fimbria (I, I’, I^″, I^‴). Scale bars are 20 μm in panels (As, Bs, C-C^″, Ds, and Fs), 10 lm in panels (C’, Es, Gs, Hs, and Is). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

FIGURE 4

mCherry retrovirus-mediated cell labeling of the LVs shows similar patterns as of CXCR4-EGFP expression. (Top panel) Scheme of mCherry retroviral labelling strategy that was used to label cells at the medial wall of the LV. mCherry retrovirus particles were injected into the LVs in adult brain of wild type and CXCR4-EGFP mice, and animals were euthanized 2 and 5 days later. The accuracy of the injection is indicated by red fluorescence at the LV in panel (A), which illustrates mCherry expression by cells of the LVs 2 days postviral injection. Five days later, streams of red mCherry- labelled cells with one or two processes were found along the aCMS pathway (B) as described for CXCR4-EGFP expression. High magnification images of boxed areas in panel (B) are shown in (C,D), showing mCherry-labeled cells (red) in the fimbria (Fi) (C) and the meninges (Me) of the hippo-campal fissure (D). In the fimbria, red labeled cells display bipolar morphology (arrows, C). In the meninges, some cells displayed multiple processes morphology resembling radial glia (arrows, D). Immunostaining for GFAP shows that cells in the fimbria stained for GFAP (arrows, C’), and seemed to lack GFAP expression along the meninges where they mainly adopt radial glia morphology (arrows, D’). E–H: Similarly, injection of a retrovirus-mCherry in the LVs of CXCR4-EGFP tg mice revealed that some mCherry-labeled cells along the aCMS migratory path (F), coexpressed GFP (E), indicating that retroviral-mCherry labeled cells are CXCR4-eGFP cells (yellow cells) (G). (G) counterstained with DAPI (blue) (H). (G’ and H’) are orthogonal projections showing the co-expression of mcherry and GFP in the same cell. DAPI counter-stain is shown in (blue, A, B). Scale bars are 250 μm in panel (A) and (B), and 2 0μm in panels (C, D, C’, D’, E, F, G, G’, H, and H’).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 5

Expression of EGFP in a previously unrecognized region with characteristics of NSCs in the adult brain of CXCR4-EGFP tg mice. A-A^″, a-a^″: Experimental protocol used to dissect the dorsal surface of the hippocampus (DSH) from the brain in adult mice. The hippocampus was dissected as shown in (A-A^″), and the DSH was displayed (a). (a’, a^″) DSH viewed at low power under an fluorescent binocular stereomicroscope equipped with a black and white CCD camera showing bright GFP fluorescence (a’), as shown by green dots in (a^″). B-B^″: Confocal reconstruction of representative whole mount preparations from areas highlighted in (a’, a^″), showing strong EGFP fluorescence in the SHZ. B: A face view of the DSH showing the location of the SHZ, also (B’). B^″: Horizontal sections through the DSH showing strong EGFP expression (green) in the SHZ (left side of image) and the DG (right side of image) with some CXCR4-EGFP cells scattered between the SHZ toward the DG (B^″), exhibiting long uni-or dual processes, suggestive of their capacity to migrate in direction to the DG (B^″). Boxed areas in panels (C, C’, and C^″) show representative higher magnification views of CXCR4-EGFP cells at the transitional area between the SHZ and the DG. C: Shows densely compacted cells in the SHZ with no apparent extracellular space. C’ and C^″: Shows streams of CXCR4- EGFP cells sometimes with long processes. D, D’, and D^″: Representative higher magnification image of these cells costained with the Tbr-2 antibody, indicating that these cells are neuronal progenitors. E–I: Whole mount preparations of the SHZ stained for EGFP antibody (green) showing CXCR4-EGFP cells also expressing a wide variety of stem cell markers (red). E–E^″: Example of a whole mount SHZ preparation stained for the stem/progenitor marker nestin (red), showing that virtually all the EGFP cells (E) costained for nestin (E’); Merged image (E^″). Whole mount SHZ preparations (F–F^″, G–G^″) and cross sections (F^‴, G^‴) showing that all SHZ cells (F, G), costained for GFAP (red, F) and the neuronal stem marker SOX-2 (red, G’); (F^″, F^‴, and G^″, G^‴) are merged images. To detect proliferating cells, animals were injected with BrdU, two times a day for 1 week, and euthanized 24 h later. Whole mount SHZ preparation (H-H^″) and cross sections (H^‴) show that virtually all EGFP cells (H, I’, green) incorporated BrdU (H’, H^″, H^‴, I, red), and also coexpressed the cell cycle marker Ki67 (I’, I^″, blue), indicating that most of the cells are slowly dividing. J: Electrophysio-logical characterization of SHZ cells showing similar functional characteristics to SGZ stem cells (Bhattacharyya et al., 2008). K: Neurospheres (NSs) obtained from cultures of SHZ whole mount preparations, indicating their self-renewal potential in cultures. Immunostaining of SHZ NSs for CXCR4 (K’, K^‴) and nestin (K^″, K^‴) demonstrates uniform expression of CXCR4 (blue) and nestin (red), further indicating the identity of SHZ cells as progenitor cells in cultures. L: Chemokines such as SDF-1 increased [Ca²⁺]_i in whole NSs, indicating the expression of functional CXCR4 receptors. (L’) Example of three cells dissociated from SHZ NSs that show responses to SDF-1. Addition of another chemokine such MCP-1 also increased [Ca²⁺]_i as did the purinergic receptor agonist ATP. Scale bars are 2 mm in panels (A-A^″), 500 μm in panels a-a^″, 250 μm in panels (B-B^″), 100 μm in panels (EE^″, F-F^″, G-G^‴, H-H^‴), 100 μm in panels (I-I^″), and 20 μm in panels (C-C^‴), and (D-D^‴’, F^‴, G^‴, and H^‴).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 6

Expression patterns of mRFP in the brain of CXCR4-EGFP/SDF-1-mRFP tg mice: (A-D) Low power confocal images of representative sections from the brain of a 2-months old SDF-1-mRFP mouse. Representative sagittal (A) and coronal sections (1A, 2A, B) through the hemispheres; C) Coronal section through the hippocampus, and D) Face view of a whole mount SHZ preparations, showing wide punctate expression of SDF-1 in the parenchyma of the fimbria (Fi), along the meninges (Me) and in the hippocampal fissures (fis). Also there is strong expression of SDF-1 in meningeal cells of the hippocampal fissures (1A, 2A, 1A’, 2A’), and in blood vessels (bv) (2A, 2A’). DAPI counterstain is shown in (blue, A, B, C, D, 1A, 2A). A’–D’: Low power confocal images of a representative sections through the brain of a 2-months old CXCR4-EGFP/SDF-1-mRFP mouse, showing aCMS CXCR4-EGFP cells in close proximity of the cells expressing SDF-1, in particular along the meninges of the fissure, indicating that they may potentially respond to SDF-1. A’: Sagittal section, (1A’, 2A’, B’) Coronal section through the hemispheres, (C) coronal section across the hippocampus, and (C’) Face view of a whole mount SHZ preparation. The inserts (1C’, 1D’ and 2D’) show high magnification views of boxed areas in panels (C’) and (D’), showing the distribution of CXCR4-EGFP cells in relation to SDF-1 expression in the fimbria (1C’), along the meninges of the hippocampal fissure (1A’), and within the transitional area between the SHZ and the DG (arrows in 1D’, 2D’). In (E-J) Chemotaxis assay showing strong chemoattractant responses induced by the addition of SDF-1 (100 nM) and its inhibition by the CXCR4 antagonist, AMD3100 (5 μM) in cultured SHZ CXCR4-EGFP cells. Representative data from four experiments showing examples of cells that migrated in control membrane inserts (E), and in response to SDF-1 (F), SDF-1+AMD3100 (G), and also to another chemokine MCP-1 (500 nM; H). I: Post hoc Ca imaging experiment illustrating examples of three migrated SHZ-EGFP cells (three different colors) responding to SDF-1, MCP-1, and ATP, indicating sustained functional receptor expression. J: Migration index in different media conditions as defined as the quantification of the number of cells that had migrated into the bottom of the membrane. Values are means +/− SEM of 6 fields/insert and four different experiments were used. Scale bars are 250μm in panels (A, A’, B, B’, C, C’), and (D, D’); 200μm in panels (1A, 2A, 1’A, 2A’); 20 μm in panels (1C’, 1D’, 2D’).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 7

HIV1-mediated inflammation of the hippocampus induced significant changes in CXCR4 and SDF-1 expression: (A) Experimental design that was used to infect the hippocampus by HIV1. CXCR4-EGFP/SDF-1-mRFP mice were injected with a solution of HXB2 virus into the DG of the hippocampus. Mice were euthanized at 24 h and 1, 2, and 3weeks later. Histological analysis of hippocampal sections at 24 h post HIV1 revealed a massive infiltration of immune cells and an upregulated expression of CXCR4 (A, A’, A^″) and SDF-1 (B, B’, B^″) in the ipsilateral hippocampi (A, A^″ and B, B^″) compared to contralateral hippocampi (A, A’, and B, B’). This was accompanied by morphological changes to the stem cell pool of the DG. In particular, the radial glia cells and their processes in the SGZ appeared dystrophic (a^″) compared to cells of the contralateral SGZ (a’). Flow cytometry analysis of the infiltrating cells showed that most of the cells were CD45 positive (C’, for CXCR4-EGFP and C^″, for SDF-1-RFP) with the majority being macrophages as defined by CD11b high and F4/80 sorting (C’ for CXCR4-EGFP, C^″ for SDF-1-RFP). In (D-D^″, E-E^″) HIV1-mediated injury of the DG induced a directed migration of cells expressing CXCR4; (D-D^″) TUNEL assays revealed frequent cell death in the upper blade of the ipsilateral DG by 1 and 2weeks post HIV. D: DAPI staining (blue), (D’) TUNEL staining (red), merged images in (D^″). E-E^″: Apparent directed migration of CXCR4-EGFP cells towards the upper blade of the DG. While TUNEL positive cells were diminishing but still present, a population of migratory CXCR4-EGFP cells (arrowheads) was found in direction of the ipsilateral DG at 1 week (E), 2 weeks (E’), and 3 weeks (E^″) post HIV. In (F), higher power view of image (E), showing a population of migratory cells in direction to the injured DG. In (F’) the same image in (F) was costained with a GFAP antibody (red), showing that these migratory cells (arrows in F) are not reactive astrocytes (arrows, F’). Scale bars are 250 μm in panels (A, B), 200 μm in panels (A’-A^″, B’-B^″, D-D^″, and E-E^″), 100 μm in panels (F-F’) and 50lm in panels (a’, a^″).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 8

mCherry retrovirus-mediated labelling of the SHZ reveals that the SHZ and its descendants in the aCMS migrate into the injured DG in response to HIV1: (Top panel) Experimental design used. mCherry retrovirus particles were injected into the LVs of the brain of CXCR4-EGFP tg mice. After 1 week, mice were injected with a solution of HXB2 virus into the DG, and were euthanized 3 weeks later. Brain sections were prepared and analyzed by confocal microscopy to look for migratory CXCR4-EGFP (green), and mCherry labeled cells (red). (A-A’) Injection of a retrovirus-mCherry into the LVs of control mice marks SHZ cells in CXCR4-EGFP tg mice and most mCherry- labelled cells were found along the aCMS migratory path, including the MDJ, intermingled with CXCR4-EGFP cells, and some coexpressed GFP (yellow cells; A) stained with DAPI (blue; A’). 3 weeks post HIV1 injection, a subset of red and yellow cells, including green cells were found along the aCMS path, including the medial wall of the LV (B, B’), in the FDJ (B^″), and along the meninges of the hippocampal fissure (C, C’). High magnification image of boxed areas in (A and B) are shown in (B’, B^″, C’, C’) illustrating yellow cells at the LV (B’), the fimbria (B^″) and along the meninges (C, C’). Importantly, some red and yellow cells were also found within the DG in the granule layer and in the hilus (D’, D^″). D: shows the migratory path taken by SHZ green cells at the MDJ towards the DG after mCherry injection for labelling followed by HIV1CD4 injection as stimulus. (E-E^″) enlarged panels of boxed area in (D) showing representative image of GFAP (E), IBA-1(E’), and CD45 (E^″) staining. These show that most mCherry positive cells have migrated away from the initial site of viral injection. Scale bars are 500 μm in panels (A, A’, D); 250μm in panels (B, E-E^″), and 20 μm in panels (B’, B^″, C, C’).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]