DEVELOPMENTAL DYNAMICS 237:1449 –1462, 2008
PATTERNS & PHENOTYPES
SPARC Is Expressed by Macroglia and Microglia in the Developing and Mature Nervous System Adele J. Vincent,1* Patricia W. Lau,1 and A. Jane Roskams1
SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein that is highly expressed during development, tissue remodeling, and repair. SPARC produced by olfactory ensheathing cells (OECs) can promote axon sprouting in vitro and in vivo. Here, we show that in the developing nervous system of the mouse, SPARC is expressed by radial glia, blood vessels, and other pial-derived structures during embryogenesis and postnatal development. The rostral migratory stream contains SPARC that becomes progressively restricted to the SVZ in adulthood. In the adult CNS, SPARC is enriched in specialized radial glial derivatives (Mu ¨ ller and Bergmann glia), microglia, and brainstem astrocytes. The peripheral glia, Schwann cells, and OECs express SPARC throughout development and in maturity, although it appears to be down-regulated with maturation. These data suggest that SPARC may be expressed by glia in a spatiotemporal manner consistent with a role in cell migration, neurogenesis, synaptic plasticity, and angiogenesis. Developmental Dynamics 237:1449 –1462, 2008. © 2008 Wiley-Liss, Inc. Key words: matricellular; extracellular matrix; glia; astrocytes; radial glia; microglia Accepted 27 January 2008
INTRODUCTION Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord enables tissue repair in an environment that is normally inhibitory to regeneration (Richter and Roskams, 2007). OECs that are derived from the peripheral olfactory system, lamina propria (LP)-OECs, can minimize glial scar and cavity formation, stimulate axon sprouting and invasion of Schwann cells, and promote directed angiogenesis (Ramer et al., 2004; Richter et al., 2005). Since the cellular mechanisms underlying these effects are not well understood,
we recently applied a proteomics screen to identify secreted factors produced by OECs that support neural tissue repair (Au et al., 2007). Our analysis revealed high levels of the glycoprotein, secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin and basement membrane protein-40 (BM-40). SPARC is a matricellular protein that belongs to a family of extracellular proteins that modulate cellular interactions with ECM molecules, and includes thrombspondins, tenascins, osteopontin, syndecans, and the SPARC-related proteins, SPARC like-1/hevin, testican,
and SMOC-1/2 (Brekken and Sage, 2000; Sage, 2001; Rocnik et al., 2006). SPARC is highly expressed during embryogenesis and is restricted in the adult to tissues undergoing remodeling, repair, and tumorigenesis (Holland et al., 1987; Sage et al., 1989a; Bradshaw and Sage, 2001; Framson and Sage, 2004). SPARC is a multifunctional protein that regulates extracellular matrix (ECM) organization and cell–ECM interactions, leading to the modulation of cell adhesion and migration, promotion of cell survival, and inhibition of cell proliferation (Brekken and Sage, 2000). SPARC null mice have a subtle pheno-
Department of Zoology, University of British Columbia, Vancouver, Canada Grant sponsor: Canadian Institutes of Health Research; Grant number: MOP-74666; Grant sponsor: Spinal Research Trust; Grant sponsor: Michael Smith Foundation for Health Research; Grant sponsor: Heart & Stroke Foundation of Canada; Grant sponsor: Canadian Stroke Network). *Correspondence to: Dr. Adele Vincent, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Rm 3320, Vancouver, B.C., V6T 1Z3 Canada. E-mail:
[email protected] DOI 10.1002/dvdy.21495 Published online 25 March 2008 in Wiley InterScience (www.interscience.wiley.com).
© 2008 Wiley-Liss, Inc.
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type that includes osteopenia (Delany et al., 2000), abnormal dermal collagen fibrils (Bradshaw et al., 2003b), cataractogenesis (Yan et al., 2002), and adiposity (Bradshaw et al., 2003a), and is exacerbated by challenge, including impaired wound healing (Basu et al., 2001; Bradshaw et al., 2002), and an impaired immune response (Kelly et al., 2007; Rempel et al., 2007). Some regions of the central nervous system (CNS) are now recognized to have a considerable capacity for repair and remodeling after injury (Carmichael, 2006; Okano et al., 2007). Although SPARC null mice have not been tested in this context, SPARC is up-regulated by astrocytes in the injured hippocampus (Liu et al., 2005), and in the amygdala after morphine exposure (Ikemoto et al., 2000). This suggests that SPARC may be involved in regulating plasticity and repair in the CNS. In support of this, we have shown that SPARC is a major component of the neurite outgrowth-promoting activity of OEC-conditioned medium in a dorsal root ganglion assay (Au et al., 2007). SPARC promotes axon outgrowth indirectly, by enhancing the ability of Schwann cells to support neurite outgrowth (Bampton et al., 2005; Au et al., 2007). When SPARC null OECs are transplanted into the injured rat spinal cord, the outgrowth of specific subsets of sensory and supraspinal axons is reduced, and the immune response is altered (Au et al., 2007). Furthermore, in the absence of OECderived SPARC, we found that SPARC is present in endogenous glia surrounding the lesion site (Au et al., 2007), which further supports a potential role for SPARC in CNS repair. Because development can provide clues about mechanisms of repair in the adult nervous system (Cramer and Chopp, 2000), and because SPARC mRNA is present in the developing nervous system (Mendis and Brown, 1994; Mothe and Brown, 2001), we used cell-specific markers to determine the expression of SPARC protein in the developing and mature nervous system of the mouse. We found that SPARC is enriched in neurogenic zones in the CNS, and is highly expressed in subpopulations of radial glia, astrocytes, Schwann cells, OECs, and microglia at distinct developmental stages. The data suggest
that SPARC is expressed by glia in a spatiotemporal manner consistent with an involvement in fundamental mechanisms of plasticity, such as cell migration, neurogenesis, angiogenesis, and peripheral myelination, and is likely to recapitulate such roles during regeneration of the nervous system.
RESULTS SPARC Is Expressed by Embryonic Radial Glia and Pial-Derived Structures in the CNS In the developing CNS, radial glia function both as neuronal progenitors in the ventricular zone and as a scaffold along which newborn neurons migrate. The cell bodies of radial glia line the brain ventricles, and their processes extend to the pial surface where they attach with endfeet (Rakic, 1972). Radial glia express the marker nestin (Hockfield and McKay, 1985), an intermediate filament protein, and subpopulations express the neurogenic radial glial markers, brain lipid binding protein (BLBP) (Feng et al., 1994; Anthony et al., 2004), and a disintegrin and metalloprotease 21 (ADAM21) (Yang et al., 2005). SPARC is first detected in regions of the developing brain undergoing neurogenesis (Fig. 1; Table 1). At embryonic day 10.5 (E10.5), SPARC is expressed in parts of the ventricular zone in all brain vesicles, for example, in the septal neuroepithelium (SN) in the telencephalon (Fig. 1A–D), and in the central canal of the neural tube (Fig. 1E). Notably, SPARC is highly enriched in brain flexures (Fig. 1F–H). At the cellular level, SPARC is expressed by nestin-positive radial glia (Fig. 1C,E,F), and overlaps with regions of BLBP-positive radial glia (Fig. 1B,I–K). SPARC is expressed with greatest intensity in the cell bodies of radial glia lining the ventricles (Fig. 1D,F, bracket), but is also present in their processes (Fig. 1B, large arrow; C, inset; F, arrow). Some SPARC immunoreactivity does not overlap with nestin or BLBP and appears to be present in the surrounding ECM, but is always segregated from neurons (Fig. 1D, Dcx-positive; !III tubulin-positive not shown). At E13.5 and E17.5, SPARC has a similar expression pattern in the
CNS. In the forebrain (Fig. 2A), SPARC is expressed in the hippocampal neuroepithelium (Fig. 2A, arrow, inset), lateral ganglionic eminence (LGE, Fig. 2A), and in the ventricular zone of the lateral ventricle (Fig. 2A,C–E). In the hindbrain, SPARC is expressed in the neuroepithelium of the forth ventricle and mesencephalic aqueduct (Fig. 2B arrows), and is prominently expressed in discreet regions of the developing cerebellum (Fig. 2B,F–H) and brainstem (Fig. 2I). In the spinal cord (Fig. 2J–N), SPARC is expressed in the central canal and floorplate. In all these regions, SPARC is expressed by nestin-positive radial glia (Fig. 2C,G,I), and overlaps with the BLBP-positive subpopulation of radial glia (Fig. 2D,F,J,M,N). In contrast, SPARC is not expressed by neurons (!III tubulin-positive) or neuroblasts (Dcx-positive) in any region of the embryonic nervous system (Fig. 2H,K,L). In addition, SPARC is highly expressed in developing blood vessels (arrowheads in Figs. 1, 2) and other pial-derived structures, including choroid plexus (CP, Fig. 2A–C) and pia mater (asterisks in Fig. 2B,G).
SPARC Is Expressed by Postnatal Radial Glia and at Brain Barriers Because SPARC is extensively expressed during embryonic development by radial glia, we next tested if SPARC is retained in postnatal radial glia. Nestin-positive radial glia are widespread throughout the brain at P1, but are restricted mainly to the cerebral hemispheres by P5, and are almost absent at P14. In early postnatal development (P1–P5), SPARC is expressed by nestin-positive radial glia in many regions of the postnatal brain and spinal cord (Fig. 3; Table 2), with high levels of expression detected in the inferior colliculus (Fig. 3A), cortex (Fig. 3B), brainstem (Fig. 3B inset), lateral ventricle (Fig. 3D,E), fimbria (Fig. 3F), and hippocampus (Fig. 3I). SPARC is also present in the rostral migratory stream (RMS) surrounding proliferating (PCNA") migratory cells (Fig. 3C). At the subcellular level, SPARC immunoreactivity is most intense in the cell bodies of radial glia lining the ventricles
SPARC EXPRESSION IN THE NERVOUS SYSTEM 1451
Fig. 1. SPARC in early embryonic radial glia and blood vessels. At E10.5/E11.5, SPARC is expressed in ventricular zones (VZ), including in the (A–D) septal neuroepithelium (SN) and (E) neural tube (NT), and is highly enriched at (F–H) brain flexures. SPARC is expressed by (C,E–H) nestin" radial glia, in their cell bodies (bracket in F) and processes (inset in C, arrow in F), and overlaps with regions of (A,B,I–K) BLBP" radial glia (arrows). SPARC is also expressed by (A,B,E,I–K) blood vessels (BV, arrowheads), but is excluded from (D) Dcx-positive immature neurons. R, rhombencephalon; DI, diencephalon; TEL, telencephalon; MZ, marginal zone; olf. pit, olfactory pit; D, dorsal; V, ventral; A, anterior; P, posterior. Directional arrows apply to A–E. Scale bars $ (A) 200 #m; (E, F) 100 #m; (I) 50 #m.
(arrows in Fig. 3A,D,F, bracket in Fig. 3E), and in their endfeet on the pial surface (barbed arrows in Fig. 3A,B). Radial glial processes are enriched for SPARC near their basal (arrow in Fig. 3E) and apical ends (arrows in Fig. 3B), usually as punctate staining that is distinct from the smooth staining associated with blood vessels (BV, arrowheads in Fig. 3A–C). A subset of ADAM21-positive radial glia lining the dorsal wall of the lateral ventricle also expresses SPARC at P5 (arrow in Fig. 3D inset). During later postnatal development (P14), SPARC is present in a small number of radial glia–like processes, most of which also express nestin (arrow in Fig. 3G) that extend from the lateral ventricle along the RMS (Fig. 3H) or into the striatum (not shown). SPARC is maintained at a low level in the adult subventricular zone (SVZ; Fig. 3G, inset).
The hippocampus displays a distinct pattern of SPARC expression that changes during postnatal development. At P1, SPARC-positive radial glial processes (nestin- and BLBP-positive) extend from the third ventricle into the CA1 region of the hippocampus (Fig. 3I), but are largely absent at later time points. The dentate gyrus contains SPARC-positive radial glia, and these cells are retained in the adult (arrows in Fig. 3I–K). Coincidental with the onset of synaptogenesis, SPARC is transiently expressed as punctate staining in the molecular layer of the dentate gyrus during postnatal development (Fig. 3I–K). The puncta are evident at a low level at P1 (Fig. 3I), and are prominent at P5 (inset in Fig. 3I) and P14 (Fig. 3J, K), but are no longer present in the adult (Fig. 3L). The SPARC-positive puncta do not appear to colocalise with nestin or BLBP (Fig. 3J, K). SPARC is enriched in components of
the blood-brain and blood-cerebrospinal fluid barriers. Ependymal cells that line the brain ventricles express SPARC during embryogenesis (Table 1) and postnatal development (barbed arrows in Fig. 3G,M). In the adult, a low level of SPARC expression is restricted to ependymal cells that line the lateral wall of the lateral ventricle (inset in Fig. 3G; Table 2). SPARC is expressed strongly by blood vessels (BV, Fig. 3A– C,K,N), choroid plexus (CP, Fig. 3D), and pia mater throughout development, but is down-regulated after P5–P14 (Table 2). Concomitantly, astrocytes in the brainstem begin to express SPARC in their endfeet surrounding blood vessels (part of the blood-brain barrier), and this is maintained in the adult (Fig. 3O). The colocalization of SPARC with S100 in astrocytic endfeet was confirmed by deconvolving Z-stacks of blood vessels in 50-#m tissue sections (not shown).
1452 VINCENT ET AL. TABLE 1. Embryonic Expression of SPARC in the Nervous System by Region
SPARC Is Maintained in Specialized Radial Glia in the Adult CNS Radial glia–like cells persist in regions of the adult CNS that retain activity-dependent plasticity (GudinoCabrera and Nieto-Sampedro, 1999). The widespread expression of SPARC by radial glia in the postnatal brain suggests that its expression may be retained in adult radial glia. Indeed, Bergmann glia in the cerebellum and Mu¨ller glia in the retina express SPARC in the adult brain (Fig. 4; Table 2). In the cerebellum, SPARC is not detectable at P1 (Fig. 4A), except in blood vessels (arrowhead) and pia mater (asterisk), but is present at P5 as punctate staining in the layer containing Bergmann glia and Purkinje cell bodies (arrows in Fig. 4B). At P14 and in the adult (Fig. 4C,D), SPARC is strongly expressed by all Bergmann glia (S100-positive, Fig. 4C–E; nestinpositive, Fig. 4F), with particular intensity in their cell bodies and endfeet on the pial surface (arrowhead and arrow in Fig. 4D, respectively). SPARC is also expressed by microglia (Iba-1-positive, Fig. 4G), but not by neurons, including Purkinje cells (Fig. 4H). In the retina, Mu¨ller glia, or their
precursors, express SPARC and nestin from early in embryonic development (Fig. 4I; Table 1) to maturity (Fig. 4J– L). SPARC staining is present in the endfeet of Mu¨ller glia at all developmental stages (arrows in Fig. 4K), and only becomes detectable in their cell bodies at P5 (Fig. 4J) and thereafter (arrowheads in Fig. 4K). SPARC is also expressed by microglia at P5 (Fig. 4M), but not by neuroblasts (Fig. 4N) or neurons (Fig. 4O).
SPARC Is Expressed by Microglia and Brainstem Astrocytes in the Mature CNS Having discovered that microglia in the cerebellum and retina express SPARC (Fig. 5), we asked if this expression is common to all microglia in the mature brain. SPARC mRNA appears to be widely expressed in the adult mouse brain (Fig. 5A–C; Allen Brain Atlas). In the cortex, SPARC immunoreactivity is found exclusively in microglia (Fig. 5D), and not astrocytes (Fig. 5E). Microglia are most densely packed in the cortex and dorsal superior and inferior colliculi, and express SPARC throughout the brain (e.g., Fig 4G,M; Fig. 5; Table 2). However, microglia are not the major source of SPARC mRNA in the
brainstem (Fig. 5A–C). SPARC is highly expressed in the brainstem (Fig. 5F–H), and in the grey matter of the spinal cord (Fig. 5J), by S100-positive astrocytes (e.g., pons, Fig. 5I). In contrast, astrocytes in more anterior structures express little or no SPARC, such as in the striatum and lateral septal nuclei (Fig. 5F), cortex (Fig. 5E) and olfactory bulbs (Fig. 5K). Although microglia are derived from the same haemopoietic lineage as macrophages, we did not detect SPARC in macrophages in any embryonic or postnatal organ. This is best illustrated in the olfactory system, where microglia in the olfactory bulb express SPARC (Fig. 5K,M arrow), but macrophages in the peripheral olfactory nerves do not (Fig. 5K,L arrow). Furthermore, SPARC is not detectable in oligodendrocyte precursors (NG2-positive) at P1, P14, or in the adult, or in oligodendrocytes (CNPase/O4-positive) at P14 (not shown).
SPARC Is Expressed by Schwann Cells and Olfactory Ensheathing Cells Because several classes of central glia express SPARC, we also examined SPARC expression in peripheral glia. A subset of Schwann cells are strongly
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Fig. 2. SPARC in mid-late embryonic radial glia. A–I: At E13.5/E17.5, SPARC is expressed in the ventricular zones (VZ) of the (A,C–E) lateral ventricle (LV), (B) mesencephalic aqueduct (MA), and fourth ventricle (V4), in the (A) lateral ganglionic eminence (LGE) and hippocampal neuroepithelium (HIPP; arrow), and in the (B,F–H) developing cerebellum (CB), and (I) brainstem (BS). J–N: SPARC is expressed in the central canal (CC) and floorplate (FL) of the spinal cord (SC). At the cellular level, SPARC is expressed by (C,F,I) nestin" radial glia (arrow in F), and by a subset of (D,H,M,N) BLBP" radial glia (arrows). SPARC is also highly enriched in pial-derived structures including (A–C) choroid plexus (CP), (B,F) pia mater (asterisks), and in blood vessels (BV, arrowheads throughout). SPARC is not expressed by (G,K) neurons (!III tubulin") or (L) neuroblasts (Dcx"). CTX, cortex; MB, midbrain; D, dorsal; V, ventral; A, anterior; P, posterior; HB, hindbrain. A–L,N are in sagittal section and M is in coronal section. Directional arrows apply to A–I. Scale bars $ (A,C,F,H–J, M) 100 #m; (K,L,N) 50 #m; (D) 25 #m.
immunopositive for SPARC at all developmental stages (Fig. 6A–G; Tables 1, 2), and in all peripheral nerves examined, including cranial nerve roots (arrows in Fig. 6A), facial nerves (Fig. 6B–F), sciatic nerves (Fig. 6G), spinal nerve roots, and cauda equina (not shown). SPARC is concentrated in the
cell soma of Schwann cells (S100-positive, Fig. 6C,D, arrow in E), and is distributed along the outer plasma membrane and/or basal lamina of myelinating Schwann cells (P0-positive, Fig. 6G), including around nodes of Ranvier (arrow in Fig. 6F). Olfactory ensheathing cells are
also intensely immunopositive for SPARC during embryogenesis (arrows in Fig. 6H,I), but appear to down-regulate SPARC postnatally (Fig. 6J; Table 2). SPARC is present in OEC processes (nestin/BLBP-positive) (Murdoch and Roskams, 2007) that surround the outside of olfac-
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Fig. 3. SPARC in postnatal radial glia and brain barriers. A–L: SPARC is expressed by radial glia during early postnatal development, including in the (A) inferior colliculus, (B) cortex (CTX), (B inset) brainstem, (C,G,H) rostral migratory stream (RMS), (D,E) subventricular zone (SVZ) of the lateral ventricle (LV), (F) fimbria, and (I–L) hippocampus. SPARC is highly enriched in radial glial (A,D–F,I–K) cell bodies (arrows, bracket in E), and (A,B) endfeet (barbed arrows), and is present in their processes (arrows in B,E). A subset expresses (D inset) ADAM21 (arrow). SPARC is also expressed in (G,M) ependymal cells (barbed arrows), (D) choroid plexus (CP), (A–C,K,N) endothelial cells in blood vessels (BV, arrowheads), and (O) astrocytic endfeet in the adult brainstem. D, dorsal; V, ventral; A, anterior; P, posterior; OB, olfactory bulb; CA1-3, hippocampal regions (cornu ammonis); DG, dentate gyrus; ML, molecular layer; GL, granule layer; H, hylus; V3, third ventricle. A-L, N are in sagittal section, M is in transverse section, and O is in coronal section. L, detection is by chromogen immunohistochemistry. Directional arrows apply to A–G,I–L. Scale bars $ (A–D,I,J) 100 #m; (E–H, K–O) 50 #m.
tory axon bundles (Fig. 6I inset is in cross-section). In the adult, SPARC is restricted to the outer sheaths of a subset of OEC processes surrounding mesaxons (S100-positive, arrows in Fig. 6J), and is not detectable in the fine processes within each mesaxon (arrowhead in Fig. 6J).
DISCUSSION The matricellular glycoprotein SPARC is a glial-derived factor that is expressed in distinct stages of nervous system development, and is retained in the adult in unique niches that retain a high degree of plasticity. SPARC is enriched in radial glia in
neurogenic zones within the developing mouse embryo, and is maintained in adult radial glia–like cells, the SVZ, and dentate gyrus, where it is restricted to regions of postnatal and adult neurogenesis (Figs. 1– 4). Microglia and all classes of macroglia in the CNS and PNS, except for oligodendro-
SPARC EXPRESSION IN THE NERVOUS SYSTEM 1455 TABLE 2. Postnatal Expression of SPARC in the Nervous Systems by Cell Type
cytes and their precursors, express SPARC in at least a subset of cells during development (Figs. 4 – 6). In the peripheral glia, Schwann cells, and OECs, SPARC is concentrated in the basal lamina that forms a boundary between glial processes and their adjacent environment (Fig. 6). These detailed cellular SPARC expression erns are largely in accord with lowpower in situ hybridization or immunohistochemistry for SPARC in the developing rodent (Mendis and Brown, 1994; Mendis et al., 1995; Mothe and Brown, 2001). However, we show for the first time that SPARC is expressed in radial glia (Figs. 1– 4), microglia (Figs. 4, 5), and Schwann cells (Fig. 6). SPARC can be secreted to regulate local growth factor, ECM, and matrix metalloproteinase (MMP) activity, to modulate cell morphology and migration responses, angiogenesis, and cell proliferation and survival (Rempel et
al., 2001; Francki et al., 2004; Barker et al., 2005a; Yan et al., 2005; Said et al., 2007a). In vitro, SPARC acts on many cell types as a deadhesive factor that induces cell rounding and inhibits cell migration (Sage et al., 1989b; Bradshaw and Sage, 2001), including on neuronal and glial cell lines (Ikemoto et al., 2000). However, SPARC is thought to act in vivo as a contextual regulator of cellular adhesion strength (Greenwood and Murphy-Ullrich, 1998), and is required for fibroblast migration during dermal wound healing (Basu et al., 2001), leukocyte infiltration after immune challenge (Kelly et al., 2007; Rempel et al., 2007), and tumor invasiveness (Rempel et al., 2001; Framson and Sage, 2004). SPARC modulates cell morphology, migration, and survival via signaling pathways that include integrin-linked kinase (ILK), focal adhesion kinase (FAK), mitogen-associated protein kinase (MAPK), and AKT/pro-
tein kinase B (Shi et al., 2004, 2007; Barker et al., 2005a; Said et al., 2007a). SPARC is highly expressed in embryonic, postnatal, and adult radial glia (Figs. 1– 4), where it has the potential to subserve several functions. The distribution of SPARC along radial glial processes may facilitate the apical migration of neuroblasts from the ventricular layer towards the pia mater (Rakic, 1972). The SPARC homolog, hevin, is expressed by cortical radial glia during embryogenesis and is necessary for cortical lamination (Gongidi et al., 2004). Hevin has deadhesive properties that terminate neuroblast migration at the cortical plate (Gongidi et al., 2004). Thus, SPARC and hevin may have some redundancy or complementarity of function, as they do in the foreign body response (Barker et al., 2005b), although overlap in their expression has not been fully determined. SPARC is not present in embry-
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Fig. 4. SPARC in Bergmann glia and Mu¨ller glia. A–H: SPARC is absent from the cerebellum (CB) (A) at P1, except in blood vessels (BV, arrowhead) and pia mater (asterisk), but is present (B) at P5 as punctate staining in the Purkinje cell layer (PCL, arrows). SPARC is expressed by Bergmann glia (S100") at (C,E) P14 and (D) in the adult, with greatest intensely in their endfeet and cell bodies (arrow and arrowhead in D, respectively). SPARC is coexpressed with (F) nestin in Bergmann glia processes, and is expressed by (G) microglia (Iba-1"), but is not expressed by (H) Purkinje cells (!III tubulin"). I–O: SPARC is expressed in the retina at (I) E13.5, (J,L–O) P5, and (K) P14 by Mu¨ller glia (nestin"), with greatest intensity in their endfeet (arrows in K) in the optic nerve fibre layer (ONFL), and reduced intensity in their cell bodies (arrowheads in K) in the inner neuronal layer (INL). At P5, SPARC is also expressed by (M) microglia (Iba-1"), but not by (N) neuroblasts (Dcx") or (O) neurons (!III tubulin"). IC, inferior colliculus; WM, white matter; GL, granular layer; ML, molecular layer; GCL, granule cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; ONL, outer neuronal layer. All images are in sagittal section. Scale bars $ (A–D) 100 #m; (E–H,I) 25 #m; (J,K) 50 #m.
onic cortical radial glia in the same time window as hevin (Fig. 2), but both matricellular proteins have a similar expression pattern in the developing cerebellum, where they are thought to regulate granule cell migration (Fig. 2) (Mendis et al., 1994; Mothe and Brown, 2001). During postnatal development, SPARC may facilitate neuronal migration along the RMS, which contains SPARC during the peak of postnatal interneuron migration to the olfactory bulb (Fig. 3) (Luskin, 1993). Prominent
SPARC expression also persists in cortical radial glial at P5 (Fig. 3), and may be involved in the axial migration of late-born projection neurons and interneurons, which utilize glial-guided migration in the embryo (Rakic, 1972; Yokota et al., 2007), during the first postnatal week (Hevner et al., 2004). In the adult brain, specialized radial glia–like cells that are associated with ongoing plasticity and cell migration continue to express SPARC, including Bergmann glia in the cerebellum (Fig. 4) (Mothe and
Brown, 2001), Mu¨ller glia in the retina (Fig. 4), and radial glia–like cells in the mature dentate gyrus (Fig. 3). Furthermore, SPARC is concentrated at brain flexures in the embryonic brain (Fig. 1) and in radial glial endfeet (Figs. 2, 3), and is thus ideally positioned to stabilize the rapidly expanding cytoarchitecture of the developing brain. Radial glial endfeet provide anchorage at the meningeal basement membrane of the pia mater to support the highly extended morphology of radial fibres in
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the developing brain. Disruption of this attachment causes defects in cortical lamination, such as in mice with genetic defects in integrins, ILK, FAK, and ECM components (Graus-Porta et al., 2001; Halfter et al., 2002; Beggs et al., 2003; Poschl et al., 2004; Niewmierzycka et al., 2005). SPARC could potentially act to anchor radial glial endfeet, either by modulating the stability of focal adhesions via integrin-mediated FAK and ILK signaling (Shi et al., 2007), or alternatively by the assembly and maintenance of ECM components in the basement membrane (Yan et al., 2003). SPARC, which remains enriched in endfeet, may continue to stabilize radial glial morphology in the adult, such as in the cerebellum and retina (Fig. 4). From its presence in other nonneuronal cell types, SPARC has the potential to play a plethora of other roles in the developing or injured nervous system. SPARC becomes progressively restricted during postnatal development to distinct neurogenic niches in the adult brain; the SVZ and ependymal cells of the lateral ventricle, and radial glial–like cells of the dentate gyrus (Fig. 3). The SVZ contains specialized structures of extravascular basal lamina, termed fractones, that contain laminin and collagen (Mercier et al., 2002). As a highly conserved base-
Fig. 5.
Fig. 5. SPARC in microglia and astrocytes. A–C: Allen Brain Atlas images of SPARC mRNA expression in the adult mouse brain (boxes represent the positions of D,E,I,K, and vertical lines represent the positions of coronal sections in B,C,F–H). D,E: SPARC is expressed in the cortex by (D) microglia (Iba-1"), but not by (E) astrocytes (S100"). F–J: SPARC is expressed throughout the (F–H) brainstem, including the hypothalamus (HYP), globus pallidus (GP), and thalamus (TH). This expression is in astrocytes (S100"), such as in the (I) pons, and is also in the (J) spinal cord. K–M: SPARC is expressed by (K,M) microglia (Iba-1", arrow in M) in the olfactory bulb (OB), but not by (K,L) macrophages (Iba-1", arrow in L) in the olfactory nerve (ON). Note that SPARC is expressed by (L) blood vessels (BV) in the ON and olfactory nerve layer (ONL), but very weakly by (M) blood vessels in the OB. STR, striatum; LV, lateral ventricle; LS, lateral septal nucleus; IC, internal capsule; FN, fornix; V3, third ventricle; HIPP, hippocampus; WM, white matter; GM, grey matter; GL, glomerular layer. A,D,E,I–M are in sagittal section and B,C,F–H are in coronal section. Scale bars $ (D,K) 100 #m; (F) 200 #m; (I,J) 50 #m.
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Fig. 6. SPARC in Schwann cells and olfactory ensheathing cells. A–G: SPARC is expressed throughout development by a subset of Schwann cells (S100", B-D), including in (A) cranial nerve roots (arrows), (B–F) facial nerves, and (G) sciatic nerves. The cellular localization is most intense in the (B–D,E) cell soma (arrow in E), and delineates the outer membrane at (F) nodes of Ranvier (arrow) and surrounding (G) myelin (P0"). SPARC is completely segregated from (E–G) axons (!III tubulin"). H–J: SPARC is expressed throughout development by olfactory ensheathing cells (arrows; nestin/BLBP/S100"), with greatest intensity at (H,I) E13.5 and weak expression in the (J) adult. SPARC is only detectable in the glial processes that surround mesaxons (inset in I, arrows in J). BS, brainstem; G, ganglion; TEL, telencephalon; OE, olfactory epithelium; LP, lamina propria; NP, nasal pit; ONL, olfactory nerve layer (of the olfactory bulb). A–I are in sagittal section and J is in coronal section. Scale bars $ (A,B,H) 50 #m; (C,E) 25 #m; (G,I,J) 10 #m.
ment membrane molecule (Fitzgerald and Schwarzbauer, 1998; Bradshaw and Sage, 2001; Martinek et al., 2002), SPARC may be involved in the assembly and maintenance of fractones by regulating the production of ECM molecules such as laminin (Kamihagi et al., 1994; Weaver et al., 2006) and collagen (Francki et al., 1999; Bradshaw et al., 2003b). Ultrastructural examination of SPARC in the SVZ will be necessary to investigate this possibility further. Ependymal cells form a part of the neural stem cell niche (Doetsch et al., 1999) that suppresses gliogenesis in favor of neurogenesis (Lim et al., 2000). SPARC, expressed by cells adjacent to candidate neural stem
cells, could potentially regulate the quiescence of the neural stem cell niche by modulating the activity of growth factors and matrix remodeling factors to inhibit cell cycle progression and regulate survival (Funk and Sage, 1991; Shi et al., 2007). SPARC negatively regulates signaling by various growth factors known to be involved in neural stem cell proliferation and differentiation, including fibroblast growth factor 2 (FGF2) (Motamed et al., 2003), epidermal growth factor (EGF) (Said et al., 2007a), insulin-like growth factor-1 (IGF-1) (Francki et al., 2003), and transforming growth factor ! (TGF!) (Schiemann et al., 2003; Francki et al., 2004). However, the
expression of SPARC is in turn regulated by each of these factors; decreased by FGF2 and EGF, increased by IGF-1, and reciprocally regulated by TGF! (Wrana et al., 1991; Chandrasekhar et al., 1994; Delany and Canalis, 1998; Shiba et al., 1998, 2001). Furthermore, SPARC can positively and negatively modulate growth factor- and adhesion-dependent cell survival signaling (Shi et al., 2004, 2007; Said et al., 2007a), with the potential to maintain neural stem cell equilibrium. Thus, SPARC may also contribute to the generation of the neural stem niche by a similar mechanism to that of the matricellular protein, tenascin-C, which orchestrates growth
SPARC EXPRESSION IN THE NERVOUS SYSTEM 1459
factor signaling to promote neural stem cell development in vitro (Garcion et al., 2004). Although tenascin-C deficiency does not cause a reduction in neural stem cells or their progeny in vivo (Kazanis et al., 2007), this may be another example of functional redundancy between matricellular proteins (Sage, 2001; Barker et al., 2005b; Puolakkainen et al., 2005). As the resident immune cell of the brain, microglia require exquisite regulation of cellular adhesion to enable constant surveillance of the surrounding neuropil, and rapid process motility and cell migration to sites of injury (Kurpius et al., 2006). The expression of SPARC by all resting microglia (Fig. 5) suggests that this deadhesive protein may play a role in maintaining microglial homeostasis in the normal brain, as it does in ovarian cancer cells (Said et al., 2007b). This may occur in a manner similar to the matricellular protein, tenascin-R, which is a constituent of perineuronal nets, and is deadhesive for activated microglia (Angelov et al., 1998). Different tenascin-R peptide fragments can either inhibit or promote microglial migration (Liao et al., 2005). SPARC can similarly be cleaved by proteases and MMPs to yield peptides with distinct functions (Lane et al., 1994; Iruela-Arispe et al., 1995; Sasaki et al., 1997), and can also regulate protease expression and activation (Brekken and Sage, 2000; McClung et al., 2007). Since many proteases are up-regulated after injury by microglia and other cells (Yong, 2005; del Zoppo et al., 2007), it is possible that cleavage of SPARC might facilitate the rapid process motility and chemotaxis of activated microglia (Davalos et al., 2005; Nimmerjahn et al., 2005; Kurpius et al., 2006). It is equally likely that resting microglia may sequester SPARC intracellularly, and, once activated, secrete the protein to enable rapid disengagement from the ECM and cell migration. This is consistent with the finding that glioma cell lines are more migratory when secreting high levels of SPARC (Rempel et al., 2001). SPARC also has the potential to regulate angiogenesis in the developing brain (Figs. 1–3) (Lane et al., 1994; Mendis and Brown, 1994), as it does in other developing tissues, and in tumors
and dermal wounds (Lane et al., 1994; Iruela-Arispe et al., 1995; Vajkoczy et al., 2000; Chlenski et al., 2002). SPARC negatively regulates signaling by several angiogenic growth factors, including platelet derived growth factor (Motamed et al., 2002) and vascular endothelial growth factor (Kupprion et al., 1998), but can also be cleaved to produce the potent pro-angiogenic peptide (K)GHK (Lane et al., 1994). Thus, SPARC may function in an anti- or proangiogenic capacity depending on the cellular and extracellular microenvironment. A role for SPARC in synaptogenesis and synaptic plasticity is suggested by its restriction to key populations of astrocytes in brainstem nuclei (Fig. 4) (Mendis et al., 1995), and in the molecular layer of the dentate gyrus during the peak period of synaptogenesis (Fig. 3). Furthermore, SPARC is upregulated in hippocampal astrocytes during deafferentiation-induced sprouting and synaptogenesis (Liu et al., 2005), and in the amygdala during morphine-induced synaptic remodeling (Ikemoto et al., 2000). Recent work suggests that SPARC antagonizes the synapse-inducing activity of hevin in cultured retinal ganglion cells (Eroglu et al., 2007), and may therefore act as a homeostatic regulator of synapse density in the developing and regenerating CNS. Finally, in the peripheral nerves, SPARC may facilitate the migration of neural crest cells, embryonic Schwann cells, and OEC precursors (Fig. 6; Table 1) (Au et al., 2007), which migrate in tandem with their developing nerves (Tennent and Chuah, 1996; Jessen and Mirsky, 2005). Later, SPARC is ideally positioned to play a role in the formation and maintenance of the basal lamina that is necessary for Schwann cell myelination (Fig. 6) (Court et al., 2006), similar to its role in basement membranes in other tissues and across multiple organisms (Fitzgerald and Schwarzbauer, 1998; Huynh et al., 2000; Bradshaw and Sage, 2001; Martinek et al., 2002). This may also explain why oligodendrocytes, whose myelin does not have a basal lamina, do not express SPARC. While OECs do not myelinate olfactory axons, they ensheathe multiple axons in large nerve bundles and have a basal lamina on their abaxial
surface that contains a low level of SPARC (Fig. 6). After bulbectomy, the upregulation of SPARC by OECs (Au et al., 2007) may help to stabilize the structure of the nerve conduits until they refill with newly growing axons (Li et al., 2005), possibly via SPARC’s interaction with collagen in the basal lamina (Brekken and Sage, 2000). SPARC is thus a dynamically regulated, glial-derived factor in the developing and mature nervous system, and is localized in radial glia, brainstem astrocytes, microglia, peripheral glia, and brain barriers. This suggests that SPARC may have multifunctional roles as a modulator of cell migration, cytoarchitecture, neurogenesis, angiogenesis, and peripheral myelination, and could be an important modulator of integrative growth factor and ECM signaling in neural development and regeneration.
EXPERIMENTAL PROCEDURES Animals Animal procedures were performed on CD-1, Thy1-YFP H-Line (Feng et al., 2000), and C57Bl6 mice (Jackson Laboratories, Bar Harbour, ME) in accordance with the guidelines of the Canadian Council for Animal Care and the University of British Columbia animal care committee. At least two mice per time point were analyzed.
Tissue Processing Mice aged from P10 to adulthood were sacrificed by lethal intraperitoneal injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) and transcardially perfused with phosphate buffered saline (PBS) and 4% phosphate-buffered formaldehyde (PFA). Mice aged from P1 to P5 were sacrificed by inhalation of isoflurane and transcardially perfused as above. Pregnant dams were sacrificed by lethal injection and embryos were removed and placed in 4% PFA overnight. Perfused tissue was post-fixed overnight in 4% PFA, cryopreserved in 30% sucrose in PBS, and frozen in Tissue-Tex optimal cutting compound (Sakura, Tokyo, Japan) in isopentane over dry ice. Frozen 14- or 20-#m sections were cut, collected on
1460 VINCENT ET AL.
Superfrost Plus slides (Fisher Scientific, Edmonton, AB, Canada), and stored at %20°C. Free-floating 50-#m coronal sections were collected from adult brain and stored in 0.01% sodium azide in PBS at 4°C.
Immunohistochemistry Frozen sections and free-floating sections were stained as previously described (Au et al., 2007) with slight modifications. For antigen retrieval to detect proliferating cell nuclear antigen (PCNA), the tissue was post-fixed in 4% formaldehyde for 10 min, and boiled in 0.01M citric acid in PBS, pH 6.0, for 10 min in the microwave. All tissue was then processed as follows. The tissue was permeabilized in 0.3% Triton X-100 (Sigma, St Louis, MO) in PBS for 30 min, washed in PBS, then blocked in 4% normal serum in PBS for 20 min, and incubated with primary antibodies in 2% normal serum at 4°C overnight. Primary antibodies included goat anti-SPARC (1:500; R&D Systems, Minneapolis, MA), monoclonal anti-PCNA (1:5,000; Sigma), rabbit anti-BLBP (brain lipid binding protein; 1:1,000; Chemicon, Temecula, CA), rabbit anti-!III tubulin (TUJ1 clone; 1:1,000; CoVance, Princeton, NJ), rabbit anti-S100 (1:1,000; Neomarkers, Waltham, MA), rabbit anti-Iba-1 (1:1,000, Wako Chemicals USA, Richmond, VA), rabbit anti-nestin (1:1,000; Covance), rabbit anti-Dcx (doublecortin; 1:1,000; Cell Signaling Technology, Danvers, MA), and rabbit anti-Adam21 (a disintegrin and metallopeptidase domain 21; 1:1,000; Chemicon). The sections were washed with 0.3% Triton X-100 in PBS and then incubated with secondary antibodies raised in donkey and conjugated to Alexa Fluor 488 or 594 for 1 hr at room temperature (1: 200; Molecular Probes, Eugene, OR). Nuclei were counterstained with 0.5 #g/ml DAPI and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Detection by peroxidase chromogen reaction was performed using biotinylated horse anti-goat secondary antibodies (1:200; Vector Laboratories), and the Vectastain ABC kit and Vector VIP kit (Vector Laboratories). Primary antibodies were omitted as a negative control.
Image Collection Images were captured using an Axioplan2 Imaging epifluorescent microscope (Zeiss, Jena, Germany) and Northern Eclipse 6.0 software (Empix Imaging, Mississauga, ON, Canada), and processed using Photoshop 7.0 software (Adobe Systems, San Jose, CA). Images were corrected for sharpness, contrast, and brightness.
ACKNOWLEDGMENTS This project was funded by the Spinal Research Trust (to A.J.V. and J.R.), Michael Smith Foundation for Health Research (to A.J.V. and J.R.), Heart & Stroke Foundation of Canada (to A.J.V.), Canadian Stroke Network (to A.J.V.), and the Canadian Institutes of Health Research (to J.R.). We thank Dr. Matt Larouche, Miranda Witheford Richter and Barbara Murdoch for valuable comments on the manuscript, and Dr. Craig Brown, Dr. Matt Larouche, Kathryn Westendorf, and James Cooke for providing tissue sections.
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