Expression of the gene encoding the extracellular matrix glycoprotein SPARC in the developing and adult mouse brain

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 24 (1994) 11-19

Research Report

Expression of the gene encoding the extracellular matrix glycoprotein SPARC in the developing and adult mouse brain D.B. Mendis, I.R. Brown * Department of Zoology, University of Toronto, Scarborough Campus, West Hill, Ont., Canada MIC 1A4

(Accepted 30 November 1993)

Abstract

The pattern of expression of the SPARC gene was examined during postnatal development of the mouse brain using in situ hybridization. At postnatal day 3 (P3), a strong signal representing SPARC mRNA was apparent in boundary layers such as the pia mater and the lining of the ventricles. By P12, increased levels of SPARC mRNA were noted in the cerebellum, midbrain and brain stem with a lower signal in more frontal areas, a pattern which was retained in the adult. This pronounced caudal versus frontal difference in SPARC mRNA levels was confirmed by Northern blot analysis. At P3, SPARC mRNA was detected in developing blood vessels in the cerebral cortex, suggesting a role for SPARC in angiogenesis. During development of the cerebellum, expression of SPARC mRNA became highly restricted to the Purkinje cellular layer and in the adult was localized to Bergmann glial cells rather than Purkinje neurons. Key words: In situ hybridization; Northern blotting; Cerebellum; Angiogenesis

I. Introduction

S P A R C / O s t e o n e c t i n is a secreted, calcium-binding phosphoglycoprotein that was initially isolated from the extracellular matrix (ECM) of developing bone, and thought to be bone-specific [51]. Subsequently, S P A R C m R N A has been shown to be present in variety of embryonic and adult tissues [3,38,55], however no studies have concentrated on the adult or developing central nervous system. The same protein was studied independently in a n u m b e r of systems, and has been referred to as (i) a 'culture shock' protein synthesized under stress conditions in bovine aortic endothelial cells [43], (ii) S P A R C (Secreted Protein Acidic and Rich in Cysteine) from parietal e n d o d e r m [27], and (iii) BM40, a component of basement m e m branes [8]. Analysis of c D N A sequences has revealed that all three proteins are identical. For the purposes of this paper, the protein will be referred to as SPARC. In platelets, S P A R C is released along with the E C M glycoprotein thrombospondin in response to thrombin induction [50] and can form a complex with throm-

* Corresponding author. Fax: (1) (416) 287-7642. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(93)E0224-N

bospondin [7], suggesting a role for S P A R C in adhesive interactions at wound sites [53]. S P A R C has recently b e e n shown to interact with other extracellular molecules such as platelet derived growth factor B [36] and type III and V collagens [28,45]. Functional properties attributed to S P A R C in vitro have suggested that this protein can have effects on cell shape [44]. In the presence of purified SPARC, cells have b e e n reported to adopt a rounded morphology, dependent on calcium [45]. S P A R C also has the ability to inhibit the entry of mitotic cells into S-phase [11], as well as antagonizing the effects of basic fibroblast growth factor on the migration of endothelial cells [13]. T a k e n together, these observations suggest that S P A R C is expressed in tissues undergoing reorganization, and appears to be involved in the modulation of cell shape, by interacting with other extracellular molecules and their ceil-surface receptors. Although S P A R C has been studied in a wide range of tissues and cell cultures, no studies have focused on the developing or m a t u r e nervous system. During the analysis of SC1, a protein showing partial sequence similarity to SPARC, we noted that significant levels of S P A R C m R N A were present in the adult brain [19]. This observation p r o m p t e d us to undertake the present

12

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19

investigation to examine the pattern of expression of the SPARC gene during postnatal development of the mouse brain. ECM molecules such as SPARC may play important roles in cell interactions resulting in morphological changes during neurogenesis. Continued expression of SPARC in the adult brain indicates a further role in the mature nervous system.

2. Materials and methods 2.1. In situ hybridization In situ hybridization was carried out essentially as described by Sprang and Brown [49]. Briefly, CD1 mice of postnatal age 3 and 12 days plus adult were anaesthetized and perfused intracardially with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were removed and mounted in OCT embedding medium. Frozen sections (8 ~zm) were cut in various planes and thawed onto gelatin-coated slides. Sections were probed with 35S-labelled antisense or sense riboprobe transcribed in vitro from Pvu II fragments of the mouse SPARC cDNA [15] inserted into the Hinc site of the vector pGEM-3Z (Promega). Following hybridization and washing, slides were exposed to X-ray film or Kodak NTB2 liquid emulsion for autoradiography. Sections were subsequently stained with Cresyl violet and viewed using both lightfield and darkfield microscopy.

adult brain a pronounced difference in SPARC mRNA levels is apparent in frontal versus more caudal regions of the mouse brain. A strong SPARC signal is observed in specific cellular layers of the adult cerebellum (Cb) and also in the brain stem (Bs) and midbrain (Mb) but is greatly decreased in frontal regions such as the cerebral cortex (Cc). In the adult brain, SPARC mRNA levels in the pia mater at the surface of the brain and the ventricular linings are decreased compared to P3 and P12. 3.2. Regional differences in SPARC mRNA in the adult mouse brain The pronounced frontal versus caudal difference in SPARC mRNA levels, observed in Fig. 1, was further analyzed by sectioning of the adult mouse brain in different planes. As shown in Fig. 2, transverse sectioning (A) and coronal sectioning (C) demonstrated abundant levels of SPARC mRNA in the cerebellum (Cb), midbrain (Mb) and thalamic regions (T), with significantly lower levels in frontal regions such as the olfac-

~'Pm 2.2. Northern blotting Northern blot analysis was carried out essentially as described by Johnston et al. [19] Total RNA was isolated from three adult brain regions. Aliquots of 5 /zg were separated on a 1.5% agarose gel containing 6% formaldehyde and blotted onto Biotrans nylon transfer membrane. Blots were prehybridized at 42°C for 4 hours in solution containing 50% formamide, 5 x SSC, 5 x Denhardt's solution, 50 mM sodium phosphate, pH 6.5, 0.1% SDS, 250 /zg/ml sheared salmon sperm DNA. Hybridization was carried out overnight in the same buffer containing 32p-labelled antisense SPARC riboprobe. Blots were washed at 70°C in 0.1XSSC, 0.1% SDS and exposed to Kodak X-OMAT film at - 70°C with a Cronex Lightning plus intensifying screen. Blots were stripped with 10 mM sodium phosphate at 100°C and reprobed with antisense cyclophilin (1B15) riboprobe according to Landry et al. [20].

Mb

P3

Cb .

'2 Bs

fPm

Cb

.i'

Cc Mb

P12 Bs

"v ,

3. Results

3.1. Expression of SPARC mRNA during postnatal development of the mouse brain Sagittal brain sections were hybridized with 35Slabelled SPARC antisense riboprobe and mRNA distribution visualized by darkfield microscopy at postnatal days 3 and 12 (P3 and P12) and in the adult (Fig. 1). At P3, a striking pattern of SPARC mRNA is detected in the pia mater (Pm) at the surface of the brain and the lining of the ventricles (V). By P12, additional signal is apparent in the cerebellum (Cb), midbrain (Mb), and brain stem (Bs) with less signal in more frontal areas such as the cerebral cortex (Cc). In the

Adult Fig. 1. In situ hybridization of SPARC gene expression during postnatal development of the mouse brain. Sagittal brain sections, taken at postnatal days 3 and 12 (P3 and P12) and adult, were hybridized with 35S-labelled SPARC antisense riboprobe as described in Methods. Photographic emulsion was exposed for 4-6 weeks and viewed using darkfield microscopy to visualize the distribution of SPARC mRNA. Bs, brain stem; Cb, cerebellum; Cc, cerebral cortex; Mb, Midbrain; Pm, Pia mater; V, ventricles. Bar = 1 mm.

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19 tory bulb (Ob) and cerebral cortex (Cc), as is clearly seen in sagittal sectioning (B). In situ hybridization with 35S-labelled S P A R C antisense riboprobe is shown in Fig. 2 A - C while sense riboprobe has b e e n used in Fig. 2D as a control for non-specific binding. 3.3. Northern blot analysis o f SPARC m R N A in the adult mouse brain Total R N A was isolated from three adult mouse brain regions and hybridized with 32p-labelled S P A R C antisense riboprobe which detects a 2.2 kb m R N A species. As shown in Fig. 3A, this regional Northern blot analysis confirmed the in situ hybridization observations of high levels of S P A R C m R N A in caudal versus frontal areas of the adult mouse brain. Abundant levels of a 2.2 kb S P A R C m R N A species were noted in cerebellum plus brain stem (lane 1) and midbrain plus thalamus (lane 3) with much lower levels in the cerebral cortex (lane 2). To verify that equal

13

amounts of R N A had been loaded in each lane, the blot was stripped and reprobed with an antisense riboprobe which recognized transcripts of the housekeeping gene cyclophilin (1B15) [20]. 3.4. Expression o f S P A R C m R N A cerebral cortex

in the developing

As shown in Fig. 1, S P A R C m R N A was detected in the pia m a t e r at the surface of the brain. In Fig. 4, this pattern was analyzed at higher magnification in the frontal cerebral cortex region (left panels - S P A R C m R N A distribution, right panels - Cresyl violet staining for cellular detail). S P A R C m R N A is detected in the surface pia m a t e r (Pm) but also in developing blood vessels (Db) at P3 which arise from the pia mater, suggesting a role for S P A R C in the process of angiogenesis. In the adult when angiogenesis has ceased, S P A R C m R N A is not detected in mature blood vessels (Bv).

:c

Ob

_ ii!!iii!iii!!!!ii!~ii~i!~ii~Ii'!~ i!ii!i!i~!i!i~i,~i~i~i~i

Fig. 2. Regional differences in SPARC mRNA in the adult mouse brain. Adult brains were sectioned at 8/~m in the transverse (A), sagittal (B,D) or coronal (C) plane. Sections were then hybridized with 35S-labelled SPARC antisense (A-C) or sense probe (D), and exposed to X-ray film for 4 weeks. Cb, cerebellum; Cc, cerebral cortex; Mb, midbrain; Ob, olfactory bulb; T, thalamus. Bar = 2 mm.

14

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19

3.5. Developmental expression o f S P A R C m R N A in the cerebellum

Analysis of the developing cerebellum (Fig. 5) revealed that SPARC mRNA is present in several cellular layers at P3 and P12 but becomes highly restricted to the Purkinje cellular layer at the adult stage. At P3, SPARC mRNA (left panel) is seen throughout the cerebellum except in the external granule cell layer (Eg). At P12, signal becomes localized to particular cerebellar layers such as the Purkinje cellular layer (Pc), the deep white matter (Dw), as well as the pia mater (Pm). In the adult, SPARC mRNA becomes highly restricted to the Purkinje cellular layer (Pc) with a scattered signal extending into the molecular layer (M1) of the cerebellum. 3.6. Distribution of SPARC m R N A in the Purkinje cellular layer of the adult mouse brain

As shown in Fig. 5, SPARC mRNA in the adult cerebellum is highly localized to the Purkinje cellular layer. Analysis at higher magnification (Fig. 6) indicates that Bergmann glial cells (Bg) and not Purkinje neurons (Pn) in this cellular layer are expressing SPARC mRNA in the adult brain as indicated by lightfield microscopy focusing on the cellular level (Fig. 6A) and on overlying silver grains (Fig. 6B). Corre-

ili!!ii!i!~ii!iiii¸¸ii!

iI

SPARC 2.2 kb'--~

1.15

I

I 1

2

3

Fig. 3. Northern blot of SPARC mRNA in the adult mouse brain. Aliquots of 5 p,g of total RNA isolatedfromthree brain regionswere analyzed by Northern blotting using 32p-labelled SPARC antisense riboprobe which detects a 2.2 kilobase(kb) mRNAspecies. The blot was subsequently stripped and reprobed with a cyclophilin(1B15) riboprobe to verifythat equal amountsof RNA were loaded in each lane. Lane 1, cerebellumand brain stem; lane 2, cerebral cortexand hippocampus; lane 3 midbrain and thalamicregions.

sponding darkfield microscopy is shown in Fig. 6C. Silver grains present in the molecular layer (M1), which were present in the adult in Fig. 5, do not localize to individual cell bodies (Fig. 6), perhaps reflecting the presence of SPARC mRNA in Bergmann glial processes that radiate through the molecular layer.

4. Discussion

SPARC is an extracellular matrix (ECM) glycoprotein that was originally isolated from bone, and then shown to be expressed in a variety of embryonic and adult tissues [3,32,46,51,55]. Although no studies have concentrated on the central nervous system (CNS), low levels of SPARC mRNA have been noted in the embryonic CNS [15,34]. Since expression of ECM glycoproteins is thought to decrease in the adult brain following the completion of developmental events [6,23,25], it was assumed that SPARC would not be expressed at high levels in the mature CNS. However, during the analysis of SC1, a protein showing partial sequence similarity to SPARC, we observed a significant level of SPARC mRNA in the adult brain by Northern blotting [19], a finding also reported by Ringuette et al. [39] Since ECM glycoproteins are thought to provide a permissive environment for migratory and proliferative events [1,17,52], we have subsequently examined the regional expression of the SPARC gene during the development of the mouse brain. The present in situ hybridization analysis revealed features of the neural expression of the SPARC gene which were masked in the previous Northern blot studies on total brain RNA [19,39]. A pronounced caudal versus frontal difference in expression of the SPARC gene was apparent in the adult mouse brain and also at postnatal day 12 (P12). A robust SPARC mRNA signal was observed in caudal regions such as the cerebellum, midbrain and brain stem while greatly reduced levels were apparent in the cerebral cortex. These observations were confirmed by the regional Northern blot analysis in the adult. A similar pattern of expression has also been shown for mRNA encoding the glutamate receptors, NMDAR2C and NMDAR2D, as well as a member of the PDGF receptor family, c-k/t and its ligand steel [31,33]. Interestingly, SPARC has been shown to inhibit ligand binding of the PDGF receptor [36]. Striking changes also were apparent in the distribution of SPARC mRNA during postnatal development of the mouse brain. At P3, a strong signal was observed in the pia mater at the surface of the brain and in developing blood vessels which arise from the pia mater. Expression of SPARC mRNA in developing but not mature blood vessels suggests a role for SPARC in

D.B. Mendis, L R. Brown/Molecular Brain Research 24 (1994) 11-19

angiogenesis. Angiogenesis involves the process of morphological change and migration of endothelial cells from the pial surface to form growth sprouts which give rise to mature blood vessels [10]. S P A R C has been reported to interact with known regulators of angiogenesis such as platelet derived growth factor ( P D G F ) and thrombospondin and can also inhibit the migrational effect of the angiogenic mitogen, basic fibroblast growth factor (bFGF) [7,12,13,21,36]. In cultures of endothelial cells undergoing in vitro angiogenesis, S P A R C has been reported to alter the synthesis of extracellular molecules such as fibronectin, thrombospondin and type-1 plasminogen activator inhibitor (PAl-l) [21]. T a k e n together with our present results

15

on the expression of S P A R C in developing blood vessels, these observations suggest that S P A R C participates, along with other E C M molecules, in morphogenetic events associated with angiogenesis. The pattern of expression of the S P A R C gene in the Purkinje cellular layer during cerebellar development suggests that Bergmann glial cells express S P A R C as granule neurons migrate inward from the outer surface of the cerebellum along Bergmann glial fibres. This migratory event is believed to be controlled by complex interactions between the migrating neuron and extracellular cues originating from the stationary Bergmann glial fibres. E C M glycoproteins such as thrombospondin and t e n a s c i n / c y t o t a c t i n are also located in

Fig. 4. Distribution of SPARC mRNA in the developing cerebral cortex. Tissue sections taken at postnatal days 3 and 12 (P3 and P12) and adult were hybridized with 3sS-labelled SPARC antisense riboprobe as described in Fig. 1. The SPARC mRNA signal was visualized by darkfield microscopy in panels on the left, while panels on the right show Cresyl violet staining for cellular detail. The adult panel is overexposed to highlight the absence of signal in blood vessels at this stage. Bv, blood vessel; Db, developing blood vessel; Pm, pia mater. Bar = 40/zm.

16

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19

this r e g i o n d u r i n g t h e m i g r a t o r y events [9,24,35]. I n t e r a c t i o n s b e t w e e n S P A R C , a s s o c i a t e d with B e r g m a n n glial cells, a n d t h r o m b o s p o n d i n , a s s o c i a t e d with t h e g r a n u l e n e u r o n s , m a y c o n t r i b u t e to this cell m i g r a t i o n process.

I n c o n t r a s t to the e x p r e s s i o n o f o t h e r E C M glycop r o t e i n s such as l a m i n i n , f i b r o n e c t i n , t e n a s c i n / cytotactin, a n d t h r o m b o s p o n d i n which d e c r e a s e as t h e b r a i n m a t u r e s [23,35,40,42], levels o f S P A R C m R N A i n c r e a s e with n e u r a l d e v e l o p m e n t a n d r e m a i n high in

P19

MI

Fig. 5. Developmental expression of SPARC mRNA in the cerebellum. Tissue sections of mouse cerebellum at the indicated stages of development were hybridized with 35S-labelled SPARC antisense riboprobe. Emulsion was exposed for 4 weeks and viewed using darkfield microscopy on the left to visualize SPARC mRNA while Cresyl violet staining on the right shows cellular detail. Dw, deep white matter; Eg, external granule cell layer; M1, molecular layer; Pc, Purkinje cell layer; Pm, Pia mater. Bar = 175 ~m.

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19

17

SPARC, is also expressed in the adult and developing mammalian brain [19,29]. During development of the cerebellum, expression of SPARC mRNA becomes highly restricted to the Purkinje cellular layer and in the adult is localized to Bergmann glial ceils rather than Purkinje neurons. The significance of the expression of this ECM molecule in Bergmann glial ceils in the adult is not known at present but this feature distinguishes SPARC from other known ECM glycoproteins. Recently, we have observed that the gene encoding SC1, a putative ECM glycoprotein related to SPARC, is also expressed in Bergmann glial cells in the adult and developing mammalian cerebellum [29]. Bergmann glial cells are a specialized form of astrocyte called radial glia which persist in the adult CNS unlike other forms of mammalian radial glia which developmentally transform into type-1 astroctyes or ependymal ceUs [2,4,5,14,47]. Radial glia have been suggested to behave like astrocytes in birds, reptiles, and amphibians where these cells do not transform during developmental [22,26,30,37,41,48]. Identification of SPARC mRNA in Bergmann glial cells agrees with previously reported expression of SPARC by astrocytes in cell culture [27,54]. In summary, pronounced developmental and regional differences in the expression of the SPARC gene were noted during postnatal development of the mouse brain. Detection of SPARC mRNA in developing blood vessels may reflect a role in angiogenesis while expression in Bergmann glial ceils in the developing cerebellum may be associated with migratory events of granule neurons. Unlike other ECM glycoproteins, the SPARC gene is expressed in the adult brain where a pronounced caudal versus frontal gradient in mRNA levels is apparent.

Acknowledgements

Fig. 6. Distribution of SPARC mRNA in the Purkinje c~llular layer of the adult mouse brain, Lightfield microscopy focusing on cells (A) and overlying silver grains (B). Corresponding darkfield microscopy is shown in C. Bg, Bergmann glial cells; MI, molecular layer; Pn, Purkinje neurons. Bar = 10/zm.

the adult, particularly in caudal regions such as the cerebellum, midbrain, and brain stem. It should be noted however, that there is evidence that some components of the neural ECM, notably proteoglycans of the relatively insoluble matrix, increase in expression over postnatal development [16,18]. In addition, our previous studies have shown that SC1, a putative ECM glycoprotein which shows partial sequence similarity to

The authors wish to thank Dr. M. Ringuette and S. Damjanovski for the subcloning of the mouse SPARC cDNA into the pGEM-3Z vector. These studies were supported by grants to I.R.B. from the Natural Sciences and Engineering Research Council (NSERC), Canada.

References [1] Baier, H. and Bonhoeffer, F., Axon guidance by gradients of a target derived component, Science, 255' (1992) 472-475. [2] Benjelloun-Touimi, S., Jacque, C., Derer, P., De Vitry, F., Maunoury, R. and Dupouey, P., Evidence that mouse astrocytes may be derived from the radial glia, J. Neuroimmunol., 9 (1985) 87-97.

18

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19

[3] Bolander, M., Young, M., Fisher, L., Yamada, Y. and Termine, J.D., Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid), Proc. Natl. Acad. Sci. USA, 85 (1988) 2919-2913. [4] Cameron, R.S, and Rakic, P., Glial cell lineage in the cerebral cortex: a review and synthesis, Glia, 4 (1991) 124-137. [5] Choi, B., Kim, R. and Lapham, L., Do radial glia give rise to both astroglial and oligodendroglial cells?, Brain Res., 8 (1983) 119-130. [6] Chun, J.M. and Shatz, C.J., A fibronectin-like molecule is present in the developing cat cerebral cortex and is correlated with subplate neurons, J. Cell Biol., 106 (1988) 857-872. [7] Clezardin, P., Malaval, L., Ehrensperger, A., Delmas, P.D., Dechavanni, M. and McGregor, J., Complex formation of human thrombospondin with osteonectin, Eur. J. Biochem., 175 (1988) 275-284. [8] Dziadek, M., Paulsson, M., Aumailey, M. and Timpl, R., Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumour, Eur. J. Bioehem., 161 (1986) 455-464. [9] Faissner A. and Kruse, J., J1/tenascin is a repulsive substrate for central nervous system neurons, Neuron, 5 (1990) 627-637. [10] Folkman, J. and Shing, Y., Minireview: Angiogenesis, J. Biol. Chem., 267 (1992) 10931-10934. [11] Funk, S.E. and Sage, E.H., The Ca2+-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells, Proc. Natl. Acad. ScL USA, 88 (1991) 2648-2652. [12] Good, D.J., Polverini, P.J., Rastinejad, F., Le Beau, M., Lemons, R.S., Frazier W.A. and Bouck, N.P., A tumour suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin, Proc. Natl. Acad. Sci. USA, 87 (1990) 6624-6628. [13] Hasselaar, P. and Sage, E.H., SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells, J. Cell. Biochem., 49 (1992) 272-283. [14] Hirano, M. and Goldman, J., Gliogenesis in the rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors, J. Neurosci. Res., 21 (1988) 155-167. [15] Holland, P.W., Harper, S.J., McVey, J.H. and Hogan, B.L.M., In vivo expression of mRNA for the Ca2+-binding protein SPARC (Osteonectin) revealed by in situ hybridization, Z Cell Biol., 105 (1987) 473-482. [16] Hockfield, S., Kalb, R.G., Zaremba, S. and Fryer, H., Expression of neural proteoglycans correlates with the acquisition of mature neuronal properties in the mammalian brain. Cold Spring Harbor Symp. Quant. Biol., 55 (1990) 505-514. [17] Husmann, K., Faissner, A. and Schachner, M., Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats, J. Cell Biol. 116 (1992) 1475-1486. [18] Iwata, M. and Carlson, S., A large chondroitin sulfate proteoglycan has the characteristics of a general extracellular matrix component of adult brain, J. Neurosci., 13, 195-207. [19] Johnston, I.J., Paladino, T., Gurd, J.W. and Brown, I.R., Molecular cloning of SCI: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/ SPARC, Neuron, 2 (1990) 165-176. [20] Landry, C.F., Ivy, G.O., Dunn, R.J., Marks, A. and Brown, I.R., Expression of the gene encoding the fl-subunit of S-100 protein in the developing rat brain analyzed by in situ hybridization, Mol. Brain Res., 6 (1989) 251-262. [21] Lane, T.F., Iruela-Arispe, M.L. and Sage, E.H., Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-l, Z Biol. Chem., 267 (1992) 16736-16745.

[22] Laterra, J., Cuerin, C. and Goldstein, G.W., Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro, J. Cell. Physiol., 144 (1990) 204-215. [23] Laurie, G.W., Stone, C.M. and Yamada, Y., Elevated 32-kDa and low laminin mRNA expression in developing mouse cerebrum, Differentiation, 46 (1991) 173-179. [24] Laywell, E.D., Dorries, U., Bartsch, U., Faissner, A., Schachner, M. and Steidler, D.A., Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury, Proc. Natl. Acad. Sci. USA, 89 (1992) 26342638. [25] Letourneau, P., Madsen, A., Palm, S. and Furcht, L., Immunoreactivity for laminin in the developing ventral longitudinal pathway of the brain, Dev. Biol., 1125 (1988) 135-144. [26] Linder, J., Geunther, J., Nick, H., Zinser, G., Antonicek, H., Schachner, M. and Monard, D., Modulation of granule cell migration by a glia-derived protein, Proc. Natl. Acad. Sci. USA, 83 (1986) 4568-4571. [27] Mason, I., Murphy, D., Munke, M., Francke, U., Elliott, R. and Hogan, B., Developmental and transformation-sensitive expression of the SPARC gene on mouse chromosome 11, EMBO J., 5 (1986) 1831-1837. [28] Mayer, U., Aumailley, M., Mann, K., Timple, R. and Engel, J., Calcium-dependent binding of basement membrane protein BM-40 (SPARC) to basement membrane collagen type IV, Eur. J. Biochem., 205 (1991) 141-150. [29] Mendis, D.B., Shahin, S., Gurd, J.W. and Brown, I.R., Developmental expression in the rat cerebellum of SC1, a putative brain extracellular matrix glycoprotein related to SPARC, Brain Res., 633 (1994) 197-205. [30] Morganti, M.C., Taylor, J., Pesheva, P. and Schachner, M., Oligodendrocyte- derived J1-160/180 extracellular matrix glycoproteins are adhesive or repulsive depending on the partner cell type and time of interaction, Exp. Neurol., 109 (1985) 98-110. [31] Motro, B., Van der Kooy, D., Rossant, J., Reith, A. and Bernstein, A., Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and SI loci, Development, 113 (1991) 1207-1221. [32] Mundlos, S., Schwahn, B., Reichert, T. and Zabel, B., Distribution of osteonectin mRNA and protein during human embryonic and fetal development, J. Histochem. Cytochem., 40 (1992) 283-291. [33] Nakanishi, S., Molecular diversity of glutamate receptors and implications for brain function, Science, 258 (1992) 597-603. [34] Nomura, S., Wills, A.J., Edward, D.R., Heath, J.K. and Hogan B.L.M., Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization, J. Cell Biol., 106 (1988) 441-450. [35] O'Shea, K.S., Rheinheimer, J.S. and Dixit, V.M., Depostion and role of thrombospondin in the histogenesis of the cerebellar cortex, J. Cell Biol., 110 (1990) 1275-1283. [36] Raines, E.W., Lane, T.F., Iruela-Arispe, M.L., Ross, R. and Sage, E.H., The extracellular glycoprotein SPARC interacts with platelet derived growth factor PDGF-AB and BB and inhibits the binding of PDGF to its receptors, Proc. Natl. Acad. Sci. USA, 89 (1992) 1281-1285. [37] Raub, T.J., Keuntzel, S. and Sawada, G., Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released form astroglioma cells, Exp. Cell Res., 199 (1992) 330-340. [38] Reichert, T., Strokel, W., Becker, K. and Fisher, L., The role of osteonectin in human tooth development: an immunohistological study, Calcif Tissue Int., 50 (1992) 468-472. [39] Ringuette, M., Damjanovski, S. and Wheeler, D., Expression of SPARC/osteonectin in tissues of bony and cartilaginous vertebrates, Biochem. Cell Biol., 69 (1991) 245-250. [40] Rogers, S.L., Letourneau, P.C. and Pech, I.V., The role of

D.B. Mendis, LR. Brown/Molecular Brain Research 24 (1994) 11-19

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

fibronectin in neural development, Dev. Neurosci., 11 (1989) 248-265. Rosen, C.L., Lianti, M.P. and Slazer, J.L., Expression of unique set of GPI-Linked proteins by different primary neurons in vitro, Z Cell BioL, 117 (1992) 617-627. Saga, Y., Tsukamoto, T., Jing, N., Kusakabe, M. and Sakakura, T., Murine tenascin: cDNA cloning, structure and temporal expression of isoforms, Gene, 104 (1991) 177-185. Sage, E.H., Johnson, C. and Bornstein, P., Characterization of a novel serum albumin-binding glycoprotein secreted by endothelial cells in culture, J. Biol. Chem., 259 (1984) 3993-4007. Sage, E.H. and Bornstein, P., Minireview: Extracellular proteins that modulate cell-matrix interactions; SPARC, tenascin, and thrombospondin, J. Biol. Chem. 142 (1991) 488-495. Sage, E.H., Vernon, R.B., Funk, S.E., Everitt, E.A. and Angello, J., SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca 2+dependent binding to the extracellular matrix, J. Cell BioL, 109 (1989) 341-356. Sage, E.H., Vernon, R.B., Decker, J., Funk, S. and Iruela-Arispe, M., Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice, J. Histochem. Cytochem., 37 (1989) 819-829. Schmechel, D.E. and Rakic, P., A golgi study of radial glia cells in the developing monkey telencephalon: morphogenesis and transformation into astrocytes, Anat. EmbryoL, 156 (1979) 115152. Sims, T.J., Gilmore, S.A. and Waxman, S.G., Radial glial give rise to perinodal processes, Brain Res., 548 (1991) 25-35.

19

[49] Sprang, G.K. and Brown, I.R., Selective induction of a heat shock gene in fibre tracts and cerebellar neurons of the rabbit brain detected by in situ hybridization, Mol. Brain Res., 3 (1987) 89-93. [50] Stenner, D.D., Tracy, R.P., Riggs, B.L. and Mann, K.G., Human platelets contain and secrete osteonectin, a major protein of mineralized bone, Proc. Natl. Acad. Sci. USA, 83 (1986) 68926896. [51] Termine, J., Kleinman, H., Whitson, S., Corm, M., McGarvey, M. and Martin, G., Osteonectin, a bone-specific protein linking mineral to collagen, Cell, 26 (1981) 99-105. [52] Tiveron, M.C., Barboni, E., River, F.B.P., Gormley, A.M., Seeley, J.P. and Grosveld, F., Selective inhibition of neurite outgrowth on mature astrocytes by thy-1 glycoprotein, Nature, 355 (1992) 745-748. [53] Tracy, R.P., Schull, S., Riggs, B.L. and Mann, K.G., The osteonectin family of proteins, Int. J. Biochem., 20 (1988) 653-660. [54] Webersinke, G., Bauer, H., Amberger, A., Zach, O. and Bauer, H.C., Comparison of gene expression of extraceUular matrix molecules in brain microvascular endothelial cells and astrocytes, Biochem. Biophys. Res. Commun., 189 (1992)877-884, [55] Young, M., Findlay, D., Dominguez, P., Burbelo, P., McQuillan, C., Kopp, J., Robey, P. and Termine, J., Osteonectin promoter: DNA sequence analysis and $1 endonuclease site potentially associated with transcriptional control in bone cells, J. Biol. Chem., 264 (1989) 450-456.