- Email: [email protected]
Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system
Brain Research 860 Ž2000. 174–177 www.elsevier.comrlocaterbres
Short communication
Developmental regulation of membrane type-5 matrix metalloproteinase ŽMT5-MMP. expression in the rat nervous system Diane M. Jaworski
)
Department of Anatomy and Neurobiology, UniÕersity of Vermont College of Medicine, GiÕen C454, Burlington, VT 05405, USA Accepted 11 January 2000
Abstract An intricate balance between extracellular matrix ŽECM. synthesis and degradation must be maintained during developmental tissue remodeling. Matrix metalloproteinases ŽMMPs. are the main mediators of ECM degradation. A subset of MMPs, referred to as membrane-type MMPs, contains a transmembrane domain that restricts protease activity at the cell surface. Membrane type-5 MMP is predominantly expressed in the brain. The present report is the first to demonstrate the temporal regulation and spatial distribution of MT5-MMP mRNA during nervous system development. q 2000 Elsevier Science B.V. All rights reserved. Keywords: CNS; Development; Extracellular matrix; Matrix metalloproteinase; MMP
To preserve tissue integrity during the structural rearrangements that occur during development, an intricate balance between extracellular matrix ŽECM. synthesis and degradation must be maintained. The matrix metalloproteinases ŽMMPs. are products of a multigene family of endopeptidases that are believed to be the main physiologically relevant mediators of ECM degradation. The control of MMP activity is complex and multifactorial w14x. Once transcriptionally induced and biochemically activated, MMPs function in the degradation of a broad spectrum of ECM and basement membrane proteins, including collagens, laminin, fibronectin, glycoproteins and proteoglycans w13x. In contrast to the majority of MMPs that are secreted into the extracellular milieu as soluble enzymes, a subfamily of recently identified MMPs possess a single membrane-spanning domain, thereby concentrating proteolytic activity at the cell surface. To date five membrane-type MMPs ŽMT-MMPs. have been identified w10,11,17–20,23x. MT5-MMP is predominantly expressed in the brain w11,17x. To begin to ascertain the function of MT5-MMP in rat nervous system development, Northern and in situ hybridization analyses were undertaken. A rat MT5-MMP cDNA was constructed by RT-PCR from P0 brain ŽF: 5X-ATGTGGCGTCCCTGAT-3X , R: 5XTGAAGTTGTGTGTCTCC-3X , nucleotides 408–931 of mouse MT5-MMP, Accession AJ10262. using cycle pa-
)
Fax: q1-802-656-8704; e-mail: [email protected]
rameters of 948 Ž45 s., 478 Ž45 s., and 728C Ž45 s. repeated 30 times. The PCR product was ligated to the dual promoter pCR II vector ŽInvitrogen; San Diego, CA. and the construct verified by automated DNA sequencing utilizing fluorescent-based dye terminators on an Applied Biosystems 373XL. Northern blot analysis and in situ hybridization were performed as previously described w8x. Combined in situ hybridizationrimmunohistochemistry was performed as previously described w9x. Autoradiograms were scanned using an Epson Expression 800 scanner with transparency adapter and imported directly into Adobe Photoshop. Photographic negatives of emulsion-coated sections were scanned with a Polaroid SprintScan and imported into Adobe Photoshop. Northern blot analysis was performed to determine the temporal regulation of MT5-MMP mRNA expression. Rat MT5-MMP recognizes a single transcript of 4.5 kb ŽFig. 1.. A message of ; 4.5 kb has been reported for both mouse w17x and human w11x MT5-MMP. Expression is first detected in embryonic day 16 ŽE16. brain, increases dramatically at E20, and peaks at postnatal day 0 ŽP0, within 24 h of birth.. The postnatal expression of MT5-MMP mRNA declines steadily to attain adult levels at P60. To characterize the spatial distribution of MT5-MMP mRNA expression over the course of early brain development, in situ hybridization was performed on parasagittal sections of embryos from E14 to E20 ŽFig. 2.. Sections were probed with a 35 S-labeled antisense cRNA probe ŽFig. 2A–D.. Hybridization with a sense probe showed no
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 3 5 - 7
D.M. Jaworskir Brain Research 860 (2000) 174–177
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MT5-MMP expression declines dramatically. Expression is enriched in the cerebellum and hippocampal formation. The adult level and pattern of MT5-MMP expression ŽFig. 3F. is attained at P21 ŽFig. 3D.. MT5-MMP mRNA is primarily expressed in the cerebellum, with a lower level
Fig. 1. MT5-MMP mRNA is developmentally regulated. Northern blot analysis was performed with 25 mg total RNA from embryonic day 14 ŽE14. whole embryo and E16, E18, E20, postnatal day 0 ŽP0, within 24 h of birth., P7, P14, P21, P60 and adult ŽAd, dams at least 6 months of age. whole brain. Following MT5-MMP hybridization, the blot was hybridized with the non-developmentally regulated gene cyclophilin Žlower panel. to verify equal loading. A single 4.5-kb mRNA transcript is first detected at E16. Expression increases to peak at P0, then gradually declines to reach adult levels at P60. RNA molecular weight standards are indicated on the left. The positions of 28S and 18S rRNA are indicated by the arrowheads.
detectable hybridization signal Ždata not shown.. Each section used for the in situ analysis was subsequently counterstained with cresyl violet to reveal histological details ŽFig. 2E–H.. At E14 ŽFig. 2A., MT5-MMP mRNA is abundantly expressed throughout the neuraxis. MT5MMP mRNA has been detected by PCR as early as E11 in the mouse w17x. MT5-MMP expression, which increases throughout embryonic development, is primarily expressed in the nervous system. While enriched in the central nervous system, expression is also detected in the peripheral nervous system, including the trigeminal ganglion ŽFig. 2B. and dorsal root ganglia ŽFig. 2C.. MT5-MMP expression is not restricted to the nervous system. MT5MMP mRNA is also enriched in the thymus, with a lower level of expression present in the aorta ŽFig. 2C.. By E20, MT5-MMP hybridization signal is clearly present in the brain parenchyma, particularly within the neocortex ŽFig. 2D.. MT5-MMP mRNA expression is enriched in regions of postnatal plasticity ŽFig. 3.. At P0 ŽFig. 3A., an intense MT5-MMP hybridization signal is detected throughout the brain parenchyma. Expression is enriched in the neocortex, differentiating cerebellar cortex and pituitary. At P7 ŽFig. 3B., although slightly diminished, MT5-MMP expression continues to be enriched in the neocortex. A diffuse signal is present throughout all brain regions. By P14 ŽFig. 3C.,
Fig. 2. MT5-MMP mRNA is enriched in the central and peripheral nervous system. In situ hybridization was performed with a 35 S-labeled cRNA probe on parasagittal sections of rat embryos ŽA–D.. Each in situ section is shown counterstained with cresyl violet ŽE–H.. ŽA. Intense MT5-MMP mRNA expression is detected throughout the neuraxis at E14, the earliest time-point examined. ŽB. In addition to expression in the central nervous system, MT5-MMP mRNA is detected in peripheral ganglia, including the trigeminal ŽTG. and dorsal root ŽDRG. ganglia. ŽC. MT5-MMP mRNA is also expressed outside of the nervous system, within the thymus and aorta. ŽD. While a strong hybridization signal is present in the olfactory bulb ŽOB., MT5-MMP expression is not detected in the olfactory epithelium. Abbreviations: CA, cerebral aqueduct; CB, cerebellum; Ctx, cortex; DRG, dorsal root ganglia; GE, ganglionic eminence; H, hippocampus; LV, lateral ventricle; M, medulla; MB, midbrain; OB, olfactory bulb; Pit, pituitary; R, rhombencephalon; SC, spinal cord; TG, trigeminal ganglion; Thl, thalamus. Scale bar s1 mm.
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D.M. Jaworskir Brain Research 860 (2000) 174–177
Since it is difficult to determine whether MT5-MMP expression observed at E14 is present in the proliferative ventricular zone or post-mitotic tissue, in situ hybridization for MT5-MMP mRNA was combined with immunohistochemical localization of nestin ŽFig. 4.. Nestin is an intermediate filament protein expressed only by radial glial cells and undifferentiated progenitor cells w5,7x. Nestin expression is enriched in the rhombencephalic ventricular zone adjacent to the cerebral aqueduct ŽFig. 4B.. In sharp contrast, the most intense MT5-MMP hybridization signal is present outside the ventricular zone ŽFig. 4A.. A weak hybridization signal is also present within the ventricular zone. The use of a proliferation marker, such as proliferating cell nuclear antigen ŽPCNA., is required to definitively resolve whether MT5-MMP mRNA is expressed in postmitotic cells. In addition, the cellular source of MT5-MMP mRNA will need to be determined. MMPs and are multifunctional proteins. MMPs can increase growth factors in the extracellular microenvironment by releasing membrane-bound w6x or proteoglycanbound w22x growth factors, and processing growth factor binding proteins w4x. In addition, the migration of a number of nervous system cell types appears to be MMP-mediated w1–3,21x. MMPs mediate the migration of neural progenitor cells by promoting neurite outgrowth and growth cone extension w12,15,16,24x. The temporal regulation and spatial distribution of MT5-MMP mRNA demonstrated here during nervous system development suggests a role in synaptic plasticity.
Fig. 3. Spatial distribution of MT5-MMP mRNA during postnatal brain development. In situ hybridization on parasagittal sections of P0 ŽA., P7 ŽB., P14 ŽC., P21 ŽD., P60 ŽE. and adult ŽF. rat brain demonstrates that MT5-MMP is significantly downregulated postnatally. MT5-MMP expression is maintained in the adult brain in regions of continued neuronal plasticity, including the olfactory bulb, hippocampus, dentate gyrus, and cerebellum. Abbreviations: AON, anterior olfactory nucleus; CB, cerebellum; CC, corpus callosum; Ctx, cortex; CPu, striatum Žcaudaterputamen.; DG, dentate gyrus; GCL, olfactory bulb granule cell layer; H, hippocampus; LV, lateral ventricle; Pit, pituitary. Scale bar s1 mm.
of expression in the hippocampus, dentate gyrus and olfactory bulb, regions of neuronal plasticity in the adult nervous system.
Fig. 4. MT5-MMP mRNA is not enriched in the ventricular zone. Following hybridization, immunohistochemical localization of the intermediate filament protein nestin was performed to determine whether the intense MT5-MMP expression observed in the rhombencephalon at E14 was present in progenitors cells in the ventricular zone. Close examination of emulsion-coated sections ŽA. reveals that the strongest hybridization signal is not present in the ventricular zone containing nestin-positive progenitors ŽB.. Rather, MT5-MMP mRNA is enriched in the post-mitotic parenchyma. Scale bar s 50 mm.
D.M. Jaworskir Brain Research 860 (2000) 174–177
Acknowledgements This work was supported by National Institutes of Health NS35874 and American Heart Association 9950039N. DNA sequencing at the VT Cancer Center DNA Analysis Facility is subsidized by NCI Grant P30CA22435. The author thanks Nicole Fager for expert technical assistance.
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w13x
References
w14x w15x
w1x V.R. Amberger, V. Avellana-Adalid, T. Hensel, B. Evercooren, M.E. Schwab, Oligodendrocyte-type 2 progenitors use a metalloendoprotease to spread and migrate on CNS myelin, Eur. J. Neurosci. 9 Ž1997. 151–162. w2x A.T.J. Belien, ¨ P.A. Paganetti, M.E. Schwab, Membrane-type 1 matrix metalloprotease ŽMT1-MMP. enables invasive migration of glioma cells in central nervous system white matter, J. Cell Biol. 144 Ž1999. 373–384. w3x M.R. Del Bigio, J.L. Tchelingerian, C.M. Jacque, Expression of extracellular matrix degrading enzymes during migration of xenografted brain cells, Neuropathol. Appl. Neurobiol. 25 Ž1999. 54–62. w4x J.L. Fowlkes, K.M. Thrailkill, D.M. Serra, K. Suzuki, H. Nagase, Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases, Prog. Growth Factor Res. 6 Ž1995. 255– 263. w5x F. Frederiksen, R.D.G. McKay, Proliferation and differentiation of rat neuroepithelial precursor cells in vivo, J. Neurosci. 8 Ž1988. 1144–1151. w6x A.J. Gearing, P. Beckett, M. Christodoulou, M. Chruchill, J.M. Clements, M. Crimmin, A.H. Davidson, A.H. Drummond, W.A. Galloway, R. Gilbert et al., Matrix metalloproteinases and processing of pro-TNF-alpha, J. Leukocyte Biol. 57 Ž1995. 774–777. w7x S. Hockfield, R. McKay, Identification of major cell classes in the developing mammalian nervous system, J. Neurosci. 5 Ž1985. 3310– 3328. w8x D.M. Jaworski, G.M. Kelly, S. Hockfield, BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to brain, J. Cell Biol. 125 Ž1994. 495–509. w9x D.M. Jaworski, G.M. Kelly, S. Hockfield, Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHABrbrevican, Exp. Neurol. 157 Ž1999. 327–337. w10x M. Kajita, H. Kinoh, N. Ito, A. Takamura, Y. Itoh, A. Okada, H. Sato, M. Seiki, Human type-4 matrix metalloproteinase ŽMT4-MMP. is encoded by a novel major transcript: isolation of complementary
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DNA clones for human and mouse mt4-mmp transcripts, FEBS Lett. 457 Ž1999. 353–356. E. Llano, A.M. Pendas, G. ´ J.P. Freije, A. Nakano, V. Knauper, ¨ Murphy, C. Lopez-Otin, Identification and characterization of hu´ man MT5-MMP, a new membrane-bound activator of progelatinase A overexpressed in brain tumors, Cancer Res. 59 Ž1999. 2570–2576. C.M. Machida, K.D. Rodland, L. Matrisian, B.E. Magun, G. Ciment, NGF induction of the gene encoding the protease transin accompanies neuronal differentiation in PC12 cells, Neuron 2 Ž1989. 1587–1596. H. Nagase, Activation mechanisms of matrix metalloproteinases, Biol. Chem. 378 Ž1997. 151–160. H. Nagase, J.F. Woessner, Matrix metalloproteinases, J. Biol. Chem. 274 Ž1999. 21491–21494. L.A. Nordstrom, J. Lochner, W. Yeung, G. Ciment, The metalloproteinase stromelysin-1 Žtransin. mediates PC12 cell growth cone invasiveness through basal laminae, Mol. Cell. Neurosci. 6 Ž1995. 56–68. L.Y.S. Oh, P.H. Larsen, C.A. Krekoski, D.R. Edwards, F. Donovan, Z. Werb, V.W. Yong, Matrix metalloproteinase-9rgelatinase B is required for process outgrowth by oligodendrocytes, J. Neurosci. 19 Ž1999. 8464–8475. D. Pei, Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP, J. Biol. Chem. 274 Ž1999. 8925–8932. X.S. Puente, A.M. Pendas, E. Llano, G. Velasco, C. Lopez-Otin, Molecular cloning of a novel membrane-type matrix metalloproteinase from human breast carcinoma, Cancer Res. 56 Ž1996. 944– 949. H. Sato, T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, M. Seiki, A matrix metalloproteinase expressed on the surface of invasive tumour cells, Nature 370 Ž1994. 61–65. T. Takino, H. Sato, A. Shinagawa, M. Seiki, Identification of a second membrane-type matrix metalloproteinase ŽMT-MMP-2. gene from a human placenta cDNA library, J. Biol. Chem. 270 Ž1995. 23013–23020. C. Vaillant, M. Didier-Bazes, ` A. Hutter, M.-F. Belin, N. Thomasset, Spatiotemporal expression patterns of metalloproteinases and their inhibitors in the postnatal developing rat cerebellum, J. Neurosci. 19 Ž1999. 4994–5004. J.M. Whitelock, A.D. Murdoch, R.V. Iozzo, P.A. Underwood, The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin and heparanases, J. Biol. Chem. 271 Ž1996. 10079–10086. H. Will, B. Hinzmann, cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment, Eur. J. Biochem. 231 Ž1995. 602–608. J. Zuo, T.A. Ferguson, Y.J. Hernandez, W.G. Stetler-Stevenson, D. Muir, Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan, J. Neurosci. 18 Ž1998. 5203–5211.
Short communication
Developmental regulation of membrane type-5 matrix metalloproteinase ŽMT5-MMP. expression in the rat nervous system Diane M. Jaworski
)
Department of Anatomy and Neurobiology, UniÕersity of Vermont College of Medicine, GiÕen C454, Burlington, VT 05405, USA Accepted 11 January 2000
Abstract An intricate balance between extracellular matrix ŽECM. synthesis and degradation must be maintained during developmental tissue remodeling. Matrix metalloproteinases ŽMMPs. are the main mediators of ECM degradation. A subset of MMPs, referred to as membrane-type MMPs, contains a transmembrane domain that restricts protease activity at the cell surface. Membrane type-5 MMP is predominantly expressed in the brain. The present report is the first to demonstrate the temporal regulation and spatial distribution of MT5-MMP mRNA during nervous system development. q 2000 Elsevier Science B.V. All rights reserved. Keywords: CNS; Development; Extracellular matrix; Matrix metalloproteinase; MMP
To preserve tissue integrity during the structural rearrangements that occur during development, an intricate balance between extracellular matrix ŽECM. synthesis and degradation must be maintained. The matrix metalloproteinases ŽMMPs. are products of a multigene family of endopeptidases that are believed to be the main physiologically relevant mediators of ECM degradation. The control of MMP activity is complex and multifactorial w14x. Once transcriptionally induced and biochemically activated, MMPs function in the degradation of a broad spectrum of ECM and basement membrane proteins, including collagens, laminin, fibronectin, glycoproteins and proteoglycans w13x. In contrast to the majority of MMPs that are secreted into the extracellular milieu as soluble enzymes, a subfamily of recently identified MMPs possess a single membrane-spanning domain, thereby concentrating proteolytic activity at the cell surface. To date five membrane-type MMPs ŽMT-MMPs. have been identified w10,11,17–20,23x. MT5-MMP is predominantly expressed in the brain w11,17x. To begin to ascertain the function of MT5-MMP in rat nervous system development, Northern and in situ hybridization analyses were undertaken. A rat MT5-MMP cDNA was constructed by RT-PCR from P0 brain ŽF: 5X-ATGTGGCGTCCCTGAT-3X , R: 5XTGAAGTTGTGTGTCTCC-3X , nucleotides 408–931 of mouse MT5-MMP, Accession AJ10262. using cycle pa-
)
Fax: q1-802-656-8704; e-mail: [email protected]
rameters of 948 Ž45 s., 478 Ž45 s., and 728C Ž45 s. repeated 30 times. The PCR product was ligated to the dual promoter pCR II vector ŽInvitrogen; San Diego, CA. and the construct verified by automated DNA sequencing utilizing fluorescent-based dye terminators on an Applied Biosystems 373XL. Northern blot analysis and in situ hybridization were performed as previously described w8x. Combined in situ hybridizationrimmunohistochemistry was performed as previously described w9x. Autoradiograms were scanned using an Epson Expression 800 scanner with transparency adapter and imported directly into Adobe Photoshop. Photographic negatives of emulsion-coated sections were scanned with a Polaroid SprintScan and imported into Adobe Photoshop. Northern blot analysis was performed to determine the temporal regulation of MT5-MMP mRNA expression. Rat MT5-MMP recognizes a single transcript of 4.5 kb ŽFig. 1.. A message of ; 4.5 kb has been reported for both mouse w17x and human w11x MT5-MMP. Expression is first detected in embryonic day 16 ŽE16. brain, increases dramatically at E20, and peaks at postnatal day 0 ŽP0, within 24 h of birth.. The postnatal expression of MT5-MMP mRNA declines steadily to attain adult levels at P60. To characterize the spatial distribution of MT5-MMP mRNA expression over the course of early brain development, in situ hybridization was performed on parasagittal sections of embryos from E14 to E20 ŽFig. 2.. Sections were probed with a 35 S-labeled antisense cRNA probe ŽFig. 2A–D.. Hybridization with a sense probe showed no
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 3 5 - 7
D.M. Jaworskir Brain Research 860 (2000) 174–177
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MT5-MMP expression declines dramatically. Expression is enriched in the cerebellum and hippocampal formation. The adult level and pattern of MT5-MMP expression ŽFig. 3F. is attained at P21 ŽFig. 3D.. MT5-MMP mRNA is primarily expressed in the cerebellum, with a lower level
Fig. 1. MT5-MMP mRNA is developmentally regulated. Northern blot analysis was performed with 25 mg total RNA from embryonic day 14 ŽE14. whole embryo and E16, E18, E20, postnatal day 0 ŽP0, within 24 h of birth., P7, P14, P21, P60 and adult ŽAd, dams at least 6 months of age. whole brain. Following MT5-MMP hybridization, the blot was hybridized with the non-developmentally regulated gene cyclophilin Žlower panel. to verify equal loading. A single 4.5-kb mRNA transcript is first detected at E16. Expression increases to peak at P0, then gradually declines to reach adult levels at P60. RNA molecular weight standards are indicated on the left. The positions of 28S and 18S rRNA are indicated by the arrowheads.
detectable hybridization signal Ždata not shown.. Each section used for the in situ analysis was subsequently counterstained with cresyl violet to reveal histological details ŽFig. 2E–H.. At E14 ŽFig. 2A., MT5-MMP mRNA is abundantly expressed throughout the neuraxis. MT5MMP mRNA has been detected by PCR as early as E11 in the mouse w17x. MT5-MMP expression, which increases throughout embryonic development, is primarily expressed in the nervous system. While enriched in the central nervous system, expression is also detected in the peripheral nervous system, including the trigeminal ganglion ŽFig. 2B. and dorsal root ganglia ŽFig. 2C.. MT5-MMP expression is not restricted to the nervous system. MT5MMP mRNA is also enriched in the thymus, with a lower level of expression present in the aorta ŽFig. 2C.. By E20, MT5-MMP hybridization signal is clearly present in the brain parenchyma, particularly within the neocortex ŽFig. 2D.. MT5-MMP mRNA expression is enriched in regions of postnatal plasticity ŽFig. 3.. At P0 ŽFig. 3A., an intense MT5-MMP hybridization signal is detected throughout the brain parenchyma. Expression is enriched in the neocortex, differentiating cerebellar cortex and pituitary. At P7 ŽFig. 3B., although slightly diminished, MT5-MMP expression continues to be enriched in the neocortex. A diffuse signal is present throughout all brain regions. By P14 ŽFig. 3C.,
Fig. 2. MT5-MMP mRNA is enriched in the central and peripheral nervous system. In situ hybridization was performed with a 35 S-labeled cRNA probe on parasagittal sections of rat embryos ŽA–D.. Each in situ section is shown counterstained with cresyl violet ŽE–H.. ŽA. Intense MT5-MMP mRNA expression is detected throughout the neuraxis at E14, the earliest time-point examined. ŽB. In addition to expression in the central nervous system, MT5-MMP mRNA is detected in peripheral ganglia, including the trigeminal ŽTG. and dorsal root ŽDRG. ganglia. ŽC. MT5-MMP mRNA is also expressed outside of the nervous system, within the thymus and aorta. ŽD. While a strong hybridization signal is present in the olfactory bulb ŽOB., MT5-MMP expression is not detected in the olfactory epithelium. Abbreviations: CA, cerebral aqueduct; CB, cerebellum; Ctx, cortex; DRG, dorsal root ganglia; GE, ganglionic eminence; H, hippocampus; LV, lateral ventricle; M, medulla; MB, midbrain; OB, olfactory bulb; Pit, pituitary; R, rhombencephalon; SC, spinal cord; TG, trigeminal ganglion; Thl, thalamus. Scale bar s1 mm.
176
D.M. Jaworskir Brain Research 860 (2000) 174–177
Since it is difficult to determine whether MT5-MMP expression observed at E14 is present in the proliferative ventricular zone or post-mitotic tissue, in situ hybridization for MT5-MMP mRNA was combined with immunohistochemical localization of nestin ŽFig. 4.. Nestin is an intermediate filament protein expressed only by radial glial cells and undifferentiated progenitor cells w5,7x. Nestin expression is enriched in the rhombencephalic ventricular zone adjacent to the cerebral aqueduct ŽFig. 4B.. In sharp contrast, the most intense MT5-MMP hybridization signal is present outside the ventricular zone ŽFig. 4A.. A weak hybridization signal is also present within the ventricular zone. The use of a proliferation marker, such as proliferating cell nuclear antigen ŽPCNA., is required to definitively resolve whether MT5-MMP mRNA is expressed in postmitotic cells. In addition, the cellular source of MT5-MMP mRNA will need to be determined. MMPs and are multifunctional proteins. MMPs can increase growth factors in the extracellular microenvironment by releasing membrane-bound w6x or proteoglycanbound w22x growth factors, and processing growth factor binding proteins w4x. In addition, the migration of a number of nervous system cell types appears to be MMP-mediated w1–3,21x. MMPs mediate the migration of neural progenitor cells by promoting neurite outgrowth and growth cone extension w12,15,16,24x. The temporal regulation and spatial distribution of MT5-MMP mRNA demonstrated here during nervous system development suggests a role in synaptic plasticity.
Fig. 3. Spatial distribution of MT5-MMP mRNA during postnatal brain development. In situ hybridization on parasagittal sections of P0 ŽA., P7 ŽB., P14 ŽC., P21 ŽD., P60 ŽE. and adult ŽF. rat brain demonstrates that MT5-MMP is significantly downregulated postnatally. MT5-MMP expression is maintained in the adult brain in regions of continued neuronal plasticity, including the olfactory bulb, hippocampus, dentate gyrus, and cerebellum. Abbreviations: AON, anterior olfactory nucleus; CB, cerebellum; CC, corpus callosum; Ctx, cortex; CPu, striatum Žcaudaterputamen.; DG, dentate gyrus; GCL, olfactory bulb granule cell layer; H, hippocampus; LV, lateral ventricle; Pit, pituitary. Scale bar s1 mm.
of expression in the hippocampus, dentate gyrus and olfactory bulb, regions of neuronal plasticity in the adult nervous system.
Fig. 4. MT5-MMP mRNA is not enriched in the ventricular zone. Following hybridization, immunohistochemical localization of the intermediate filament protein nestin was performed to determine whether the intense MT5-MMP expression observed in the rhombencephalon at E14 was present in progenitors cells in the ventricular zone. Close examination of emulsion-coated sections ŽA. reveals that the strongest hybridization signal is not present in the ventricular zone containing nestin-positive progenitors ŽB.. Rather, MT5-MMP mRNA is enriched in the post-mitotic parenchyma. Scale bar s 50 mm.
D.M. Jaworskir Brain Research 860 (2000) 174–177
Acknowledgements This work was supported by National Institutes of Health NS35874 and American Heart Association 9950039N. DNA sequencing at the VT Cancer Center DNA Analysis Facility is subsidized by NCI Grant P30CA22435. The author thanks Nicole Fager for expert technical assistance.
w11x
w12x
w13x
References
w14x w15x
w1x V.R. Amberger, V. Avellana-Adalid, T. Hensel, B. Evercooren, M.E. Schwab, Oligodendrocyte-type 2 progenitors use a metalloendoprotease to spread and migrate on CNS myelin, Eur. J. Neurosci. 9 Ž1997. 151–162. w2x A.T.J. Belien, ¨ P.A. Paganetti, M.E. Schwab, Membrane-type 1 matrix metalloprotease ŽMT1-MMP. enables invasive migration of glioma cells in central nervous system white matter, J. Cell Biol. 144 Ž1999. 373–384. w3x M.R. Del Bigio, J.L. Tchelingerian, C.M. Jacque, Expression of extracellular matrix degrading enzymes during migration of xenografted brain cells, Neuropathol. Appl. Neurobiol. 25 Ž1999. 54–62. w4x J.L. Fowlkes, K.M. Thrailkill, D.M. Serra, K. Suzuki, H. Nagase, Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases, Prog. Growth Factor Res. 6 Ž1995. 255– 263. w5x F. Frederiksen, R.D.G. McKay, Proliferation and differentiation of rat neuroepithelial precursor cells in vivo, J. Neurosci. 8 Ž1988. 1144–1151. w6x A.J. Gearing, P. Beckett, M. Christodoulou, M. Chruchill, J.M. Clements, M. Crimmin, A.H. Davidson, A.H. Drummond, W.A. Galloway, R. Gilbert et al., Matrix metalloproteinases and processing of pro-TNF-alpha, J. Leukocyte Biol. 57 Ž1995. 774–777. w7x S. Hockfield, R. McKay, Identification of major cell classes in the developing mammalian nervous system, J. Neurosci. 5 Ž1985. 3310– 3328. w8x D.M. Jaworski, G.M. Kelly, S. Hockfield, BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to brain, J. Cell Biol. 125 Ž1994. 495–509. w9x D.M. Jaworski, G.M. Kelly, S. Hockfield, Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHABrbrevican, Exp. Neurol. 157 Ž1999. 327–337. w10x M. Kajita, H. Kinoh, N. Ito, A. Takamura, Y. Itoh, A. Okada, H. Sato, M. Seiki, Human type-4 matrix metalloproteinase ŽMT4-MMP. is encoded by a novel major transcript: isolation of complementary
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