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Developmental changes in expression of small GTPase RhoG mRNA in the rat brain
Molecular Brain Research 106 (2002) 145–150 www.elsevier.com / locate / bres
Short communication
Developmental changes in expression of small GTPase RhoG mRNA in the rat brain Yukio Ishikawa, Hironori Katoh, Kazuhiro Nakamura, Kazutoshi Mori, Manabu Negishi* Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606 -8502, Japan Accepted 30 July 2002
Abstract We have recently reported that RhoG, a member of Rho family small GTPases, is involved in neurite outgrowth in cultured neuronal cells. Here, we report the expression of RhoG mRNA in the developing rat brain by in situ hybridization analysis. At embryonic day 16, RhoG expression was observed throughout the ventricular zone, but was down-regulated in the region at birth. On the other hand, RhoG expression at postnatal day 20 was highly enriched in white matter tracts, including the corpus callosum, the anterior commissure, and the cerebellar white matter, and double-labeling experiments demonstrated that major RhoG-expressing cells in white matter tracts were oligodendrocytes. These results suggest distinct pre- and postnatal roles of RhoG in the development of the central nervous system. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Rho family GTPase; In situ hybridization; Development; Oligodendrocyte
The Rho family of small GTPases has been implicated in the reorganization of the actin cytoskeleton and subsequent morphological changes in various cell types [3,5]. Like other GTPases of the Ras superfamily, they serve as molecular switches by cycling between an inactive GDPbound state and an active GTP-bound state, and once activated, they can interact with their specific effectors, leading to a variety of biological functions. Activation of the Rho family proteins requires GDP–GTP exchange catalyzed by various guanine nucleotide exchange factors, and is regulated by GTPase-activating proteins that stimulate the intrinsic GTPase activities of the G proteins. Presently, at least 14 mammalian Rho family proteins have been identified: Rho (A, B and C), Rac (1, 2 and 3), Cdc42, Rnd (1, 2, and 3), RhoD, TC10, RhoH / TTF, and RhoG. Among them, the functions of RhoA, Rac, and Cdc42 have been extensively characterized, and recent evidence implicates these Rho GTPases in the regulation of neuronal morphological changes, including axon growth *Corresponding author. Tel.: 181-75-753-4547; fax: 181-75-7537688. E-mail address: [email protected] (M. Negishi).
and guidance, and dendrite formation [9]. Furthermore, the distribution of Rho, Rac and Cdc42 in the brain has also been studied [13,14]. We have recently reported that RhoG is involved in the nerve growth factor-induced neurite outgrowth acting upstream of Rac and Cdc42 in rat pheochromocytoma PC12 cells [6]. Therefore, it is possible that RhoG also participates in the regulation of neuritogenesis during the development of the central nervous system. As an initial approach to address this issue, we have studied the distribution of mRNA for RhoG during the development of rat brain by in situ hybridization analysis. Rat RhoG cDNA [6] was subcloned into pBluescript SK(1) (Stratagene). Antisense and sense riboprobes were synthesized and digoxigenin (DIG)-labeled by in vitro transcription with T7 and T3 RNA polymerases and DIG RNA labeling mix (Roche) from the BamHI- and HindIIIdigested plasmid, respectively. Pregnant Wistar rats at defined gestational stages were used in this study. Timed pregnant rats were ether-anesthetized deeply and decapitated. Collected embryos were decapitated, and the brains were removed and fixed by incubating with a fixative containing 4% paraformaldehyde in 0.1 M phosphate
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00413-8
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buffer (pH 7.4) at 4 8C overnight. Postnatal rats were anesthetized with chloral hydrate and perfused transcardially with the same fixative. The brains were removed and postfixed in the fixative at 4 8C overnight. Embryonic and postnatal brains were then saturated with 0.1 M phosphate buffer containing 30% sucrose at 4 8C. The brains were cut into 60-mm thick (embryonic brains) or 40-mm thick (postnatal brains) coronal sections on a cryostat at 218 8C, and free-floating sections were acetylated in acetic anhydride / triethanolamine–HCl. The sections of postnatal rats were treated with 2.5 mg / ml proteinase K (Roche) for 10 min at room temperature before hybridization. After prehybridization in hybridization buffer (50% deionized formamide, 53 SSC, 53 Denhardt’s solution, 250 mg / ml salmon sperm DNA, 250 mg / ml yeast tRNA) for 2 h, the sections were incubated overnight at 55 8C with 500 ng / ml DIG-labeled sense or antisense probe for rat RhoG in the hybridization buffer. The sections were then washed in 23 SSC at 55 8C three times for 1 h each, and were blocked with 1% blocking reagent (Roche) in 0.1 M Tris–HCl (pH 7.5) and 0.15 M NaCl for 1 h at room temperature. They were incubated with alkaline phosphatase-conjugated antiDIG antibody (1:1000 dilution, Roche) at 4 8C overnight, and then reacted with chromogenic substrates, 5-bromo-4chloro-3-indolylphosphate and nitroblue tetrazolium in 0.1 M Tris–HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl 2 . For Northern blot analysis, whole brains from embryonic or postnatal rats were removed, frozen immediately in liquid nitrogen, and then stored at 280 8C until use. Purification of total RNA and Northern blotting were performed as described previously [6]. For normalization, the membrane was rehybridized with a cDNA probe for the mouse glyceraldehyde 3-phosphate dehydrogenase. The probes were labeled using Ready-To-Go DNA Labeling Beads (Amersham Pharmacia Biotech). For double-labeling experiments, the sections after in situ hybridization were incubated at 4 8C overnight with a monoclonal antibody to adenomatous polyposis coli protein (CC-1, Oncogene Research Products, 1:100 dilution), used as an oligodendrocyte marker, a monoclonal antibody to an astrocyte marker glial fibrillary acidic protein (GFAP) (Chemicon, 1:500 dilution), or a monoclonal antibody to a neuronal marker NeuN (Chemicon, 1:100 dilution) in phosphate-buffered saline containing 0.3% Triton X-100, 0.25% l-carrageenan, and 0.5% normal donkey serum. The detection of the antibodies was performed as described previously [11]. We also used a monoclonal antibody to vimentin as a marker for radial glial cells (Sigma, 1:40 dilution). Northern blot analysis of rat brain RNA from embryonic day 14 (E14) to the adult indicated that RhoG mRNA was detected at all developmental stages, and high expression levels were observed during the E14–16 and the postnatal day 20 (P20)–28 (Fig. 1). To reveal the distribution of RhoG in the developing rat brain, we performed in situ hybridization using a DIG-labeled RhoG antisense ribop-
Fig. 1. Expression of RhoG mRNA in pre- and postnatal rat brain. (A) Total RNA (20 mg) isolated from embryonic (E14, E16, E20) or postnatal (P0, P4, P8, P14, P20, P28, adult) rat brain was subjected to Northern blot analysis with a cDNA probe for RhoG. The same membrane rehybridized with GAPDH cDNA probe is shown below as the internal control. (B) Quantification of the RhoG mRNA level from E14 to the adult rat brain. The level of RhoG mRNA was normalized to that of GAPDH mRNA and expressed as fold increases over the value for the adult brain.
robe. In the E16 brain, expression of RhoG mRNA was observed throughout the ventricular zone (Fig. 2A,B; the immunostaining with an antibody to the radial glial marker vimentin [12,16] shown in Fig. 2C). However, RhoG expression was down-regulated in the ventricular zone at P0 (Fig. 2D,E). We next examined the expression of RhoG mRNA at P20. In contrast to the expression at E16, expression of RhoG mRNA was detected throughout the brain, particularly in white matter tracts including the corpus callosum (Fig. 3A; the control sense strand hybridization shown in Fig. 3B), the anterior commissure (Fig. 3C), the hippocampal fimbria, the external capsule (Fig. 3D), the cerebellar white matter (Fig. 3E), and the tectospinal tract (Fig. 3F). These areas are rich in glial cells but do not contain neuronal cell bodies, and RhoG mRNA-positive cells formed linear arrays, which are a characteristic feature of oligodendrocytes (Fig. 4A,B). Immunostaining with the monoclonal antibody CC-1, a marker for oligodendrocytes [1,8] after in situ hybridization to RhoG demonstrated that RhoG mRNA expression was detected in CC-1 antibody-positive cells (Fig. 4C). We also detected the expression of RhoG mRNA in myelin proteolipid protein-positive cells, another marker for oligodendrocytes [2] (data not shown). Astrocytes are also distributed in some portions of white matter tracts [7], but expression of RhoG mRNA did not overlap with that of
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Fig. 2. Distribution of RhoG mRNA in the E16 and P0 rat brain. (A) In situ hybridization on a coronal section of the E16 cortex shows the expression of RhoG mRNA throughout the ventricular zone. (B) A higher magnified image of the expression of RhoG mRNA at E16. (C) Immunoperoxidase staining on a coronal section of the E16 cortex with an antibody to the radial glial marker vimentin. (D) In situ hybridization for RhoG mRNA on a coronal section of the P0 cortex. (E) Immunoperoxidase staining on a coronal section of the P0 cortex with an antibody to vimentin. Ctx, cerebral cortex; LV, lateral ventricle; GE, ganglionic eminence; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate; WM, white matter. Scale bars: (A) 500 mm; (C) 50 mm; (E) 100 mm.
GFAP, an astrocyte marker (Fig. 4D). Furthermore, RhoG mRNA expression did not overlap with that of a neuronal marker NeuN (Fig. 4E). Expression of RhoG mRNA at P20 was also found in scattered cells throughout the brain (data not shown). These results indicate that major RhoGexpressing cells in the P20 rat brain are oligodendrocytes. The expression of RhoG mRNA in white matter tracts was also detected in the adult brain but weak compared to that of the P20 brain (data not shown). In the present study, we found that there are at least two phases of the RhoG expression in the developing rat brain, and they are not only temporally but may also be functionally distinct. At E16, expression of RhoG mRNA is observed throughout the ventricular zone. During the embryonic days, proliferation of neuronal and glial precursors occurs in the ventricular zone. Since RhoG was first identified as a growth-stimulated gene and has been shown to be involved in the regulation of cell proliferation in fibroblasts [17,19], it is inferred that regulated expression of RhoG may play a role in the regulation of cell proliferation in the ventricular zone during the embryonic
days. RhoA and Cdc42 are also expressed in the ventricular zone during the neurogenetic period [14], and they play essential roles in the cell cycle progression [15]. Considering that RhoG acts as an upstream regulator for Cdc42 [4,6], RhoG may regulate neurogenesis through the activation of Cdc42 in the ventricular zone. We also found that RhoG mRNA expression was down-regulated at P0. A previous study shows that mRNA contents of Rho, Rac and Cdc42 in the rat cortex did not change between E16 and P0 [18], suggesting that the down-regulation of RhoG mRNA observed at P0 is specific. On the other hand, expression of RhoG mRNA at P20 is highly enriched in white matter tracts, in which the development of brain myelination is observed [20], and double-labeling experiments demonstrated that major RhoG-expressing cells in white matter tracts are oligodendrocytes. Oligodendrocytes play an essential role in the mature nervous system through their ability to myelinate axon fascicles, thereby allowing efficient transmission of the electrical signals, and typical differentiated oligodendrocytes extend highly branched processes that wrap
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Fig. 3. Distribution of RhoG mRNA in the postnatal rat brain. In situ hybridization on coronal sections of the P20 brain shows the expression of RhoG mRNA in white matter tracts, including the corpus callosum (A), the anterior commissure (C), the hippocampal fimbria and external capsule (D), the cerebellar white matter (E), and the tectospinal tract (F). The control sense strand hybridization on the tissue section adjacent to (A) is shown in (B). Ctx, cerebral cortex; cc, corpus callosum; Cpu, caudate putamen; ac, anterior commissure; fi, hippocampal fimbria; ec, external capsule; wm, white matter; Gr, granular layer; ts, tectospinal tract. Scale bar: 100 mm.
around the axons to form an insulating myelin sheath [10]. We have recently reported that expression of RhoG induces neurite outgrowth in cultured neuronal cells [6]. Taken together, we speculate that RhoG may have an important role in the process formation in oligodendrocytes during the development of brain myelination. The functions of Rho family small GTPases in oligodendrocytes
have not yet been understood, but a previous study has reported that p190 RhoGAP, a GTPase-activating protein for Rho family GTPases, is involved in the regulation of oligodendrocyte differentiation [21]. Further studies are necessary to elucidate the mechanisms involving RhoG and other Rho family GTPases leading to oligodendrocyte differentiation and myelination.
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Fig. 4. Expression of RhoG mRNA in oligodendrocytes. (A) A higher magnification view of RhoG mRNA expression at P20 in the corpus callosum by in situ hybridization (visualized as purple color) shows linear arrays of cell bodies (indicated by arrows), which are a characteristic feature of oligodendrocytes. (B) Immunoperoxidase staining with the CC-1 antibody (visualized as brown color) shows linear arrays of oligodendrocytes. (C) Immunoperoxidase staining with the CC-1 antibody after in situ hybridization for RhoG shows that the majority of RhoG mRNA-expressing cells are colabeled with the CC-1 antibody (dark brown color, indicated by arrows). (D) Immunoperoxidase staining with an antibody to GFAP, an astrocyte marker (visualized as brown color), after in situ hybridization for RhoG. (E) Immunoperoxidase staining with an antibody to NeuN, a neuronal marker (visualized as brown color), after in situ hybridization for RhoG. Scale bar: 10 mm.
Acknowledgements This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (13041029, 13210068, 13780571, 13480256).
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[7] C.F. Landry, G.O. Ivy, I.R. Brown, Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization, J. Neurosci. Res. 25 (1990) 194–203. [8] Q.R. Lu, D.- in Yuk, J.A. Alberta, Z. Zhu, I. Pawlitzky, J. Chan, A.P. McMahon, C.D. Stiles, D.H. Rowitch, Sonic hedgehog-regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system, Neuron 25 (2000) 317–329. [9] L. Luo, Rho GTPases in neuronal morphogenesis, Nat. Rev. Neurosci. 1 (2000) 173–180. [10] R.H. Miller, Oligodendrocyte origins, Trends Neurosci. 19 (1996) 92–96. [11] K. Nakamura, T. Kaneko, Y. Yamashita, H. Hasegawa, H. Katoh, M. Negishi, Immunohistochemical localization of prostaglandin EP3 receptor in the rat nervous system, J. Comp. Neurol. 421 (2000) 543–569. [12] S.C. Noctor, A.C. Flint, T.A. Weissman, W.S. Wong, B.K. Clinton, A.R. Kriegstein, Dividing precursor cells of the embryonic cortical ventricular zone have morphological and molecular characteristics of radial glia, J. Neurosci. 22 (2002) 3161–3173. [13] C. Olenik, H. Barth, I. Just, K. Aktories, D.K. Meyer, Gene expression of the small GTP-binding proteins RhoA, RhoB, Rac1, and Cdc42 in adult rat brain, Mol. Brain Res. 52 (1997) 263–269. [14] C. Olenik, K. Aktories, D.K. Meyer, Differential expression of the small GTP-binding proteins RhoA, RhoB, Cdc42u and Cdc42b in developing rat neocortex, Mol. Brain Res. 70 (1999) 9–17. [15] M.F. Olson, A. Ashworth, A. Hall, An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1, Science 269 (1995) 1270–1272. [16] S.K. Pixley, J. de Vellis, Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin, Brain Res. 317 (1984) 201–209. [17] P. Roux, C. Gauthier-Rouviere, S. Doucet-Brutin, P. Fort, The small
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GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells, Curr. Biol. 7 (1997) 629–637. [18] R. Threadgill, K. Bobb, A. Ghosh, Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42, Neuron 19 (1997) 625– 634. [19] S. Vincent, P. Jeanteur, P. Fort, Growth-regulated expression of rhoG, a new member of the ras homolog gene family, Mol. Cell. Biol. 12 (1992) 3138–3148.
[20] J.A. Weiner, J.H. Hecht, J. Chun, Lysophosphatidic acid receptor gene vzg-1 /lpa 1 /edg-2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain, J. Comp. Neurol. 398 (1998) 587–598. [21] R.M. Wolf, J.J. Wilkes, M.V. Chao, M.D. Resh, Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation, J. Neurobiol. 49 (2001) 62–78.
Short communication
Developmental changes in expression of small GTPase RhoG mRNA in the rat brain Yukio Ishikawa, Hironori Katoh, Kazuhiro Nakamura, Kazutoshi Mori, Manabu Negishi* Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606 -8502, Japan Accepted 30 July 2002
Abstract We have recently reported that RhoG, a member of Rho family small GTPases, is involved in neurite outgrowth in cultured neuronal cells. Here, we report the expression of RhoG mRNA in the developing rat brain by in situ hybridization analysis. At embryonic day 16, RhoG expression was observed throughout the ventricular zone, but was down-regulated in the region at birth. On the other hand, RhoG expression at postnatal day 20 was highly enriched in white matter tracts, including the corpus callosum, the anterior commissure, and the cerebellar white matter, and double-labeling experiments demonstrated that major RhoG-expressing cells in white matter tracts were oligodendrocytes. These results suggest distinct pre- and postnatal roles of RhoG in the development of the central nervous system. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Rho family GTPase; In situ hybridization; Development; Oligodendrocyte
The Rho family of small GTPases has been implicated in the reorganization of the actin cytoskeleton and subsequent morphological changes in various cell types [3,5]. Like other GTPases of the Ras superfamily, they serve as molecular switches by cycling between an inactive GDPbound state and an active GTP-bound state, and once activated, they can interact with their specific effectors, leading to a variety of biological functions. Activation of the Rho family proteins requires GDP–GTP exchange catalyzed by various guanine nucleotide exchange factors, and is regulated by GTPase-activating proteins that stimulate the intrinsic GTPase activities of the G proteins. Presently, at least 14 mammalian Rho family proteins have been identified: Rho (A, B and C), Rac (1, 2 and 3), Cdc42, Rnd (1, 2, and 3), RhoD, TC10, RhoH / TTF, and RhoG. Among them, the functions of RhoA, Rac, and Cdc42 have been extensively characterized, and recent evidence implicates these Rho GTPases in the regulation of neuronal morphological changes, including axon growth *Corresponding author. Tel.: 181-75-753-4547; fax: 181-75-7537688. E-mail address: [email protected] (M. Negishi).
and guidance, and dendrite formation [9]. Furthermore, the distribution of Rho, Rac and Cdc42 in the brain has also been studied [13,14]. We have recently reported that RhoG is involved in the nerve growth factor-induced neurite outgrowth acting upstream of Rac and Cdc42 in rat pheochromocytoma PC12 cells [6]. Therefore, it is possible that RhoG also participates in the regulation of neuritogenesis during the development of the central nervous system. As an initial approach to address this issue, we have studied the distribution of mRNA for RhoG during the development of rat brain by in situ hybridization analysis. Rat RhoG cDNA [6] was subcloned into pBluescript SK(1) (Stratagene). Antisense and sense riboprobes were synthesized and digoxigenin (DIG)-labeled by in vitro transcription with T7 and T3 RNA polymerases and DIG RNA labeling mix (Roche) from the BamHI- and HindIIIdigested plasmid, respectively. Pregnant Wistar rats at defined gestational stages were used in this study. Timed pregnant rats were ether-anesthetized deeply and decapitated. Collected embryos were decapitated, and the brains were removed and fixed by incubating with a fixative containing 4% paraformaldehyde in 0.1 M phosphate
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00413-8
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buffer (pH 7.4) at 4 8C overnight. Postnatal rats were anesthetized with chloral hydrate and perfused transcardially with the same fixative. The brains were removed and postfixed in the fixative at 4 8C overnight. Embryonic and postnatal brains were then saturated with 0.1 M phosphate buffer containing 30% sucrose at 4 8C. The brains were cut into 60-mm thick (embryonic brains) or 40-mm thick (postnatal brains) coronal sections on a cryostat at 218 8C, and free-floating sections were acetylated in acetic anhydride / triethanolamine–HCl. The sections of postnatal rats were treated with 2.5 mg / ml proteinase K (Roche) for 10 min at room temperature before hybridization. After prehybridization in hybridization buffer (50% deionized formamide, 53 SSC, 53 Denhardt’s solution, 250 mg / ml salmon sperm DNA, 250 mg / ml yeast tRNA) for 2 h, the sections were incubated overnight at 55 8C with 500 ng / ml DIG-labeled sense or antisense probe for rat RhoG in the hybridization buffer. The sections were then washed in 23 SSC at 55 8C three times for 1 h each, and were blocked with 1% blocking reagent (Roche) in 0.1 M Tris–HCl (pH 7.5) and 0.15 M NaCl for 1 h at room temperature. They were incubated with alkaline phosphatase-conjugated antiDIG antibody (1:1000 dilution, Roche) at 4 8C overnight, and then reacted with chromogenic substrates, 5-bromo-4chloro-3-indolylphosphate and nitroblue tetrazolium in 0.1 M Tris–HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl 2 . For Northern blot analysis, whole brains from embryonic or postnatal rats were removed, frozen immediately in liquid nitrogen, and then stored at 280 8C until use. Purification of total RNA and Northern blotting were performed as described previously [6]. For normalization, the membrane was rehybridized with a cDNA probe for the mouse glyceraldehyde 3-phosphate dehydrogenase. The probes were labeled using Ready-To-Go DNA Labeling Beads (Amersham Pharmacia Biotech). For double-labeling experiments, the sections after in situ hybridization were incubated at 4 8C overnight with a monoclonal antibody to adenomatous polyposis coli protein (CC-1, Oncogene Research Products, 1:100 dilution), used as an oligodendrocyte marker, a monoclonal antibody to an astrocyte marker glial fibrillary acidic protein (GFAP) (Chemicon, 1:500 dilution), or a monoclonal antibody to a neuronal marker NeuN (Chemicon, 1:100 dilution) in phosphate-buffered saline containing 0.3% Triton X-100, 0.25% l-carrageenan, and 0.5% normal donkey serum. The detection of the antibodies was performed as described previously [11]. We also used a monoclonal antibody to vimentin as a marker for radial glial cells (Sigma, 1:40 dilution). Northern blot analysis of rat brain RNA from embryonic day 14 (E14) to the adult indicated that RhoG mRNA was detected at all developmental stages, and high expression levels were observed during the E14–16 and the postnatal day 20 (P20)–28 (Fig. 1). To reveal the distribution of RhoG in the developing rat brain, we performed in situ hybridization using a DIG-labeled RhoG antisense ribop-
Fig. 1. Expression of RhoG mRNA in pre- and postnatal rat brain. (A) Total RNA (20 mg) isolated from embryonic (E14, E16, E20) or postnatal (P0, P4, P8, P14, P20, P28, adult) rat brain was subjected to Northern blot analysis with a cDNA probe for RhoG. The same membrane rehybridized with GAPDH cDNA probe is shown below as the internal control. (B) Quantification of the RhoG mRNA level from E14 to the adult rat brain. The level of RhoG mRNA was normalized to that of GAPDH mRNA and expressed as fold increases over the value for the adult brain.
robe. In the E16 brain, expression of RhoG mRNA was observed throughout the ventricular zone (Fig. 2A,B; the immunostaining with an antibody to the radial glial marker vimentin [12,16] shown in Fig. 2C). However, RhoG expression was down-regulated in the ventricular zone at P0 (Fig. 2D,E). We next examined the expression of RhoG mRNA at P20. In contrast to the expression at E16, expression of RhoG mRNA was detected throughout the brain, particularly in white matter tracts including the corpus callosum (Fig. 3A; the control sense strand hybridization shown in Fig. 3B), the anterior commissure (Fig. 3C), the hippocampal fimbria, the external capsule (Fig. 3D), the cerebellar white matter (Fig. 3E), and the tectospinal tract (Fig. 3F). These areas are rich in glial cells but do not contain neuronal cell bodies, and RhoG mRNA-positive cells formed linear arrays, which are a characteristic feature of oligodendrocytes (Fig. 4A,B). Immunostaining with the monoclonal antibody CC-1, a marker for oligodendrocytes [1,8] after in situ hybridization to RhoG demonstrated that RhoG mRNA expression was detected in CC-1 antibody-positive cells (Fig. 4C). We also detected the expression of RhoG mRNA in myelin proteolipid protein-positive cells, another marker for oligodendrocytes [2] (data not shown). Astrocytes are also distributed in some portions of white matter tracts [7], but expression of RhoG mRNA did not overlap with that of
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Fig. 2. Distribution of RhoG mRNA in the E16 and P0 rat brain. (A) In situ hybridization on a coronal section of the E16 cortex shows the expression of RhoG mRNA throughout the ventricular zone. (B) A higher magnified image of the expression of RhoG mRNA at E16. (C) Immunoperoxidase staining on a coronal section of the E16 cortex with an antibody to the radial glial marker vimentin. (D) In situ hybridization for RhoG mRNA on a coronal section of the P0 cortex. (E) Immunoperoxidase staining on a coronal section of the P0 cortex with an antibody to vimentin. Ctx, cerebral cortex; LV, lateral ventricle; GE, ganglionic eminence; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate; WM, white matter. Scale bars: (A) 500 mm; (C) 50 mm; (E) 100 mm.
GFAP, an astrocyte marker (Fig. 4D). Furthermore, RhoG mRNA expression did not overlap with that of a neuronal marker NeuN (Fig. 4E). Expression of RhoG mRNA at P20 was also found in scattered cells throughout the brain (data not shown). These results indicate that major RhoGexpressing cells in the P20 rat brain are oligodendrocytes. The expression of RhoG mRNA in white matter tracts was also detected in the adult brain but weak compared to that of the P20 brain (data not shown). In the present study, we found that there are at least two phases of the RhoG expression in the developing rat brain, and they are not only temporally but may also be functionally distinct. At E16, expression of RhoG mRNA is observed throughout the ventricular zone. During the embryonic days, proliferation of neuronal and glial precursors occurs in the ventricular zone. Since RhoG was first identified as a growth-stimulated gene and has been shown to be involved in the regulation of cell proliferation in fibroblasts [17,19], it is inferred that regulated expression of RhoG may play a role in the regulation of cell proliferation in the ventricular zone during the embryonic
days. RhoA and Cdc42 are also expressed in the ventricular zone during the neurogenetic period [14], and they play essential roles in the cell cycle progression [15]. Considering that RhoG acts as an upstream regulator for Cdc42 [4,6], RhoG may regulate neurogenesis through the activation of Cdc42 in the ventricular zone. We also found that RhoG mRNA expression was down-regulated at P0. A previous study shows that mRNA contents of Rho, Rac and Cdc42 in the rat cortex did not change between E16 and P0 [18], suggesting that the down-regulation of RhoG mRNA observed at P0 is specific. On the other hand, expression of RhoG mRNA at P20 is highly enriched in white matter tracts, in which the development of brain myelination is observed [20], and double-labeling experiments demonstrated that major RhoG-expressing cells in white matter tracts are oligodendrocytes. Oligodendrocytes play an essential role in the mature nervous system through their ability to myelinate axon fascicles, thereby allowing efficient transmission of the electrical signals, and typical differentiated oligodendrocytes extend highly branched processes that wrap
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Fig. 3. Distribution of RhoG mRNA in the postnatal rat brain. In situ hybridization on coronal sections of the P20 brain shows the expression of RhoG mRNA in white matter tracts, including the corpus callosum (A), the anterior commissure (C), the hippocampal fimbria and external capsule (D), the cerebellar white matter (E), and the tectospinal tract (F). The control sense strand hybridization on the tissue section adjacent to (A) is shown in (B). Ctx, cerebral cortex; cc, corpus callosum; Cpu, caudate putamen; ac, anterior commissure; fi, hippocampal fimbria; ec, external capsule; wm, white matter; Gr, granular layer; ts, tectospinal tract. Scale bar: 100 mm.
around the axons to form an insulating myelin sheath [10]. We have recently reported that expression of RhoG induces neurite outgrowth in cultured neuronal cells [6]. Taken together, we speculate that RhoG may have an important role in the process formation in oligodendrocytes during the development of brain myelination. The functions of Rho family small GTPases in oligodendrocytes
have not yet been understood, but a previous study has reported that p190 RhoGAP, a GTPase-activating protein for Rho family GTPases, is involved in the regulation of oligodendrocyte differentiation [21]. Further studies are necessary to elucidate the mechanisms involving RhoG and other Rho family GTPases leading to oligodendrocyte differentiation and myelination.
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Fig. 4. Expression of RhoG mRNA in oligodendrocytes. (A) A higher magnification view of RhoG mRNA expression at P20 in the corpus callosum by in situ hybridization (visualized as purple color) shows linear arrays of cell bodies (indicated by arrows), which are a characteristic feature of oligodendrocytes. (B) Immunoperoxidase staining with the CC-1 antibody (visualized as brown color) shows linear arrays of oligodendrocytes. (C) Immunoperoxidase staining with the CC-1 antibody after in situ hybridization for RhoG shows that the majority of RhoG mRNA-expressing cells are colabeled with the CC-1 antibody (dark brown color, indicated by arrows). (D) Immunoperoxidase staining with an antibody to GFAP, an astrocyte marker (visualized as brown color), after in situ hybridization for RhoG. (E) Immunoperoxidase staining with an antibody to NeuN, a neuronal marker (visualized as brown color), after in situ hybridization for RhoG. Scale bar: 10 mm.
Acknowledgements This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (13041029, 13210068, 13780571, 13480256).
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