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• W1972504544 abstract "Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain. Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain. The generation of neurons is regulated by several diffusible signals including the fibroblast growth factor-2 (FGF-2), 1The abbreviations used are: FGF-2, fibroblast growth factor-2; G protein, heterotrimeric guanine nucleotide-binding regulatory protein; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PTX, pertussis toxin; ET, endothelin; E, embryonic day; LPA, lysophosphatidic acid; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; PBS, phosphate-buffered saline; SFM, serum-free medium. neurotrophin-3, and the brain-derived neurotrophic factor, which bind receptor-tyrosine kinases (1Ghosh A. Greenberg M.E. Neuron. 1995; 15: 89-103Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 2Temple S. Qian X. Neuron. 1995; 15: 249-252Abstract Full Text PDF PubMed Scopus (136) Google Scholar). FGF-2 is a potent mitogen present in the cerebral cortex throughout neurogenesis (3Powell P.P. Finklestein S.T. Dionne C.A. Jaye M. Klagsbrun M. Mol. Brain Res. 1991; 11: 71-77Crossref PubMed Scopus (82) Google Scholar, 4Weise B. Janet T. Groth C. J. Neurosci. Res. 1993; 34: 442-453Crossref PubMed Scopus (94) Google Scholar) and in the neural precursor cells isolated from the embryonic telencephalon (5Maric D. Maric I. Chang Y.H. Barker J.L. J. Neurosci. 2003; 23: 240-251Crossref PubMed Google Scholar). Other signals caused by binding of an agonist for the muscarinic acetylcholine receptor, a G protein-coupled receptor (GPCR) (6Li B-S. Ma W. Zhang L. Barker J.L. Stenger D.A. Pant H.C. J. Neurosci. 2001; 21: 1569-1579Crossref PubMed Google Scholar), as well as gp130 ligands (7Hatta T. Moriyama K. Nakashima K. Taga T. Otani H. J. Neurosci. 2002; 22: 5516-5524Crossref PubMed Google Scholar), have been also shown to stimulate DNA synthesis in neural precursor cells in the ventricular zone. Receptor-tyrosine kinases and GPCRs can both activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and there is evidence that FGF-2 utilizes a G protein to transmit signals to the MAPK pathway (8Fedorov Y.V. Jones N.C. Olwin B.B. Mol. Cell. Biol. 1998; 18: 5780-5787Crossref PubMed Scopus (49) Google Scholar). Heterotrimeric G proteins function as signal transducers from receptors in the cell membrane to intracellular effectors. They consist of three subunits (α, β, γ) and are classified into four subfamilies, Gs, Gi, Gq, and G12 (9Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The α-subunits of Gi family G proteins, including Gi, Go, transducin, and gustducin, are specifically ADP-ribosylated by pertussis toxin (PTX) to become unable to couple with GPCRs. Therefore, PTX serves as a useful tool to explore Gi-mediated signal transduction both in vitro and in vivo. The α- and/or βγ-subunits of Gi and Go directly regulate adenylyl cyclase, phospholipase Cβ, phosphatidylinositol 3-kinase, and K+ and Ca2+ channels. βγ-subunits released from Gi/o indirectly stimulate ERK (9Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 10Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar, 11Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). In early embryonic brains, Gαi2 and Gγ5 are highly expressed in the ventricular zone but their levels decrease with development (12Morishita R. Shinohara H. Ueda H. Kato K. Asano T. J. Neurochem. 1999; 73: 2369-2374Crossref PubMed Scopus (32) Google Scholar, 13Asano T. Shinohara H. Morishita R. Ueda H. Kawamura N. Katoh-Semba R. Kishikawa M. Kato K. J. Neurochem. 2001; 79: 1129-1135Crossref PubMed Scopus (15) Google Scholar). In contrast, levels of Gαo and Gγ2, which are limited to the marginal zone, increase with the addition of newly generated neuronal cells from the ventricular zone (12Morishita R. Shinohara H. Ueda H. Kato K. Asano T. J. Neurochem. 1999; 73: 2369-2374Crossref PubMed Scopus (32) Google Scholar, 14Schmidt C.J. Zubiaur M. Valenzuela D. Neer E.J. Drager U.C. J. Neurosci. Res. 1994; 38: 182-187Crossref PubMed Scopus (14) Google Scholar). In the adult brain, Gαo and Gγ2, as well as Gαi1 and Gγ3, are major isoforms of G proteins (15Worley P.F. Baraban J.M. Van Dop C. Neer E.J. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4561-4565Crossref PubMed Scopus (130) Google Scholar, 16Asano T. Semba R. Kamiya N. Ogasawara N. Kato K. J. Neurochem. 1988; 50: 1164-1169Crossref PubMed Scopus (93) Google Scholar, 17Asano T. Shinohara H. Morishita R. Kato K. J. Biochem. (Tokyo). 1990; 108: 988-994Crossref PubMed Scopus (28) Google Scholar, 18Asano T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar) but the expression of Gαi2 and Gγ5 persists in the neural stem cells in the ventricular zone at the rostral part of lateral ventricle and progeny cells migrating toward the olfactory bulb (13Asano T. Shinohara H. Morishita R. Ueda H. Kawamura N. Katoh-Semba R. Kishikawa M. Kato K. J. Neurochem. 2001; 79: 1129-1135Crossref PubMed Scopus (15) Google Scholar). Therefore, the expression of Gi2 (Gαi2βγ5) seems to be consistently involved in neurogenesis, both in embryonic and postnatal brains. To clarify the role of Gi2 in the developing brain, we first injected PTX into the lateral ventricle of mouse embryos, and found suppression of neuroepithelial cell proliferation. Next, an in vitro survey of several Gi-coupled receptor agonists revealed that endothelins (ETs) have potent mitogenic activity for neural progenitor cells via their action on ET-B receptors, and such an effect of ET was confirmed in vivo. Animal Preparation—Each female mouse (Jcl: ICR) or rat (Sprague-Dawley) was mated with a male overnight and noon of the day to confirm a vaginal plug was designated as embryonic day (E) 0.5. All the treatments of animals and the experimental procedures were conducted following the guidelines for care and use of laboratory animals, Mie University School of Medicine and the Institute for Developmental Research, Aichi Human Service Center. Exo-utero Microinjection—PTX or the vehicle was injected into the ventricular cavity of developing mouse embryos at E14.5 with the exo-utero microinjection method, as previously reported (7Hatta T. Moriyama K. Nakashima K. Taga T. Otani H. J. Neurosci. 2002; 22: 5516-5524Crossref PubMed Google Scholar, 19Muneoka K. Wanek N. Bryant S.V. J. Exp. Zool. 1986; 239: 289-293Crossref PubMed Scopus (80) Google Scholar). In short, pregnant mice were anesthetized with pentobarbital (70 mg/kg body weight) and injected with 1.4 mg of ritodrine (Sigma) dissolved in saline solution (0.1 ml) to relax the myometrium. The abdominal wall was cut, and the uterus gently pulled out of the peritoneal cavity. The uterine walls were cut longitudinally and 4-6 embryos were selected for injection, while other embryos were removed. By using glass pipettes (60-80-μm in diameter) under a binocular microscope, 1 μl of PTX (0.1 μg, Calbiochem) or the vehicle was injected into the ventricular cavity of the experimental and the control groups, respectively. After the uterus was returned to within the body, ∼2 ml of warmed Hank's solution were gently injected, to prevent unexpected adhesions within the peritoneal cavity, and the abdominal wall was sutured. Then the animals were returned to the mouse room after confirmation of recovery from the anesthesia. The embryos were fixed at E18.5 for counting total cells in the cerebral cortices. To label proliferating cells, intraperitoneal injection of bromodeoxyuridine (BrdUrd, 50 mg/kg of body weight) was performed on pregnant females at E16.5, and the animals were sacrificed 2 h after BrdUrd injection to collect embryos. For examination of the effect of ET, 1 μl of ET-3 (10 μm), BQ788 (1 mm), or vehicle was injected into the ventricle of embryos at E14.5, BrdUrd was injected at E15.5, and the animals were sacrificed 2 h later. To evaluate whether PTX could affect cell migration of newly generated neural cells, BrdUrd was administered 3-4 h prior to the injection of PTX or vehicle at E14.5, followed by the fixation of embryos at E16.5. Tissue Preparation for Histology—The embryos were fixed in toto in a mixture of 10% formalin and 70% methanol overnight. After observation for external malformations, their brains were resected and processed for embedding in paraffin. Serial coronal sections (6-μm thick) were prepared and every tenth section was stained with cresyl violet (Nissl staining). BrdUrd Immunohistochemistry—Sections of the parietal cortex (at the level of the supraoptic nucleus) were deparaffinized and treated with 1% H2O2 in methanol to block endogenous peroxidase activity, and with 2 n HCl for 2 h at room temperature to denature the DNA. After the sections were treated with 10% horse serum in 50 mm Tris-HCl (pH 7.6) and 150 mm NaCl (TBS) for 10 min, they were incubated with mouse anti-BrdUrd antibodies (1:100, Roche Diagnostics) in 50 mm Tris-HCl, pH 7.6 with 0.5 m NaCl at 4 °C overnight, followed by visualization of binding sites with diaminobenzidine through the ABC method (Elite ABC system, 1:100, with biotinylated horse anti-mouse IgG antibodies, 1:200; Vector Laboratories). Cell Counts—For cell counts, sections of parietal cortex (at the level of the supraoptic nucleus) were photographed and developed at the final magnification of ×280. For each cerebral cortex, Nissl stain-positive cells and BrdUrd-positive cells were counted in ten different columnar zones (about 35-μm wide) extending from the ventricular lumen to the pial surface of the cerebral cortex. The choice of imaging areas and cell counts were performed blindly. Detection of Apoptotic Cells—Sections close to those used for BrdUrd immunostaining were subjected to terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) assay, which was performed with an in situ Cell Death Detection Kit (Roche Diagnostics). Samples were analyzed by following immunostaining with peroxidase conjugated anti-fluorescein antibody and reaction with diaminobenzidine. Primary Culture of Neural Progenitor Cells of Rats—Neural progenitor cells were cultured according to the method described previously (20Zhu G. Mehler M.F. Zhao J. Yung S.Y. Kessler J.A. Dev. Biol. 1999; 215: 118-129Crossref PubMed Scopus (86) Google Scholar) with modifications. Forebrains of rat embryos at E13.5 were dissected and dissociated by incubation with 0.2% trypsin for 15 min followed by repeated trituration. To remove contaminating fibroblasts tightly attached to the dishes, dissociated cells were seeded onto 100-mm uncoated culture dishes in the presence of 10% fetal bovine serum in serum-free medium (SFM). After 4 h incubation, cells floating or lightly attaching to fibroblasts were collected for seeding to culture dishes coated with poly(2-hydroxy-ethyl methacrylate) (Sigma, 1.6 mg/cm2) (21Torii M. Matsuzaki F. Osumi N. Kaibuchi K. Nakamura S. Casarosa S. Guillemot F. Nakafuku M. Development. 1999; 126: 443-456Crossref PubMed Google Scholar) and cultured in SFM containing 20 ng/ml FGF-2 (Pepro Tech EC) and 2 μg/ml heparin (Sigma) for 6 days to generate proliferative progenitor neurospheres. Refreshment with a half volume of the medium was performed at 3 days. The SFM consisted of DMEM/F12 (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and nutrient additives including 0.01% bovine serum albumin (Fraction V), 30 nm selenium, 15 nm triiodo-l-thyronine, 40 nm biotin, 25 μg/ml apo-transferrin, 1 nm hydrocortisone, and 25 μg/ml insulin (all of these reagents were from Sigma). Neurospheres were dissociated by initial incubation with 0.05% trypsin for 10 min followed by repeated trituration. Dissociated cells were plated onto dishes precoated with 100 μg/ml poly-l-lysine (Sigma) plus or minus 10 μg/ml fibronectin (Nacalai Tesque), propagated in SFM with 10 ng/ml FGF-2 under an atmosphere of 5% CO2, 95% air and used for the various experiments. [3H]Thymidine Incorporation Assay—This was performed essentially as described previously (6Li B-S. Ma W. Zhang L. Barker J.L. Stenger D.A. Pant H.C. J. Neurosci. 2001; 21: 1569-1579Crossref PubMed Google Scholar). Briefly, dissociated progenitor cells (1 × 105/well) were seeded to 12-well plates and cultured for 48 h in SFM in the presence of 10 ng/ml FGF-2 and then for 24 h in SFM without FGF-2 to decrease the basal levels of proliferation. PTX (20 ng/ml) was added for the last 6 h. Exposure to agonists was for 24 h, with 0.2 μCi/well [methyl-3H]thymidine introduced for the last 6 h of the incubation at 37 °C. Cells were washed three times with ice-cold phosphate-buffered saline (PBS), and 5% trichloroacetic acid was added for 20 min at 4 °C. The cells were then washed once with 5% trichloroacetic acid, and a mixture of 0.1 m NaOH, and 1% SDS was added for 10 min. Samples were transferred to scintillation vials, and the radioactivity was counted with a Beckman LS6000IC scintillation counter. The agonists and antagonists used were as follows: ET-1 and ET-3 (Peptide Institute), lysophosphatidic acid (LPA, Cayman Chemical), carbachol (Sigma), dopamine (Wako Pure Chemical Industries), U-69593 (Biomol Research Laboratories), BQ123 (Research Biochemicals International), BQ788 (Sigma), and U0126 (Calbiochem). Cell Proliferation—Dissociated progenitor cells (4 × 105/dish) were plated to 35-mm dishes and cultured for 24 h in SFM in the presence of 10 ng/ml FGF-2 and then for 24 h in SFM without FGF-2. PTX (20 ng/ml) was added for the last 6 h. Various agonists were then introduced with incubation for a further 3 days. Cells were washed twice with PBS, incubated with 0.2% trypsin for 15 min at 37 °C, and counted using a hematocytometer. Phosphorylation of ERK—Dissociated progenitor cells (5 × 105/dish) were plated to 35-mm dishes and cultured for 48 h in SFM in the presence of 10 ng/ml FGF-2 and then for 24 h in SFM without FGF-2. PTX (20 ng/ml), and inhibitors were added for the last 6 h and the last 30 min, respectively. At various times after addition of agonists, cells were washed twice with ice-cold PBS and lysed with 1% SDS in 20 mm Tris-HCl (pH 8.0) and 1 mm EDTA. The amount of protein in each lysate was quantified using a micro bicinchoninic acid protein assay kit (Pierce), with bovine serum albumin as the standard. The lysates (15 μg of proteins) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblotted with rabbit polyclonal antibody to phosphorylated or unphosphorylated ERK1/2 (1:1000, Cell Signaling Technology). Immunoblotting was performed employing a chemiluminescence reagent (PerkinElmer Life Sciences), and densitometry was achieved using a LAS-1000 (Fujifilm). Immunofluorescence Staining—Dissociated progenitor cells were plated onto poly-l-lysine-coated coverslips within 35-mm dishes and cultured in SFM with 10 ng/ml FGF-2 for 48 h (undifferentiated cells) or for 24 h followed by further incubation in SFM without FGF-2 for 7 days (differentiated cells). To examine ET-induced differentiation, progenitor cells were plated onto coverslips coated with poly-l-lysine plus fibronectin and cultured for 48 h in SFM in the presence of 10 nm ET-3, with 10 μm BrdUrd introduced for the last 24 h of the incubation. Cells were further cultured without ET-3 for 3 or 6 days. Then cells were fixed in 4% paraformaldehyde in PBS for 30 min, washed with PBS, permeabilized in 0.2% Triton X-100 in PBS for 2 min, and blocked in 10% goat serum in TBS for 1 h. Fixed cells were incubated with antibodies to Gαi2 (rabbit, 5 μg/ml) (22Morishita R. Kato K. Asano T. Eur. J. Biochem. 1988; 174: 87-94Crossref PubMed Scopus (46) Google Scholar), Gγ5 (rabbit, 10 μg/ml) (18Asano T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar), nestin (mouse, 1:2000, BD Pharmingen), β-tubulin type III (mouse, 1:400, Chemicon), glial fibrillary acidic protein (mouse, 1:600, Sigma), α-smooth muscle actin (mouse, 1:2000, Sigma), and BrdUrd (rat, 1:500, Harlan Sera-Lab) for 3 h at room temperature. After washing with TBS containing 1% goat serum, cells were incubated with secondary antibodies, Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 568 anti-mouse IgG, Alexa Fluor 488 anti-mouse IgG, or Alexa Fluor 568 anti-rat IgG (1:400, Molecular Probes), for 1 h at room temperature. For differentiated cells after removal of FGF-2, incubation was with propidium iodide (10 μg/ml) for 5 min. After applying the coverslip, slides were examined under a laser-scanning microscope (FLUOVIEW, Olympus) equipped for fluorescence or an inverted fluorescence microscope (Axiovert 200 m, Carl Zeiss). Evaluation of PTX-mediated ADP-Ribosylation of G Proteins in the Cerebral Cortex of Mouse Brains or Cultured Cells—Two days after injection of PTX or vehicle solution at E14.5, embryos were collected from dams and whole brains without the cerebellum were dissected. Cultured neural progenitor cells were incubated with 20 ng/ml PTX at 37 °C for 6 h and washed with PBS. These samples were kept frozen at -80 °C until analysis. Frozen brains were homogenized at 0 °C in a Potter-Elvehjem homogenizer in 20 volumes (v/w) of 20 mm Tris-HCl (pH 8.0), 1 mm EDTA, and 100 mm NaCl, and centrifuged at 4 °C at 20,000 × g for 20 min to obtain the membrane fraction. The brain membranes or neural progenitor cells were solubilized in 20 mm Tris-HCl (pH 8.0) containing 1 mm EDTA, 100 mm NaCl, and 1% sodium cholate, sonicated and then centrifuged at 4 °C at 100,000 × g for 40 min. The supernatant, referred to as the cholate extract, was incubated for 60 min at 30 °C in 20 mm Tris-HCl (pH 7.5), 2 μm [32P]NAD, 10 mm thymidine, 1 mm EDTA, and 1 mml-α-dimyristoyl phosphatidylcholine with 10 μg/ml PTX, which had been preactivated by incubation with 50 mm Tris-HCl (pH 7.5), 10 mm dithiothreitol and 1 mm ATP. The reaction was terminated by the addition of Laemmli sample buffer (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar), then aliquots (1.5 μg of protein) were subjected to SDS-PAGE, with subsequent autoradiography using x-ray film (RX-U, Fujifilm). The levels of Gαi2, Gαo, and Gβ proteins in cholate extracts were determined by immunoblotting with antibodies to Gαi2 (22Morishita R. Kato K. Asano T. Eur. J. Biochem. 1988; 174: 87-94Crossref PubMed Scopus (46) Google Scholar), Gαo (16Asano T. Semba R. Kamiya N. Ogasawara N. Kato K. J. Neurochem. 1988; 50: 1164-1169Crossref PubMed Scopus (93) Google Scholar), and Gβ (22Morishita R. Kato K. Asano T. Eur. J. Biochem. 1988; 174: 87-94Crossref PubMed Scopus (46) Google Scholar), respectively. Statistical Analysis—All results are given as mean ± S.E. values. The significance of group differences was analyzed with the Student's t test for single comparisons, and with one-way analysis of variance followed by the Bonferroni correction for multiple comparisons. A p value of < 0.05 was considered significant. Effects of PTX on the Developing Mouse Brain—To block the function of Gi2 in the ventricular zone, PTX was injected into the lateral ventricle of mice at E14.5. When the embryos were fixed at E18.5, there was no significant decrease in body weight and crown-rump length of PTX-injected embryos compared with controls (Fig. 1, A and C). However, PTX-injected embryos showed a decrease in the head vault compared with those of controls (Fig. 1A) and the cerebral cortices taken from the PTX-injected embryos were smaller than those of control embryos (Fig. 1, B and C). When they were taken out at E19.5 (full term), most PTX-injected mice could hardly start respiration and died. To confirm the effect of PTX, ADP-ribosylation of endogenous Gi/o proteins was evaluated in cholate extracts from PTX- or vehicle-injected brains at E16.5 using [32P]NAD and exogenous PTX. The cholate extracts from PTX-injected brains showed much weaker 32P-labeled bands (34 ± 5% of the control, n = 6) than those from control brains, indicating that a large proportion of Gi/o proteins had already been ADP-ribosylated by the injection of PTX at E14.5 (Fig. 1D). Immunoblot analyses of the same extracts revealed levels of total Gαi2 and Gαo proteins in PTX-injected brains to be somewhat decreased (Gαi2, 72 ± 3% of the control; Gαo, 81 ± 4% of the control, n = 6; Fig. 1D), suggesting that the degradation of ADP-ribosylated Gα subunits is faster than that of unmodified ones. In contrast, the levels of Gβ (Fig. 1D, 101 ± 4% of the control, n = 6) and PTX-insensitive Gαq/11 proteins (101 ± 9% of the control, n = 6) were unchanged by PTX treatment. Brains of PTX-injected Embryos Have Thinner Cerebral Cortices with Fewer Constituent Cells and Fewer BrdUrd-positive Cells—Histological studies revealed that the cerebral cortex from PTX-injected embryos at E18.5 was thinner than those from controls (Fig. 2A) and the total cell number in this zone was significantly reduced (Fig. 2B). To label proliferating cells, BrdUrd was administrated 2 days after PTX injection, and embryos were fixed 2 h later. Immunostaining with an antibody against BrdUrd showed a decreased number of BrdUrd-positive cells in the cerebral cortex of PTX-injected embryos (Fig. 2, C and D). TUNEL staining of close sections revealed that injection of PTX did not significantly increase cell death in the cerebral cortex (Fig. 2, E and F). Moreover, PTX seems not to affect the migration of neural cells, because there was no significant decrease in the number of neural cells labeled with BrdUrd before PTX injection and migrated from the ventricular zone into the cortical plate (control, 39.8 ± 8.7; PTX, 35.5 ± 9.4 in the area shown by box in Fig. 2F, n = 4). These results suggest that Gi2 mediates signaling from GPCRs to maintain proliferation of neural progenitor cells in the developing brain. To investigate which receptors are coupled to Gi2 to stimulate neurogenesis, we next performed in vitro experiments using neural progenitor cells dissociated from rat embryonic brains. Characterization of Cultured Neural Progenitor Cells—Proliferative progenitor neurospheres were generated from forebrains of E13.5 rats, dissociated, and cultured in the presence of FGF-2. The majority of cells were nestin-positive, indicating that they retained properties of undifferentiated cells (Fig. 3A). These cells also expressed Gαi2 and Gγ5, as observed previously in the ventricular zone of embryonic brain (Fig. 3A) (12Morishita R. Shinohara H. Ueda H. Kato K. Asano T. J. Neurochem. 1999; 73: 2369-2374Crossref PubMed Scopus (32) Google Scholar). Among other PTX-sensitive Gα subunits, Gαo was expressed at a low level in these cells, but Gαi1 and Gαi3 were not detectable by immunoblot analysis (data not shown). After withdrawal of FGF-2 for 7 days, many cells expressed neuron-specific antigen (β-tubulin type III) or an astrocyte marker (glial fibrillary acidic protein) (Fig. 3B), in good agreement with the previously established observation that neural progenitor cells can differentiate either into neurons or glial cells. ETs Increase Proliferation of Neural Progenitor Cells through Gi2—Several Gi-coupled receptors have been found to be expressed in the ventricular zone of developing brains, including ET-B (24Tsaur M.-L. Wan Y.-C. Lai F.-P. Cheng H.-F. FEBS Lett. 1997; 417: 208-212Crossref PubMed Scopus (18) Google Scholar), LPA (25Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar), muscarinic acetylcholine (6Li B-S. Ma W. Zhang L. Barker J.L. Stenger D.A. Pant H.C. J. Neurosci. 2001; 21: 1569-1579Crossref PubMed Google Scholar), D3 dopamine (26Diaz J. Ridray S. Mignon V.N.G. Schwartz J.-C. Sokoloff P. J. Neurosci. 1997; 17: 4282-4292Crossref PubMed Google Scholar), and κ-opioid receptors (27Leslie F.M. Chen Y. Winzer-Serhan U.H. Can. J. Physiol. Pharmacol. 1998; 76: 284-293Crossref PubMed Scopus (37) Google Scholar). Therefore, we examined whether agonists for these receptors might stimulate the incorporation of [3H]thymidine in neural progenitor cells, and compared the results with those for FGF-2, known to be a major mitogen which binds tyrosine kinase receptors. Among the agonists for Gi-coupled receptors, ET-3 was most effective, while LPA also significantly increased the incorporation of [3H]thymidine (Fig. 4). However, such stimulatory effects were not observed with the other agonists such as carbachol, dopamine, and U-69593 (an agonist for the κ-opioid receptor). In these experiments, we also found that ET-3-stimulated [3H]thymidine incorporation was greatly enhanced in the presence of fibronectin, but the FGF-2-mediated effect was not so augmented (Fig. 4). Laminin, another extracellular matrix component, was similarly effective (data not shown). Therefore, cell culture was carried out on dishes coated with poly-l-lysine plus fibronectin unless otherwise described. ET-1 and ET-3 revealed a similar dose-dependence for stimulation of [3H]thymidine incorporation with submaximal effects at 1 nm (Fig. 5A). The stimulatory effects were blocked by an ET-B receptor antagonist BQ788, but not by an ET-A receptor antagonist BQ123 (28Douglas S.A. Meek T.D. Ohlstein E.H. Trends Pharmacol. Sci. 1994; 15: 313-316Abstract Full Text PDF PubMed Scopus (81) Google Scholar) (Fig. 5B). These results indicate that the effects of ETs are mediated by the ET-B receptor. Because ET receptors are known to couple not only with Gi but also with Gq/11 and G13 (29Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar, 30Kitamura K. Shiraishi N. Singer W.D. Handlogten M.E. Tomita K. Miller R.T. Am. J. Physiol. 1999; 276: C930-C937Crossref PubMed Google Scholar), we next examined whether Gi is indeed involved in stimulation of DNA synthesis. PTX treatment of neural progenitor cells partially decreased ET-3-enhanced [3H]thymidine incorporation (Fig. 5C), whereas the toxin completely ADP-ribosylated endogenous Gi/o proteins (Fig. 5C, inset), suggesting that both Gi2 and PTX-insensitive G proteins mediate ET-stimulated DNA synthesis. Pertussis toxin did not influence FGF-2-stimulated [3H]t" @default.
• W1972504544 created "2016-06-24" @default.
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• W1972504544 date "2004-09-01" @default.
• W1972504544 modified "2023-09-29" @default.
• W1972504544 title "Gi2 Signaling Enhances Proliferation of Neural Progenitor Cells in the Developing Brain" @default.
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