Gi2 Signaling Enhances Proliferation of Neural Progenitor Cells in the 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. 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 (7Hatta T. Moriyama K. Nakashima K. Taga T. Otani H. J. Neurosci. 2002; 22: 5516-5524Crossref PubMed Google Scholar, K. N. J. 1986; PubMed Scopus Google Scholar). In were with and injected with of in to the The was and the of the The were and embryos were for embryos were in a of PTX or the vehicle was injected into the ventricular cavity of the experimental and the the was to the of were to the and the was the animals were to the mouse of from the The embryos were at for cells in the cerebral To injection of of was on at and the animals were injection to of the effect of ET, of or vehicle was injected into the ventricle of embryos at was injected at and the animals were To PTX cell of newly generated neural was administered to the injection of PTX or vehicle at by the of embryos at for embryos were in in a of and for their brains were and for in were and was with of the cortex the of the were and with in to and with for at to the the were with in and for were with mouse in with at by of binding with the method with cell of cortex the of the were and at the of cerebral cortex, cells and cells were in from the ventricular to the of the cerebral The of and cell were of to used for were to terminal dUTP nick labeling which was with an in were by following with and with of of progenitor cells were cultured to the method J. Biol. 1999; PubMed Scopus Google Scholar) with of rat embryos at were and by with for by To to the cells were in the presence of in serum-free cells or to were for to with M. N. K. S. S. M. 1999; PubMed Google Scholar) and cultured in FGF-2 and for to progenitor with a of the was at The of and including and of were from were by with for by cells were with or in with FGF-2 an of and used for the was as (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). progenitor cells were to and cultured for in in the presence of FGF-2 and for in FGF-2 to decrease the levels of proliferation. PTX was for the to agonists was for with for the of the at were three with phosphate-buffered and was for at The cells were with and a of and was for were to and the was with a The agonists and used were as and lysophosphatidic and progenitor cells were to and cultured for in in the presence of FGF-2 and for in PTX was for the agonists were with for a were with PBS, with for at and a of progenitor cells were to and cultured for in in the presence of FGF-2 and for in PTX and were for the and the addition of cells were with and with in and The of protein in was a protein with as the The of were to and with to or was a and was a progenitor cells were and cultured in with FGF-2 for or for by in FGF-2 for To progenitor cells were with and cultured for in in the presence of with for the of the were cultured for or cells were in in for with PBS, in in for and in in for cells were with to Gαi2 R. Kato K. Asano T. J. Biochem. 1988; PubMed Scopus Google Scholar), Gγ5 T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar), protein and for at with cells were with or for at cells of FGF-2, was with for the were a for or an of of G in the of or injection of PTX or vehicle at embryos were from and brains the were neural progenitor cells were with PTX at for and with These were at brains were at in a in of and and at at for to the membrane The brain or neural progenitor cells were in and and at at for The to as the was for at in and with PTX, which had been by with and The was by the addition of PubMed Scopus Google Scholar), of were to with The levels of and proteins in were by with to Gαi2 R. Kato K. Asano T. J. Biochem. 1988; PubMed Scopus Google Scholar), Gαo T. Semba R. Kamiya N. Ogasawara N. Kato K. J. Neurochem. 1988; 50: 1164-1169Crossref PubMed Scopus (93) Google Scholar), and R. Kato K. Asano T. J. Biochem. 1988; PubMed Scopus Google Scholar), are as The of was with the for and with of by the for of was of PTX on the the function of Gi2 in the ventricular zone, PTX was injected into the lateral ventricle of at the embryos were at there was decrease in and of embryos with and embryos showed a decrease in the with of and the cerebral cortices from the embryos were of embryos and were at most and To confirm the effect of PTX, of Gi/o proteins was evaluated in from or brains at and The from brains showed of the from brains, that a of Gi/o proteins had been ADP-ribosylated by the injection of PTX at E14.5 of the revealed levels of Gαi2 and Gαo proteins in brains to be decreased of the of the suggesting that the of ADP-ribosylated subunits is that of In contrast, the levels of of the and proteins of the were by PTX of with and revealed that the cerebral cortex from embryos at was thinner from and the cell in this zone was To was PTX and embryos were with an showed a decreased of cells in the cerebral cortex of embryos and of revealed that injection of PTX increase cell in the cerebral cortex and PTX seems to the of neural there was decrease in the of neural cells with PTX injection and from the ventricular zone into the PTX, in the shown by in These that Gi2 mediates signaling from GPCRs to maintain proliferation of neural progenitor cells in the developing brain. To which receptors are to Gi2 to stimulate neurogenesis, we in vitro neural progenitor cells from rat embryonic brains. of progenitor were generated from of and cultured in the presence of The of cells were that of cells These cells also expressed Gαi2 and as in the ventricular zone of embryonic brain (12Morishita R. Shinohara H. Ueda H. Kato K. Asano T. J. Neurochem. 1999; 73: 2369-2374Crossref PubMed Scopus (32) Google Scholar). Among Gαo was expressed at a in but Gαi1 and were by of FGF-2 for cells expressed or an in with the that neural progenitor cells can into neurons or cells. of Gi-coupled receptors have been found to be expressed in the ventricular zone of developing brains, including ET-B 1997; PubMed Scopus Google Scholar), J. J. Biol. PubMed Scopus 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), J. S. J. Neurosci. 1997; 17: PubMed Google Scholar), and receptors J. Pharmacol. 1998; PubMed Scopus (37) Google Scholar). Therefore, we agonists for receptors stimulate the incorporation of [3H]thymidine in neural progenitor and the with for FGF-2, known to be a major mitogen which kinase Among the agonists for Gi-coupled receptors, was most also increased the incorporation of [3H]thymidine such effects were with the agonists such as and agonist for the In we also found that [3H]thymidine incorporation was in the presence of fibronectin, but the effect was extracellular was Therefore, cell was on with and revealed a for of [3H]thymidine incorporation with effects at The effects were by an ET-B receptor antagonist but by an receptor antagonist Trends Pharmacol. Sci. 1994; 15: Full Text PDF PubMed Scopus Google Scholar) These indicate that the effects of are by the ET-B receptor. ET receptors are known to couple with Gi but also with and Pharmacol. Rev. 1994; Google Scholar, K. N. M.E. K. J. 1999; PubMed Google Scholar), we Gi is involved in of DNA PTX of neural progenitor cells partially decreased [3H]thymidine incorporation whereas the toxin ADP-ribosylated Gi/o proteins suggesting that both Gi2 and G proteins DNA toxin [3H]thymidine suggesting that Gi is involved in DNA synthesis To increase cell cell numbers were shown in increased cell numbers in the presence of but effect was in proliferation was partially inhibited by with PTX, that ET cell proliferation at in part via PTX of on proliferation of neural progenitor cells. progenitor cells were with or and cultured for in in the presence of FGF-2 and for in PTX was for the or FGF-2 was to for and cells were are from four in To ET is as a mitogen for the in the ventricular an agonist and an antagonist for the ET-B receptor were injected into the ventricle of at by injection increased a of cells in the cerebral cortex and injection decreased and of ERK Gi is known to stimulate the MAPK that DNA synthesis and cell proliferation J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar), we this pathway is involved in proliferation of neural progenitor cells. DNA synthesis was inhibited by an of MAPK kinase Next we the of and FGF-2 to activate ERK by phosphorylation of is a major in neural progenitor cells and phosphorylation is that of phosphorylation of was in the present increased phosphorylation of and and the levels In contrast, FGF-2 increased phosphorylation of and a was for a the of ERK phosphorylation by both and phosphorylation at was in the presence of that in suggesting that signaling levels of ERK phosphorylation phosphorylation of was by with PTX and an in with the for DNA synthesis PTX ERK phosphorylation The effect of on phosphorylation was also by the ET-B receptor antagonist but by the receptor antagonist of the of the cells ET progenitor cells were cultured with and BrdUrd, which were from the to for or were with to and cell proteins including showed neural progenitor cells to into cells J. Neurosci. PubMed Google Scholar, K. S. J. 2002; Scopus Google Scholar). that cells were to become neurons and but also cells. cells were also neural progenitor cells were cultured for in the presence of ET the have shown that Gαi2 and Gγ5 are expressed in where neurons are generated in the embryonic and postnatal brains of G proteins are in (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 the present we the role of Gi2 by injection of PTX into the lateral ventricle of developing mouse embryos and found that of the Gi2 pathway to decreased levels of proliferation of neuroepithelial cells in the ventricular zone. The injection of PTX effectively ADP-ribosylated the Gi/o in the brains of mouse This is in to the with adult rat brains in which most Gi/o proteins were ADP-ribosylated of limited of PTX into the brain from the ventricular cavity I. A. K. B.B. 1991; PubMed Scopus Google Scholar). injection of PTX into the ventricular cavity of embryos seems to be useful to the of Gi family G proteins in the developing brain. of Gi-coupled receptors for their effects on the incorporation of [3H]thymidine in cultured neural progenitor cells revealed ET to be the most of the agonists DNA synthesis was stimulated also by to but by including in with an (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). the for the is in the procedures for cell generation of have the were found to be potent from the cells of M. H. S. M. K. T. 1988; PubMed Scopus Google Scholar, A. M. S. T. K. T. Proc. Natl. Acad. Sci. U. S. A. 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The effects of on mitogenic activity of neural progenitor cells in the present study with this the early ERK in to potent effect on DNA to to maintain ERK phosphorylation at levels for a showed of signaling to ERK and DNA was shown to DNA synthesis in by the ERK pathway via G proteins, and DNA synthesis was whereas ERK was of suggesting at to DNA synthesis S. J. Neurosci. 1997; 17: PubMed Google Scholar). In contrast, receptor signaling to ERK is highly on cell in cells J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the the that ET increased cell numbers in the presence of to be as a cells in the presence of fibronectin, neural progenitor cells were cultured with ET for day and ET for in the presence or of is well known that neural progenitor cells can into neurons or cells and both and cells into both of of In that progenitor cells also into cells J. Neurosci. PubMed Google Scholar, K. S. J. 2002; Scopus Google Scholar). In the present we showed cells to into cells. was also in the presence of ET the suggesting that ET proliferation and into cells in the of neural progenitor cells. These findings that ET maintain progenitor cells in an In ET stimulate proliferation in to to that neural progenitor cells be in vitro by In the present we showed proliferation of neural progenitor cells to be stimulated by and FGF-2, by and receptor kinase, previous study that PTX of ERK and proliferation of suggesting of Gi in signals (8Fedorov Y.V. Jones N.C. Olwin B.B. Mol. Cell. Biol. 1998; 18: 5780-5787Crossref PubMed Scopus (49) Google Scholar). this was found to be the for neural progenitor PTX DNA proliferation and ERK In effects were by fibronectin, in with and proliferation of neural progenitor by signaling be embryonic