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Immunohistochemical localization of phospholipase D2 in embryonic rat brain
Neuroscience Letters 357 (2004) 147–151 www.elsevier.com/locate/neulet
Immunohistochemical localization of phospholipase D2 in embryonic rat brainq Jeong-Sun Choia, Hyun-Jung Parka, Young Chan Joa, Myung-Hoon Chuna, Jin-Woong Chunga, Joon Mo Kimb, Do Sik Minb,1, Mun-Yong Leea,1,* a
b
Department of Anatomy, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, 137-701 Seoul, South Korea Department of Physiology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, 137-701 Seoul, South Korea Received 10 October 2003; received in revised form 3 December 2003; accepted 3 December 2003
Abstract The present study has characterized the cellular and temporal localization of the phospholipase D2 (PLD2) protein in the embryonic rat brain, using immunohistochemistry. PLD2 immunoreactivity was first observed in the choroid plexus and in the most ventricular zone of the lateral and third ventricles at embryonic day 15 (E15), followed by gradual restriction to the limited zone of ventricles at E20. In addition, PLD2 expression was high in the developing cerebral cortex and hippocampus. In the cortex, PLD2 expression was observed in the marginal zone from the earliest stage (E15) and then declined and had completely disappeared by E20. Double-labelling studies demonstrated coexpression of the anti-class III b-tubulin antibody in most of the PLD2 immunoreactive cells. Therefore, our findings suggest that PLD2 may be involved in early developmental processes of some neuronal progenitors. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Phospholipase D2; Ventricular zone; Development; Differentiation; Immunohistochemistry
Phospholipase D (PLD), which produces the lipid signalling messenger phosphatidic acid by the hydrolysis of phosphatidylcholine, is activated by diverse extracellular stimuli such as growth factors, hormones and neurotransmitters (for reviews, refs. [4,8]). Two isozymes of mammalian PLD, PLD1 and PLD2, have been cloned and characterized [3,5]. Information about the potential role of PLD in the developing nervous system is growing. In situ hybridization studies have also demonstrated that the expression of PLD1 and PLD2 in the developing brain decreases with maturation [2,11]. In particular, transient in situ localization of PLD2 mRNA in the grey matter of the developing rat brain suggests that PLD2 may play an essential role in neuronal differentiation. To date, however, there has been no information regarding the distribution patterns of PLD2 protein in the developing brain. In the present study, immunohistochemical examination q This work was supported by the Basic Research Program of the Korea Science and Engineering Foundation (Grant Number: 2000-0-213-001-2). * Corresponding author. Tel.: þ 82-2-590-1108; fax: þ82-2-536-3110. E-mail address: [email protected] (M.Y. Lee). 1 These authors contributed equally to this study.
using monoclonal and polyclonal antibodies specific to PLD2 was performed to characterize the cellular and temporal localization of PLD2 protein in the embryonic brain, which was correlated to the TuJ1 (anti-class III btubulin antibody) specific staining of differentiating and postmitotic neurons during neurogenesis [9]. Pregnant Sprague– Dawley rats, with the exact date of conception known, were purchased from BioKorea (Osan, Korea). Twenty rats at different stages of development (embryonic days E15, E17, E18, E20; n ¼ 5 per group) were used. Embryonic pups were collected following caesarean sections; the mother rats were anaesthetized with 4% chloral hydrate (1 ml/100 mg body weight, intraperitoneally). The heads were immersed in fixative containing 4% paraformaldehyde buffered with 0.1 M phosphate buffer (PB, pH 7.2). Dissected brains were equilibrated with 30% sucrose in 0.1 M PB and frozen until required. All experimental procedures performed on the animals were conducted with the approval of the Ethics Committee of the Catholic University of Korea and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996).
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.054
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J.-S. Choi et al. / Neuroscience Letters 357 (2004) 147–151
Monoclonal or polyclonal antibodies specific to PLD2 were generated. For a polyclonal antibody, antisera were raised against the N-terminal peptide of PLD2 corresponding to amino acid residues 1– 19 of human PLD2 sequence (MTATPESLFPTGDELDSSQ) and affinity-purified using peptide-conjugated Affi-Gel 15 (BioRad, Hercules, CA, USA) as previously described using the New Zealand white rabbit [10]. The production of a monoclonal antibody specific to PLD2 was also completed as detailed previously [1]. Specificity of monoclonal and polyclonal antibodies was confirmed as described previously [1,10]. For PLD2 immunohistochemistry, free-floating cryostat sections (25 mm) were incubated overnight at 4 8C with a mouse monoclonal or a rabbit polyclonal antibody to PLD2. Primary antibody binding was visualized using a peroxidase-labelled donkey anti-mouse or peroxidase-labelled donkey anti-rabbit antibody (1:200, Jackson ImmunoResearch Laboratories, PA, USA), with 0.05% 3,30 -diamino-
benzidine tetrahydrochloride and H2O2 as substrate. The specificity of PLD2 immunoreactivity was described in a recent report [10] and was confirmed by the absence of immunohistochemical staining in sections from which the primary antibody was omitted or in which it was substituted with non-specific rabbit IgG. For double-immunofluorescence histochemistry, sections were incubated with a combination of a rabbit polyclonal antibody to PLD2 and a monoclonal anti-neuron specific class III b-tubulin TuJ1 (Sigma, MO, USA) overnight at 4 8C. The sections were then incubated for 2 h at room temperature with a mixture of Cy3-conjugated anti-rabbit IgG (Jackson; diluted at 1:150) and FITCconjugated anti-mouse IgG (Jackson; diluted at 1:150), respectively. Control sections were prepared as described above. Slides were viewed using a confocal microscope (MRC-1024, Bio-Rad). Images were converted to TIFF format and contrast levels of images were adjusted using
Fig. 1. PLD2 immunoreactivity in E15 (A –C) and E17 (D –J) brain. (A –C) E15 forebrain. Specific immunoreactivity for PLD2 using affinity-purified antiPLD2 antibody was observed in the ventricular zone of the lateral ventricle (LV) and of the third ventricle (3V), the marginal zone (MZ) in the cerebral cortex and the choroid plexus (chp; arrowheads in C). (B, C) Higher magnification views of the boxed areas in A. (D –J) E17 forebrain. PLD2 immunoreactivity was observed in a restricted zone of the lateral ventricle (D) and the hippocampus (F). (E, G) Higher magnification views of the boxed areas in D and F, respectively. (H) E17 diencephalon. (I) Higher magnification view of the boxed area in H. (J) E17 brainstem. ah, Ammon’s horn primordium; Aq, the cerebral aqueduct; CP, cortical plate; dg, dentate gyrus primordium; hi, hippocampal neuroepithelium; IZ, intermediate zone; SP, subplate; and su, subiculum. Scale bars ¼ 300 mm for A, D, H, J; 100 mm for C, F, I; 50 mm for E, G; 25 mm for B.
J.-S. Choi et al. / Neuroscience Letters 357 (2004) 147–151
Adobe Photoshop v. 6.0 (Adobe Systems Inc., San Jose, CA, USA). Immunohistochemical experiments on tissue sections from embryonic rat brains localized specific PLD2 signals in distinct regions at defined embryonic stages. Both polyclonal and monoclonal antibodies specific to PLD2 showed similar immunohistochemical features. The results reported below are based on experiments employing affinity-purified anti-PLD2 polyclonal antibody. At the earliest stage studied (E15), PLD2 immunoreactive cells appeared along most of the ventricular zone of the lateral and the third ventricles, but clusters of immunoreactive cells were observed in the ventricular zone of the medial wall of the lateral ventricle, the area destined to form the hippocampus (Fig. 1A). In addition, PLD2 expression was observed in tangentially oriented cells in the marginal zone of the cerebral cortex (Fig. 1B) and
149
within a limited epithelium of the choroid plexus (Fig. 1C). At E17, PLD2 immunoreactive cells, grouped in discrete patches, were present in the ventricular zone of the lateral ventricles (Fig. 1D). In the cortex, intense immunoreactivity was present both in the subplate and in the marginal zone, which contained horizontal neurons that appeared to be actively migrating, and occasionally in the cortical plate (Fig. 1E). Scattered immunoreactive cells were observed in the developing hippocampus (Figs. 1F,G). In addition, intense immunoreactivity was observed in the somata located in the ventricular zone of the dorsal part of the third ventricle, with radially oriented processes, and in clusters of cells in successive migrating waves from the ventricular zone (Figs. 1H,I). More caudally, the expression of PLD2 was observed within the limited ventricular zone of the cerebral aqueduct and in actively migrating cells in the developing brainstem (Fig. 1J).
Fig. 2. PLD2 immunoreactivity in E18 (A–G) and E20 (H –J) brain. (A–G) E18 forebrain. PLD2 immunoreactivity using affinity-purified anti-PLD2 antibody was observed within a limited zone of the ventricular zone of the lateral ventricle (LV in A), developing cortex (B) and hippocampus (C, D). (D) Higher magnification view of the boxed area in C. (E) E18 diencephalon. (F) Higher magnification view of the boxed area in E. (G) E18 brainstem. (H –J) E20 brain. (I) Higher magnification view of the boxed areas in H. Arrows in H indicate the border between the Ammon’s horn and the dentate gyrus (DG). ah, Ammon’s horn primordium; Aq, the cerebral aqueduct; CP, cortical plate; dg, dentate gyrus primordium; D3V, dorsal third ventricle; SP, subplate; and su, subiculum. Scale bars ¼ 400 mm for E; 300 mm for A; 200 mm for G, H, J; 100 mm for C, F; 50 mm for B, D, I.
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In the E18 brain, PLD2 was expressed within the more limited ventricular zone of the lateral ventricle (Fig. 2A). In addition, the number of PLD2 immunoreactive cells in the cerebral cortex decreased significantly and only a few cells in the marginal zone were immunoreactive for PLD2 (Fig. 2B). In the hippocampus, PLD2 immunoreactivity was observed in randomly oriented cells in the developing dentate gyrus and the Ammon’s horn (Figs. 2C,D). The labelled cells were also observed within the limited ventricular zone of the third ventricle and the cerebral aqueduct (Figs. 2E –G). At E20, very faint staining appeared within the ventricular zone of the lateral and third ventricles and in the cerebral cortex (data not shown). In the hippocampus, most of the labelled cells were located in the subiculum and in the CA1 bordering the dentate gyrus, but were rare in the dentate gyrus (Figs. 2H,I). By this stage, the intensity of the PLD2 immunoreactivity had been reduced in the cerebral aqueduct, but the labelling pattern was maintained (Fig. 2J). The phenotype of PLD2-expressing cells was investigated by co-labelling with PLD2 and TuJ1 antibodies. Most PLD2 immunoreactive cells in the developing cerebral cortex were also immunoreactive for TuJ1 (Figs. 3A – C). In addition, the double labelling of PLD2 and TuJ1 was observed in somata within the limited ventricular zone of the lateral ventricle with processes extending radially (Figs. 3D – F). These immunohistochemical data are the first to show the developmental regulation and distribution of PLD2 protein in the embryonic rat brain. The distinct and selective staining of well-defined cellular populations seen in the developing rat brain indicates that the affinity-purified antibody to PLD2 used in this study
Fig. 3. Confocal laser microscopic imaging of immunofluorescence for PLD2 (A, D) and TuJ1 (B, E) in the developing cortical plate (A– C) and in the ventricular zone of the lateral ventricle (D –F) in E17 brain. (C, F) Superimposed images of Cy3 (A, D) and FITC (B, E). Note that PLD2 immunoreactive cells that were located in the subplate (SP) and in the marginal zone (MZ) of the developing cerebral cortex, and in the ventricular zone of the lateral ventricle (LV) were co-labelled with TuJ1. CP, cortical plate; and IZ, intermediate zone. Scale bar ¼ 50 mm for A–F.
specifically detected PLD2 under the conditions used. Moreover, we have found that the affinity-purified antibody to PLD2 recognized specifically rat PLD2 by Western blot and immunofluorescent analysis [10]. PLD2 expression appeared in epithelial cells of the choroid plexus and along most of the ventricular zone of the lateral and third ventricles as early as E15, followed by gradual restriction to the limited zone of ventricles by E20. These results agree closely with the in situ hybridization studies of Colley et al. [2] and Saito et al. [11]. However, PLD2 immunoreactivity is not generally associated with the proliferation of neuronal progenitors, as the labelling has been observed only in the selected ventricular zone. The functional significance of the transient immunolabelling in this ventricular zone remains speculative, but the finding does suggest that PLD2 may perform specific roles in the proliferation of selected neuronal progenitors in the embryonic brain. Jang et al. [6] reported recently that the direct interaction of phospholipase C-g 1 with phospholipase D2 is important for epidermal growth factor (EGF) signalling. The EGF receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C-g 1 is regarded as critical for EGF-induced mitogenesis. Moreover, PLD2 is strongly tyrosine phosphorylated by c-Src and stimulates c-Src kinase activity, thereby inducing cell proliferation [1]. These findings suggest that PLD2 plays a role in neuronal proliferation. Another significant issue is that distinct populations of differentiating neurons expressed substantial PLD2 immunoreactivity during embryonic development. The morphology and distribution pattern of PLD2 immunoreactive cells suggest that many of them could be migrating neuroblasts. The co-expression of TuJ1, a differentiating and postmitotic neuronal marker, in most of the PLD2 immunoreactive cells supports this hypothesis. Considering that over-expression of PLD2 enhances cell protrusion, which is implicated in cell motility, and that this phenomenon is dependent on both PLD2 and v-Src [7,12], PLD2-mediated signalling pathways may play a role in neuronal migration and differentiation. Our findings provide evidence that PLD2 is expressed in selected ventricular zones and in cells that appear to be migrating cells in the embryonic rat brain, and that most of the PLD1 immunoreactive cells are also immunoreactive for TuJ1. These results suggest that PLD2 may be involved in early developmental processes such as the proliferation, migration and differentiation of some neuronal populations.
Acknowledgements The authors thank Hee-Duck Rho and Byoung-Ouk Hong for their excellent technical assistance.
J.-S. Choi et al. / Neuroscience Letters 357 (2004) 147–151
References [1] B.H. Ahn, S.Y. Kim, E.H. Kim, K.S. Choi, T.K. Kwon, Y.H. Lee, J.S. Chang, M.S. Kim, Y.H. Jo, D.S. Min, Transmodulation between phospholipase D and c-Src enhances cell proliferation, Mol. Cell. Biol. 23 (2003) 3103–3115. [2] W.C. Colley, Y.M. Altshuller, C.K. Sue-Ling, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, K.D. Branch, S.E. Tsirka, R.J. Bollag, W.B. Bollag, M.A. Frohman, Cloning and expression analysis of murine phospholipase D1, Biochem. J. 326 (1997) 745– 753. [3] J.H. Exton, Regulation of phospholipase D, Biochim. Biophys. Acta 1439 (1999) 121 –133. [4] J.H. Exton, Phospholipase D – structure, regulation, and function, Rev. Physiol. Biochem. Pharmacol. 144 (2002) 1–94. [5] M.A. Frohman, A.J. Morris, Phospholipase D structure and regulation, Chem. Phys. Lipids 98 (1999) 127–140. [6] I.H. Jang, S. Lee, J.B. Park, J.H. Kim, C.S. Lee, E.M. Hur, I.S. Kim, K.T. Kim, H. Yagisawa, P.G. Suh, S.H. Ryu, The direct interaction of phospholipase C-1 with phospholipase D2 is important for epidermal growth factor signaling, J. Biol. Chem. 278 (2003) 18184–18190.
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[7] D.A. Lauffenburger, A.F. Horwitz, Cell migration: a physically integrated molecular process, Cell 84 (1996) 359–369. [8] M. Liscovitch, M. Czarny, G. Fiucci, X. Tang, Phospholipase D: molecular and cell biology of a novel gene family, Biochem. J. 345 (2000) 401–415. [9] J.R. Menezes, M.B. Luskin, Expression of neuron-specific tubulin defines a novel population in the proliferative layers of the developing telencephalon, J. Neurosci. 14 (1994) 5399–5416. [10] G.R. Ryu, M.J. Kim, C.H. Song, S.S. Sim, D.S. Min, D.J. Rhie, S.H. Yoon, S.J. Hahn, M.S. Kim, Y.H. Jo, Site-specific distribution of phospholipase D isoforms in the rat pancreas, Acta Histochem. Cytochem. 36 (2003) 51–60. [11] S. Saito, H. Sakagami, H. Kondo, Localization of mRNAs for phospholipase D (PLD) type 1 and 2 in the brain of developing and mature brain, Dev. Brain Res. 120 (2000) 41 –47. [12] Y. Shen, Y. Zheng, D.A. Foster, Phospholipase D2 stimulates cell protrusion in v-Src-transformed cells, Biochim. Biophys. Res. Commun. 293 (2002) 201 –206.
Immunohistochemical localization of phospholipase D2 in embryonic rat brainq Jeong-Sun Choia, Hyun-Jung Parka, Young Chan Joa, Myung-Hoon Chuna, Jin-Woong Chunga, Joon Mo Kimb, Do Sik Minb,1, Mun-Yong Leea,1,* a
b
Department of Anatomy, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, 137-701 Seoul, South Korea Department of Physiology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, 137-701 Seoul, South Korea Received 10 October 2003; received in revised form 3 December 2003; accepted 3 December 2003
Abstract The present study has characterized the cellular and temporal localization of the phospholipase D2 (PLD2) protein in the embryonic rat brain, using immunohistochemistry. PLD2 immunoreactivity was first observed in the choroid plexus and in the most ventricular zone of the lateral and third ventricles at embryonic day 15 (E15), followed by gradual restriction to the limited zone of ventricles at E20. In addition, PLD2 expression was high in the developing cerebral cortex and hippocampus. In the cortex, PLD2 expression was observed in the marginal zone from the earliest stage (E15) and then declined and had completely disappeared by E20. Double-labelling studies demonstrated coexpression of the anti-class III b-tubulin antibody in most of the PLD2 immunoreactive cells. Therefore, our findings suggest that PLD2 may be involved in early developmental processes of some neuronal progenitors. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Phospholipase D2; Ventricular zone; Development; Differentiation; Immunohistochemistry
Phospholipase D (PLD), which produces the lipid signalling messenger phosphatidic acid by the hydrolysis of phosphatidylcholine, is activated by diverse extracellular stimuli such as growth factors, hormones and neurotransmitters (for reviews, refs. [4,8]). Two isozymes of mammalian PLD, PLD1 and PLD2, have been cloned and characterized [3,5]. Information about the potential role of PLD in the developing nervous system is growing. In situ hybridization studies have also demonstrated that the expression of PLD1 and PLD2 in the developing brain decreases with maturation [2,11]. In particular, transient in situ localization of PLD2 mRNA in the grey matter of the developing rat brain suggests that PLD2 may play an essential role in neuronal differentiation. To date, however, there has been no information regarding the distribution patterns of PLD2 protein in the developing brain. In the present study, immunohistochemical examination q This work was supported by the Basic Research Program of the Korea Science and Engineering Foundation (Grant Number: 2000-0-213-001-2). * Corresponding author. Tel.: þ 82-2-590-1108; fax: þ82-2-536-3110. E-mail address: [email protected] (M.Y. Lee). 1 These authors contributed equally to this study.
using monoclonal and polyclonal antibodies specific to PLD2 was performed to characterize the cellular and temporal localization of PLD2 protein in the embryonic brain, which was correlated to the TuJ1 (anti-class III btubulin antibody) specific staining of differentiating and postmitotic neurons during neurogenesis [9]. Pregnant Sprague– Dawley rats, with the exact date of conception known, were purchased from BioKorea (Osan, Korea). Twenty rats at different stages of development (embryonic days E15, E17, E18, E20; n ¼ 5 per group) were used. Embryonic pups were collected following caesarean sections; the mother rats were anaesthetized with 4% chloral hydrate (1 ml/100 mg body weight, intraperitoneally). The heads were immersed in fixative containing 4% paraformaldehyde buffered with 0.1 M phosphate buffer (PB, pH 7.2). Dissected brains were equilibrated with 30% sucrose in 0.1 M PB and frozen until required. All experimental procedures performed on the animals were conducted with the approval of the Ethics Committee of the Catholic University of Korea and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996).
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.054
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J.-S. Choi et al. / Neuroscience Letters 357 (2004) 147–151
Monoclonal or polyclonal antibodies specific to PLD2 were generated. For a polyclonal antibody, antisera were raised against the N-terminal peptide of PLD2 corresponding to amino acid residues 1– 19 of human PLD2 sequence (MTATPESLFPTGDELDSSQ) and affinity-purified using peptide-conjugated Affi-Gel 15 (BioRad, Hercules, CA, USA) as previously described using the New Zealand white rabbit [10]. The production of a monoclonal antibody specific to PLD2 was also completed as detailed previously [1]. Specificity of monoclonal and polyclonal antibodies was confirmed as described previously [1,10]. For PLD2 immunohistochemistry, free-floating cryostat sections (25 mm) were incubated overnight at 4 8C with a mouse monoclonal or a rabbit polyclonal antibody to PLD2. Primary antibody binding was visualized using a peroxidase-labelled donkey anti-mouse or peroxidase-labelled donkey anti-rabbit antibody (1:200, Jackson ImmunoResearch Laboratories, PA, USA), with 0.05% 3,30 -diamino-
benzidine tetrahydrochloride and H2O2 as substrate. The specificity of PLD2 immunoreactivity was described in a recent report [10] and was confirmed by the absence of immunohistochemical staining in sections from which the primary antibody was omitted or in which it was substituted with non-specific rabbit IgG. For double-immunofluorescence histochemistry, sections were incubated with a combination of a rabbit polyclonal antibody to PLD2 and a monoclonal anti-neuron specific class III b-tubulin TuJ1 (Sigma, MO, USA) overnight at 4 8C. The sections were then incubated for 2 h at room temperature with a mixture of Cy3-conjugated anti-rabbit IgG (Jackson; diluted at 1:150) and FITCconjugated anti-mouse IgG (Jackson; diluted at 1:150), respectively. Control sections were prepared as described above. Slides were viewed using a confocal microscope (MRC-1024, Bio-Rad). Images were converted to TIFF format and contrast levels of images were adjusted using
Fig. 1. PLD2 immunoreactivity in E15 (A –C) and E17 (D –J) brain. (A –C) E15 forebrain. Specific immunoreactivity for PLD2 using affinity-purified antiPLD2 antibody was observed in the ventricular zone of the lateral ventricle (LV) and of the third ventricle (3V), the marginal zone (MZ) in the cerebral cortex and the choroid plexus (chp; arrowheads in C). (B, C) Higher magnification views of the boxed areas in A. (D –J) E17 forebrain. PLD2 immunoreactivity was observed in a restricted zone of the lateral ventricle (D) and the hippocampus (F). (E, G) Higher magnification views of the boxed areas in D and F, respectively. (H) E17 diencephalon. (I) Higher magnification view of the boxed area in H. (J) E17 brainstem. ah, Ammon’s horn primordium; Aq, the cerebral aqueduct; CP, cortical plate; dg, dentate gyrus primordium; hi, hippocampal neuroepithelium; IZ, intermediate zone; SP, subplate; and su, subiculum. Scale bars ¼ 300 mm for A, D, H, J; 100 mm for C, F, I; 50 mm for E, G; 25 mm for B.
J.-S. Choi et al. / Neuroscience Letters 357 (2004) 147–151
Adobe Photoshop v. 6.0 (Adobe Systems Inc., San Jose, CA, USA). Immunohistochemical experiments on tissue sections from embryonic rat brains localized specific PLD2 signals in distinct regions at defined embryonic stages. Both polyclonal and monoclonal antibodies specific to PLD2 showed similar immunohistochemical features. The results reported below are based on experiments employing affinity-purified anti-PLD2 polyclonal antibody. At the earliest stage studied (E15), PLD2 immunoreactive cells appeared along most of the ventricular zone of the lateral and the third ventricles, but clusters of immunoreactive cells were observed in the ventricular zone of the medial wall of the lateral ventricle, the area destined to form the hippocampus (Fig. 1A). In addition, PLD2 expression was observed in tangentially oriented cells in the marginal zone of the cerebral cortex (Fig. 1B) and
149
within a limited epithelium of the choroid plexus (Fig. 1C). At E17, PLD2 immunoreactive cells, grouped in discrete patches, were present in the ventricular zone of the lateral ventricles (Fig. 1D). In the cortex, intense immunoreactivity was present both in the subplate and in the marginal zone, which contained horizontal neurons that appeared to be actively migrating, and occasionally in the cortical plate (Fig. 1E). Scattered immunoreactive cells were observed in the developing hippocampus (Figs. 1F,G). In addition, intense immunoreactivity was observed in the somata located in the ventricular zone of the dorsal part of the third ventricle, with radially oriented processes, and in clusters of cells in successive migrating waves from the ventricular zone (Figs. 1H,I). More caudally, the expression of PLD2 was observed within the limited ventricular zone of the cerebral aqueduct and in actively migrating cells in the developing brainstem (Fig. 1J).
Fig. 2. PLD2 immunoreactivity in E18 (A–G) and E20 (H –J) brain. (A–G) E18 forebrain. PLD2 immunoreactivity using affinity-purified anti-PLD2 antibody was observed within a limited zone of the ventricular zone of the lateral ventricle (LV in A), developing cortex (B) and hippocampus (C, D). (D) Higher magnification view of the boxed area in C. (E) E18 diencephalon. (F) Higher magnification view of the boxed area in E. (G) E18 brainstem. (H –J) E20 brain. (I) Higher magnification view of the boxed areas in H. Arrows in H indicate the border between the Ammon’s horn and the dentate gyrus (DG). ah, Ammon’s horn primordium; Aq, the cerebral aqueduct; CP, cortical plate; dg, dentate gyrus primordium; D3V, dorsal third ventricle; SP, subplate; and su, subiculum. Scale bars ¼ 400 mm for E; 300 mm for A; 200 mm for G, H, J; 100 mm for C, F; 50 mm for B, D, I.
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In the E18 brain, PLD2 was expressed within the more limited ventricular zone of the lateral ventricle (Fig. 2A). In addition, the number of PLD2 immunoreactive cells in the cerebral cortex decreased significantly and only a few cells in the marginal zone were immunoreactive for PLD2 (Fig. 2B). In the hippocampus, PLD2 immunoreactivity was observed in randomly oriented cells in the developing dentate gyrus and the Ammon’s horn (Figs. 2C,D). The labelled cells were also observed within the limited ventricular zone of the third ventricle and the cerebral aqueduct (Figs. 2E –G). At E20, very faint staining appeared within the ventricular zone of the lateral and third ventricles and in the cerebral cortex (data not shown). In the hippocampus, most of the labelled cells were located in the subiculum and in the CA1 bordering the dentate gyrus, but were rare in the dentate gyrus (Figs. 2H,I). By this stage, the intensity of the PLD2 immunoreactivity had been reduced in the cerebral aqueduct, but the labelling pattern was maintained (Fig. 2J). The phenotype of PLD2-expressing cells was investigated by co-labelling with PLD2 and TuJ1 antibodies. Most PLD2 immunoreactive cells in the developing cerebral cortex were also immunoreactive for TuJ1 (Figs. 3A – C). In addition, the double labelling of PLD2 and TuJ1 was observed in somata within the limited ventricular zone of the lateral ventricle with processes extending radially (Figs. 3D – F). These immunohistochemical data are the first to show the developmental regulation and distribution of PLD2 protein in the embryonic rat brain. The distinct and selective staining of well-defined cellular populations seen in the developing rat brain indicates that the affinity-purified antibody to PLD2 used in this study
Fig. 3. Confocal laser microscopic imaging of immunofluorescence for PLD2 (A, D) and TuJ1 (B, E) in the developing cortical plate (A– C) and in the ventricular zone of the lateral ventricle (D –F) in E17 brain. (C, F) Superimposed images of Cy3 (A, D) and FITC (B, E). Note that PLD2 immunoreactive cells that were located in the subplate (SP) and in the marginal zone (MZ) of the developing cerebral cortex, and in the ventricular zone of the lateral ventricle (LV) were co-labelled with TuJ1. CP, cortical plate; and IZ, intermediate zone. Scale bar ¼ 50 mm for A–F.
specifically detected PLD2 under the conditions used. Moreover, we have found that the affinity-purified antibody to PLD2 recognized specifically rat PLD2 by Western blot and immunofluorescent analysis [10]. PLD2 expression appeared in epithelial cells of the choroid plexus and along most of the ventricular zone of the lateral and third ventricles as early as E15, followed by gradual restriction to the limited zone of ventricles by E20. These results agree closely with the in situ hybridization studies of Colley et al. [2] and Saito et al. [11]. However, PLD2 immunoreactivity is not generally associated with the proliferation of neuronal progenitors, as the labelling has been observed only in the selected ventricular zone. The functional significance of the transient immunolabelling in this ventricular zone remains speculative, but the finding does suggest that PLD2 may perform specific roles in the proliferation of selected neuronal progenitors in the embryonic brain. Jang et al. [6] reported recently that the direct interaction of phospholipase C-g 1 with phospholipase D2 is important for epidermal growth factor (EGF) signalling. The EGF receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C-g 1 is regarded as critical for EGF-induced mitogenesis. Moreover, PLD2 is strongly tyrosine phosphorylated by c-Src and stimulates c-Src kinase activity, thereby inducing cell proliferation [1]. These findings suggest that PLD2 plays a role in neuronal proliferation. Another significant issue is that distinct populations of differentiating neurons expressed substantial PLD2 immunoreactivity during embryonic development. The morphology and distribution pattern of PLD2 immunoreactive cells suggest that many of them could be migrating neuroblasts. The co-expression of TuJ1, a differentiating and postmitotic neuronal marker, in most of the PLD2 immunoreactive cells supports this hypothesis. Considering that over-expression of PLD2 enhances cell protrusion, which is implicated in cell motility, and that this phenomenon is dependent on both PLD2 and v-Src [7,12], PLD2-mediated signalling pathways may play a role in neuronal migration and differentiation. Our findings provide evidence that PLD2 is expressed in selected ventricular zones and in cells that appear to be migrating cells in the embryonic rat brain, and that most of the PLD1 immunoreactive cells are also immunoreactive for TuJ1. These results suggest that PLD2 may be involved in early developmental processes such as the proliferation, migration and differentiation of some neuronal populations.
Acknowledgements The authors thank Hee-Duck Rho and Byoung-Ouk Hong for their excellent technical assistance.
J.-S. Choi et al. / Neuroscience Letters 357 (2004) 147–151
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