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Expression of the Gap Junction Protein Connexin43 in Human Telencephalon Microvessels
Microvascular Research 62, 435– 439 (2001) doi:10.1006/mvre.2001.2345, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Expression of the Gap Junction Protein Connexin43 in Human Telencephalon Microvessels Daniela Virgintino,* ,1 David Robertson,† Mariella Errede,* Vincenzo Benagiano,* Mirella Bertossi,‡ Glauco Ambrosi,* and Luisa Roncali* *Department of Human Anatomy and Histology, University of Bari School of Medicine, I-70124 Bari, Italy; †Institute of Cancer Research, Haddow Laboratories, SM2 5NG, Sutton, United Kingdom; and ‡University of Foggia School of Medicine, Foggia, Italy Received March 22, 2001; published online August 6, 2001
Key Words: connexin43; gap junctions; microvessels; endothelium; human telencephalon; immunoconfocal microscopy.
INTRODUCTION The gap junction protein connexin43 (Cx43) is believed specific to interastrocytic gap junctions in the adult brain and is expressed by cells of the astroglial lineage in the developing brain (Dermietzel and Spray, 1993; Nadarajah et al., 1997; Nagy and Rash, 2000). The presence of Cx43 as well as Cx37 and Cx40 has also been demonstrated in the blood vessels of different organs and, in particular, in the endothelial cells, where the expression of these connexins seems to be species, organ, and vessel specific (Little et al., 1995; Hillis et al., 1997; Janssen-Bienhold et al., 1998; Ko et al., 1 To whom correspondence should be addressed at the Department of Human Anatomy and Histology, University of Bari School of Medicine, Piazza Giulio Cesare, I-70124 Bari, Italy. Fax: ⫹39 080 5478309. E-mail: [email protected].
0026-2862/⫺1900 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
1999; van Kempen and Jongsma, 1999). So far, there are no data on Cx43 expression in human brain microvessels during prenatal development, when the endothelial cells acquire morphological and biochemical barrier features (blood– brain barrier). Here we present findings on the expression of Cx43, investigated by confocal microscopy, in the human fetus telencephalon at 18 weeks of gestation, a developmental age characterized by significant progression of vessel growth and differentiation (Bertossi et al., 1999; Virgintino et al., 2000).
MATERIAL AND METHODS Four fetuses, at 18 weeks of gestation, were obtained after spontaneous abortion under informed consent and approval by the local ethics committee. The gestation age was estimated on the basis of the crown– rump length and/or pregnancy records of the gestational age. The telencephalon dorsolateral wall was dissected, immersed in the Bouin’s fixative for 2 h at 4°C, and embedded in paraffin wax. Five-micrometer
435
436
sections were cut perpendicular to the telencephalon surface and collected on Vectabond (Vector Laboratories)-treated slides.
Cx43 Immunohistochemistry The immunolabeling for Cx43 was preceded by a heat-mediated antigen retrieval by microwaving the sections immersed in 0.01 M citrate buffer (pH 6.0) for 10 min at 750 W. Sections were then incubated with (1) blocking buffer (phosphate-buffered saline (PBS), 0.8% BSA, 5% FCS) for 30 min at room temperature (RT), (2) mouse monoclonal antibody anti-connexin43 (diluted 1:200; Zymed Laboratories) overnight at 4°C, (3) horse antibody anti-mouse IgG (diluted 10 g/ml; Vector Laboratories) for 1 h at RT, and (4) fluorescein isothiocyanate-labeled avidin D (diluted 30 g/ml; Vector Laboratories) for 1 h at RT. The sections were washed 10 min ⫻ 3 in PBS between each step and finally drained and mounted in Vectashield (Vector Laboratories). Control sections of human fetal telencephalon, submitted to the same immunohistochemical procedure with the primary antibody being either omitted or preadsorbed with an excess (20 nmol ml ⫺1) of the pure antigen (Zymed Laboratories), showed no immunolabeling.
Confocal Laser Scanning Microscopy The sections were viewed under a confocal laser scanning microscope (Leica TCS SP) using 20⫻ and 63⫻ objective lenses. Optical sections were taken at 200-nm intervals through their z axis covering in total 5 m in depth and digitally recorded as single sections and as projection images assembled from a series of optical sections. Images were stored as TIFF files and analyzed by Adobe PhotoShop (Adobe Systems, CA).
RESULTS At 18 weeks the telencephalon is crossed by bundles of Cx43-immunofluorescent fibers of the radial glia that extend as far as the pia between rows of unlabeled neuroblasts. The radial glial fibers are distinctly re-
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
vealed by a fine pattern of labeled puncta, either isolated or, more frequently, aligned in short chains (Figs. 1A–1D). The vascular network is formed by immunofluorescent radially oriented large microvessels and their small, orthogonal and oblique, collaterals. In the thin wall of the radial vessels, the endothelial cells show a punctate Cx43 labeling; chains of puncta decorate the endothelial basal profile, which is encircled by unlabeled pericytes (Figs. 1A and 1B). Cx43-immunoreactive strands, formed by fused puncta, mark discrete regions of the endothelial layer corresponding to lateral contacts between adjoining endothelial cells (Figs. 1A and 1B). In the small collaterals of the radial vessels, fluorescent puncta diffusely label the endothelial cells and decorate their luminal and basal profiles (Figs. 1C–1F). Many Cx43-immunoreactive glial fibers are seen adjacent to the microvessel walls and some of them make contact with the endothelial lining (Figs. 1C and 1D).
DISCUSSION The results confirm that confocal laser scanning microscopy greatly improves the analysis of immunofluorescent structures on thick paraffin sections; on both single optical plane and projection images of the 18week human telencephalon, Cx43 is revealed at the glial and vascular compartments as a punctate labeling, and this is characteristic of gap junctions. The detection of Cx43-labeled radial glia close to unlabeled neuroblasts supports the view that communication via heterotypic gap junctions controls neuronal migration along the radial fibers in the developing brain (Nadarajah et al., 1997). As far as the microvascular compartment is concerned, radial vessels, which first penetrate the telencephalon from 8 weeks of gestation, and their collaterals, which develop at 18 weeks (Norman and O’Kusky, 1986), show a Cx43 punctate labeling in the endothelial cells. This indicates that the interendothelial communication via homotypic gap junctions is active in the growing microvessels of the 18-week human telencephalon. Furthermore, there are noticeable differences in Cx43 expression between the main trunks and the collaterals of the radial vessels. In
Brief Communication
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FIG. 1. Cx43 expression by confocal microscopy in a radial vessel (A, B) and collaterals of radial vessels (C–F) of 18-week telencephalon. In the single optical section (A) and projection image (B) isolated immunofluorescent puncta are detectable in endothelial cells, and chains of puncta label their basal profile (arrowheads), which is paralleled by the pericyte layer (p). Immunofluorescent strands are seen in discrete endothelial regions (arrows), corresponding to lateral contacts between adjoining cells. Inset: A fluorescent spot between an endothelial cell (e) and a pericyte. Single image (C) and projection image (D) of a collateral vessel crossed by radial glia fibers (f). The endothelial cells show a diffuse, punctate immunofluorescence and isolated or packed puncta are aligned along the endothelial luminal and basal profiles (arrowheads). The radial glial fibers are also revealed by a fine, punctate immunofluorescence; glial fibers adhere to the endothelial cells (arrows) and to an unlabeled neuroblast (n). Single image (E) and projection image (F) of tangentially cut collaterals showing the Cx43 punctate pattern on the endothelial basal front. Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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the collaterals, Cx43 punctate labeling is detectable everywhere in the endothelial cells, whereas in the radial vessels the labeling is mainly restricted to discrete regions corresponding to interendothelial contacts. It is well known that the adult brain endothelial cells lack gap junctions and are sealed by extensive tight junctions, which are part of the blood– brain barrier (Dermietzel, 1975; Nagy et al., 1984). It has also been demonstrated that transient gap junctions are present between immature brain endothelial cells and disappear as their differentiation progresses (Delorme et al., 1970; Wijsman and Shivers, 1998). Compared with the collaterals the localized expression of Cx43 in the endothelial cells of the radial vessels indicates an advanced degree of endothelial differentiation. Demonstration that endothelial differentiation of the radial vessel main trunks is under way in the 18-week human telencephalon has been given by studies showing the presence of some blood– brain barrier devices at this stage, such as tight interendothelial junctions, albumin barrier, and glucose transporter expression (Bertossi et al., 1999; Virgintino et al., 2000). The Cx43 immunofluorescent puncta on the basal endothelial front overlying unlabeled pericytes could correspond to heterotypic gap junctions between endothelial cells and pericytes. Endothelium–pericyte gap junctions are present in the developing brain microvessels and persist in the mature ones, being involved respectively in the regulation of vessel growth and functioning (Cuevas et al., 1984; Shepro and Morel, 1993; Bertossi et al., 1995; Fujimoto, 1995; Balabanov and Dore-Duffy, 1998). The detection of Cx43 radial glial fibers in close contact with labeled microvessels suggests the existence of gap junctions also between perivascular processes of astroglial cells and vascular cells. Though never before documented in situ at the histological level, the existence of this coupling has been evidenced by studies carried out in a blood– brain barrier culture model that shows passage of waves of Ca 2⫹ ions from endothelial cells to astrocytes and vice versa (Leybaert et al., 1998). During brain angiogenesis, this coupling might be responsible for the role played by astroglial cells in endothelial cell differentiation (Holash et al., 1993; Bauer and Bauer, 2000). In conclusion, the research gives the first demonstration of Cx43 expression during human telencephalon
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
angiogenesis. The expression of the same connexin in endothelial cells and perivascular astroglial cells suggests that these cell types communicate and are mutually influenced by direct signaling via homotypic gap junctions.
ACKNOWLEDGEMENT This work was supported in part by Grant 98-505-04, 1998 (to L.R.), from the Consiglio Nazionale delle Ricerche (CNR), Italy.
REFERENCES Balabanov, R., and Dore-Duffy, P. (1998). Role of the CNS microvascular pericyte in the blood– brain barrier. J. Neurosci. Res. 53, 637– 644. Bauer, H.-C., and Bauer, H. (2000). Neural induction of the blood– brain barrier: Still an enigma. Cell. Mol. Neurobiol. 20, 13–28. Bertossi, M., Riva, A., Congiu, T., Virgintino, D., Nico, B., and Roncali, L. (1995). A compared TEM/SEM investigation on the pericytic investment in developing microvasculature of the chick optic tectum. J. Submicrosc. Cytol. Pathol. 27, 349 –358. Bertossi, M., Virgintino, D., Errede, M., and Roncali, L. (1999). Immunohistochemical and ultrastructural characterization of cortical plate microvasculature in the human fetus telencephalon. Microvasc. Res. 58, 49 – 61. Cuevas, P., Gutierrez-Diaz, J. A., Reimers, D., Dujovny, M., Diaz, F. G., and Ausman, J. I. (1984). Pericyte endothelial gap junctions in human cerebral capillaries. Anat. Embryol. 170, 155–159. Delorme, P., Gayet, J., and Grignon, G. (1970). Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken. Brain Res. 22, 269 –283. Dermietzel, R. (1975). Junctions in the central nervous system of the cat. IV. Interendothelial junctions of cerebral blood vessels from selected areas of the brain. Cell Tissue Res. 164, 45– 62. Dermietzel, R., and Spray, D. C. (1993). Gap junctions in the brain: Where, what type, how many and why? Trends Neurosci. 16, 186 –192. Fujimoto, K. (1995). Pericyte– endothelial gap junctions in developing rat cerebral capillaries: A fine structural study. Anat. Rec. 242, 562–565. Hillis, G. S., Duthie, L. A., Mlynski, R., McKay, N. G., Mistry, S., MacLeod, A. M., Simpson, J. G., and Haites, N. E. (1997). The expression of connexin 43 in human kidney and cultured renal cells. Nephron 75, 458 – 463. Holash, J. A., Noden, D. M., and Stewart, P. A. (1993). Re-evaluating the role of astrocytes in blood– brain barrier induction. Dev. Dyn. 197, 14 –25.
Brief Communication
Janssen-Bienhold, U., Dermietzel, R., and Weiler, R. (1998). Distribution of connexin43 immunoreactivity in the retinas of different vertebrates. J. Comp. Neurol. 396, 310 –321. Ko, Y.-S., Yeh, H.-I., Rothery, S., Dupont, E., Coppen, S. R., and Severs, N. J. (1999). Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy. J. Histochem. Cytochem. 47, 683– 692. Leybaert, L., Paemeleire, K., Strahonja, A., and Sanderson, M. J. (1998). Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells. Glia 24, 398 – 407. Little, T. L., Beyer, E. C., and Duling, B. R. (1995). Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am. J. Physiol. 268, 729 –739. Nadarajah, B., Jones, A. M., Evans, W. H., and Parnavelas, J. G. (1997). Differential expression of connexins during neocortical development and neuronal circuit formation. J. Neurosci. 17, 3096 –3111. Nagy, J. I., and Rash, J. E. (2000). Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS. Brain Res. Rev. 32, 29 – 44.
439 Nagy, Z., Peters, H., and Huttner, I. (1984). Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions. Lab. Invest. 50, 313–322. Norman, M. G., and O’Kusky, J. R. (1986). The growth and development of microvasculature in human cerebral cortex. J. Neuropathol. Exp. Neurol. 45, 222–232. Shepro, D., and Morel, N. M. (1993). Pericyte physiology. FASEB J. 7, 1031–1038. van Kempen, M. J., and Jongsma, H. J. (1999). Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals. Histochem. Cell Biol. 112, 479 – 486. Virgintino, D., Robertson, D., Benagiano, V., Errede, M., Bertossi, M., Ambrosi, G., and Roncali, L. (2000). Immunogold cytochemistry of the blood– brain barrier glucose transporter GLUTI and endogenous albumin in the developing human brain. Dev. Brain Res. 123, 95–101. Wijsman, J. A., and Shivers, R. R. (1998). Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium. In Vitro Cell Dev. Biol. 34, 777–784.
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
BRIEF COMMUNICATION Expression of the Gap Junction Protein Connexin43 in Human Telencephalon Microvessels Daniela Virgintino,* ,1 David Robertson,† Mariella Errede,* Vincenzo Benagiano,* Mirella Bertossi,‡ Glauco Ambrosi,* and Luisa Roncali* *Department of Human Anatomy and Histology, University of Bari School of Medicine, I-70124 Bari, Italy; †Institute of Cancer Research, Haddow Laboratories, SM2 5NG, Sutton, United Kingdom; and ‡University of Foggia School of Medicine, Foggia, Italy Received March 22, 2001; published online August 6, 2001
Key Words: connexin43; gap junctions; microvessels; endothelium; human telencephalon; immunoconfocal microscopy.
INTRODUCTION The gap junction protein connexin43 (Cx43) is believed specific to interastrocytic gap junctions in the adult brain and is expressed by cells of the astroglial lineage in the developing brain (Dermietzel and Spray, 1993; Nadarajah et al., 1997; Nagy and Rash, 2000). The presence of Cx43 as well as Cx37 and Cx40 has also been demonstrated in the blood vessels of different organs and, in particular, in the endothelial cells, where the expression of these connexins seems to be species, organ, and vessel specific (Little et al., 1995; Hillis et al., 1997; Janssen-Bienhold et al., 1998; Ko et al., 1 To whom correspondence should be addressed at the Department of Human Anatomy and Histology, University of Bari School of Medicine, Piazza Giulio Cesare, I-70124 Bari, Italy. Fax: ⫹39 080 5478309. E-mail: [email protected].
0026-2862/⫺1900 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
1999; van Kempen and Jongsma, 1999). So far, there are no data on Cx43 expression in human brain microvessels during prenatal development, when the endothelial cells acquire morphological and biochemical barrier features (blood– brain barrier). Here we present findings on the expression of Cx43, investigated by confocal microscopy, in the human fetus telencephalon at 18 weeks of gestation, a developmental age characterized by significant progression of vessel growth and differentiation (Bertossi et al., 1999; Virgintino et al., 2000).
MATERIAL AND METHODS Four fetuses, at 18 weeks of gestation, were obtained after spontaneous abortion under informed consent and approval by the local ethics committee. The gestation age was estimated on the basis of the crown– rump length and/or pregnancy records of the gestational age. The telencephalon dorsolateral wall was dissected, immersed in the Bouin’s fixative for 2 h at 4°C, and embedded in paraffin wax. Five-micrometer
435
436
sections were cut perpendicular to the telencephalon surface and collected on Vectabond (Vector Laboratories)-treated slides.
Cx43 Immunohistochemistry The immunolabeling for Cx43 was preceded by a heat-mediated antigen retrieval by microwaving the sections immersed in 0.01 M citrate buffer (pH 6.0) for 10 min at 750 W. Sections were then incubated with (1) blocking buffer (phosphate-buffered saline (PBS), 0.8% BSA, 5% FCS) for 30 min at room temperature (RT), (2) mouse monoclonal antibody anti-connexin43 (diluted 1:200; Zymed Laboratories) overnight at 4°C, (3) horse antibody anti-mouse IgG (diluted 10 g/ml; Vector Laboratories) for 1 h at RT, and (4) fluorescein isothiocyanate-labeled avidin D (diluted 30 g/ml; Vector Laboratories) for 1 h at RT. The sections were washed 10 min ⫻ 3 in PBS between each step and finally drained and mounted in Vectashield (Vector Laboratories). Control sections of human fetal telencephalon, submitted to the same immunohistochemical procedure with the primary antibody being either omitted or preadsorbed with an excess (20 nmol ml ⫺1) of the pure antigen (Zymed Laboratories), showed no immunolabeling.
Confocal Laser Scanning Microscopy The sections were viewed under a confocal laser scanning microscope (Leica TCS SP) using 20⫻ and 63⫻ objective lenses. Optical sections were taken at 200-nm intervals through their z axis covering in total 5 m in depth and digitally recorded as single sections and as projection images assembled from a series of optical sections. Images were stored as TIFF files and analyzed by Adobe PhotoShop (Adobe Systems, CA).
RESULTS At 18 weeks the telencephalon is crossed by bundles of Cx43-immunofluorescent fibers of the radial glia that extend as far as the pia between rows of unlabeled neuroblasts. The radial glial fibers are distinctly re-
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
vealed by a fine pattern of labeled puncta, either isolated or, more frequently, aligned in short chains (Figs. 1A–1D). The vascular network is formed by immunofluorescent radially oriented large microvessels and their small, orthogonal and oblique, collaterals. In the thin wall of the radial vessels, the endothelial cells show a punctate Cx43 labeling; chains of puncta decorate the endothelial basal profile, which is encircled by unlabeled pericytes (Figs. 1A and 1B). Cx43-immunoreactive strands, formed by fused puncta, mark discrete regions of the endothelial layer corresponding to lateral contacts between adjoining endothelial cells (Figs. 1A and 1B). In the small collaterals of the radial vessels, fluorescent puncta diffusely label the endothelial cells and decorate their luminal and basal profiles (Figs. 1C–1F). Many Cx43-immunoreactive glial fibers are seen adjacent to the microvessel walls and some of them make contact with the endothelial lining (Figs. 1C and 1D).
DISCUSSION The results confirm that confocal laser scanning microscopy greatly improves the analysis of immunofluorescent structures on thick paraffin sections; on both single optical plane and projection images of the 18week human telencephalon, Cx43 is revealed at the glial and vascular compartments as a punctate labeling, and this is characteristic of gap junctions. The detection of Cx43-labeled radial glia close to unlabeled neuroblasts supports the view that communication via heterotypic gap junctions controls neuronal migration along the radial fibers in the developing brain (Nadarajah et al., 1997). As far as the microvascular compartment is concerned, radial vessels, which first penetrate the telencephalon from 8 weeks of gestation, and their collaterals, which develop at 18 weeks (Norman and O’Kusky, 1986), show a Cx43 punctate labeling in the endothelial cells. This indicates that the interendothelial communication via homotypic gap junctions is active in the growing microvessels of the 18-week human telencephalon. Furthermore, there are noticeable differences in Cx43 expression between the main trunks and the collaterals of the radial vessels. In
Brief Communication
437
FIG. 1. Cx43 expression by confocal microscopy in a radial vessel (A, B) and collaterals of radial vessels (C–F) of 18-week telencephalon. In the single optical section (A) and projection image (B) isolated immunofluorescent puncta are detectable in endothelial cells, and chains of puncta label their basal profile (arrowheads), which is paralleled by the pericyte layer (p). Immunofluorescent strands are seen in discrete endothelial regions (arrows), corresponding to lateral contacts between adjoining cells. Inset: A fluorescent spot between an endothelial cell (e) and a pericyte. Single image (C) and projection image (D) of a collateral vessel crossed by radial glia fibers (f). The endothelial cells show a diffuse, punctate immunofluorescence and isolated or packed puncta are aligned along the endothelial luminal and basal profiles (arrowheads). The radial glial fibers are also revealed by a fine, punctate immunofluorescence; glial fibers adhere to the endothelial cells (arrows) and to an unlabeled neuroblast (n). Single image (E) and projection image (F) of tangentially cut collaterals showing the Cx43 punctate pattern on the endothelial basal front. Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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the collaterals, Cx43 punctate labeling is detectable everywhere in the endothelial cells, whereas in the radial vessels the labeling is mainly restricted to discrete regions corresponding to interendothelial contacts. It is well known that the adult brain endothelial cells lack gap junctions and are sealed by extensive tight junctions, which are part of the blood– brain barrier (Dermietzel, 1975; Nagy et al., 1984). It has also been demonstrated that transient gap junctions are present between immature brain endothelial cells and disappear as their differentiation progresses (Delorme et al., 1970; Wijsman and Shivers, 1998). Compared with the collaterals the localized expression of Cx43 in the endothelial cells of the radial vessels indicates an advanced degree of endothelial differentiation. Demonstration that endothelial differentiation of the radial vessel main trunks is under way in the 18-week human telencephalon has been given by studies showing the presence of some blood– brain barrier devices at this stage, such as tight interendothelial junctions, albumin barrier, and glucose transporter expression (Bertossi et al., 1999; Virgintino et al., 2000). The Cx43 immunofluorescent puncta on the basal endothelial front overlying unlabeled pericytes could correspond to heterotypic gap junctions between endothelial cells and pericytes. Endothelium–pericyte gap junctions are present in the developing brain microvessels and persist in the mature ones, being involved respectively in the regulation of vessel growth and functioning (Cuevas et al., 1984; Shepro and Morel, 1993; Bertossi et al., 1995; Fujimoto, 1995; Balabanov and Dore-Duffy, 1998). The detection of Cx43 radial glial fibers in close contact with labeled microvessels suggests the existence of gap junctions also between perivascular processes of astroglial cells and vascular cells. Though never before documented in situ at the histological level, the existence of this coupling has been evidenced by studies carried out in a blood– brain barrier culture model that shows passage of waves of Ca 2⫹ ions from endothelial cells to astrocytes and vice versa (Leybaert et al., 1998). During brain angiogenesis, this coupling might be responsible for the role played by astroglial cells in endothelial cell differentiation (Holash et al., 1993; Bauer and Bauer, 2000). In conclusion, the research gives the first demonstration of Cx43 expression during human telencephalon
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
angiogenesis. The expression of the same connexin in endothelial cells and perivascular astroglial cells suggests that these cell types communicate and are mutually influenced by direct signaling via homotypic gap junctions.
ACKNOWLEDGEMENT This work was supported in part by Grant 98-505-04, 1998 (to L.R.), from the Consiglio Nazionale delle Ricerche (CNR), Italy.
REFERENCES Balabanov, R., and Dore-Duffy, P. (1998). Role of the CNS microvascular pericyte in the blood– brain barrier. J. Neurosci. Res. 53, 637– 644. Bauer, H.-C., and Bauer, H. (2000). Neural induction of the blood– brain barrier: Still an enigma. Cell. Mol. Neurobiol. 20, 13–28. Bertossi, M., Riva, A., Congiu, T., Virgintino, D., Nico, B., and Roncali, L. (1995). A compared TEM/SEM investigation on the pericytic investment in developing microvasculature of the chick optic tectum. J. Submicrosc. Cytol. Pathol. 27, 349 –358. Bertossi, M., Virgintino, D., Errede, M., and Roncali, L. (1999). Immunohistochemical and ultrastructural characterization of cortical plate microvasculature in the human fetus telencephalon. Microvasc. Res. 58, 49 – 61. Cuevas, P., Gutierrez-Diaz, J. A., Reimers, D., Dujovny, M., Diaz, F. G., and Ausman, J. I. (1984). Pericyte endothelial gap junctions in human cerebral capillaries. Anat. Embryol. 170, 155–159. Delorme, P., Gayet, J., and Grignon, G. (1970). Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken. Brain Res. 22, 269 –283. Dermietzel, R. (1975). Junctions in the central nervous system of the cat. IV. Interendothelial junctions of cerebral blood vessels from selected areas of the brain. Cell Tissue Res. 164, 45– 62. Dermietzel, R., and Spray, D. C. (1993). Gap junctions in the brain: Where, what type, how many and why? Trends Neurosci. 16, 186 –192. Fujimoto, K. (1995). Pericyte– endothelial gap junctions in developing rat cerebral capillaries: A fine structural study. Anat. Rec. 242, 562–565. Hillis, G. S., Duthie, L. A., Mlynski, R., McKay, N. G., Mistry, S., MacLeod, A. M., Simpson, J. G., and Haites, N. E. (1997). The expression of connexin 43 in human kidney and cultured renal cells. Nephron 75, 458 – 463. Holash, J. A., Noden, D. M., and Stewart, P. A. (1993). Re-evaluating the role of astrocytes in blood– brain barrier induction. Dev. Dyn. 197, 14 –25.
Brief Communication
Janssen-Bienhold, U., Dermietzel, R., and Weiler, R. (1998). Distribution of connexin43 immunoreactivity in the retinas of different vertebrates. J. Comp. Neurol. 396, 310 –321. Ko, Y.-S., Yeh, H.-I., Rothery, S., Dupont, E., Coppen, S. R., and Severs, N. J. (1999). Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy. J. Histochem. Cytochem. 47, 683– 692. Leybaert, L., Paemeleire, K., Strahonja, A., and Sanderson, M. J. (1998). Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells. Glia 24, 398 – 407. Little, T. L., Beyer, E. C., and Duling, B. R. (1995). Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am. J. Physiol. 268, 729 –739. Nadarajah, B., Jones, A. M., Evans, W. H., and Parnavelas, J. G. (1997). Differential expression of connexins during neocortical development and neuronal circuit formation. J. Neurosci. 17, 3096 –3111. Nagy, J. I., and Rash, J. E. (2000). Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS. Brain Res. Rev. 32, 29 – 44.
439 Nagy, Z., Peters, H., and Huttner, I. (1984). Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions. Lab. Invest. 50, 313–322. Norman, M. G., and O’Kusky, J. R. (1986). The growth and development of microvasculature in human cerebral cortex. J. Neuropathol. Exp. Neurol. 45, 222–232. Shepro, D., and Morel, N. M. (1993). Pericyte physiology. FASEB J. 7, 1031–1038. van Kempen, M. J., and Jongsma, H. J. (1999). Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals. Histochem. Cell Biol. 112, 479 – 486. Virgintino, D., Robertson, D., Benagiano, V., Errede, M., Bertossi, M., Ambrosi, G., and Roncali, L. (2000). Immunogold cytochemistry of the blood– brain barrier glucose transporter GLUTI and endogenous albumin in the developing human brain. Dev. Brain Res. 123, 95–101. Wijsman, J. A., and Shivers, R. R. (1998). Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium. In Vitro Cell Dev. Biol. 34, 777–784.
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.