developmental expression of zo-1 antigen in the mouse blood–brain barrier

9
Ž . Developmental Brain Research 114 1999 161–169 Research report Developmental expression of ZO-1 antigen in the mouse blood–brain barrier Beatrice Nico a, ) , Fabio Quondamatteo d , Rainer Herken d , Andrea Marzullo b , Patrizia Corsi c , Mirella Bertossi a , Giangiuseppe Russo a , Domenico Ribatti a,1 , Luisa Roncali a a Institute of Human Anatomy, Histology and Embryology, UniÕersity of Bari, Piazza G. Cesare, 11, Policlinico, I 70124 Bari, Italy b Institute of Pathology, UniÕersity of Bari, I 70124 Bari, Italy c Department of Human Physiology, UniÕersity of Bari, I 70124 Bari, Italy d Zentrum Anatomie, Abteilung Histologie, UniÕersitat Gottingen, D-37075 Gottingen, Germany Accepted 22 December 1998 Abstract Ž . Tight junction biogenesis during blood–brain barrier development BBB in mesencephalon microvessels of mouse embryos of day 9, Ž . foetuses of day 15 and 19 and new-born 2-day-old mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasmamembrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Blood–brain barrier; Mouse; ZO-1 protein 1. Introduction Ž . The homeostasis of the Central Nervous System CNS environment is maintained by the blood–brain barrier Ž . BBB which separates the brain from the systemic circula- tion and is structurally localized in the blood capillaries w x 33 . The endothelial cells lining the lumina of the brain capillaries possess specific morphological and biochemical barrier properties, owing to intercellular tight junctions Ž . TJ , paucity of cytoplasmic vesicles, and specialised w x transport systems and enzymatic activities 2,4,8,29,30,47 . The TJ is composed of a complex belt-like zonula occlu- w x dens close to the capillary lumen 24 . It is considered to be the major structure responsible for the restriction of the ) Corresponding author. Fax: q39-80-5478309; E-mail: [email protected] 1 E-mail: [email protected]. compound paracellular escape across the cerebral endothe- lium, as well as for its polarity and high electrical resis- w x tance in vivo 9,10,21,32 . TJs have been morphologically located by freeze frac- ture and transmission electronic microscopy between en- w x w x dothelial 3,6,33 and epithelial cells 20 . The fusion points between the opposing plasmamembrane external leaflets, viewed by electron microscopy, were seen as networks of intramembranous fibrils on the fractured P- w x faces, with complementary grooves on the E-faces 43 . The biochemical composition of the junctional fibrils and their functional properties are not completely known. Some researches think that either lipids or proteins partici- pate in fibril formation, while others have shown that both lipidic and proteinic components contribute to the fibril w x molecular composition 13,15,31 . Several associated pro- wx teins have been identified, including cingulin 5 , ZO-1 w x w x 44 and ZO-2 14 . Important progress in the biochemical characterization of the TJ has been achieved through the 0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0165-3806 99 00008-5

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Ž .Developmental Brain Research 114 1999 161–169

Research report

Developmental expression of ZO-1 antigen in the mouse blood–brainbarrier

Beatrice Nico a,), Fabio Quondamatteo d, Rainer Herken d, Andrea Marzullo b,Patrizia Corsi c, Mirella Bertossi a, Giangiuseppe Russo a, Domenico Ribatti a,1,

Luisa Roncali a

a Institute of Human Anatomy, Histology and Embryology, UniÕersity of Bari, Piazza G. Cesare, 11, Policlinico, I 70124 Bari, Italyb Institute of Pathology, UniÕersity of Bari, I 70124 Bari, Italy

c Department of Human Physiology, UniÕersity of Bari, I 70124 Bari, Italyd Zentrum Anatomie, Abteilung Histologie, UniÕersitat Gottingen, D-37075 Gottingen, Germany

Accepted 22 December 1998

Abstract

Ž .Tight junction biogenesis during blood–brain barrier development BBB in mesencephalon microvessels of mouse embryos of day 9,Ž .foetuses of day 15 and 19 and new-born 2-day-old mice was examined by light and electron microscopy, using monoclonal antibodies

recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the lightmicroscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasmamembrane leafletsunder the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when theendothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout thecytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at thecytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developingbrain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation. q 1999 Elsevier ScienceB.V. All rights reserved.

Keywords: Blood–brain barrier; Mouse; ZO-1 protein

1. Introduction

Ž .The homeostasis of the Central Nervous System CNSenvironment is maintained by the blood–brain barrierŽ .BBB which separates the brain from the systemic circula-tion and is structurally localized in the blood capillariesw x33 . The endothelial cells lining the lumina of the braincapillaries possess specific morphological and biochemicalbarrier properties, owing to intercellular tight junctionsŽ .TJ , paucity of cytoplasmic vesicles, and specialised

w xtransport systems and enzymatic activities 2,4,8,29,30,47 .The TJ is composed of a complex belt-like zonula occlu-

w xdens close to the capillary lumen 24 . It is considered tobe the major structure responsible for the restriction of the

) Corresponding author. Fax: q39-80-5478309; E-mail:[email protected]

1 E-mail: [email protected].

compound paracellular escape across the cerebral endothe-lium, as well as for its polarity and high electrical resis-

w xtance in vivo 9,10,21,32 .TJs have been morphologically located by freeze frac-

ture and transmission electronic microscopy between en-w x w xdothelial 3,6,33 and epithelial cells 20 . The fusion

points between the opposing plasmamembrane externalleaflets, viewed by electron microscopy, were seen asnetworks of intramembranous fibrils on the fractured P-

w xfaces, with complementary grooves on the E-faces 43 .The biochemical composition of the junctional fibrils

and their functional properties are not completely known.Some researches think that either lipids or proteins partici-pate in fibril formation, while others have shown that bothlipidic and proteinic components contribute to the fibril

w xmolecular composition 13,15,31 . Several associated pro-w xteins have been identified, including cingulin 5 , ZO-1

w x w x44 and ZO-2 14 . Important progress in the biochemicalcharacterization of the TJ has been achieved through the

0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-3806 99 00008-5

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169162

Ž . Ž .Fig. 1. Day 9 embryos. a No labelling of microvessel wall is recognizable after immunolabelling with anti-ZO-1 antibodies. b The terminal ends ofŽ . Ž .contiguous endothelial cells show points of plasmamembrane drawing closer together arrow . Bars: a, 5 mm; b, 0.25 mm; c, 0.1 mm. c An irregular

Ž . Ž .endothelium with alternating thick arrow and thin cytoplasmic regions arrowhead surrounds the vessel lumen.

identification of ZO-1, a phosphoprotein of 225 kDa asso-ciated with the cytoplasmic domain of the TJ in both

w xepithelial and endothelial cells 1,17,40,44 , though not yetlocated in embryonic endothelial cells. It has still not beendemonstrated whether the protein ZO-1 is a molecularcomponent of the fibril, although a strict correlation hasbeen found between ZO-1 expression, density of the junc-

w xtion fibrils and cellular resistance 6,7,17,45 .A further much debated question about the BBB con-

cerns the time course of morphological and biochemicaldifferentiation of the TJ joining the endothelial cells of thebrain microvasculature during embryonic development.There is evidence that the TJ are mature already during

w xearly development in the human brain 22,23 , whereas inthe brain of other species, such as chicken, they reachcomplete differentiation only at the end of development

w xand during post-natal life 25–27,36–38 .

In the present study, the ultrastructure of the TJ and thehistochemical expression of ZO-1 antigen have been inves-tigated in developing and mature cerebral murine mi-crovasculature, by means of light and electron microscopy.The aim was to better understand the chemical nature ofTJs in embryonic, foetal and in post-natal stages, and toprovide further information on the mechanisms involved inthe BBB development.

2. Materials and methods

2.1. Animals and preparation

Ž .New-born 2-day-old and pregnant C57 Bl mice werekilled by cervical dislocation on day 9, 15, and 19 of

Ž . Ž . Ž .Fig. 2. Day 15 foetuses. Microvessels showing a spot-like staining pattern a, arrow and diffuse immunolabelling b, arrow with anti-ZO-1 antibodies. cŽ . Ž .The microvessel wall, built up of a more regular endothelium and pericyte, is not completely surrounded by isolated glial endfeet arrowhead . d The

Ž . Ž .overlapping expansions of endothelial cells are ‘tightly’ joined at several points arrowhead , strengthened by dense cytoplasmic material. eŽ .Ultrastructural localization of ZO-1 on ultrathin sections: gold particles are scattered throughout the cytoplasm of the endothelial cells E ; note some

Ž .particles arrowhead near to two interendothelial junctions. Bars: a–b, 5 mm; c, 0.4 mm; d, 0.1 mm; e, 0.2 mm.

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169 163

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169164

gestation and embryos and foetuses were removed fromthe uterus.

2.2. Fixation and preparation of tissue

Small pieces from mesencephalon of embryos, foetuses.and new-born mice were fixed: i in 0.1 M phosphate-

buffered 3% glutaraldehyde solution for conventional elec-.tron microscopy; ii in a modified acetate-free Bouin fluid

for immunohistochemistry investigation at the light micro-.scopical level; iii in 0.1 M phosphate buffered 0.5%

glutaraldehyde for immunoelectron microscopic study.

2.3. ConÕentional electron microscopy

The specimens were post-fixed with 1% osmium tetrox-ide, dehydrated in an ascending ethanol series and embed-ded in Epon 812. Semithin sections were cut for orienta-tion purposes and stained with toluidine blue. Thereafter,ultrathin sections of 60 nm were cut with a diamond knifeon an LKB V ultratome, stained with uranyl acetate andlead citrate and examined with a Zeiss EM109 electronmicroscope.

2.4. Immunohistochemistry

2.4.1. Light microscopyFive-micrometer microtome coronal sections collected

on poly-lysine coated slides were deparaffinized and stainedwith a three-step alkaline phosphatase-anti-alkaline phos-

Ž .phatase APAAP technique. The sections were rehydratedin a graded alcohol series and immersed in tris-buffered

Ž .saline TBS for 10 min. Thereafter, the sections weretreated with 2.5% ficin in TBS, pH 7 for 30 min and then

.sequentially exposed to: i primary rat monoclonal antiŽZO-1 antibody diluted 1:100 in TBS Chemicon Intern.,

. .S.I.C., Rome, Italy overnight at 48C; ii biotinylated rabbitanti-rat antibody diluted 1:25 in TBS for 30 min at room

. Žtemperature, and iii APAAP complexes Dako Italia, Mi-.lan, Italy . Each step was followed by a 10 min wash in

TBS. The immunocytochemical reaction product was de-veloped in Fast Red chromogen containing 0.01% lev-amisole, for 20 min at room temperature.

Negative controls were obtained by substituting normalrat serum for the primary antibodies.

2.4.2. Electron microscopyThe fixed specimens were rinsed with 10 mM ammo-

nium chloride in 0.1 M PBS for 45 min, dehydrated in an

ascending ethanol series up to 70% ethanol and embeddedŽ .in the acrylic resin LR-Gold Biorad with 0.8% benzil.

The resin was hardened at y258C under the light of aŽ .halogen lamp 500 W . Thin sections were cut with an

LKB V ultramicrotome and collected on formvar-coatednickel grids. The grids were incubated for 10 min at roomtemperature with TBS buffer and then treated for 4 minwith 2.5% ficin in TBS, pH 7. After having been rinsedwith TBS, the grids were treated with 1% BSA-TBS, pH7.4 for 10 min at room temperature to block unspecificreactions. The sections were incubated at room tempera-ture overnight with primary antibody, washed with TBS,and incubated for 1 h at room temperature with the second

Ž .antibody goat anti-rat IgG coupled to 6 and 15 nm goldŽ .particles Chemicon Intern. . After being washed with

TBS, the grids were stained with 1% uranyl acetate,followed by 1% lead citrate and examined with a ZeissEM 109 electron microscope.

3. Results

3.1. Mouse embryo day 9

On day 9 no capillaries nor neural cells were labelledŽ .by the anti-ZO 1 antibody Fig. 1a . At this developmental

stage microvessels appeared large, thick walled, and linedby an irregularly thick endothelium. Its cytoplasm showedmany organelles, well preserved Golgi areas, numerousvesicles and mitochondria. The terminal ends of the en-dothelial cells were simply apposed in a regular way, orcloser at some points, with reduction of the intercellularspace. In these areas, a cytoplasmic dense material wasadherent to the endothelial plasmamembranes. No basallamina and pericytes were found beneath the endothelial

Ž .cells Fig. 1b,c .

3.2. Mouse foetus day 15

On day 15, the mesencephalon anlage was greatlyincreased in size. Capillaries showed a light microscopi-cally visible irregular staining pattern. Some vessels werecharacterized either by a faint diffuse immunolabelling orby a spot-like labelling, others were unlabelled. The neu-

Ž .ropile remained completely unstained Fig. 2a,b .Ultrastructurally, microvessels also changed. The capil-

lary lumen became more rounded and the endothelium wasmore regular in thickness. The peripheral expansions ofcontiguous endothelial cells were overlapped over longer

Ž . Ž . Ž .Fig. 3. Day 19 foetuses. a Numerous immunostained microvessels with continuous arrow and discontinuous staining arrowhead are visible afterŽ . Ž .immunohistochemical localization of ZO-1 at the light microscopical level. b A microvessel showing endothelial cells E backed by basal lamina

Ž . Ž . Ž . Ž .arrow , pericyte and a continuous layer of glial endfeet G . c Ultrastructural localization of ZO-1 on ultrathin sections: isolated gold particles arrowŽ .and clusters of particles arrowhead are located on the plasmamembranes of a tight junction and in the endothelial cytoplasm near the junction. Bars: a, 25

mm; b, 0.003 mm; c, 0.025 mm.

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169 165

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169166

Ž . Ž .Fig. 4. New-born mice. a A microvessel showing discrete lines of staining along the cell-to-cell contacts arrow ; no ZO-1 staining is detectable outsideŽ . Ž . Ž .the vessels. b An extensive tight junction with points and lines of fusions of the plasmamembrane external leaflets arrow seals two endothelial cells. c

Ultrastructural localization of ZO-1 on ultrathin sections: gold particles decorate the cytoplasmic surface of the tight junction, close to points and lines ofŽ .fusion between the plasmamembranes arrowhead . Bars: a, 5 mm; b–c, 0.035 mm.

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169 167

tracts and having approached each other, they now became‘tight’ in some places. At these fusion points, a discontinu-ous and dense cytoplasmic material was detected on thefused plasmamembranes. Pericytes were discontinuouslyassociated with the endothelial cells that were backed by adiscontinuous basal lamina and isolated endfeet of glial

Ž .cells Fig. 2c,d .ZO-1 immunoreactivity was ultrastructurally localized

with a diffuse pattern in the cytoplasm of endothelial cells.Only in a few vessels, were isolated gold particles presentalong the cytoplasmic aspects close to endothelial junc-

Ž .tions Fig. 2e .

3.3. Mouse foetus day 19

On day 19, the immunoreactive capillaries increased innumber; the ZO-1 staining still varied, being continuous in

Ž .some vessels, discontinuous in others Fig. 3a .At this stage the capillary endothelial layer was very

thin and backed by a continuous basal lamina and peri-cytes. Besides pericytes, astroglial foot processes almost

Ž .completely enveloped the capillary wall Fig. 3b . Moreextensive TJ sealed the regularly faced plasmamembranesof the endothelial cells.

After postembedding immunogold treatment of ultrathinsections, in capillaries of day 19 foetuses the gold particleswere mainly localised at the TJ level, where they weregathered along the cytoplasmic surface of the junctions,often close to the points and lines of fusion of the plas-

Ž .mamembranes Fig. 3c .

3.4. New-born mouse

In the mesencephalon of new-born mice all the capillar-ies displayed a ZO-1 labelling which appeared as discrete

Ž .lines along the cell-to-cell contacts Fig. 4a .The ultrastructural features of the mesencephalic mi-

crovessels were essentially not different from those of the19-day embryos. TJs with numerous points and lines offusion of the plasmamembrane external leaflets completely

Ž .occluded the interendothelial spaces Fig. 4b . Afterpostembedding immunogold treatment, capillaries showedreactivity which was mainly localised at TJ level. Goldparticles appeared gathered along the cytoplasmic surfaceof the junctions, often close to the points and lines of

Ž .fusion of the plasmamembranes Fig. 4c .

4. Discussion

The results of this study demonstrate that the morpho-logical maturation of the TJ occurs progressively duringthe pre-natal development of the murine cerebral microves-sels and involves the expression of the ZO-1 protein at theendothelial tight junctional sites. In fact, fusion pointsbetween the plasmamembranes join the endothelial cells

on day 15 of pre-natal life, and mature TJ with severalpoints and lines of fusion between the opposing plas-mamembranes are recognizable only at the end of thefoetal stage and onwards. The ZO-1 protein expressionparallels the morphological maturation of the junction.ZO-1 is lacking in the day 9 embryonic stage, when thebrain microvessels lack junctional complexes and is ex-pressed by day 15, when the ultrastructural differentiationof the interendothelial TJ has begun. Moreover, the lightmicroscopic ZO-1 expression changes from a spot-like,diffuse pattern in the embryonic and foetal vessels to alinear and discontinuous one post-natal.

The immunogold electron microscopic observationsprovide the study with further, more precise details on theZO-1 localisation. At embryonic and foetal stages, theanti-ZO-1 gold particles are found in endothelial cells,both spread throughout their cytoplasm and concentratednear the developing junctional complexes. In the new-bornanimals, the anti-ZO-1 gold particles are more numerousthan at the end of foetal development, and they mainlydecorate the cytoplasmic domains of the TJ, close to thefusion points and lines of the joined plasmamembranes.

Overall, these data indicate that the endothelial cells ofthe murine cerebral microvessels express the ZO-1 antigenat first unordered in the cytoplasm and then, when thedifferentiation of the TJ has been achieved, according tothe specific arrangement on the endothelial plasmamem-branes. Therefore, it might be that the establishment oftight contacts determines the induction of the ZO-1 assem-bly protein on the membrane.

Developmental changes in the ZO-1 expression patternhave been demonstrated in the pre-implanted mouse em-

w xbryo 11,12 and correlated to the acquisition of a specificcellular polarity, according to cell–cell contact, whichcould cause an antigen redistribution on the plasmamem-branes. Recent studies have demonstrated that ZO-1 is amember of a family of protein kinases, namely mem-

Ž .brane-associated guanilate kinase homologues MAGUKs ,thought to be important in signal transduction at sites ofcell–cell contact during growth and differentiationw x16,46,48 . A strict correlation between ZO-1 degree ofphosphorilation, and its functional role has been demon-strated for MDCK cells treated with a protein kinase

w xinhibitor 28 . The lack of ZO-1 phosphorilation inhibits itsassemblage on the membrane, suggesting that the proteinphosphorilation plays an important role in TJ development.It is, therefore, likely that the ZO-1 protein is first ex-pressed during the BBB differentiation as a non-phos-phorilated molecule, in a diffuse manner and that thesubsequent establishment of tight contacts between en-dothelial cells might activate the protein kinase with phos-phorilation and assemblage of the protein on the mem-brane.

Little is known about the functional meaning of ZO-1.Recent data indicate that the phosphorilation of junctionalproteins, both ZO-1 and cytoplasm catenins-anchoring

( )B. Nico et al.rDeÕelopmental Brain Research 114 1999 161–169168

molecules associated to junctional systems and cyto-skeleton may play a regulatory role in the cellular perme-

w xability 18,19,39,42 . Therefore, the final assemblage ofthe ZO-1 protein on the endothelial membrane, probablymediated by phosphorilative processes, could modulate thetightness of the junctional areas and be involved in thereduction of the vascular permeability that also occurs

w xduring murine prepartal stages 34,35,41 .In conclusion, these results indicate a close relation

between development of the interendothelial TJ and ex-pression of ZO-1 protein. In particular, the faint immuno-staining of the capillaries in the embryo, when the devel-opment of the junction begins, and the specific immuno-gold labelling, when the TJ is completely formed, seem toindicate that the cell-to-cell contacts regulate the ZO-1antigen biosynthesis during development, and in turn theformation of the blood–brain barrier.

Acknowledgements

The authors are grateful to Elke Heyder and BertiManshausen for technical support and to Rold Dungan andMarisa Ambrosi for photographic help. The authors alsowish to thank Cyrilla Maelicke and Clelia Roca for editingthe manuscript. This work was supported in part by a grant

Ž . Ž .from MURST 40%—1996 and 60%—1997 , Rome Italyto Luisa Roncali and Beatrice Nico.

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