Characterization of ependymal cells in hypothalamic and choroidal primary cultures

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h’euroscience Vol. 24, No. 3, pp. 993-1007, 1988 Printed in Great Britain

Pergamon Press plc IBRO

CHARACTERIZATION OF EPENDYMAL CELLS IN HYPOTHALAMIC AND CHOROIDAL PRIMARY CULTURES J. GABRION,‘~ S. PERALDI,* A. FAIVRE-BAUMAN,~ C. KLOTZ,~ M. S. GHANWUR,II D. PAULIN,~ I. ASSENMACHER+ and A. TIXIER-VIDAL$ *UA CNRS 1197, Laboratoire de Neurobiologie Endocrinologique, Universite des Sciences et Techniques du Languedoc, F-34060 Montpellier CXdex, France; #UA CNRS 1115, Groupe de Neuroendocrinologie Cellulaire et Moleculaire, College de France, F-75231 Paris Cedex 05, France; !jCentre de GWtique MolCulaire, CNRS, F-91190 Gif-sur-Yvette, France; 1lCentre de Neurochimie, CNRS, F-67084 Strasbourg, France; fLaboratoire de G&nttique Cellulaire, Institut Pasteur, F-75015 Paris, France

Abstract-Long-term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus were obtained in serum-supplemented and chemically defined media. In order to identify and characterize cell types growing in our cultures, we used morphological features provided by phase-contrast, scanning and transmission electron microscopy. Immunological criteria were recognized, using antibodies against intermediate filament proteins (vimentin, gliofibrillar acid protein, cytokeratin, desmin, neurotllament proteins), actin, myosin, ciliary rootlets, laminin and fibronectin in single or double immunostaining, and monoclonal antibodies known to detect epitopes of ependymal or endothelial cells. Minor cell types such as astrocytes, fibroblasts and endothelial cells were distinguished. Ependymal cells, which exceeded 75% of the cultured cells, were identified by their cell shape and epithelial organization revealed by phase-contrast and transmission electron microscopy, by their apical differentiation evidenced by scanning and transmission electron microscopy, and by certain molecular markers (e.g. gliofibrillar acid or ciliary rootlet proteins) detected by immunofluorescence. Four ependymal cell types were recognized: choroidal ependymocytes, ciliated and unciliated ependymal cells, and tanycytes. All these cultured ependymal cell types showed a remarkable resemblance to in vivo ependymocytes, in tenns of marker expression and ultrastructural features.

The cell biology of the ependymal wall is of interest for both the study of neuron-glia interactions in general, and the transcellular tralhc between the brain extracellular space and the ventricles. In particular, the recent detection of more and more hormones, neurotransmitters and other neuroactive substances of systemic and/or neural origin in the cerebrospinal fluid (CSF) has raised questions about the possible involvement of ependymal cells in the transfer of chemical signals.3*4*‘9Special consideration has been given to the ependymal cells which line the third ventricle and its related circumventricular organs (CVOs).” Ependymal cells form a continuous but heterogeneous “epithelial” layer, composed of several cell types, which are variably modified in the neurohemal areas of the CVOS.*~,*~*‘~~~~

tTo whom correspondence should be addressed. Abbreviations: CSF, cerebrospinal fluid; CVO, circumventricular organs; DMEM, Dulbecco’s minimum essential medium; EDTA, ethylenediaminetetra-acetic acid; FITC, fluorescein isothiocyanate; Fib, fibronectin; GFAP. aliofibrillar acid urotein: ItiH + L). immunoglobulin-heavy and lighi chains; &un, la∈ NF, neurofilament proteins; PBS, phosphate-buffeted saline; SEM, Scanning electron microscope; SFM, serum-free medium; SSM, serum-supplemented medium; TEM, transmission electron microscope; TRITC, trimethylrhodamin isothiocyanate; Vim, vimentin.

In vivo, ependymal cells have essentially been characterized by morphological and immunological features. Typically polarized, these cells can be distinguished by differences in their junctional complexes and apical or basolateral membranes and by their specific localization at the surface of the third ventricle.“*u Choroidal ependymocytes are identified by their cuboidal shape, their numerous apical, long, club-shaped microvilli, the tightness of their junctions and by their occurrence only in the choroid plexuses.B*37Other discrete populations of specialized ependymal cells constitute, for instance, the subcommissural organ, a secretory CVO which overlies the posterior commissure at the dorsal junction of the third ventricle, and the aqueduct4’ or the subfornical organ, protruding from the roof of the diencephalon into the third ventricle.* Along the ventrolateral walls of the third ventricle, small ciliated cells are numerous. They are mingled with small unciliated cells bearing only irregular microvilli at their apical surface. Some of the unciliated cells occasionally develop a basal pole that contacts neuropil vessels.i8@ This first category of “tanycytes” observed in the ventrolateral walls of the third ventricle is characterized by long cytoplasmic shafts but differs from the typical kind of tanycytes in the median eminence. The latter project their basal processes towards the hypophyseal portal vessels, 993

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they frequently display spherical protrusions at their apical surface1.‘4,30,46 and are associated by small, tight junctions.13 Apart from these morphological criteria, immunological reactivities have been described in several ependymal cell types. Positive reactions were obtained with antibodies directed against either glial fibrillary acidic protein (GFAP)‘0~“~26*42or Slm protein 9.“. More recently, the occurrence of keratin was reported in adult choroid plexus and ependyma in man and rat and in human tumoral cells derived from these organs. 3’ Ad~tiona~y, positive reactions have also been noted on ependymal cells in vim, using several monoclonal antibodies raised against cerebellar (Cl”, H8, H1916) or ependymal antigens (Epen145). The present study was designed to develop a diffe~ntiat~ in vitro system to gain deeper in~gh~ into the cell biology of ependyma. In this line of research, long-term primary cultures of hypothalamus and choroid plexus were raised in conditions allowing in vitro differentiation of ependymocytes. From earlier work it was known that ependymal cells can be found in primary cultures of fetal hypothalamic cells where small areas containing two types of ependymal cells-ciliated and unciliated cells-have been described by conventional electron microscopy. w Enriched ependymal cell cultures were obtained from cerebral hemispheres28*“8which contained mainly ciliated ependymal c&s with a positive reaction to GFAP antibodies.48 As ciliated cells constitute only part of the ependymal interface between neuropil and CSF, we decided to develop efficient methods for culturing ependymal cells from fetal hypothalamus or choroid plexus in order to select and enrich ependymal oells, and we characterized the various types of ependymal, glial and non-glial cells growing in these cultures. For this purpose, we used a series of polyclonal and monoclonal antibodies directed against glial or nonglial molecular markers, and ultrastructural criteria.

BXPERSMBNTALPR~URD3 Ceil cultures Hypothalami and choroid plexuses were dissected from

15to M-day-old mow fetuses (OFi) or 17 to 18-&y-old rat fetuses (OFA, IFFA CREDO, France). After removing the mertinges, fragments were mineed with scissors and meehanie&y disse&ated by PesDagethrough a Pasteur pig&tee, tapered in a Bunsen flame. Sometimes, a It&nin in&&ion in EDTA (Versene 1:SOOO,aibee Laborator&, U.S.A.) was performed before meehanieal disso&tiim. A&r centrifu8ation (500 x g. S mm), dissociated cells were suspended in various culture media and distributed into Petri dishes to obtain a final concentration of 0.25-0.5 bypothalami per ml or 2.55 choroid pkxuses per ml. Cultures were also developed on glass covers&s under the same conditions. Cultured cells were incubated at 37°C in a 95% air-5% CO, atmosphere under 90% humidity. In all cases, the culture medium was replaced after 2 days and subsequently twice a week. C&s were maintained for as long as 6 months in culture.

Culture media and substrates

Testing the advantages of several culture media and substrates, we focused our study on three methods: (1) Serum-supplemented media (SSM) were composed of 1: I mixtures of Dulbesco’s modified essential medium (DMEM) and Ham’s Fl2, (Gibco Laboratories, U.S.A.), supplemented with 10% fetal calf serum. Twelve batches of fetal calf serum were tested (Boehringer, Flow, Seromed, Irvine and Gibco). Three of them, obtained from Irvine (U.S.A.) or Be&ringer (Mannheim, F.R.G.), provided improved growth and differentiation of ependymal cells. The same serum was also used to coat petri dishes, after ~latin-poly-L-lysine treatment, according to a method previously described for h~tha~c neuron cultures. u Sometimes, serum coating of the culture dishes was replaced by a fibronectin-coating (3 h with serum-free medium containing 10-50 yg/ml human fibronectin, Institut J. Boy, Reims, France). Under these conditions. cells remained alive and grew for 226 months. (2) Serum-free chemically defined media (SFM), previously used for glial cultures,2i~3M were also tested. Growth and differ~tiation of ependymal cells were only obtained with the method of Weibel et al.” Accordingly, cultures were raised for a first week in Dh4EM supplemented with bovine insulin (5 pg/ml, I-5500, Sigma, U.S.A.), fatty-acidfree bovine serum albumin (0.5 mg/ml, A-6003, Sigma, U.S.A.), human transfer& (lOpg/ml, Institut J. Boy, Reims, France) and thrombin (0.5 U/ml, T-6759, Sigma, U.S.A.). and subseouentlv in Wa~ou~s MD 70511(Flow Laboratories, u.K,jsupplemented with insulin, bovine serum albumin and transferrin. Dishes were always coated with fibronectin, as described above. (3) Finally, some cultures were raised with a composite method (SSM-SFM). In brief, cells were allowed to attach for 2-3 days on dishes coated with serum or fibronectin, in a burn-supplements medium, then shifted to Waymou~s MD 705/I supplemented with insulin, bovine serum albumin and transferrin and cultured for several weeks. Phase-contrast microscopy

Growth and differentiation of cultured cells were monitored by phase-contrast microscopy, using an Olympus

0MT2 inverted mieroseepe. Attempts to quantify various eel1 types hong in our cultures were developed by counting numbers of cells present in epithelioid and non-epithelioid measuring the surfaces respectively occupied.

areas

and

Transmission and scarming electron microscopy

Cell cultures were fixed by qlacing the culture medium with 2% ~u~~hyde in 0.1 M cacodylate or phosphate buffer (pH 7.4). After a 30&n &&ion at room temperature, cells still attached to petri dishes were rinsed in buffer and postfixed in 1% osmiumtetroxide but&red with 0.05M sodium barbital at pH 7.5. CZultures wem then dehydrated in an ethanol series. For scanning electron mieroseopy (SEM) processing, culture dishes were desiccated with CO, at the critical point in a Balzrs device, sputtered with gold or platinum, uader a rarefied argon atmosphere and observed with a scanning electron microscope (Cambridge Instruments, U.K.), at 15 or 25 kV accelerating voltages. For transmission electron mk==oPY (TEM) pnnrsoin& dehydrated cells were embedded in Epon resin 6, sitn in the dishes. After Epon poIymerixation at 69°C for 24h, cell areas were selected with an inverted pbase+om.rast microscope, punched out and m-embedded either PergenbiaJar to or parallel with the bottom of the culture dishes. Blocks containing selected areas were cut and sections were contrasted with many1 acetate and lead citrate before observation with a Jeol200 CX electron microscope, under a NO-kV accelerating voltage.

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Fig. I. Cultured hypothalamic cells, observed by phase-contrast microscopy. (a, b) In 8-day-old cultures epithelioid cells were frequent. Some were dark-lined @uge arrow). Others were organized in round areas with mitoses (smaU arrow). These areas (E) were identifrad as ependymal and were surrounded by glial cells (G). (c) In 15day-old cultures, multiple epithelioid areas (E) had started to grow. They were inserted in a glial population (G). (d) AtIer 21 days in culture, hypothalamic cells were organized in large areas of ependymal cells (F$ and strands of glial ~4s (G). Neurons, still recognizable in (a). had completely disappeared in (c) and (d). (a. b) x 276; (c) x 138; (d) x 92.

Even tbougb a few neurons were occasionally observed on the surface of younger cultures, they bad completely disappeared within IS days. Cell counts in 21-, 45- and 6O-day-old cultures in SSM, SFM or SSMSFM conditions displayed large variations, which made it difficult to obtain reliable growth rates of the d#erent cell types. The only conclusion was that epitbelioid areas always contained more than 75% of the cultured cells, in all conditions considered. hmnnocy~ochemicfzl characterization of ependymal and non-ependywud cells Very few specific markers have been so far described in oiuo in ependymal cells of rat and mouse brain. In the same line of research, we used, in cultured hypothalamic and cboroidal cells, firstly antibodies raised against cytoskektal proteins contamed in intermediate filaments, ciliary structures, or microfilaments (Figs 2 and 3), then a series of monoclonal antibodies against cerebellar epitopes pre-

viously reported to stain ependymal cells (Figs 4 and 5). and, finally, a few antibodies that recognize fibroblasts, endotbelial cells and oligodendrocytes. No staining was obtained with anti-MBP antibodies,2* which demonstrated a lack of myelinproducing oligodendrocytes in our cultures. Cytoskeletal markers Intermediate filament proteins. These were investigated by immunofluorea~~~. whereas no positive staining was obtained with antibodies against desmin or neurotilament proteins, and cytokeratin was weakly detected in both hypothalamic and cboroidal ependymocytes, many cells displayed poaitive reactions with antibodies raised against vimentin and GFAP (Fig. 2). Using both markers in doublestaining reactions, we observed tbe expression of both proteins in small cell aggregates as early as 2 days after the seeding. Vimentin then decmased gradually while GFAP increased in most cells. In lkiay-old cultures, astroglial ails were distinguished by their

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Fig. 2. Double immnnostaining of intermediate filament proteins (GFAP in a, c; vimentin in b, d) in h~~~~ cnhnres. (a, b) After 21 days in cultnre, GFAP and vimentin were sirn~~~~~ly detected in small populations of ependymal cells considered to be tanycytes because of their small cell bodies (filled arrow) and long positive shafts (open arrow). Neighboring celis were mainly vimentin-positive. (cc)After 51 days in culture, GFAP was strongly stained in astroghal cell populations (asterisk), whereas ependymal cells were weakly stained (star). (d) Vimentin was poorly or not expressed in the same cell populations (asterisks, astrocytes; stars, ependymal cells). (a, b) x 274; (c, d) x 470.

strong enrichment in GFAP as well as their typical morphology seen in phase-contrast microscopy (Figs 3c and 4d,e). However, in addition to the GFAPcontaining protoplasmic and fibrous astrocytes, GFAP-positive cells were also observed in the areas of epithelioid cells. Most of these GFAP-positive epitheloid cells had a small “perikaryon” and a long basal process, supported by a dense GFAPcontaining cytoskeleton. These uneiliated cells, which also expressed vimentin, were concentrated in small areas, with their basal cytoplasmic shafts arranged in parallel (Fig. 2a and b). They were identified as “tanycytes”. After 50 days in culture, vimentin was poorly revealed in astroglial cells and in most ependymal cells, whereas the tanycyte shafts remained vimentinpositive. GFAP was still detectable in both astroglial and tanycyte processes. The latter cells were identified by their single positive process and by their epithelial arrangement Observed by phase-contrast microscopy. Ciliated and unciliated ependymal cells displayed a

weaker reactivity to anti-GFAP antibodies and no staining at all with anti-vimentin antibodies (Fig. Zc and d). Other epithelioid cells, resembling flat endothelial cells, and dispersed round cells, resembling macrophages, were vimentin-positive and GFAPnegative. On the other hand, neither anti-vim~tin nor antiGFAP antibodies stained choroidal ependymocytes. Other cytoskeletal proteins (Fig. 3). Proteins presumably involved in the highly organized structures of ependymal cells were also investigated. Actin and myosin were found in all epithelioid cell types, whether ependymal or non~nd~al (Fig. 3a and b). Whereas actin was preferentially localized near junctional complexes and displayed a polygonal pattern, myosin was found with the same peripheral distribution and close to the ciliary structures of ciliated ependymocytes. Mon~lonal antibodies directed against ciliary rootlet proteins (CC3 103 were used in a doublestaining method with the anti-GFAP antibodies (Fig.

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Fig. 3. Immunodetection of some cytoskeletal components in i-month-old hypothalamic cuhures (a, actin; b, myosin; d, f, g, ciliary rootlets; c, e, h, double”stainjng with GFAP antibodies). {a) Xn the ependymal cells. as in most epithelial cells, ~Iy~~on~ antibodiesdire&d against actin reacted with cytop~smi~ structures related to intercellular contacts and with the apical surface, when focused upon. (b) Myosin, detected by the 2F 12 monoclonal antibody, was also present in the junctional areas and in the ciliary complexes of ciliated ependymal celk (stars). (cl d) CC3 IO monoclonal antibody which detects a cihary rootlet protein (in d), along with anti-GFAP polyclonal antibodies (in c), was used in double-stai~ng reactions to &arty identify the ciliated ependymaf cells @tars). GFAP was weakly detected in the same cells next to the rootlets detected by CC3 IO. The astrogliai cell population (asterisk) was strongly stained by anti-GFAP antibodies, whereas CC3 10 only reacted with single points in these cells. (e, f) Some ependymaI cehs reacted strongly with both CC3 IO and anti-GFAP antibodies. The eel1 (indicted by arrow}, with its negative nucleus (n), shows a network of GFAP-containing intermediate filaments, which fills its long cytoplasmic shaft (in e), and cihary rootlets above the nucleus (in f). (g, h) CC3 10 also demonstrated the occurrence of small “follicular” structures (arrows) formed by ciliated ependymai cells (in g) embedded in astroglia cell components (in ~)~~) x436; (b) x 407; (c, d) x 388; (e, f) x 698; (g, b)

Choroidal and hy~thaIamjc ~d~~yt~

in culture

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341). CC3 10 twxted with an abundant network of react with the ependyma of the third and fourth B Cl,* HI9 and H8 antibodies.*” These ~~~~ in the apex of ciliated a~nd~~~ and ventricles, i,” provided, themfore, an excellent marker for this c&l antibodies do not cross-react with GFAP. type* CC3 t&positive cells often ~~~~~pie~ In numerous epithelioid cells of hypothahxmic cuiGF~-~~vc or contained scarce GF~-~~ti~ tures, Cl labeled c~op~~~c ‘matrix ~rn~en~, structures_ By contrast, GFAPqositive cells, es- probably related to the cytoske~eton (Fig. 4). CI also pecially ~tr~~cs and tanycytes, poorly reacted to reacted with axonema in ciliated ependymal c&s CC3 i0. A few c&h had both ciiiary rootlets and lang (Fig. 4a) and with the apical surface of uncitiated GF~~n~~~ basal shafts (Fig. 3e and f), 1x1 ependymal c&Is (Fig. 4b). Findly, the epitope recogaddition, CC3 10 detected ciliary rootlets at the c&e nized by Cl was also found in astroglial cells~ In of smail “Follicles” embedded in the astroglial corn- agreement with those obtained &Jv&o by Sommcr et pment of the cultures. These ciliated cavities, &.,* these results confirm that Cl presumably detects confirmed by phase-contrast observation, were simi-cytoskeletal cpitopes of unknown nature prestmt in lar to those dwxibed in ependyma in five under both @aI and ependymai s&s. H19, an antibody known to recognize a few minor normal and pathological ~ndjtia~s.~ proteins (23,oocr(ro,~ mol. wt) and a major 43,0(w) ~~~~Q~~ mtibodies ii.9 ~~~~~ markers mol. wt protein in Western blotting, strongly st&ned cc&. The endear We have ex#ored the reac~~ty of the cultured a fibrous structure in stroke c&s to three monoclonal antibodii raised against cell areas of h~~~arn~G cultures weakly reacted mouse cerebellum, and which have been shown to (Fig. 4c), while a strong reaction was obtained with

Fig. 4. ~on~~~nal ant%odies as ~~nd~~ ceil markers in i-month-old h~~arn~~ and ~h~r~~ vultures (a, b, Cl; c, HIP; d, anti-GFAF antibodies; e, phase-contrast). (a) cf mon&.mal antibody reacted with cytoskeletat ~rn~u~~ts of dIiated ependymal c&s. The axowma themselves raw&d strongly to this antibody.@)In uncifiatedependymocytes, Cl ma&d with the apical region of the cell, in a dotted pattern. (c, d, e). Double irnrnun~~~ng and phaswwtrast observation of apcndymat (star) and astroglial cells (asterisk) showed a su~~m~osition of structuns recognized by W19 and anti-OF@, WI9 produced no staining in the ciliated ependymal population. (a, b) x 532; (c?d, e) x 352.

J. GABRIOh-

loo0

cl

ui

Fig. 5. identification of endothelial cells by the MESA 1monoclomtf antibody (a, b) and immunodetection of fibronectin (c, d) in 1-month-old hypothalamic cukures. (a) The MESA 1monoclonal antibody strongly bound to surface epitopcs of the flat, poiygonal endothelial cells, (b) whereas it did no: react with ependymal cells. (c) In fibroblastic areas, fibronectin was organized in fibroi structures distributed over GFAP-negative spreading alls. (d) Under T&on X100-perme&iiized ependymocytcs, fibronectin was detected as a diffuse component, also present in the lateral interalluiar spaces. (a) x 301; (b) x 376; (c) x282; (d) x376.

ependymocytes. Astroglial structures stained by HI9 (Fig. 4~) coincided closely with GFAP-positive reactions (Fig. 4d). suggesting that, at least in astrocytes, the recognized epitopes may be associated with GF~-inte~ia~ @amen% However, the H19 reactivity of choroidai ceils that did not express GFAP, is unclear. H8, an antibody known to recognize 58,000 and 68,000 mok wt proteins in adult rat and mouse brains and a 62,000 mol. wt protein in new-born brains, did not react with any cultured cell types, apart from an extracelhdar component present on astrocytes and ependymal cells.

choroidal

&dothelial and extracellular matrix markers MESA i, a monoclonal antibody marker for the endotheiiaf cell~,‘~~~’ co&rmed the smali extent of this cell type noted in hypothalamic and choroidal cultures (Fig. 5a). even though the positive areas were larger in choroidal than in hypothalamic cultures. As expected, the staining corresponded to the flat polygonal cells which are outhned by dark intercellular

contacts when observed by phase-contrast microscopy. No other cell type reacted with this antibody (Fig. Sb). Anti-fibronectin antibodies were used to reveal the presenoe of fibroblasts. Positive extracelhdar g&s were observed on the surface of typical fibrobiasts, which appeared dispersed in both Rbronectin- or serum-coated cultuqzs. Scarce in I)-day-old cultures, positive fibers amply covered the few flattened fibrobiasts occurring in the SO-day-old cultures (Fig. SC), A positive staining was also observed on endothebal ceIis, as evidenced with a MESA l/anti-Fib double staining. Surprisingly, whatever the culture conditions, a positive reaction was noted under epithciial layers consisting of ciliated and unciiiated ependymocytes. There, fibronectin displayed a diffuse honeycomb rather than a fibrous pattern (Fig. 5d). Even when the fibers attached to fibroblasts increased in older cultures, a diffuse arrangement of nonfibrous fibronectin was observed under the ependymal cells. It was also interesting to note that the basal sides of the ciliated ependyrnal cells appeared

Choroidal and hypothalamic ependymocytes in culture surprisingly poorer in fibronectin than those of unciliated cells, suggesting possible differences in fibronectin receptor amounts between both cell types. Finally, fibronectin-positive cells were never observed within, or under, the ependymal layers themselves. In choroidal cultures, fibronectin was always extensively detected, either as fibers associated with fibroblasts or as a diffuse layer under the ependymal cells. Contrary to hypothalamic cultures, the amounts of fibronectin increased markedly with time. This could be related to a higher ratio of fibroblasts in choroidal cultures. Laminin was detected with specific antibodies in hypothalamic and choroidal cultures after 8 days in vitro. However, whereas positive reactions disappeared in all cell types in older hypothalamic cultures, laminin remained visible under ependymocytes of choroidal 3%day-old cultures, suggesting a persistent production of laminin by the choroidal cultured cells. Scanning electron microscope observations of the apical; surfaces In addition to clearcut ciliated ependymal cells displaying a central bundle of 20-50 long kinocilia (Fig. 6a-c), several types of unciliated or poorly ciliated cells were identified in hypothalamic cultures. Three types of “epithelial” cells were thus recognized according to their polygonal contacts and different sorts of microvilli. Some unciliated cells were only covered with long and slim apical microvilli, while others bore small spherical extensions, in addition to microvilli and l-2 kinocilia (Fig. 6d and e). These differentiated surfaces resembled those described on tanycytes in rat or human median eminence.‘4J” All the ependymal cells were easily distinguished from the endothelial cells, which appeared as flattened cells with small and scarce microvilli, and from the irregularly shaped fibroblasts (Fig. 6f). A few macrophages were recognized by their round shape and rufIled membranes (Fig. 6f). Choroidal ependymocytes were frequently clustered in irregular folds and covered by a number of bulbous and irregular apical microvilli. Some cells tended to spread over the culture substrate at the periphery of the aggregates. In general, microvilli were scarce on these cells (Fig. 6g and h). Fibroblasts, endothelial cells and macrophages were also identified in the choroidal cultures. Transmission electron microscope observations on cultured ependymal cells Epithelioid areas of cultured hypothalamic and choroidal cells selected by phase-contrast microscopy were studied by TEM, using transverse sections to observe apical and basolateral differentiations. As early as 8 days of culture, a number of “epithelial” cells were discernible in the hypothalamic cultures (Fig. 7a). They were characterized by junctional complexes, apical microvilli, and possibly ciliary processes (basal bodies and axonema). After 15 days,

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ciliated ependymocytes were easily recognized (Fig. 7b). Several types of unciliated cells were also observed in hypothalamic cultures. Most of them resembled unciliatedependymal cells, with typical apical microvilli and junctional complexes (Fig. 7~). Some dispersed unciliated cells had spherical extensions on their apical surface similar to those described in vivo on the surface of tanycytes,’ whereas their basolateral cytoplasm formed long processes under an ependymal layer (Fig. 7e and f). Choroidal ependymocytes were clearly identified by their typical bulbous microvilli, their cuboidal shape and their junctional complexes (Fig. 7g and h). Very few ciliated cells were observed here in cultures, as is the case in intact choroid plexus.” Ependymal cells were always connected by highly differentiated junctional complexes (Fig. 7c,d and h). Their nature differed considerably according to the cell types. Typical epithelial junctions were found between choroidal ependymocytes (Fig. 7h). Large zonulae aakentes and gap junctions distributed along interdigitated and shingled intercellular clefts were frequently observed between several ciliated cells, as previously described by Benda et al.’ and Tier-Vidal et ai.& Moreover, small tight junctions were discernible between ciliated and/or unciliated ependymal cells in hypothalamic cultures (Fig. 7d). They resembled those described in vivo between ependymal cells of the infundibular floor of the third ventricle. Ependymal cells were often directly attached to serum- or fibronectin-coated surfaces, without any interposed cells, in both hypothalamic and choroidal cultures (Fig. 7h). However, some ependymal cell areas organized in monolayers were occasionally detached from the culture substrate, which was itself covered by a flat and irregular sheet of spread cells (Fig. 7a and e). The ‘occurrence of such spaces under ependymal monolayers may suggest the probable tightness of junctional complexes in some areas, the functional aspects of which are now under investigation. To sum up, most of the results and all of the selected criteria are given in Table 2. Cell shape, as revealed by phase-contrast microscopy, molecular markers of differentiation and ultrastructural features were used to distinguish five cell types which displayed a typical appearance of simple non-stratified “epithelial” cells. One of these types, essentially identified by MESA 1, was found to be endothelial, and the others were ependymal. Choroidal ependymocytes, ciliated ependymocytes and tanycytes could be distinguished from undifferentiated ependymal cells using several markers and/or morphological features. DISCUSSION

Morphological and immunocytochemical criteria were used to characterize the different cell types

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Table 2. Criteria for the characterization of hypothalamic and choroidal cell types in l-month-old cultures Cell shape Astrocytes Endothelial cells Fibroblasts Macrophages Ependymocytes ciliated unciliated “Tanycytes” Choroidal

xVim

xGFAP

Fibrous Epithelial* Fibrous Round

+ + + +

+

Epithelial* Epithelial* Epithelial’ Epithelial*

+ + -

+ + + -

-

CC3 10 -

MESA 1

xFib

+

+ + -

xLam -

+ + +

+

+ -

-

Surface t&odes ruiiies Cilia, mv mv mv, blebs mvt

*According to morphological rather than embryological criteria. tTypical club-shaped microvilli (Maxwell and Ped.

obtained in cultures derived from mouse or rat fetal hypothalami or choroid plexuses. Several types of (i.e. glial, ependymal and non-ependymal fibroblastic, endothelial) cells were evidenced in our culture systems, which reflects the heterogeneity described in uivo in both brain regions.20*“*37~39 A first result concerned the total lack of myelin-producing oligodendrocytes in these cultures, which is in agreement with earlier in uiuo observations showing that both the hypothalamus and the choroid plexus are poorly or not myelinated.12J9 The immunological and morphological criteria, summarized in Table 2, enables us to distinguish different cell types. Some of them provided an easy identification for certain cell types, e.g. the CC3 lo-positive reaction for ciliated ependymal cells, and the MESA l-positive reaction for endothelial cells. However, most cell types required two or three criteria for identification, e.g. tanycytes appeared as GFAP-positive epithelial cells with apical microvilli and spherical bulbs, and astrocytes as GFAP-positive non-epithelial cells. As described by Schnitzer ef ~1.4~in other brain primary cultures, hypothalamic ependymal cells displayed an early expression of intermediate filament markers. Vimentin was expressed well before the onset of GFAP and decreased in older cultures. On the other hand, GFAP was more strongly expressed after 15 days in culture. Moreover, differences were noted between ependymal and glial cells. Only tanycytes and astrocytes displayed a strong reactivity to anti-GFAP antibodies. Keratin which was recently detected in normal and tumoral ependyma,31 was

weakly expressed in our cultures. Cl monoclonal antibodiesU reacted with cultured ependymal cells, but further investigations are required to clarify the nature of the component detected by this antibody and its specificity. On the other hand, Hl9- and HS-detected epitopes16 were missing in cultured ependymal cells. The interactions between substrates and ependymal cells raise a series of interesting problems. Two situations were observed by TEM: hypothalamic and choroidal ependymal cells were either directly attached to, or detached from the culture substrate, which in the latter case was then covered by an additional cell layer. This additional cell type could be characterized by none of the immunolabels used. In cerebellar ependymal cultures, such cells have been identified as astroglial components on the basis of morphological criteria,* but this appears very unlikely in hypothalamic ependymal cultures. Since fibronectin markedly improves the initial steps of ependymal cultures’* (and the present study), its presence under the ependymal sheets suggests a direct role in the epithelial organization and in the stabilization of cultured ependymal cells. However, it was not clear whether the detected fibronectin originated essentially from the coating of the dishes (calf serum or human fibronectin) rather than from the cultured cells themselves, as shown for cultured astrocyte# or cultured epithelial non-ependymal cells.” The occurrence of contacts between the basolateral membrane domains of ependymal cells and the basal lamina has been postulated in vioo ’ and in oitro * but no clearcut evidence along these lines was ob-

Fig. 6. Surface of cultured hypothalamic and choroidal ependymal cells observed by scanning electron microscopy, after 21 days in culture. (a, b) Different types of epithelioid areas were observed. Some ependymocytes were ciliated @led circle), bearing a central brush of kinocilia (filled arrow) or were unciliated (asterisk), bearing a single kinocilium (open arrow). (c) About twenty to fifty cilia were present at the apex of the ciliated ependymocytes. (d, e) Unciliated ependymocyte surfaces (asterisk) were covered with microvilli, sometimes intermingled with small and spherical protrusions and possibly with one or two cilia. (f) Fibroblasts were irregularly delineated (asterisk) and macrophages (large arrow) were scarcely observed. (g, h) Choroidal ependymocytes, still organized in folds or caulifiower-shaped structures (filled circle), presented numerous and irregular microvilli at their convex apical surface. At the periphery of the choroidal aggregate, the cells tended to spread (asterisk). (a, g) Bars = 50 pm; (b. d, f) bars = 10 pm; (c, e, h) bars = 2 pm.

^,

Fig. 7.

Choroidal and hypothalamic ependymocytes in culture tained in our cell cultures. The progressive disappearance of laminin in hypothalamic cultures was noted from the first days in vitro. Its complete absence after 15 days confirms that laminin was expressed transiently, as previously found for hypothalamic” or brait?’ culture astrocytes. This suggests that laminin may be needed only for the first stages of culture. On the other hand, both laminin and fibronectin were detected under choroidal ependymocytes throughout culturing, which is in agreement with in vivo observations.29.37s47 We were unable to quantify the growth of the various cell types that were morphologically or immunologically identified. Whatever the culture conditions, it appeared unreliable to measure the growth rates of any cell type. A large variation was noted in the number of astrocytes, endothelial cells, fibroblasts and macrophages in the different cultures, but the amount of ependymal cells, as appreciated by cell numbers of epithelioid areas, always exceeded 75% of the cells in SSM, SFM or SSM-SFM cultures and only some of these were ciliated. In SFM cultures derived from cerebral hemispheres of new-born rats, Weibel et al.@’found that more than 90% of the cells were ciliated ependymocytes. These differences may be related to differences in the amounts of ciliated cells in the walls of intact lateral ventricleP compared to the walls of intact third ventricle.“.39 The presence of tanycytes in hypothalamic cultures, in

1005

addition to ciliated ependymocytes, has previously been suggested by Tixier-Vidal et aL6 on the basis of ultrastructural features. In spite of their variability, the cultured hypothalamic and choroidal ependymocytes shown in this study display a remarkable resemblance to ependymocytes observed in situ, in terms of specific marker expression and of specific ultrastructural features. In particular, they were able to re-express ependymal cell polarity and differentiations. Morphological and immunological criteria enabled us to identify several ependymal cell types and, consequently, such suitable models

cultures may constitute simplified to explore specific ependymal cell

functions. Acknowledgemenfs-Mrs B. Nguyen-Than-Da0 and V. Chalvon (UA 1197, USTL, Montpellier) are gratefully acknowledged for their skillful technical assistance. We also thank Mr Nabias (Ecole de Chimie, Montpellier) and Mr Selzner (SCME, USTL, Montpellier) for their help in scanning and transmission electron microscopies. Antibodies used in this work were kindly provided by A. Giraud (anti-fibronectin), E. Karsenti (anti-a&i), M. Schachner (Cl), G. Tramu and A. Delacourte (anti-GFAP), M. Vigny (anti-laminin). We would like to thank them for their generous gifts. This work was supported by grants from Institut National de la Sante et de la Recherche Mbdicale (CRE 85-6012) and from the French “Fondation pour la Recherche Medicale” and from the CNRS (UA 1I1 5 and 1197).

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Fig. 7. Transverse sections of hypothalamic and choroidal ependymal layers in culture, observed by transmission electron microscopy. (a) After 15 days in culture, epithelioid cells (E) with apical regions associated by junctional complexes (j), formed a continuous layer detached from the substrate by a sub-epithelial space @es). The substrate (S) was covered with a monolayer of very flattened cells. Apical differentiations of ependymal cells were still scarce, comprising only long microvilli protruding into the culture medium (M) and a centriolar complex (c). (b) Ciliated ependymocytes (E), which were frequently observed in 21-day-old hypothalamic cultures, were characterized by numerous kinocilia (k) and microvilli (mv), protruding into the medium (M) and by typical junctional complexes (j). (c, d) At the apex of ciliated or unciliated ependymal cells, highly differentiated junctional complexes consisted of zonulae adherentes (za) and gap junctions (g) distributed along folds forming intercelhtlar contacts. Some small “tight” contacts (arrow) were also observed between ciliated cells (k, kinocilimn; mv, microvilh; M, medium). (e, f) In this u&hated ependymal layer, a tanycyte-like cell (T) could be distinguished between unciliated cells (E) by its long basolateral process @) and the numerous irregular blebs (b) on its apical surface (Ses, sub-epithelial space; S, substrate; j, intercellular junctions). (g, h) After 21 days in’culture, aggregates of cultured choroidal ependymocytes (CE) are characterized by their cuboidal shape and their numerous and irregular microvilh (mv) protruding into the culture medium (M). At the primary of the aggregate, spreading ependymocytes were in close contact with the substrate (S), with no other cell layer interposed. (a, c, d, f, h) Bar = 1 pm; (b, e, g) bar = 2 Mm.

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