Neuroseience Letters, 30 ( | 982) ! 79-182 EIseder/North-Holland Scientific Publi~ters Ltd.

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CELL TYPE-SI~'EC"~'ICITY OF EPIDERMAL GROWTIt FACTOR (F~ F) IlINDING,IN!PRIMAIRY C|:' ~LTURES ~ " OF EARLY POSTNATAL MOUSECERF~ELLUM~ . . . . . . . .

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ACHIM LEUTZ and MELI2"TA SCHACHNER*

Department o f Neurobiology, Urdversity o f Heide!be~, I.,, r. ,o ~,,,~ or ... P ~ n .... m. . Feld 504, 6900 Heidelberg 1 (F.R.G.)

(Received January 25th, 1982; Revised! version received March 12th, 1982; Accepteo March 16th, 1982)

Key ~vords: epidermal growth factor - hormone r,:ceptors - ceil type:specifi:ity - a~;trocytes - primary cultures - mouse cerebellum

Binding of 125I~labeled epidermal growth factor IEGF) to cells ofcultured early postnatal mouse cerebellar cells was investigated by autoradiography in conjunction with cell type-specific immuno- labeling of neurons, astrocytes and oligodendrocyt,::s. By use of tetanus to×in for recognition of neurons and glia~ fibrillary acidic (GFA) protein and 04 ar~i~n as markers for astrocytes and oligodendrocytes, respectively, it could be .shown that oligodendro~:~::~ do not express receptors for EGF within ttae limit~, of sensitivity of the autoradiographic method, and ~i-~a t !ess than 1% of all sma!I neurons (mo.,dty granule cells) and 50-90% of all GFA protein-positive astrocytes show detectable levels of EGF binding.

In a recent report we have shown that astrocytes in primary cultures of early postnatal mouse eereMllUm express receptors for epidermal ~ growth factor (EGF) and EGF-induc~; increasedlevels o f DNA syntiiesis [5]~ :Other neural cell types in these cultures did:n0t:show enhanced synthesis of::i~NAi ~ e present study was undertaken to investigate I. Whethe:r neurons and!bligodendrocytesl bind EGF and whether all glial fibriil~ry acidic (GFA) pr0tein-pOsitive cerebeUar astrocytes or only a subpopulation of these are capable of binding EGF.

Primary rnonolayer cultures of 4-5-day-old C57BL/6J mouse cerebella were mainzained in vi t rofor 3 days as described previously [5, 7|. For immunofiuore- scence of cultures incombination with autoradiography, the procedure of Leutz and Schachner [5] was used, Tetanus toxin was ,~sed to label neurons, g~ia! f~briHary acidic (GFA) protein to mark astrocytes, 04 antigen t<'~ recognize oligo.~endro.ytcs, fibronectin to m~rk fibroblasts or fibroblast-likc ceils, and vimc~~ti~ ~:o iabe~ astrc.ytes and fibroblasts [I, 3, 6,10]. Iodination of EGF with ~2_~ was pe~:formed

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*To w~mm correspondence should-be sent.

0304.3940/82/0000-O0~/$ 02.75 (~) Elsevier/North-Holland Scientific Publishers Ltd.

as described previously [5]. The ~ 25 !-labeled EGF had a specific activity of t50-20~

#Ci/~g. GFA protein-positive astrecyte~ which express more EGF receptors than other~

ar~; shown m Figo la and b. Tt~e ~ercentage of EGF binding astrocytes among a~ GFA protein-vos~t~ve ce~s ~ 5 0 - ~ . ~t ~ not known why t ~ s ~ r c e n ~ g e v " " between experiments. Fibronectin-pes~five fibroblasts or fibroblast-~ke cells express receptors (not ~hown) as has been shown previously for other types of fibreblasts in vitro [2]. Vim::ntin-posifive cel~.s comprising fibroblasts and more and less mature astrocytes [6] also bind EGF. A very small percentage of tetanus toxin-positive neurons (,less than 1%) carry EGF receptors (Fig. lc and d). O4 antigen-positive oligodendrocytes comprising the more and tess mature oligodendrocytes [9] have

Fig. I. Combined autoradiography for [12Sl]EGF binding and immuaofluorescencc for GFA protein and tetanus toxin receptors. Cells were maintained as described previously [5, 7]. After 3 days in culture cells ~n ~overslips were washed and incabated with culture medh~m comaining 1 x lO-S-:; x 10 -s M 112: i]EGF at 37 °C for 60 mix h~ the C ~ incubator. Ce~ls were tben washed and processed for indirect immunofluorescence as described previously [5]. Competition experiments with a lO0-fold excess of ,antabeled EGF showed only background leve!s of ~2~1 binding, a: i~nunofluorescence in~ge of GFA protein-positive cells (arrows~. b: phase-contrast autoradiomicrograph of the same fiekt as ~hown in a. Two heavi!y [~2H]EGF-labeled as~ro~'~es {~arge arrows). Astrocy'~e marked by small arrow h, ve~ weakly labeled° c: immunofluorescence image of tetanus toxin receptor-positive cells with small ceil bodies, d: phase-comras~ au~oradiomicrograph of the field as shown in c. Note the |~zSl]EGF-labeling of cell bodies and some processes. In b and d, due ¢~ the presence of pho~ographic emulsion on the cultured cei~s~ the phase-contrast image appeals le~s dea~. a and b, x 340; c and d, y, 420°

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never b ~ n found to react with EGF (Fig. 2a and b) under the culture conditions used and within the levels of sensitivity of the autoradiographic method.

The present study has shown that, among ~he neural cells of early postna~at mouse cerebellum, some, but not all, astrocytes and a very small percentage of neurons brad EGF. This is the first evidence that a small fraction of neurons are able to bind EGF,: which, by the methods of binding assays with radioactive EGF on bulk-enriched neural populations, would have been missed in background levels. The demonstration of cellular heterogeneity within the popalatiom of astrocytes and neurons is a novel finding also and raises several quest:ions. Does this heterogeneity reflect a particular state during the cell cycle or distinct differences in the differentiated state between the subpopulations? These possibilities can be investigated once labeling indices for the proliferating cell types are known for the present culture conditions and when a correlation between astrocytic subpopula- tions can be established using the markers vimentin [6], and M1 and C1 antigens [4, 8]. A correlation with the subpopulations o~' cerebellar neurons will be more difficult, however, since markers for the neuronal cell types of ~he undifferentiated cerebellar cortex in vitro are yet to be found. I[~. is hoped that this question will be answered by autoradiographic evidence using EGF in the intact tissue. The possibility to recognize differences in cell surface receptors among neural sub° :)opulations opens new avenues in the l~reparative separation and isolation of more restrictea cellular subsets.

Fig. 2. Combined autoradiography for [~25t]EGF binding and immunofluorescence for 04 antigen. Cetl cu!ture, immunofluorescence and autoradiography were performe(! a~ de~.r~bed in Fig. I a: immuno- fluore~ence irosage of 04 antigen-positive cell b: phaseocomras~_ aumradiograph of the ;ame fie~d as

shown in a. x 720.

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