ABILITY OF A CELL-SURFACE PROTEIN PRODUCED BY FIBROBLASTS TO MODIFY TISSUE AFFINITY BEHAVIOUR OF CARDIAC MYOCYTES

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1 J. Cell Set. 44, (198) 263 Printed in Great Britain Company of Biologist! Limited ig8o ABILITY OF A CELL-SURFACE PROTEIN PRODUCED BY FIBROBLASTS TO MODIFY TISSUE AFFINITY BEHAVIOUR OF CARDIAC MYOCYTES PETER B. ARMSTRONG Department of Zoology, University of California, Davis, CA 95616, U.S.A. SUMMARY When fragments of 2 dissimilar embryonic tissues are placed in contact in organ culture, cells of one fragment migrate over the surface of the second to envelop it. Holtfreter proposed that this behaviour was in response to 'tissue affinities'. He proposed that these also play important roles in the control of morphogenetic cell movement during development. The present study demonstrates that the heart fibroblast, present as a minority cell type in heart ventricle, can modify the affinity behaviour of heart tissue. The fibroblast effect appears to be mediated by a factor that can be extracted from living fibroblast monolayers by 1 M urea. The factor is a cellsurface protein since it is absent in monolayers which had been treated with trypsin prior to extraction. INTRODUCTION When fragments of 2 dissimilar embryonic tissues are placed in contact in organ culture, cells of one fragment migrate over the surface of the other to cover it partially or completely. For most of the combinations of tissues that have been studied, the spreading of one tissue over its partner fragment is regular and reproducible in regard to which tissue does the spreading and which tissue ends up located internally (Steinberg, 197). Based on studies of cell behaviour in organ culture, Holtfreter (1939) formulated the concept of' tissue affinities' by which was meant the factors that direct the cell movements that result in spreading and the establishment of preferred associations of tissues. Since the spreading behaviour displayed in organ culture mimicked the spreading of cell sheets during embryonic morphogenesis, Holtfreter further proposed that the tissue affinities revealed in vitro play a role in the control of morphogenetic cell movements in vivo (Holtfreter, 1939; Townes & Holtfreter, 1955)- One situation where the tissue spreading patterns are not always uniform is in combinations involving chick embryo heart as one of the tissues (Armstrong & Niederman, 1972; Lesseps, 1973; Lesseps & Brown, 1974; Lesseps & Glowacki, 1974; Wiseman, Steinberg & Phillips, 1972). For a given pairing of tissues, heart sometimes envelops its partner tissue; in other individual pairs the partner tissue covers heart. This behaviour has been described for heart-liver (Wiseman et al. 1972), heart-limb chondroblast and heart-pigmented retina (Armstrong & Niederman, 1972)

2 264 P. B. Armstrong combinations. Heart aggregates that have been produced by reaggregation of dispersed cells and fragments tested immediately following excision from the embryo tend to envelop the partner tissue, whereas fragments maintained in organ culture for 2-5 days prior to construction of fragment pairs tend to be enveloped by the partner tissue (Wiseman et al. 1972). In pairings in which both fragments are ventricle tissue, freshly excised ventricle spreads over ventricle that had been cultured 2-5 days prior to fusion (Phillips, Wiseman & Steinberg, 1977). The phenomenon has been designated as 'position reversal' by Wiseman et al. (1972) and has been ascribed to increases in adhesiveness of heart cells during residence in organ culture (Phillips et al. 1977)- Since chick embryo heart ventricle contains several cell types (the most abundant are the myocytes (about 7-8% of heart cells) and fibroblasts (about 2-3%) (De Haan, 1967)), it is important to ascertain which cell type is responsible for the irregular spreading behaviour. The present study analysed the behaviour of heartheart aggregate pairs in which one of the members had been modified. Based on my observations, it is proposed that the position-reversal phenomenon results from activities of the fibroblasts, and that these activities are mediated by a cell-surface protein that can be extracted from fibroblast monolayers using a procedure similar to one designed to extract the cell-surface and extracellular matrix protein, fibronectin (Yamada, Yamada & Pastan, 1975). MATERIALS AND METHODS Preparation of tissue fragments Ten-day chick embryo heart ventricle was disaggregated in 1% trypsin (Difco 1:25) in Ca-, Mg-free phosphate-buffered saline with 2 mm Na s EDTA as described previously (Armstrong, 197). Suspensions of myocytes were freed of most of the fibroblasts by plating cell suspensions onto tissue culture grade plastic Petri plates (Falcon Plastics) for 9 min at 37 C. During this period the fibroblasts attach to the plastic dish whereas the myocytes can be removed when the medium is decanted (Armstrong, 1978; Armstrong & Armstrong, 1978, 1979). The culture medium at this and all succeeding stages was Dulbecco-modified Eagle's medium + 1% heat-inactivated chicken serum. Myocyte aggregates were prepared by gentle centrifugation of myocyte cell suspensions in 16-ml screw-cap test tubes (Pyrex no. 9826) followed by stationary incubation for 3 6 h at 37 C. The coherent pellets were then chopped into smaller fragments which were allowed to round up during an overnight incubation in shaker-flask culture. Myocytes were labelled by including 2 ftci/ml [*H]leucine in the culture medium during the overnight incubation of the myocyte aggregates in shaker flask culture. Although the specific activity of the labelled leucine was low (the culture medium contained o-8 mm unlabelled leucine), this regimen produced adequate cytoplasmic labelling for cell identification by autoradiography. Fusion of fragment pairs Pairs of spherical aggregates (one labelled, one unlabelled) were placed in hanging drops of culture medium. The aggregates came in contact at the bottom of the hanging drop and within 3-6 h adhered tightly enough to allow the aggregate pairs to be removed to shaker-flask culture. Histology Aggregate pairs were cultured for 2 days in shaker-flask culture, then fixed in Bouin's fixative, embedded in paraffin and sectioned at 5 fim. During embedding, each aggregate pair

3 Surface proteins and tissue affinities 265 was oriented so that the plane of section would pass approximately perpendicular to the plane of initial adhesion between partners. All aggregates were serially sectioned so that aggregates that were not so oriented could be identified. Sections were mounted on slides and coated with Kodak NTB-2 liquid emulsion. After a 3-week incubation at 4 C, the emulsion was developed in Dektol as recommended by Kopriwa & Leblond (1962) and the sections stained through the emulsion with haematoxylin and eosin. The section passing midway through the aggregate pair was identified by inspection at low magnification and the identity and degree of spreading of the superficial tissue were scored at high magnification. Slides were scored blind to avoid prejudicing the results. Preparation of fibroblast factor A soluble factor that affected tissue affinity behaviour of myocyte aggregates was prepared by treating confluent heart fibroblast monolayers with a freshly prepared solution containing 1 M urea (Schwarz/Mann, ultrapure), 2% DMSO and 1 mm PMSF (phenylmethyl-sulphonylfluoride, Sigma), dissolved in phosphate-buffered saline, ph 7-3, The monolayers were first washed extensively in phosphate-buffered saline (PBS), then extracted for 2 h. The extract was centrifuged and dialysed at 4 C for 1-2 days against 3 or more changes of phosphate-buffered saline. In some cases, the monolayers were subjected to mild trypsinization (io/ig/mlworthington 2X crystallized trypsin, 243 units/ mg. code TRL, 15 min, 22 C) prior to urea extraction. The dialysed extracts were diluted 1/1 v/v in culture medium and used immediately. Controls were prepared using urea-pmsf-pbs that was treated exactly as above save that serumcoated culture dishes that contained no cells were extracted rather than fibroblast monolayers. The extraction procedure is nearly identical to that used by Yamada et al. (1975) to extract fibronectin from chick embryo fibroblast monolayers. (DMSO which apparently was not included in the extraction medium of Yamada et al. (1975) was added to solubilize the PMSF.) At least a portion of the urea extracted cells were alive, as shown by their ability to grow if replated in fresh medium. RESULTS The goal of the present study was to identify factors important in tissue position reversal. In pursuit of this goal, aggregate pairs were prepared in which both aggregates were heart tissue. Cells of the 2 aggregates were distinguished by autoradiography of tissue sections using phjleucine as a cell label. In most of the trials, 3 classes of aggregate pairs were prepared: unlabelled control aggregates + labelled control aggregates; unlabelled experimental aggregates + labelled controls; and labelled experimental aggregates + unlabelled controls. Involvement of fibroblasts The influence of fibroblasts on spreading behaviour of heart tissue was assessed by fusing myocyte aggregates with either whole ventricle fragments or 'reconstituted ventricle' (a mixed aggregate containing 8% myocyte derived from freshly dissociated and fractionated ventricle and 2% fibroblasts prepared by trypsinizing confluent monolayers). When myocyte aggregates were paired, spreading occurred in fewer than half of the pairs and occurred with approximately equal frequency with the labelled and unlabelled members of the aggregate pairs (Fig. 1, Table 1, row 1). When a myocyte aggregate was combined with an aggregate containing fibroblasts (e.g. either ventricle fragments or reconstituted ventricle), the myocyte aggregate spread over the partner aggregate (Table 1, rows 2-5). This observation shows that fibroblasts, although present only as a minority population in heart tissue, can affect l8 CEL 44

4 266 P. B. Armstrong Fig. I. Planar interface (arrows) between the 2 members of a myocy te myocy te aggregate pair. The cells of the lower member of the pair (/) were labelled with fhjleucine and were identified by autoradiography of the tissue section. 2 days in culture, x 116. Table 1. Modulation of tissue affinity behaviour of heart tissue by fibroblasts No. of Identity of the: Superficial tissue sepa- ^ ^ rate Row Labelled aggregate Unlabelled aggregate Label- Flat Un- experiled inter- label- ments face" led I % Myocyte" Myocyte Ventricle Myocyte Myocyte + fibroblast Myocyte Ventricle' Myocyte Myocyte + fibroblast' Myocyte 7o» 18 o "Identity of each of the 2 partners of the fragment pairs. 'Labelled cells spread over the surface of the unlabelled aggregate. "The interface between labelled and unlabelled aggregate was planar (e.g. no spreading occurred, see Fig. 1). " Aggregates consisting almost entirely of myocytes. 'Fragments of ventricle freshly dissected from the heart. 'Aggregates containing 8% myocytes and 2% fibroblasts. "No. of aggregate pairs

5 Surface proteins and tissue affinities 267 the tissue affinity behaviour, producing a tissue that is enveloped by cells of a pure myocyte aggregate. Basis offibroblastactivity Fibroblasts conceivably might affect tissue affinity behaviour of heart aggregates directly, by segregating to the surface of mixed myocyte-fibroblast aggregates where they would come into direct contact with cells of the partner aggregate, or indirectly, by the production of extracellular components that in turn affect affinity properties. Previous observations that fibroblasts were present in only small numbers at the surfaces of mixed myocyte-fibroblast aggregates militate against the first suggestion (Armstrong, 1978). Support for the second suggestion is the observation that a urea extract of heart fibroblast monolayers could substitute for fibroblasts (Table 2). Row i Table 2. Modulation of tissue affinity behaviour of myocyte aggregates by urea extracts offibroblastmonolayers Treatment of the: Labelled aggregate Unlabelled aggregate Control medium 6 Urea extract" Urea extract Control medium Control Urea extract medium of trypsinized cells' 1 Urea extract of Control medium trypsinized cells Labelled n8« Superficial tissue A Flat interface i Unlabelled No. of separate experiments "Treatment of each of the 2 partners of the fragment pairs. 'One partner consisted of myocytes aggregated in control culture medium. "One partner consisted of myocytes aggregated in medium containing the urea extract of fibroblast monolayers. ''One partner consisted of myocytes aggregated in medium containing the urea extract of fibroblast monolayers that had been trypsinized prior to extraction. "No. of aggregate pairs. 5 Myocyte aggregates were treated with the urea extract during the initial preparation of aggregates; fresh culture medium was used for preparation of aggregate pairs in hanging-drop culture and their subsequent culture in shaker flasks. Aggregates prepared in control culture medium* spread over aggregates which had been exposed to fibroblast extract (Fig. 2). The active component apparently is a cell-surface protein, since the urea extract of pre-trypsinized fibroblast monolayers (1 /tg/ml Worthington 2X crystallized trypsin, 15 min, 22 C) was inactive. If anything, aggregates treated with the urea extracts of pre-trypsinized fibroblasts spread over the control aggregates (Table 2, rows 3 and 4). Control culture medium contained an equivalent volume of dialysed urea-pmsf-pbs that had been used to extract a cell-free, serum-coated culture dish.

6 P. B. Armstrong DISCUSSION Elucidation of the factors that can modulate the tissue affinity behaviour of a tissue is of interest to the study of embryonic morphogenesis since it appears that factors governing the establishment of tissue relations in vitro operate also in vivo in the control of morphogenetic cell movements (Holtfreter, 1939; Moscona, 1962; Steinberg, 197; Townes & Holtfreter, 1955). The present study presents evidence that fibroblasts, which are a minority cell population in heart ventricle, affect the associative properties of heart tissue, producing a tissue that is spread over by cells of a pure myocyte aggregate. The fibroblast effect apparently is produced by factors exterior to U 2A Fig. 2. Fused pairs of heart myocyte aggregates, one member of which was aggregated in control medium prior to fusion and one member of which was aggregated in the presence of the urea extract of fibroblast cultures. Cells of the aggregate prepared in control medium spread over the surface of the aggregate prepared in medium containing the urea extract. Both fragment pairs were prepared on the same day. In A, the control aggregate is labelled (/) with [*H]leucine and the extract-treated aggregate is unlabelled (u); in B, the control aggregate is unlabelled (u) and the extract-treated aggregate is labelled (/). 2 days in culture, x 116. the cell surface, since exposure of myocyte aggregates to a factor extracted from confluent heart fibroblast monolayers alters their affinity properties such that they are spread over by myocyte tissue exposed only to control culture medium. The factor is absent in extracts of monolayers that had been subjected to mild trypsinization prior to extraction, suggesting that the factor is a protein that is located external to the plasma membrane.

7 Surface proteins and tissue affinities 269 The extraction procedure is similar to one developed by Yamada et al. (1975) for extraction of fibronectin from chick embryo fibroblast monolayers. According to Yamada et al. (1975), 6-75 % f tne material in the extract is fibronectin. Based on the similar extractability by urea solutions, similar tissue source, and extreme sensitivity to trypsin, it is possible that the fibroblast factor active in altering affinity behaviour is fibronectin. This suggestion is plausible in light of the proposal that tissue affinities are based on adhesive interaction of cells (Holtfreter, 1939; Steinberg, 1963, 197; Townes & Holtfreter, 1955) and the evidence that fibronectin is involved in cellcell adhesion (Chen, Gallimore & McDougall, 1976; Chen et al. 1978; Hedman, Vaheri & Wartiovaara, 1978), and cell-substrate adhesion (Ali, Mantner, Lanza & Hynes, 1977; Furcht, Mosher & Wendelschafer-Crabb, 1978; Grinnell, 1978; Pearlstein, 1976; Pena & Hughes, 1978; Yamada, Olden & Pastan, 1978; Zetter, Martin, Birdwell & Gospodarowicz, 1978). I plan to pursue this suggestion by employing other extraction procedures for fibronectin (Hynes & Destree, 1977) and by determining whether fibronectin purified by gelatin-affinity chromatography (Ali & Hynes, 1978; Dessau, Adelmann, Timpl & Martin, 1978; Engvall & Ruoslahti, 1977; Vuento & Vaheri, 1978) is active in this system. The observation that fibroblasts can modulate the spreading behaviour of ventricle tissue suggests that their activity may be involved in the alteration of behaviour of ventricle tissue produced by maintenance in organ culture (Wiseman et al. 1972). For example, freshly excised fragments of ventricle tissue spread over fragments that have been maintained in organ culture for 2-5 days prior to establishment of fragment pairs (Phillips et al. 1977). These effects may be the result of the accumulation of surface and/or matrix proteins secreted by ventricle fibroblasts into the extracellular matrix during residence in tissue culture, with the increased content of fibroblast product producing fragments that are spread over by freshly excised ventricle fragments. In summary, it was observed that a cell-surface protein of fibroblasts can alter the tissue affinity behaviour of the majority cell type of an organ. Although the effect observed above has been described only for heart tissue, it may also be important in a number of the in vitro studies of cell recognition and tissue affinity behaviour where organs are used that contain admixtures of epithelial and mesenchymal components (Armstrong, 1978; Steinberg, 1963, 197). For example, the ability of amphibian embryo mesoderm to prevent de-adhesion of embryonic endoderm and ectoderm in cultured aggregates (Holtfreter, 1939; Townes & Holtfreter, 1955) may be mediated by secreted products of the mesoderm similar to those revealed by the present study. I thank Dr R. D. Grey for a careful reading of the manuscript. This work was supported by Cancer Research Funds of the University of California and NSF grants nos. PCM and PCM

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