“ECM”: Its nature, origin and function in cell aggregation

Experimental Cell Research 30, 257-279 (1963)

“ECM”:

ITS NATURE,

ORIGIN AND FUNCTION AGGREGATIONI M.

Department

of Biology,

257 IN CELL

S. STEINBERG

Johns

Hopkins

Received

University, May

Baltimore,

Md.,

U.S.A.

28, 1962

THE component processes of development are growth, differentiation and morphogenesis. Among these the last occurs, in animals, at the most complex level of organization, involving units which range from individual cells to extensive structural complexes of cells. As a result, the mechanisms of morphogenesis have yielded only slowly and in small measure to experimental analysis. With the discovery first that the mutual adhesiveness of embryonic tissues is selective [20], and later that the varied cells of dissociated vertebrate embryonic tissues and organs are able to convene again and re-establish their characteristic histological relationships [30, 31, 43, 441, it has become apparent that the fine details of vertebrate tissue and organ structure are in appreciable measure a function of the differentiated adhesive and locomotor properties of the individual cells themselves. For this reason further progress in our understanding of morphogenetic processes will be dependent upon the accuracy of our knowledge of the mechanisms which orient and limit the locomotion of cells in populations, and which provide for and render selective the mutual adhesion of cells. In recent years the investigations of Abercrombie and his colleagues [l-6] have shown that vertebrate tissue cells limit one another’s locomotion by a mutual, local paralysis of surface motility, mediated by intimate contact. In cell populations of appreciable density, upon solid substrata, a statistical orientation of locomotion is a secondary consequence of this phenomenon. At the same time Moscona [32] has proposed that the aggregation of dissociated embryonic cells of higher vertebrates is brought about through the agency of a secreted, mucoidal, extracellular matrix, referred to as “ECM”. This visible matrix, homologized with “tissue ground substance” and reportedly elaborated in vitro by aggregating cells, is provisionally credited with a variety of functions, including the physical support of cells, the orientation of their movements along its structural configurations, the physical binding of cells 1 Supported 17-

631810

in part

by Grants

G-10896

and G-21466

from

the National

Science

Experimental

Foundation. Cell

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30

258

M. S. Sfeinberg

into tissue fabrics, and the transmission among them of information as a communications network [32]. These two accounts of the details of social intercourse among cells are not readily harmonized. Furthermore our own studies, using a system closely comparable with that employed by Moscona, have shown aggregation to proceed normally, in the absence of any demonstrable ECM, by means of the progressive formation of intimate adhesions, seemingly among the cells themselves [42]. Electron microscope observations of aggregating cells, made in this laboratory [23], have confirmed the intimacy of these mutual adhesions at all stages of aggregation. In view of these conflicting reports, we have elected to conduct an investigation of the nature and origin of ECM as obtained by the methods utilized by Moscona. Because of the variety of techniques which we have employed, methods will be given in the appropriate sections. Incubation for any purpose was always at 37°C. EXPERIMENTS

AND

RESULTS

Eflect of DNase upon Slime in Cd2 Suspensions It has previously been observed [8] that dissociation of embryonic cells with trypsin causes the appearance of a slimy material which entrains the cells, and that this slime is digested by some component of pancreatin. We have utilized pancreatic digestion in our previous study [42], in which it was found that ECM could not subsequently be demonstrated. The present observations were made upon heart and liver cells of 5-day chick embryos. The tissues were minced with fine, steel knives fashioned from sewing needles, and incubated, for dissociation, in the supernatant extract of 3 per cent trypsin I-250 plus 1 per cent pancreatin 4X in calcium- and magnesium-freeTyrode’s solution (CMF) containing 0.1 per cent ethylene diamine tetraacetic acid, disodium (EDTA), and adjusted to pH 7.6. After 15-20 min of incubation the tissues were subjected to a standard shearing force for 2 min with the apparatus described by Auerbach and Grobstein [8], to bring about the separation of the loosened cells. Different commercial preparations of trypsin l-250 were found to vary greatly in their efficacy in producing dissociation under these conditions. The sliminess of the resulting suspensions was also found to be a function of the source of the crude trypsin, but no correlation existed between these two different properties of the trypsin preparations. They are therefore referable to different components of the preparations. Addition of appreciably greater amounts of EDTA was found to prevent Experimental

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“ECM”:

Its nature, origin and function

259

the digestion of slime by those preparations which were otherwise effective in this respect. It therefore appeared that the enzyme responsible for digesting the slime was activated by one or more polyvalent cations present in the enzyme preparations. Analysis of Difco trypsin l-250, lot No. 448630, by EDTA titration with Eriochrome Black T indicator at pH 10 [ 191 revealed the presence of 4.2 X 1O-4 moles of Ca2+ plus Mg2+ per gram. The 0.1 per cent EDTA (3 x 10-3M) in our trypsin-pancreatin solution therefore chelates no more than 24 per cent of the Mg2+ and Ca2+ present, leaving a combined concentration of these ions of no less than 9.6 X 10-3M in solution. Because of the greater affinity of EDTA for Ca 2+, the proportional reduction of the Mg2+ in the solution will be less than that of the Ca2+. Since pancreatic deoxyribonuclease (DNase) might be expected to occur in crude trypsin and pancreatin preparations, is activated by Mg2+, and acts upon a substrate the viscosity and gel-like properties of which are well known, the effects of this enzyme upon trypsin-produced slime were examined in cell suspensions from 5-day heart and liver. It was found that incubation for 5 min in 0.1 mg/ml crystalline DNasel in Tyrode’s solution at pH 7.6 resulted in the total digestion of slime produced by either trypsin 1:250 or crystalline trypsin.l Identical results were obtained with slime produced during dissociation of S-day epidermis in EDTA at pH 10 [42]. Accordingly all preparations of trypsin l-250 and the pancreatin 4X used in this study1 were I. DNase content of crude enzyme preparations compared with their effectiveness in dissociating chick embryonic heart and liver and in digesting slime produced during dissociation. TABLE

Effectiveness in dissociating cells Difco trypsin l-250, No. 450837 Difco trypsin l-250, No. 448630 Difco trypsin l-250, No. 443968 NB Co trypsin l-250 Pancreatin 4X a Contains

3 per cent trypsin

r Crystalline trypsin Nutritional Biochemicals

l-250

Residual slime after standard dissociation

ccc! DNWg

pg DNase/ml dissociation solution

++

++

20

2.03’

++

++

22

2.0ga

+++ +

+I-

90 263 143

4.13a 9.32a

plus

1 per cent pancreatin

and DNase: Worthington Corp.; paucreatin 4X:

Biochemicals N. B. Corp.

4X. Corp.;

trypsin

Experimenfal

l-250:

Difco

and

Cell Research

30

M. S. Steinbeq assayed for their DSase content after the method of Kurnick [45], the observed activities being referred to that of an appropriately diluted standard sample of crystalline DNase. The results, expressed in equivalents by weight of the standard, are presented in Table I along with those relating to the effectiveness of the trypsin preparations in producing dissociation and in digesting slime when used in conjunction with the pancreatin as described above. It is clear that the effectiveness of pancreatin in bringing about the digestion of slime when added to crude trypsin preparations is attributable to the DNase which it contains. The sample which we have used, and several others as well, contains insufficient DNase to bring about complete digestion of the slime under our defined conditions. However, when the trypsin preparation to which it is added contains sufficient DNase to bring the total amount to a value approximating 9 ,ug/ml of dissociation solution, the slime is totally digested under our standard conditions. Corollary to these observations is the conclusion that the slime under investigation possesses a structural backbone of DNA. Effects

of Serum and DNase

upon ECM

Because cell suspensions produced in the presence of or subsequently treated with sufficient DNase to digest all slime do not display, during aggregation, detectable ECM, it appeared probable that the DNA-containing slime is identical with ECM. Since it is claimed, however, that ECM is produced metabolically during culture [33, 341 rather than by artificial means during tissue dissociation, we considered it desirable to investigate the characteristics of ECM obtained from the tissues and by the methods previously used for its preparation. Aggregation in a framework of ECM has been described for the cells of chick limb buds [32] and of 7-day chick embryonic retinas [35]. We have therefore conducted experiments with material from both sources, the limb buds being taken from 4-day embryos and largely denuded of their epidermis with a solution of crystalline trypsin. The procedures employed in the present study were those of Moscona [3G], in which crystalline trypsin is used for dissociating the tissues, and in which the standard culture medium is Eagle’s basal medium with 1 per cent glutamine, 10 per cent horse serum and 2 per cent embryo extract, plus penicillin and streptomycin, both at 50 units per ml of medium. Tissue dispersates, produced by pipetting, were dispensed into custom-made 10 ml Erlenmeyer flasks as described previously [42], each containing 2 ml of medium. Aggregation was then brought about in a 95 per cent air: 5 per cent CO, atmosphere saturated with water vapor, by the method of Moscona ([32], p. 52; [36]), Experimental

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30

“ECM”:

Its nature, origin

and function

261

using a thermostated, gyratory water bath shaker with gassing hood, modified by the manufacturer’ to operate in the range from 50-150 rpm with l/2 inch diameter of gyration. Limb buds from 24 embryos were minced and subjected to the dissociation procedure. Immediately upon the addition of trypsin, the tissue fragments adhered to one another, held together by the slime described above. Such slime is not removed during the washes which follow incubation in trypsin, but is still present when the fragments are transferred to culture medium to be dissociated by means of rapid flushing through a pipette. With the addition of culture medium, however, the slime loses its stickiness, so that after dissociation is complete, it is no longer detectable as such. In this experiment one half of the cell suspension was placed directly into a culture flask (flask A) while the remaining half was washed and resuspended in a solution of 3 per cent trypsin l-250 (Difco No. 443968) plus 1 per cent pancreatin 4X in CMF, in which the cells were incubated for 10 min. They were then washed twice in culture medium and transferred to a second flask (flask B), after which both flasks were incubated for 30 min while being gyrated at 70 rpm. Figs. 1 a and b show the resulting aggregates, photographed within the culture flasks. The suspension which was not subjected to post-digestion with the DNase-containing crude trypsin-pancreatin preparation showed, in addition to many free cells, large masses of cells held together by an extraneous material, while the preparation subjected to such post-digestion showed, in addition to free cells, only very small aggregates which might possibly have been assembled on residual traces of such material which may have escaped digestion. Upon return to the shaker for 14 hr, the cells in both flasks formed large numbers of small, independent aggregates which secondarily fused to produce very large aggregates (Figs. 1 c and d). In flask A there also developed two large, spheroidal aggregates of unique appearance, which proved to contain the bulk of the extraneous material present in the flask. It could thus be established that pancreatin-digestible, structured materials produced during dissociation are carried over into the culture flasks, where cells adhere to and aggregate upon them. At no time, however, were the cultures seen to pass through a condition in which the cells were enmeshed in a mucoidal framework. We therefore turned to the study of dispersates of neural retina from ‘i-day embryos. Minced retinas were dissociated in the same manner as the limb 1 New

Brunswick

Scientific

Co., New

Brunswick,

NJ. Experimental

Cell

Research

30

262

hf. S. Steinberg

Fig. l.-Limb bud preparation dissociated (70 rpm, 37”C, 30 min) in serum-containing A), showing extraneous material; (b) with (flask B), showing little or no extraneous indicate aggregates containing extraneous Experimental

Cell Research

30

with crystalline trypsin and cultured on shaker Eagle’s medium: (a) without other treatment (Flask intervening incubation in crude trypsin-pancreatin material. c, Flask A after 14 hr of culture. Arrows material. d, Flask B after 14 hr of culture. x 3.5.

“ECM”:

Ifs nature, origin

and function

buds discussed above, it being observed that here also the tissue fragments became slimy and cohered immediately upon contact with the trypsin solution. The dispersate was divided into several equal portions, one of which was dispensed directly into culture medium (flask A). A second aliquot was postincubated in our standard trypsin l-250 (No. 443968)-pancreatin-EDTA solution for 10 min and washed with culture medium before being dispensed into fresh medium (flask B). A third portion was post-incubated for 10 min in 0.1 mg/ml crystalline DNase in Tyrode’s solution at pH 7.5, washed and dispensed as above (flask C). All flasks were gyrated at 70 rpm for 30 min at 37”C, after which their contents were examined and photographed. Flask A contained, in addition to many free cells, a large strand of extraneous material and a number of smaller fragments of the same, all coated with cells (Fig. 2 a). Flasks B (Fig. 2 b) and C (Fig. 2 c) contained exclusively free cells. It is therefore clear that the conclusions set forth above with respect to dispersates derived from limb buds apply equally well to those of neural retinal origin; that the enzyme which destroys the precursor of this extraneous material is DNase; and that the extraneous material is merely a modification of the DNA-containing slime produced during dissociation and discussed in the first section of this paper. Again, however, further maintenance of these cultures failed to provide any suggestion of the presence of a mucoidal framework enveloping the cells. At this juncture it was suggested to us by Dr. Moscona that we should withhold the addition of serum to the culture solution until after the ECM framework had become fully established. The following experiment was performed with this structure in mind. Finely minced neural retinas were dissociated as before, Tyrode’s solution being substituted, however, for the culture medium. It was immediately apparent that the absence of macromolecular constituents from the medium allowed the slime produced during dissociation to persist unaltered. The cells were entirely embedded in a viscous mass. One aliquot was dispensed into a culture flask containing 2 ml of Tyrode’s solution (flask A). A second aliquot was incubated in 0.05 mg/ml crystalline DNase in Tyrode’s solution for 10 min, at the end of which time all of the slime had been digested. The cells were washed in Tyrode’s solution and dispensed as above (flask B). The slime in a third aliquot was totally digested after incubation in our standard trypsin I-250 (No. 443968)-pancreatin-EDTA solution for 25 min, after which period this suspension was washed and dispensed as above (flask C). The appearance of these three dispersates before incubation is shown in Figs. 3 a-c. Flask A, containing the cells embedded in slime, was then gyrated Fig. 2.-Retina cells prepared and cultured as in Fig. 1: (a) without other treatment, showing extraneous material; with intervening incubation in crude trypsin-pancreatin (b), and in crystalline DNase (c), showing absence of extraneous material. All photographs after 30 min of culture. x 3.5. Experimental

Cell

Research

30

AI. S. Steinberg

264

Fig. 3.-Retina cells dissociated with crystalline trypsin and dispersed in Tyrode’s solution: (u) without other treatment, showing cells embedded in viscous slime or ECM (flask A); with intervening incubation in crystalline DNase (flask B) (b), and in crude trypsin-pancreatin (flask C) (c), showing absence of ECM. d, contents of flask A after 30 min gyration at 80 rpm, 37”C, showing relaxation of DNA-containing ECM. Upon addition of Eagle’s medium with serum, ECM in flask A underwent pronounced contraction and syneresis, forming “extraneous material” and liberating many cells (e). After 12 hr of culture (80 rpm, 37°C) DNA-containing “extraneous material” in flask A is largely contained within the distinctive aggregates indicated by the arrows (f). x 3.5. Experimental

Cell

Research

30

“ECM”:

Ifs nature, origin

and function

265

at 80 rpm for 30 min (37°C) at which time the structure of the slime had relaxed, giving the appearance of a flat veil, as shown in Fig. 3d. Two ml of standard culture medium was then added, and the flask was returned to the gyratory shaker for l-314 hr. At the end of this period examination showed that the slime had undergone pronounced contraction and syneresis and had broken up to form a number of strands which were coated with cells (Fig. 3 e). The origin of the “extraneous material” coated with cells, illustrated in Figs. 1 a and 2a, is now more readily understood. Maintenance under the same conditions of culture for an additional 12 hr resulted in the formation of a large number of small aggregates plus two large ones of unique appearance (Fig. 3f). The latter contained the bulk of the contracted slime. We have thus been able to confirm Moscona’s finding that cells dissociated with crystalline trypsin and dispersed in protein-free medium are bound together in a viscous extracellular matrix (ECM). The present experiments show, however, that this material is a product of the tryptic digestion of tissues rather than of cellular metabolism, and that it owes its structural integrity to the DNA which it contains. Crystalline DNase digests it, and the addition of serum causes it to contract greatly and lose its viscous properties, probably because of the formation of a complex between the ECM and components of the serum.

Physical

Chemical

Properties

of ECM

Chemical analysis of ECM was not attempted because of the impossibility of separating it from the cells which it entrains, while avoiding with certainty the formation of complexes between the ECM and macromolecules liberated from cells broken during the separation process. In order to characterize ECM more fully a study was therefore made of certain of its physical chemical properties. Ultraviolet absorption spectrum of ECM.-Forty-eight neural retinas were minced, equally distributed in two 5 ml centrifuge tubes, and the two portions dissociated in identical manner by the method of Moscona, the final dispersion being accomplished in 0.4 ml of Tyrode’s solution. A large amount of ECM was thereby obtained. To one tube (experimental) was added 0.6 ml of Tyrode’s solution containing 0.6 mg crystalline DNase, while to the other tube (control) 0.6 ml of Tyrode’s solution was added. Both tubes were incubated for 5 min, at which time dissolution of the ECM in the DNase-containing tube was complete. 0.2 ml of a suspension of thoroughly washed Celite was added to each tube to facilitate the sedimentation of the viscous ECM in the control tube, following which the tubes were centrifuged for 5 min at 800 g. 0.35 ml of supernate was transferred from each tube to a stoppered vial and the proper amounts of DNase and Tyrode’s solutions added to equalize all additions Experimental

Cell Research

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266

M. S. Steinberg

to the vials, which were then further incubated for 75 min to allow DNA degradation to go to completion. The volume of each solution was brought to 3.35 ml with Tyrode’s solution, and an appropriate blank was prepared. Ultraviolet absorption spectra of these solutions were obtained with a Bausch and Lomb Spectronic 505 automatic recording spectrophotometer, using quartz cells 1 cm in diameter.

WAVELENGTH

Text-fig. 3. Text-fig. 1. Text-fig. 2. Text-fig. l.-Ultraviolet absorption spectrum of experimental solution containing cell supernate and DNase-digest of ECM. Text-fig. 2.-Ultraviolet absorption spectrum of control solution identical with that scanned in Text-fig. 1 except for the absence of digested ECM. Text-fig. 3.-Ultraviolet absorption spectrum of digested ECM, obtained by using control solution (Text-fig. 2) as blank for experimental solution (Text-fig. 1). Spectrum is indistinguishable from that of a typical nucleoprotein.

The experimental and control solutions now differed only by virtue of the presence in the former of digested ECM, the ECM in the latter having been sedimented prior to the removal of the sample. Text-figs. 1 and 2 show the absorption spectra of the experimental and control solutions, respectively. The difference spectrum, obtained by using the latter as the blank for the former, is the absorption spectrum of digested ECM. It is shown in Text-fig. 3 and is indistinguishable from a typical nucleoprotein absorption spectrum. While firm conclusions concerning the proportions of nucleic acid and protein in ECM cannot be drawn, the spectrum indicates at least that ECM contains appreciably more protein than nucleic acid on a weight basis. Further Experimental

Cell Research

30

“ECM”:

Ifs nafure,

origin

and function

267

studies were therefore conducted to determine whether ECM possesses certain properties which are highly characteristic of deoxyribonucleoproteins. Effects of NaCl concentration upon ECM.-Deoxyribonucleoprotein (DNP) at very low ionic strength is soluble, due to intermolecular repulsive forces, and is non-viscous. As the ionic strength of the solution is increased, the solubility of DNP decreases, the minimum being reached in isotonic solutions (0.14 M NaCl). At higher ionic strengths (e. g. 1 M NaCl) the protein and nucleic acid tend to dissociate to yield highly viscous solutions [ll]. ECM-containing cell suspensions were obtained from minced neural retinas as described above, and aliquots were dispensed in distilled water, in 0.14 IM NaCl, and in 1 M NaCl. In distilled water the ECM passed quickly into solution, yielding a preparation of low viscosity. In 0.14 M NaCl the ECM was insoluble, even after 2 days. In 1 A4 NaCl the entire mass of ECM+ cells immediately transformed into a thick, opaque, viscous gel which seemed slowly to be passing into solution. Thus the behavior of ECM as a function of salt concentration is that of a typical deoxyribonucleoprotein. Effects ofpH upon ECM.-A DNP from rat liver nuclei [29] hydrates poorly at pH values below neutrality. In alkaline solutions it forms a gel which at pH 7.5 tends to be opaque, but at pH 8.5 is clear. The gel in alkaline solution swells to till the provided volume and is elastic, exhibiting a pronounced recoil or unwinding tendency after stirring. A similar DNP precipitates from alkaline solution in 0.14 M NaCl when the pH is lowered below 10.3 [27]. ECM was obtained from neural retinas as above, washed as free of cells as possible with 0.14 M NaCl, and suspended in the same. It was characteristically opaque and cohesive, forming a small, discretely bounded mass. The pH was then progressively raised by the addition of dilute NaOH solution. As the pH increased, the ECM lost its opacity and swelled until in the pH range of 10.6-11.6 it invisibly tilled the volume of approximately 3 ml in which it was distributed. The solution was viscous; and transparent, elastic strands could be lifted for considerable distances from its surface before they snapped back into it. When stirred and released, the solution responded by “unwinding” in the counter direction. Dilute HCl solution was then gradually added, with stirring, until the first precipitation was observed. The pH at this point was 10.2. Further lowering of the pH resulted in the formation of an opaque mass indistinguishable from the original ECM. DNase digestion of this mass destroyed its gel-forming ability but not its physical integrity, indicating that the extreme pH to which the ECM had been subjected had caused denaturation of proteins to occur. The behavior of ECM as a function of pH is thus also seen to be that of a deoxyribonucleoprotein. Experimental

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M. S. Steinberg

268

Effects of Cd+ and Mg2+ upon EC,‘.-Both DNA (401 and DNP form complexes with calcium and magnesium ions. The presence of as little as 1O-5 M Mg”+ causes some aggregation and a decrease in the viscosity of DNP solutions [47]. Low concentrations of Ca”+ or Mg*+ depress the hydration of DKP and cause it to precipitate from solution [29]. Evidence also exists for the formation of stable complexes between Ca”+ and intact chromosomes [41]. The lampbrush chromosomes of amphibian oocyte nuclei isolated in dilute CaCl, or MgCl, solutions contract markedly and settle out of the nucleoplasm [9].

10-s 0.01

1.0

0.1

TIME

Text-fig. 4.-Time required and Mg2+ concentration.

for total

dissolution

IN

IO.0

HOURS

of equal

aliquots

of ECM

as a function

of Ca2+

0.2 ml aliquots of ECM prepared as above and washed several times with CMF were dispensed into 10 ml volumes of the following solutions, constantly stirred during the observation period: Tyrode’s solution, CMF, 0.14 M NaCl, iO-a M CaCl,, lO+ M CaCl,, lO-6 M CaCl,, 1O-3 M MgCl,, 1O-4 M MgCl,, and 1O-5 M MgCl,. In 0.14 M NaCl, Tyrode’s solution, and CMF the ECM remained insoluble for the 40 hr duration of the experiment. The effects of Ca2+ and Mg2+ upon the rate of solution of ECM are shown in Text-fig. 4. Both ions impede the dissolution of ECM at 1O-4 M, Mg3+much more effectively than Ca2+. They act to retard the progressive hydration of the ECM, which eventually fills the entire provided volume. It is clear from these and the foregoing results that ECM is basically a deoxyribonucleoprotein. Confirmation by independent means and the revelation of other possible constituents were sought through the application of histochemical tests. Histochemical Staining Reactions of ECM ECM was produced from neural retinas in the routine manner, washed several times in CMF to remove as many cells as possible, spread out on slides and allowed to dry at 42°C. Because of the solubility of ECM, at least Experimental

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“ECM”:

Its nature, origin

and function

269

in its original state, in distilled water, 0.14 M NaCl was used in place of water wherever necessary in the staining procedures, which were applied to the unfixed or, where specified by the procedure, to formalin-fixed preparations. That ECM is very highly hydrated may be surmised from the enormous reduction in volume which it undergoes upon the addition of serum. It was therefore anticipated that rather little material might be left after drying, and that staining reactions might for this reason be difficult to demonstrate. This proved to be the case. The best visualization of ECM was often obtained near the edges of the preparations, where the receding aqueous front had carried the ECM with it for a distance, piling up additional amounts of material. Fig. 4 shows a preparation of cells entrained in ECM stained by the Feulgen reaction for DNA. ECM also stained with pyronin after formalin fixation, but control slides incubated in RNase showed little or no decrease in the staining (Fig. 5). In other control slides incubated in DNase, staining of the ECM was prevented (Fig. 6). Pyronin-staining of DNP after formalin fixation or moderate heating has been described before [46]. Aqueous toluidine blue stained one preparation metachromatically, as observed macroscopically. However, the metachromasia was quickly lost upon transfer of the slides to alcohol, indicating that it was in all likelihood due not to ester sulfates but to a high density of more weakly acidic groups [22]. A second preparation, mounted in water, hardly stained with toluidine blue, metachromatically or orthochromatically; yet the ECM could be seen to be present, largely by the optical refraction which it caused (Figs. 7 and 8). It would appear that acidic macromolecular materials erratically liberated during preparation of the ECM may complex with it, thereby conferring upon it their staining properties. That such acidic substances may include acid mucopolysaccharides is shown by the staining of ECM with alcian blue in 3 per cent acetic acid (Fig. 9); and that these substances are indeed complexed with the DNP is shown by the observation that DNase treatment prevents subsequent staining by alcian blue. The presence in ECM of polysaccharide, probably as mucoprotein, was indicated by its positive reaction with the periodic acid-Schiff stain (PAS), a reaction which was not abolished by pretreatment of the slides with DNase (Fig. IO). That the physical integrity of ECM is not due to mucopolysaccharide, however, is shown by its dissolution upon digestion with DKase. These observations further corroborate the primacy of DNA in the structure of ECM. They indicate that the fundamental nucleoprotein gel may complex not only with components of serum, as was shown earlier, but also with other macromolecular materials which may be present in cell suspensions. Experimental

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Al. S. Steinberg

270

Fig. Fig. Fig. Fig. Fig. Fig. Fig.

4.-Feulgen-staining of ECM. x 400. 5.-Pyronin-staining of ECM after RNase dligestion. x 90. 6.-Absence of pyronin-staining of ECM aft .er DNase digestion. x 90. 7.-ECM preparation showing little or no sl .aining by toluidine blue. Water mount. S.-Portion of preparation shown in Fig. 7‘, photographed at higher magnification, 9.-Acidic Alcian blue-staining of ECM. x I IO. IO.-PAS-staining of ECM after DNase dig estion. x 400.

Experimental

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30

x 90. x 400.

“ECM”:

Iis nafure, origin

In doing so they point up the danger inherent by histochemical criteria alone.

Origin

and funcfion in the identification

271 of a material

of ECM

ECM, the deoxyribonucleoprotein interaction product of trypsin with cellular DNP, might originate during tissue dissociation in one or more of three ways. Its precursor might conceivably leach out from the nuclei of intact cells; it might arise from nuclei broken during the dissociation process; or it might derive from the ruptured cells which bound all of the cut edges of the minced tissue fragments. All of these possibilities have been examined, as reported below. Effects of trypsin and DNase upon nuclear mass.--The experiment to be described was performed with the kind cooperation of Dr. Ronald Cowden. Five-day chick embryonic hearts were dissociated with trypsin-pancreatin-EDTA as described in an earlier section. The resulting cells were washed in Tyrode’s solution, allowed to adhere to the upper coverslip of a Sykes-Moore tissue culture chamber,l and examined in white light with an AO-Baker shearing-type interference microscope adjusted to maximize color contrast between nuclei and cytoplasm. Loss of nuclear masswould be seen as a drift in the apparent color of the nuclei toward that of the cytoplasm. DNase and trypsin solutions were separately introduced through one side of the chamber and removed through the opposite side. In neither case was there any detectable lossof nuclear massover the course of about 30 min at 23°C. A few isolated nuclei which were present also showed no change in mass. It is concluded that ECM does not originate from the intact nuclei, at least in the caseof heart cells. Cell breakage during dissociation.-Neural retinas from 5-day chick embryos were dissociated by the method of Moscona after a standard 15 min incubation in 0.5 per cent crystalline trypsin in CMF at pH 7.2. The ECM was digested with 0.1 per cent DNase for 5 min at 37”C, after which the cell suspensionwas brought to a fixed volume. After thorough mixing to ensure the uniform distribution of cells, a small aliquot was removed, diluted ten fold, and the cells in a standard volume counted by means of a hemocytometer. The original suspensionwas centrifuged gently, the cells resuspended in trypsin, and the entire dissociation and counting procedure precisely repeated. Then this processwas repeated for a third time.

It was found that the dissociation procedure is responsible for very little cell breakage. Because of the necessity for speed in the counting of the cells, it was not expedient to discriminate free nuclei or enucleated cells from intact cells, although their presence was observed. Therefore cell breakage would be recorded as an increase in the number of “cells” recorded, since the separated nucleus and cytoplast of a ruptured cell are both counted. The 1 Bellco

Glass

Co., Vineland,

N.J. Experimental

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34. S. Steinberg

272

results are presented in Table II. These data indicate may be produced by the dissociation procedure does a few percent of the cells. Such breakage is low in inflicted by the original mincing of the tissue fragments, bute only slightly to the ECM produced, since not only themselves must apparently be ruptured in order for

II. CeU breakage during dissociation.

TABLE Because

free

nuclei

Number

and

that such breakage as not involve more than comparison with that and likely to contrithe cells but the nuclei EChI to be obtained.

enucleated cytoplasts are included in the enumeration, be reflected in an increase in the count.

of cells (including After initial dissociation

isolated

nuclei

and cytoplasts)

After 1st repetition of dissociation procedure

would

volume.

After 2nd repetition of dissociation procedure

1901

1877

per standard

breakage

1950

It should be noted here that although, for purposes of standardization, DNase treatment was employed in each repetition of the procedure above, ECM made its appearance only during the initial dissociation. Once digested, it did not reappear in noticeable amounts, even after repetition of the trypsinization procedure. These results made it appear that ECM must derive in greatest measure from the cut cells bounding the tissue fragments. The experiments reported in the next three sections were designed to determine whether this is indeed the case. Effect

of preincubation

of tissue fragments

in DNase

upon

quantity

of ECM.-

Neural retinas from 7-day embryos were minced and divided into two equal portions. One portion was incubated in 0.1 mg/ml crystalline DNase in Tyrode’s solution at pH 7.2 for 15 min, while the other portion was treated similarly except for the omission of the enzyme from the solution. Both preparations were then incubated for 10 min in CMF followed by 15 min of incubation in 0.5 per cent crystalline trypsin in CMF at pH 7.2. Each was washed three times in Eagle’s basal medium without serum and dissociated by rapid flushing through a pipette as described earlier. The resulting dispersates, enmeshed in ECM, were dispensed into culture flasks, gyrated at 70 rpm for IO min at 37°C to collect together the ECM-embedded cells, and photographed. The dispersate derived from the DNase-pretreated tissue fragments (Fig. 11 a), while containing an appreciable amount of ECM, did not contain nearly so much as that derived from the control fragments (Fig. 11 b). Since the Experimental

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conditions used for preincubation of the tissue fragments in DNase would have been more than adequate to allow the total digestion of already-produced ECM, it is concluded that the DNA which may potentially be incorporated into ECM is not wholly accessible to DNase digestion until the action of a proteolytic enzyme, in this case trypsin, renders it so. Effect of reducing cut surfaces upon quantity of ECM.-A number of neural retinas from 7-day embryos equal to that used per portion in the preceding experiment was excised, but in this case the retinas were not minced. A reasonable estimate would place the cut surface area of these “intact” retinas at approximately 7-10 y0 that of the cut surface area of the minced retinas in the above experiment. These retinas were then dissociated, dispensedand gyrated in precisely the manner described above.

Comparison of the quantity of ECM in this preparation (Fig. 12a) with that in its pre-minced counterpart (Fig. 11 b) shows that a large reduction in the cut surface area of the tissue is reflected in a comparably large reduction in the amount of ECM obtained from the tissue after its subsequent incubation in trypsin. While this observation strongly indicates that ECM is derived largely from ruptured cells at the cut surfaces of the tissue fragments, the conclusive demonstration of this origin is contained in the final experiment reported on below.

Lack of Function

of ECM

The experiments described above demonstrate that ECM is a deoxyribonucleoprotein to which other materials may be complexed, and indicate that it originates, at least in large part, by the hydration of DNP from ruptured cells as a consequence of the action of trypsin upon the exposed nucleoprotein. Since this material can hardly be imagined to play a normal role in the mediation of cell adhesions, it should be possible to obtain cell aggregation in a solution containing an active enzyme (DNase) capable of digesting ECM but harmless to living cells [25]. Furthermore, if the greatest part of the ECM derives from broken cells at the cut edges of tissue fragments, with only a minor contribution from the free cells broken during the dissociation process, the dissociation with crystalline trypsin of already-formed aggregates should yield very little ECM, since such aggregates have no cut edges. The following experiment was conducted to evaluate these conclusions. The starting material was the preparation of neural retina cells plus ECM shown in Fig. 12a (in 2 ml of Eagle’s basal medium without serum). To the flask was added 1 mg of crystalline DNase, bringing the concentration of that enzyme to 0.05 per cent. Incubation for 15 min resulted in the complete digestion of the ECM, as shown in 18 - 631810

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Fig. Il.-Neural retina preparation dissociated with crystalline trypsin after preincubation of tissue fragments in DNase (a), showing decrease in amount of ECM obtained as compared with identical preparation not subjected to DNase pretreatment (b). Dispensed in Eagle’s medium without serum. 30 min at 70 rpm and 37°C. x 3.5. Fig. 12.-(a) Neural retina dispersate prepared precisely as in Fig. lib, except that the retinas were not minced prior to dissociation. Reduction of cut surfaces comparably reduces amount of ECM obtained. (b) Same preparation after incubation in crystalline DNase, showing total digestion of ECM. (c) Same preparation after addition of serum and gyration at 90 rpm and 37°C for 5 hr, showing aggregates formed in the presence of DNase. (d) Same preparation after additional culture period of 15 hr at 100 rpm and 37”C, showing more advanced state of aggregation. (e) Same preparation after dissociation by procedure initially used, showing trace amount of ECM obtained in the absence of cut tissue surfaces. x 3.5. Erperimental

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Fig. 12b. 0.2 ml of sterile horse serum1 was then added to the flask, which was returned to the shaker and gyrated at 90 rpm and 37°C for 5 hr, at which time its contents were observed and photographed. As shown in Fig. 12c, aggregation was at this time well under way despite the presence in the culture solution of active DNase. Fig. 12 d showsthe samepreparation in a more advanced state of aggregation after an additional 15 hr of culture at 100 rpm. During this period the modal diameter of the aggregates increased from 0.13 mm to 0.23 mm. The aggregates were recovered from the culture flask, washed in CMF, and incubated for 10 min in CMF at pH 7.2. They were then suspendedin 0.5 per cent crystalline trypsin in CMF at pH 7.2 and incubated for 20 min. At the end of this time there was noted a slight tendency for the aggregatesto stick together, indicating that a trace of ECM was present at their surfaces. The aggregateswere washedthree times in Eagle’s basal medium without serum and dissociated by pipetting in the same manner as were the original tissue fragments in the first stage of this experiment. The dispersate was transferred to a flask containing 2 ml of the samemedium and gyrated at 70 rpm for 10 min at 37°C. Fig. 12e showsthe cell suspensionand the trace amount of ECM obtained through this procedure. It seemslikely that the surfaces of the aggregates may have suffered slight damage during recovery from the original flask, washing, incubation in CMF and in trypsin, etc., accounting for even that mote of ECM which was produced. Our expectations are therefore borne out. It is shown that ECM originates in greatest part, if not exclusively, from broken cells. That it represents partially deproteinized, enormously swollen chromosomes to which other materials may adsorb seems beyond reasonable question. It is further shown that cell aggregation occurs in the presence of active DNase and therefore in the total absence of ECM. The conclusion seems justified that “ECM” does not normally function in the process of cell aggregation.

DISCUSSION

ECM, as originally characterized [32], was described as having the following physical and chemical properties: it stained metachromatically with toluidine blue: it “reacted with periodic acid-Schiff stain, though not consistently; it gave positive biuret and Molisch tests; it swelled and increased in viscosity at elevated pH and in absence of calcium and magnesium ions; it was not distintegrated by crystalline trypsin, but its stickiness to glass, elastic recoil, and solubility in dilute alkali were increased by this enzyme; exposure to crude pancreatic preparations usually caused its destruction;” and it “showed linearly oriented, incidental or inherent configurations, with cells frequently aligned among them.” On the basis of these criteria it was 1 Horse serum: Cappel, sterile, pooled. Experimental

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proposed that this material contained or consisted of a mucous substance, probably a mucoprotein, and that it was a variant of intercellular ground substance. The properties listed above will be recognized as comparable or identical with some of those which we have outlined in the present contribution. We have demonstrated the presence of protein by means other than the biuret test, as described above; and a positive PAS reaction demonstrates the presence of polysaccharide more certainly than does the Molisch test, which is also positive with various aldehydes and acids. Since we prepared “ECM” following the methods previously employed in its preparation, and the resulting material possessed the same properties originally attributed to it, it is clear that we are dealing with the same material. The formation of viscous, hydrated gels by DNP has been described after partial removal of the protein by molar NACl [12], by heparin, which appears to compete with DNA for the protein [7], and by incubation in the presence of chymotrypsin or trypsin [as]. The last-cited observations were made upon intact chromosomes. The finding [21] that chromosomes within living, intact cells are not altered by trypsin, to which they are seemingly inaccessible, supports our conclusion that “ECM” is derived from ruptured cells. It has been proposed [47] that gel formation by DNP is due to the establishment of cross linkages between portions of the histone from different DNP molecules. If this is so, a portion of the protein remaining in “ECM” after tryptic digestion must include histone. It may be that failure of DNase pretreatment completely to prevent the subsequent appearance of “ECM” upon tryptic digestion is due to the effective enclosure of portions of the DNA by attached protein. Upon sufficient erosion of the protein barrier by trypsin, the DNA core of the resulting hydrated and expanded nucleoprotein (“ECM”) would be opened to attack by DNase. It has been shown [34] that the ability of dissociated chick embryonic cells to adhere to one another or to glass is markedly dependent upon temperature. This observation was provisionally interpreted as denoting that “at low temperatures synthesis of extracellular materials is impaired resulting in failure of dissociated cells to become effectively attached.” The extracellular materials to which reference is apparently made have been shown in the present communication to be artificial. In addition, we have recently demonstrated [42] that the temperature-dependent process involved in the aggregation of dissociated chick embryonic cells appears to operate solely during the initiation of an adhesion. Pre-warmed cells, contrary to a previous suggestion ‘([331, p. 167), d o not in our experience retain their adhesiveness in the cold; nor is warmth required for the maintenance of already-established adhesions. Experimental

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Coupled, these two facts militate against the assumption that the thermosensitivity of c,ellular adhesiveness denotes the involvement of adhesive cements. There remains, to our knowledge, no substantial evidence in support of the theory that the aggregation of dissociated cells operates through the agency of secreted intercellular cements distinguishable from the surfaces of the cells themselves. The appearance of a slimy material in cell suspensions treated with proteolytic enzymes has been noted before. It has been observed during dissociation of mouse embryonic cells with trypsin [S] and after treatment of ascites cells with elastase, trypsin, chymotrypsin, pepsin or papain, but not after treatment with any of six non-proteolytic enzymes [14]. Boyse [lo] and Madden and Burk [26] have independently described both the appearance of such slime during tryptic dissociation of solid tumors and its digestion by DNase. In fact the persistent sliminess of tissue fragments and dispersates n the presence of crystalline proteolytic enzymes and before introduction into culture medium is their most striking feature. Evidence has been presented above indicating that the DNP under discussion in this paper forms complexes with some component of serum, probably protein in nature, and with other macromolecular materials present in cell suspensions. The formation of complexes between DNP and other proteins has been previously studied. While complex formation between DNA and added proteins occurs only at pH values below the isoelectric point of the protein [13], (and therefore, with most proteins, below physiological pH), DNP has been found to combine with a number of added proteins at pH 7.3, a reaction which is favored by the presence of salts in approximately physiological concentrations [38]. Formation of such complexes at pH 7.3, however, does not proceed so far as it does at lower pH values. It is therefore likely that they are in equilibrium with dissociated DNP and protein; in other words that the complex may readily dissociate. In the case of “ECM” the materials available for complexing with the DNP liberated in a dispersate will of course be a function of the source and developmental state of the tissue from which the dispersate is prepared. Transfer of the “ECM” to a fresh solution could readily result in exchanges by which the previously complexed materials are set free into the medium, enriching it with factors which reflect the chemistry and metabolism of the cells of derivation. Some of these adventitious materials may then exert more or less specific influences upon the behavior of cells cultured in their presence; influences which differ with the qualities of the responding cells. It would be expected that similar influences might be demonstrated, in the absence of Experimental

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“ECM”, through the use for subsequent cultures of medium conditioned by the prior maintenance in it of specific kinds of cells. Such “conditioned medium” effects are well known [16, 17, 18, 24, 371 and may account for the promotion of aggregation of monolayer-modified [32] or other cells by ECM-containing supernatant fluids. Our own observations and those of Abercrombie and his colleagues, as mentioned in the Introduction, are consistent with the assumption that cells adhere to one another directly, without the interposition of extraneous cementing materials. The electron microscope observations of Lesseps [23], made in this laboratory, show the initial, localized zones of adhesion between aggregating cells to be identical in appearance with the zones of cell contact in established tissues, the unit membranes being separated by a narrow “gap” of lower electron density. Recent evidence [15] indicates that the density peaks measured on low angle X-ray diffraction patterns of myelin correspond with the location of the phosphate groups of the phospholipid, much but not all of the protein- and polysaccharide-containing outer layer lying external to the outer density peak. The “gap substance” [39] may thus represent not extracellular material, but rather the outermost layer of the cell surface complex; a viewpoint which we have tentatively adopted, while bearing in mind that this distinction may have no operational value, and that the border between cell and environment may be more a territory than a boundary line and may reflect the properties of both phases.

SUMMARY

Evidence is presented to show that a viscous material referred to elsewhere as “ECM”, reportedly elaborated in vitro by aggregating embryonic cells and suggested to possess a variety of functions in the processes of cell aggregation and tissue reconstruction, is in reality a highly hydrated deoxyribonucleoprotein gel. The gel appears to be formed by the chromosomes of ruptured cells as a result of the enzymatic removal of a portion of the chromosomal protein by the trypsin employed for tissue dissociation. It originates in greatest part, in the material studied, from the broken cells at the cut surfaces of the tissue fragments, and it appears to form complexes with a variety of macromolecular materials present in cell suspensions or culture media. Cells aggregate normally in its total absence, by processes, it is assumed, involving intimate contact of the cell surfaces themselves. The possible role of cell products in promoting cell aggregation in vitro is discussed. Experimental

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REFERENCES 1. ABERCROMBIE, 2. ABERCROMBIE, 3. ABERCROMBIE,

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4. __ ibid. 6, 293 (1954). 5. __ Nature 174, 697 (1954). 6. ABERCROMBIE, M., HEAYSMAN, 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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