Newly synthesized extracellular ribonucleic acids in the amphibian gastrula

Cell Differentiation, 13 (1983) 149-157

149

Elsevier ScientificPublishers Ireland, Ltd.

Newly synthesized extracellular ribonucleic acids in the amphibian gastrula * Sara S. S/mchez, M a r c e l o O. C a b a d a a n d F r a n c i s c o D. Barbieri Departamento de Biologia del Desarrollo, lnstituto Superior de lnvestigaciones Biolbgicas, Consejo Nacional de Inoestigaciones Cientlficas y T$cnicas and Universidad Nacional de Tucum~n, 4000 San Miguel de Tucum&n, Argentina

(Accepted 13 June 1983)

The possible synthesis of RNA located in the extracellular compartment of Bufo arenarum gastrula was studied using a biochemical method. [3H]adenosine was microinjected into the blastocoei of late blastulae or early gastrulae, which were then dissociated at advanced gastrula stage. RNA was extracted from both, the cellular supernatant and the disaggregated cells, by the Kirby-phenol procedure. Most of the ethanoi-precipitable radioactivity was sensitive to RNase and alkaline treatment. The partial characterization of these molecules indicate that the radioactive pattern of total RNA, found in sucrose gradients, the ratio Poly(A) + R N A / P o l y ( A ) - RNA as well as the radioactive pattern of Poly(A) fraction in acrylamide gels were different in samples from cellular and from extracellular origin. Although not conclusive, these results are proposed as a new argument for the existence of an extracellular RNA in the amphibian embryo. extracellular RNA; amphibian gastrulation; neural induction

Introduction The study of the extracellular matrix h a s received considerable attention during the past years, especially on account of its probable involvement in a variety of cellular events, including adhesion, migration and communication. In the particular case of the amphibian gastrula, cytochemical observations have shown an accumulation of materials between the cells implicated in the processes of morphogenesis (Kosher and Searles, 1973; Johnson, 1977a,b) and neural induction (Brachet, 1960). Concerning the latter event, it has been proposed * Preliminary results of this investigationwere reported at the IXth Congress of the International Society of Developmental Biologist, Basel, Switzerland, 1981. Correspondence to: Francisco D. Barbieri, Departamento de Biologia del Desarrollo, Chacabuco 461, 4000 San Miguel de Tucum~m,Argentina.

that induction of the central nervous system by the invaginating chordamesoblast (Spemann and Mangold, 1924) is mediated by a transfer of RNA (Brachet, 1940, 1942). Notwithstanding the great amount of research inspired by this .hypothesis, no attempt has been made, as far as we are aware, to characterize this extracellular RNA. Probably, the relatively small amount of its pool as compared to that of whole cells, as well as the difficulty to obtain uncontaminated preparations have discouraged its analysis by biochemical procedures. The only attempt to demonstrate the presence of extracellular R N A in the early amphibian embryo was that of Curtis (1958), who analyzed the materials removed after cell disaggregation of gastrulae of X e n o p u s laevis in the presence of EDTA. The results of these experiments, indicating the presence of RNA in the extracellular fraction, have been questioned, however, because the possibility cannot be excluded that it was the

0045-6039/83/$03.00 © 1983 ElsevierScientificPublishers Ireland, Ltd.

150 artifactual result of cell lysis (Brachet, 1960). Consequently, a more thorough investigation of extracellular RNA in the amphibian gastrula has been undertaken paying particular attention to the procedure of cell dissociation in connection to an eventual contamination of extracellular extracts by cell breakage products. Notwithstanding the fact that the danger of an artifact due to contamination by intracellular R N A cannot be definitely excluded, the data obtained may be interpreted as a better supported preliminary evidence for the synthesis of an extracellular RNA in the amphibian gastrula.

Material and Methods

Biological Oocytes and spermatozoa were obtained from the toad Bufo arenarum. Animals were collected in the province of Tucumhn and kept at 15-20°C until used. Females were spawned by means of body cavity injection of a homoplastic hypophysis suspension (Houssay, 1947). Sperm suspensions were PrePared by mincing testes in 10% Ringer solution. In vitro fertilization was carried out in the conventional way, and embryos were allowed to develop in 10% Ringer solution at 17°C. The egg jelly was removed with 1% thioglycolic acid solution neutralized with NaOH in the presence of phenolphthalein.

Radioactive labeling Aliquots of [2,8-3H]adenosine solution (34.4 Ci/gmol, New England Nuclear Corp., Boston, MA) were lyophilized, redissolved in Barth solution and microinjected into the blastocoel of late blastulae or early gastrulae (stages 9-10, according to Del Conte and Sirlin, 1951). Microinjections were performed by means of a Leitz micromanipulator as described elsewhere (Manes and Barbieri, 1976). The volume of adenosine solution injected was 55 + 15 nl with a concentration of 15 mCi/ml. Batches of 100-200 embryos were injected in each experiment.

Extraction of extracellular materials After injection the embryos were incubated at 17°C for about 20 h until reaching the middle or late gastrula stages (stages 11 or 12). The fertilization envelope was mechanically removed. Embryos were left undisturbed for a few minutes and then submitted to two succesive washes: first, with 10% Barth solution and then with the medium for cell dissociation proposed by Barth and Barth (1959) consisting of 2 mM EDTA in double 'A' NiuTwitty solution from which calcium and magnesium have been omitted. Polyvinyl sulfate (Sigma) (40 #g/ml) was added to the latter solution as RNase inhibitor. After 60-90 rain of incubation in this medium at room temperature without agitation, in order to minimize cell damage, the cells were completely dissociated. The supernatant was carefully removed with a fine pipette and, to ensure maximal extraction, the cells were rinsed with an additional small volume of fresh dissociating medium. The combined solutions were then centrifuged at 100 × g for 5 rain, and the supernatant was used directly for analysis. This solution will be subsequently referred to as the extracelhilar extract.

Estimation of cytolysis Lysis and cell damage during extraction of extracellular material were routinely assessed by the morphology of the cells after embryo disaggregation, as well as the presence of a sticky jelly between the cells. Control experiments showed a good correlation between the proportion of disrupted cells and the amount of this material in the medium. Yolk platelets pelleted after centrifugation of the extracts proved also to be a reliable marker of cell disruption. Determination of lactate dehydrogenase activity was used as an additional parameter for the estimation of cell lysis. The enzyme was assayed by the procedure of Kornberg (1955), and the method was standardized by measuring enzyme activity of an extracellular extract containing increasing concentrations of homogenized cells. For this purpose gastrula cells were broken in a motor-driven Potter-Elvehjem homogenizer in Barth solution, and after centrifugation

151

0.4.

0.3,

02.,

<~ 0.1

contamination 96 Fig. 1. Lactate dehydrogenase activity is plotted against the proportion of homogenized gastrulae added to a preparation of extracellular materials.

for 20 min at 17,750 x g, increasing amounts of the supernatant were mixed with the extracellular extract. Samples of 0.2 ml were used for enzyme determination in a final volume of 3.0 ml. As shown in Fig. 1, there is a linear correlation between the proportion of disruPted gastrula cells and lactate dehydrogenase activity. Cellular integrity was also assayed by the blue trypan exclusion test for cell viability. This test was carried out according to Phillips (1973). Sibling batches of embryos were dissociated and used either for obtaining the extracellular extract or added of trypan blue. Extraction and characterization of R N A The extracellular extract was mixed with the modified Kirby buffer (Kirby, 1965) followed by phenol-chloroform extractions and ethanol precipitations as described elsewhere (Cabada et al., 1977). Aliquots (0.1 ml) of the extracellular extract mixed with the Kirby solution were processed in two ways. One set was loaded onto G F / C (What-

man) glass fiber filters, air dried, immersed in a solution containing 0.8% 2-(4't-butylphenyl)-5(4"-biphenylyl)-l,3,4-oxadiazole (Butyl PBD) (Sigma) in toluene and radioactivity was estimated in a liquid scintillation counter. The remaining set (0.1 ml) was added to 0.9 ml of 2 x SSC solution, 0.5 ml of sodium acetate (pH 5.0) containing 1 m g / m l of yeast RNA, and precipitated with 0.5 ml of a 4% cetyltrimethylammonium bromide (CTAB) (Sigma) solution. The precipitate was collected on G F / C filters and assayed for radioactivity. To extract intracellular RNA, the dissociated gastrulae used for the preparation of the extracellular extract were homogenized in Kirby buffer and processed in the same way. All glassware was sterilized at 200°C for at least 3 h. Solutions were sterilized with diethyl pyrocarbonate (Sigma), and plastic materials in contact with RNA were made RNase-free by immersing in N a O H 0.3 N for 1 h and thoroughly rinsing with sterilized distilled water. When extracting RNA from the extracellular compartment, total RNA from Escherichia coli (100 # g / m l of extraction medium) was added as carrier. Aliquots of RNA from E. coil submitted separately to the same procedure proved to be responsible for < 3% of the radioactivity found in the ethanol-precipitable fraction from the extracellular extract. Practically no spurious radioactivity can be ascribed to the carrier after RNase or N a O H 0.3 N digestions. Enzymatic digestion of RNA was carried out in 2 × SSC solution containing 40 # g / m l of RNase (Sigma) for 30 rain at 37°C. RNase-resistant macromolecules were then ethanol-precipitated and collected on G F / C filters for radioactivity estimation. For alkaline hydrolysis, aliquots of total RNA were vacuum dried, redissolved in 1 ml of 0.3 N N a O H and incubated overnight at 37°C. After incubation, the samples were ethanol-precipitated, redissolved in 0.1 ml of distilled water, loaded onto G F / C filters, air dried and counted. Sucrose gradient analysis of RNA was carried out in 5-30% sucrose gradients prepared in NETS solution (0.1 M NaC1, 0.01 M EDTA, 0.01 M Tris-HC1 (pH 7.4), 0.2% SDS) and run for 3.5 h at

152 35,000 rpm in a L Beckman ultracentrifuge (rotor SW 39). Fractions of 10 drops each were CTABprecipitated and radioactivity assayed as detailed above. Total RNA from B. arenarum oocytes was run in a parallel gradient and, after gradient fractionation, A 260 was determined in the fractions in order to establish the position of 28, 18 and 4-5 S specimens. Prior to loading, RNA samples were heated at 65°C for 1 rain. Obtention of Poly(A) + RNA and Poly(A)- RNA by affinity chromatography with oligo(dT)-cellulose columns, digestion of total RNA with ribonuclease T 1, and 10% polyacrylamide gel electrophoresis of Poly(A) were carried out according to Cabada et al. (1977). The length of the Poly(A) tracts were assigned on the basis of the calibration curve published by Cabada et al. (1977).

3

H)-ADENOSINE

BLASTULA

1

O GASTRULA

Results

l

[ 3H]adenosine incorporation into extracellular RNA The experimental design used in the present study for extracellular RNA extraction is outlined in Fig. 2. After complete cell dissociation the cells and the clear supernatant were separately extracted by the phenol-Kirby procedure. Table I summarizes the results of three experiments showing the incorporation of radioactivity into macromolecules as compared to total recovered radioactivity. In most cases, the extracellular precipitable radioactivity was above 1% of cell precipitable radioactivity. Since an essential point in the rationale of this study is the prevention of contamination by products derived of cell breakage, special care has been taken to control the extent of cell damage. On the basis of the parameters chosen (see Methods), contamination of the extracellular fraction with materials of cell origin was reduced to a minimum. Dissociated cells exhibited a normal morphology, very few, if any, yolk platelets were seen after extracts centrifugation and there was no jelly sticking the cells together nor were stained cells observed under the microscope when dissociated gastrulae were treated with trypan blue. Cell contamination estimated by lactate dehy-

EMBRYO

DISSOCIATION

j

J

CELLS

"-..

EXTRACELLULAR

EXTRACT

Fig. 2. Extraction scheme for materials of the extracellular compartment.

drogenase activity in the extracellular extracts was also very low in most of the experiments here described. Those experiments in which contamination was high enough to account for the extracellular radioactivity found, were disregarded. RNA characterization When the ethanol-precipitable radioactivity was treated with RNase, < 6% of the extracellular

153 TABLE I [3H]Adenosine uptake and incorporation into extracellular (E) and intracellular (I) RNAa Expt. No.

1 2 3

Total radioactivity uptake ~

CTAB-precipitable radioactivity

Ethanol-precipitable radioactivity

E

I

E

I

E/I. 100

E

I

E/I- 100

138,390 18,079 613,744

5,860,530 1,331,961 3,434,567

12,060 9,363 77,904

939,090 644,671 906,800

1.3 1.5 8.6

38,631 8,760 48,480

1,960,745 952,380 2,060,760

2.0 0.9 2.4

a Radioactivity is expressed as cpm/100 embryos. b Samples obtained during Kirby extraction. TABLE II RNase digestion and alkaline hydrolysis of the ethanol-insoluble radioactivity Expt. No.

NaOH

RNase Extracellular

1 2 3 4 5

Intracellular

Extracellular

cpm

%

cpm

%

cpm

433 1,014 325 339 .

1.1 1.0 5.6 3.9

345,873 80,000 25,825 23,495 .

17.6 6.0 9.2 2.5

. . . 13 87

.

.

Intracellular %

. . .

cpm . . .

0.15 1,40

%

. . . 723 208,260

0.08 6.60

Results are expressed in counts/rain per 100 embryos and percentages of ethanol-precipitable radioactivity after each treatment.

r a d i o a c t i v i t y initially p r e s e n t was resistant to the e n z y m a t i c t r e a t m e n t ( T a b l e II). A l k a l i n e h y d r o l ysis lead to a recovery of < 2% of the original r a d i o a c t i v i t y ( T a b l e II).

Size distribution of newly synthesized R N A I n o r d e r to characterize the R N A e x t r a c t e d f r o m the extracellular c o m p a r t m e n t as well as that e x t r a c t e d f r o m the cells, aliquots of total R N A f r o m b o t h sources were s e p a r a t e l y r u n in 5 - 3 0 % linear sucrose gradients. R a d i o a c t i v i t y of the ext r a c e l l u l a r f r a c t i o n e x h i b i t e d disperse d i s t r i b u t i o n t h r o u g h o u t the g r a d i e n t as shown in the sedim e n t a t i o n profile r e p r e s e n t e d in Fig. 3, where r a d i o a c t i v e p e a k s at 3.5, 7.7, 18, 35, 45, a n d 82 S are a p p a r e n t . A p e a k o f r a d i o a c t i v i t y was also consistently o b s e r v e d n e a r the b o t t o m o f the gradient.

Synthesis of Poly(A) +R N A and Poly(A) - RNA These R N A molecules are d i f f e r e n t i a t e d b y their c a p a c i t y of b i n d i n g the oligo(dT)-ce.Uulose. A t high salt c o n c e n t r a t i o n P o l y ( A ) ÷ R N A is r e t a i n e d b y the column, while P o l y ( A ) - R N A elutes f r o m the c o l u m n in the s a m e c o n d i t i o n (for discussion see C a b a d a et al., 1977). T o t a l R N A o b t a i n e d f r o m the cells a n d the extracellular extract o f g a s t r u l a e was a p p l i e d to an oligo(dT)-cellulose c o l u m n a n d the newly synthesized Poly(A)+RNA and P o l y ( A ) - R N A were e s t i m a t e d b y assaying the rad i o a c t i v i t y in the b o u n d a n d void fractions. T h e results are s u m m a r i z e d in T a b l e III. Poly(A) ÷ R N A is p r e s e n t in b o t h R N A s from cells a n d the ext r a c e l l u l a r extract. T h e r a t i o P o l y ( A ) + R N A / P o l y ( A ) - R N A × 100 varies f r o m 8 to 35 ( m e a n + S E = 23.2 + 6.2; n = 4) in the case of extracellular extracts, a n d f r o m 4.2 to 15 ( m e a n _ SE = 7.2 +

154 28

18

IL

5-4

2.7; n = 4) in the case of R N A from the cells. This difference is significative from a statistical p o i n t of view ( P < 0.02). D a t a n o t shown indicate that the ratio of newly synthesized Poly(A) + R N A / P o l y ( A ) - R N A f o u n d i n the gastrula cells a n d in the extracellular extracts of those gastrulae, varies d e p e n d i n g o n the period of labeling a n d the stage of d e v e l o p m e n t of the embryo. Experiments are b e i n g carried out to further investigate this point.

1 .15

i

\ \ .10

II

I

/,

Sedimentation pattern of the newly synthesized Poly(A) +RNA

I

T6

t .5

E o. u

!

io

,,~,~

t~

2b Tub*" fxJml~*'r

Fig. 3. Sucrose gradient analysis of the RNAs extracted from extracellular extracts (e o) and cell homogenate (O . . . . . . O). The figure shows the counts/rain of [3H]adenosine incorporated into 100 embryos.

Newly synthesized P o l y ( A ) ÷ R N A f o u n d in gastrulae cells a n d in extracellular extracts differ in the a m o u n t detected a n d in its ratio to newly synthesized P o l y ( A ) - R N A , in each case. However, the s e d i m e n t a t i o n p a t t e r n of P o l y ( A ) + R N A obtained from a n oligo(dT)-cellulose column, from both, cells a n d extracellular extracts are n o t drastically different (Fig. 4). I n both cases, a heterodisperse d i s t r i b u t i o n is observed a n d only a few scattered points are n o t coincident. This similarity in the s e d i m e n t a t i o n p a t t e r n of Poly(A) ÷ R N A was observed in four separate experiments.

Size distribution of the newly synthesized Poly(A) sequences T o estimate the size d i s t r i b u t i o n of the newly s y n t h e s i z e d P o l y ( A ) sequences, s a m p l e s of

TABLE III Newly synthesized Poly(A)+ RNA and Poly(A)- RNA in extracellular extracts and cells from Bufo arenarum gastrula Expt. No.

Sample

Poly(A)- RNA

Poly(A)+ RNA

Poly(A) +RNA × 100 Voly(g) RNA

1

ECE Cells ECE Cells ECE Cells ECE Cells

2,920 835,111 5,578 1,282,914 15,239 3,396,307 14,038 4,276,781

222 36,421 1,680 197,280 2,975 163,567 5,017 179,542

7.6 4.4 30.1 15.4 19.5 4.8 35.7 4.2

2 3 4

ECE = extracellular extract. Results are expressed in cpm/100 embryos for each fraction and sample. Total RNA from ECE and cells were fractionated by oligo(dT)-cellulosecolumn as described in Material and Methods. RNA of each fraction was precipitated by adding RNA from yeast as carrier and CTAB, filtered through GF/C, filtered through GF/C filters. Radioactivity was estimated in a liquid scintillation counter.

155 ~nR

111

5sl i Die

31t

5.-4

/i l

211

9

2

6

T

I

M

?

K

H u

ID-O-.O-.O" 0

10

20

30

40

SO

~O-,O 80

Tube n u m b e r

0

10

20

Tube number

Fig. 4. SDS sucrose gradient centrifugation of Poly(A) + RNA from extracellular extracts ( e e ) and gastrula cells ( O . . . . . . O) corresponding to 100 embryos. Total RNA was fractionated by oligo(dT)-cellulose column and the bound fraction analyzed in 5-30% sucrose gradient. Each fraction of the gradient was CTAB precipitated and radioactivity estimated according to Material and Methods.

Poly(A)+RNA extracted from the cells and the extracellular extracts were electrophoresed in 10% polyacrylamide gels after digestion with ribonuclease T 1. As seen in Fig. 5 the patterns of distribution of these molecules in the gel differ in both cases. The size distribution of newly synthesized Poly(A) from cells fits a bimodal curve of distribution, being the modal figures of about 70 and 120 nucleotides. On the other hand, Poly(A) from extracellular extracts exhibits a different pattern, being the mean size longer than in the cells.

Discussion

The data presented in this report may be interpreted at least in two ways. There might be an

Fig. 5. Size distribution of Poly(A) sequences newly synthesized in extracellular extracts ( e e ) and cells ( O . . . . . . O) from gastrulae. Amounts of Poly(A) + RNA equivalent to 100 embryos were digested with ribonuclease T 1. Poly(A) sequences obtained were applied onto 10% SDS-polyacrylamide gels according to Material and Methods.

incorporation of [3H]adenosine into RNA molecules located in the ~Ftracellular compartment according to the working hypothesis. This possibility is supported by the insolubility of labeled molecules extracted with phenol, in ethanol and CTAB, combined with RNase digestion and alkaline hydrolysis. One question of considerable interest, the risk of contamination as a result of damage or breakage of cells, may be considered irrelevant on the basis of the morphological aspect of dissociated cells, the absence in the medium of such a good marker of cytolysis as yolk platelets, the absence of stained cells in the presence of trypan blue and the lack of a significant lactate dehydrogenase activity in extracellular extracts. The different nature of the RNAs extracted from the supernatant of dissociated cells from that extracted from the remaining cells may be considered as an additional reason against the possibility

156

of an artifactual result. In fact, the profiles of label distribution in sucrose gradients exhibited a different pattern for the RNAs obtained from each extract, as well as a different size distribution of the Poly(A)÷RNA and different values of Poly(A) length. It deserves to be noted by the way that the radioactive profile of the RNA extracted from the cells is in good agreement with those obtained for similar stages in X. laevis (Brown and Littna, 1964; Woodland and Gurdon, 1968). On the other hand, the size distribution of newly synthesized Poly(A) sequences differs from those reported by Sagata et al. (1980) for Xenopus gastrula. The peak of about 120 nucleotides in length is detected, but not so the peak of about 70 nucleotides in length. This difference can be ascribed to different labeling periods: 1.5 h for Xenopus and 16 h for Bufo. The high molecular weight RNA peak does not seem to be an artifactual result produced by molecular aggregation since it remained unchanged after heat treatment. Nevertheless, control experiments to exclude the possibility that it consists of an extracellular DNA, which has been detected in other cell systems (reviewed by Reid, 1979) had to be postponed to the next breeding season. Present results could also be explained by assuming that even in the case no relevant cell damage has occurred, a leakage of cytoplasmic R N A has taken place since the radioactivity profile of such a fraction is not expected to reflect that of total cell RNA. It must be recognized that despite the various contamination controls carried out, this question remains open. The correlation of present results with earlier histochemical observations lends further support to the assumption that the RNA analyzed here might be really localized in the intercellular space. Taking into account that in the experiments here described cells were separated by a very mild procedure, not involving the use of hydrolytic enzymes, the possibility that cell surface components were removed under present conditions is unlikely. Therefore, the RNA extracted was probably located in the extraceUular matrix as previously suggested for amphibian embryos (Brachet, 1959) as well as embryonic tooth primordia (Slavkin et al., 1969). Along this line, it is also reasona-

ble to assume that the extracellular RNA here described might be associated to the particulate material reported to occur between the neuroectoblast and mesoblast of Xenopus gastrula. Electron microscopy and some histochemical tests have shown that the space between these layers is occupied by particles of ribonucleoprotein similar to ribosomes together with glycogen granules and a fibrillar material (Kelley, 1969, Tarin, 1973). The heterogeneity of the RNA sedimentation pattern, the high proportion of Poly(A)÷RNA and the presence of long tracts of Poly(A) in the extracellular extract support the hypothesis that we are dealing with a particulate form of mRNA. From this standpoint it would be interesting to examine whether we are dealing with ribonucleoprotein particles of the type of informosomes according to Spirin (1966). In this respect it is interesting to point out that the different ratio of Poly(A) ÷ R N A / P o l y ( A ) - R N A newly synthesized observed in RNA from extracellular extracts and cells not only argue against the possibility of extracellular extract contamination by cell products, but also indicates that the extracellular RNA is enriched in Poly(A)÷RNA if compared to cellular RNA. It is also interesting to note in this context that the R N A from extracellular extracts contains tracts of newly synthesized Poly(A) longer than those detected in RNA from cells. This point might be of interest since long Poly(A) tracts in mRNA are associated with long-time translatable molecules (Marbaix et al., 1975, Huez et al., 1975). The observation that the removal of the granules of the ectomesoblasticjunction does not affect neurogenesis (Tarin, 1973) has cast some doubt on the involvement of these structures in neural induction (Tarin, 1973, 1977). The technique developed in the present work for extracellular RNA preparation with a minimum of intracellular RNA contamination may provide a promising advantage to test its neural inducing capacity by using an experimental design similar to that assayed by Niu and Twitty (1953). Whatever the role of the RNA here described, the presence of such a molecule in the extracellular compartment of the embryo at a stage when cell determination is at work deserves major attention.

157

Acknowledgements Appreciation is expressed to Mr. Roberto D. Ord6fiez for his assistance in the preparation of the manuscript, to Mr. Ra~l E. Barbieri for execution of the drawings, and Hugo G6mez for the typing. This work was supported in part by grants awarded by UNESCO (RLA 78/024 PNUD-UNESCO) to Marcelo O. Cabada and the Fundacibn Lucio Cherny. The authors are members of the Career Investigator of the CONICET.

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Johnson, K.E.: J. Cell Sci. 25, 323-334 (1977b). Kelley, R.O.: J. Exp. Zool. 172, 153-180 (1969). Kirby, K.S.: Biochem. J. 96, 266-269 (1965). Kornberg, A.: In: Methods in Enzymology, eds. S.P. Colowick and N.O. Kaplan, 1 (Academic Press, New York) pp. 441-443 (1955). Kosher, R.A. and R.L Searles: Dev. Biol. 32, 50-68 (1973). Manes, M.E. and F.D. Barbieri: Dev. Biol. 53, 138-141 (1976). Marbaix, G., G. Huez, A. Burny, Y. Cleuter, E. Hubert, M. Leelercq, H. Chantrenne, H. Soreq, U. Nudel and U.S. Littauer: Proc. Natl. Acad. Sci. U.S.A. 72, 3065-3067 (1975). Niu, M.C. and V.C. Twitty: Proc. Natl. Acad. Sci. U.S.A. 39, 985-989 (1953). Phillips, H.J.: In: Tissue Culture Methods and Applications, eds. P.F.I. Kxuse and M.K. Patterson, Jr. (Academic Press, New York) pp. 406-408 (1973). Reid, B.L.: Int. Rev. Cytol. 60, 27-52 (1979) Sagata, N., K. Shiokawa and K. Yamana: Dev. Biol. 77, 431-448 (1980). Sax6n, L., M. Karkinen-J~skel~nen, E. Lehtonen, S. Nordling and J. Wartiovaara: In: The Cell.Surface in Animal Embryogenesis and Development, eds. G. Poste and G. L. Nicolson (North-Holland, Amsterdam) pp. 331-407 (1976). Slavkin, H.C., P. Bringas and L.A. Bavetta: J. Cell. Physiol. 73, 179-190 (1969). Spemarm, H. and H. Mangold: Arch. Mikrosk. Anat. Entwicklungsmech. 100, 599-638 (1924). Spirin, A.S.: Dev. Biol. 1, 1-32 (1966). Tarin, D.: Differentiation 1, 109-126 (1973). Tarin, D.: In: Cell Interactions in Differentiation, eds. M. Karkinen-J~skel~iinen, L. Saxbn and L. Weiss (Academic Press, London) pp. 227-247 (1977). Woodland, H.R. and J.B. Gurdon: J. Embryol. Exp. Morphol. 19, 363-385 (1968).