Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

14 (1984) 171-177 Elsevier Scientific Publishers Ireland, Ltd.

171

Cell Differentiation,

CDF 00220

Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii Thierry Darrib6re 1, Dominique Boucher 2, Jean-Claude Lacroix 2 and Jean-Claude Boucaut 1 I Laboratoire de Biologie Expbrimentale, Universitb Renb Descartes, 45, rue des Saints- Pbres, 752 70 Paris Cedex 06, and 2 Laboratoire de Gbnbtique du Dbveloppement, Universitb Pierre et Marie Curie, 4, Place Jussieu, 75230 Paris Cedex 05, France

(Accepted 27 March 1984)

Fibronectin (FIN) synthesis during oogenesis and early embryogenesis of the amphibian Pleurodeles waltlii was investigated. The isotopically labelled amino acids [3H]leucine and [3SSlmethionine were incubated with oocytes or microinjected into embryos. Newly synthesized FN was analysed by polyacrylamide gel electrophoresis, using the high resolution two-dimensional gel system described by O'Farrell. With this method and fluorography we demonstrate that FN synthesis begins during oogenesis. De novo synthesized FN appears during cleavage and gastrulation. Using actinomycin D we show the presence of maternal messenger RNA coding for FN. It is translated during the cleavage and gastrulation stages. fibronectin; synthesis; oogenesis; early development; amphibian

Introduction Morphogenetic movements occurring in early development involve important changes both in cell morphology and cell adhesion (Perry and Waddington, 1966; Johnson, 1977; Fraser and Zalik, 1977; K u b o t a and Durston, 1978). At the molecular level, several lines of evidence suggest that these changes are mediated by cell surface glycoproteins. Recently, much interest has been focused on associated cell surface glycoproteins, especially fibronectin (FN) (Yamada, 1983). In amphibian embryos, FN is present during early development and important for cell movements. At the blastula stage, the entire inner surface of the blastocoele is covered with a FN-containing extracellular matrix (Boucaut and Darribere, 1983). During gastrulation, F N fibrils are present between invaginating mesodermal cells and the overlaying ectodermal cells. Furthermore, microinjection of anti-FN monovalent antibodies has dem-

onstrated that FN is necessary for the invagination and migration of mesodermal cells (Boucaut et al., 1984). So far, despite its key role, very little is known about FN synthesis during oogenesis and the early stages of development. In the present study, we have used radioactive amino acid labelling and two-dimensional gel electrophoresis (2-D electrophoresis) to show that F N synthesis in P l e u r o d e l e s w a l t l i i can be detected in oocytes, fertilized eggs and cleaving embryos. Our study shows clearly that stored maternal messenger R N A for FN contributes to this synthesis.

Material and Methods Oocytes

Adult females of P. w a l t f i i were anaesthetized with 1%o MS 222 (Sandoz). Pieces of ovaries were

0045-6039/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland, Ltd.

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surgically excised. Free oocytes were obtained with collagenase treatment (1 m g / m l , SIGMA, grade I). Follicular cells were then completely removed by washing with calcium-magnesium-free (CMFfree) Steinberg solution containing E D T A (1 mM). Oocytes were selected according to their diameter and appearance (Bonnanfant-Ja'is and Mentr6, 1983).

Fertifized eggs and embryos P. waltlii fertilized eggs and embryos were obtained by natural mating. They were staged according to Gallien and Durocher (1957). Selected embryos were manually dejellied and reared in sterile Steinberg solution. [ 3~S]methionine and [-~H]leucine-labelling Incorporation of [35S]methionine in oocytes was carried out at 18°C for 24 h with saline Barth's medium supplemented with [35S]methionine (Amersham, specific activity > 800 C i / m M , 200 # C i / m l ) . Then to inhibit protein synthesis, oocytes were washed and incubated at 0 ° C with cycloheximide (Calbiochem, grade B, 100 /~g/ml) in saline Barth's medium. [35S]methionine (Amersham, specific activity > 800 C i / m M , 200-1000 ~ C i / m l ) or [3H]leucine (AmerSham, specific activity 50 C i / m M , 1000 /~Ci/ml) precursors were microinjected (10 nl) in fertilized eggs and embryos subsequently reared for 4, 6 or 18 h at 18°C. As the eggs are impermeable to exogenous amino acids, the injections were performed in the blastomeres for the initial stages of development or in the blastocoele for the others.

A ctinomycin treatment Inhibition of transcription was achieved by microinjection of actinomycin D (SIGMA, grade III, 10 ~ g / m l , 10 nl) followed by [35S]methionine or [3H]leucine-labelling in the presence of actinomycin. To preserve amphimixy, fertilized eggs were microinjected 4 h after fertilization. Each experiment was repeated three times with different batches. Finally, only those embryos that

developed normally and reached a similar stage of development as the controls (microinjected with 10 nl of Barth's or Steinberg's solutions) were used for analysis.

2-D electrophoresis Oocytes or eggs were homogenized with lysis buffer containing 9.5 M urea, 2% Nonidet P 40, 2% ampholines and 5% 2-mercaptoethanol and centrifuged at 10000 g for 10 min. Isoelectric focusing gels were prepared as described by O'Farrell (1975) and run at 300 V for 18 h. Second dimension separation was carried out on exponential gradient (8-15%) or on continuous (10%) SDS-polyacrylamide gels. Proteins of known molecular weight were run in a slot at the side of each gel. Molecular weights of standard proteins were: bovine plasma fibronectin (220 kd), galactosidase (130 kd), phosphorylase (96 kd), bovine serum albumin (69 kd), aldolase (subunit 40 kd), and chymotrypsinogen (25 kd). 2-D gels were stained with Coomassie blue and destained in 45% methanol, 10% acetic acid. Silver staining was carried out as described by Oakley et al. (1980). Fluorography was performed according to Laskey and Mills (1975) and gels were exposed to Kodak XO mat films.

Immunoblotting Proteins were transferred to nitrocellulose sheets (Towbin et al., 1979) which were then overlaid with purified immunoglobulin (IgG) directed against Ambystoma mexicanum plasma FN (Boucaut and Darrib6re, 1983). IgG binding to FN was identified with peroxidase conjugated sheep antirabbit IgG (Institut Pasteur, 1 / 2 5 0 ° ) . Specificity was demonstrated by using non-immune rabbit IgG for the first antibody. Results

Identification of FN in amphibian oocytes and embryos All 2-D electrophoretic separations of proteins corresponding to early stages of development dis-

173

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Fig. 1. Identification of the spot corresponding to FN (arrow). (a) Two-dimensional gel electrophoresis of soluble proteins from 2-cell stage of P. waltlii eggs (Coomassie blue staining). F N is identified in the region of molecular weight 220 kd by comigration with h u m a n and bovine plasma FN. (b) Immunoblotting on nitrocellulose sheet of the two-dimensional gel

Fig. 2. Two-dimensional autoradiograms of [35S]methioninelabelled de novo-synthesized oocyte proteins of P. waltlii obtained from various stages. In each case, oocytes were incubated with [35S]methionine. For each oocyte stage, the first dimension isoelectric focusing gel was loaded with 20000 cpm. The proteins were separated according to their apparent molecular weight by electrophoresis on a gradient 8-15% acrylamide SDS gel. The two-dimensional dry gels were exposed on X-ray Kodak XO mat film for 2 wk. (a) Stage II oocyte; (b) stage VI oocyte. From stage II to stage VI FN synthesis was found. During this period the radiolabelled spot corresponding to FN increased in mass. M.W.: molecular weights of proteins. electrophoresis of soluble proteins from the 2-cell stage. Blotted proteins were incubated with purified rabbit antibodies against Ambystoma mexicanum plasma FN. Specific binding was revealed with peroxidase-labelled rabbit IgG (1/250). A spot of 220 kd molecular weight and 5.7 isoelectric point corresponding to cellular FN is clearly visible. (c) Two-dimensional silverstained gel of stage II oocytes. Second dimension separation was made on a gradient 8-15% in SDS polyacrylamide. The spot corresponding to FN appears stained in stage II oocytes. M.W.: molecular weights of proteins.

174

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25! Fig. 3. (a) Embryos were microinjected with [3H]leucine 4 h after fertilization and labelled proteins were isolated 6 h later (stage 4). About 50000 cpm were loaded on the first dimension isoelectric focusing gel and the fluorograph was exposed for 6 wk. No FN synthesis could be detected in these conditions. (b) Eggs were microinjected with [3H]leucine at mid-blastula stage (stage 6) and proteins were extracted at late blastula stage (stage 7) (6 h later). Approximately 50000 cpm were loaded, and the fluorography was

175 p l a y e d a C o o m a s s i e b l u e s t a i n e d c o m p o n e n t with 220 k d a p p a r e n t m o l e c u l a r weight a n d 5.7 isoelectric p o i n t (Fig. l a ) . A s p r e v i o u s l y r e p o r t e d , this 220 k d c o m p o n e n t c o m i g r a t e d with either h u m a n o r b o v i n e p l a s m a F N ( D a r r i b r r e et al., 1982). B l o t t i n g e x p e r i m e n t s p e r f o r m e d with p u r i f i e d antiF N I g G clearly i n d i c a t e d the presence of F N in P. waltlii as soon as eggs were fertilized (Fig. l b ) . M o r e o v e r , the high r e s o l u t i o n silver staining p r o c e d u r e showed t h a t F N was first d e t e c t a b l e in o o c y t e s at stage II o f oogenesis (Fig. lc).

F N synthesis Protein synthesis d u r i n g oogenesis a n d early stages of d e v e l o p m e n t was s t u d i e d using [3H]leucine a n d (or) [35S]methionine as precursors. T h e i n c o r p o r a t i o n of [35S]methionine could be clearly seen in o o c y t e s (Fig. 2). M a j o r p r o t e i n s such as t u b u l i n a n d fl-actin were heavily labelled for all stages ( I - V I ) . T h e F N s p o t b e c a m e r a d i o a c tive at stage II. F u r t h e r m o r e , c o m p a r i s o n b e t w e e n different stages i n d i c a t e d that the level of F N synthesis i n c r e a s e d d u r i n g oogenesis (Fig. 2a, b). T h e labelling of fertilized eggs a n d early emb r y o s with [3H]leucine e n a b l e d 2 5 - 5 0 r a d i o a c t i v e s p o t s to b e i d e n t i f i e d (Fig. 3a, b). F r o m fertilization to the 32-cell stage, F N was n o t d e t e c t a b l e (Fig. 3a). However, F N - l a b e l l i n g was evident at the late b l a s t u l a stage (Fig. 3b). I n a d d i t i o n , when [3H]leucine was i n c o r p o r a t e d i n t o early a n d m i d dle gastrulae, F N was d e a r l y labelled. U s i n g high c o n c e n t r a t i o n s o f [35S]methionine as a p r e c u r s o r (see M a t e r i a l a n d M e t h o d s ) , F N - s y n t h e s i s c o u l d b e d e t e c t e d as soon as fertilization h a d o c c u r r e d (Fig. 3c). A u t o r a d i o g r a m s showed that the r a t e of

F N - s y n t h e s i s , as a n a l y s e d with [35S]methionine inc o r p o r a t i o n , increased f r o m the early b l a s t u l a stage to the end of g a s t r u l a t i o n (Fig. 3d). I n o r d e r to ascertain the origin of F N - s y n t h e s i s in fertilized eggs, t r a n s c r i p t i o n was i n h i b i t e d b y m i c r o i n j e c t i n g a c t i n o m y c i n D a n d [35S]methionine p r i o r to the first cleavage. F o l l o w i n g a c t i n o m y c i n t r e a t m e n t , e m b r y o s d i s p l a y e d n o r m a l cleavage b u t failed to gastrulate, a n d then were dissociated. A n a l y s i s o f labelling using 2-D e l e c t r o p h o r e s i s a n d a u t o r a d i o g r a p h y s h o w e d that m a j o r spots were c o n t i n u o u s l y labelled. F o r example, F N synthesis was o b s e r v e d in the presence o f a c t i n o m y c i n D. R a d i o a c t i v e spots c o r r e s p o n d i n g to F N were identiffed f r o m fertilized eggs to g a s t r u l a e (Fig. 3e, f). O u r results thus show that F N synthesis occurs a n d increases d u r i n g oogenesis. T h e a p p e a r a n c e of F N - l a b e l l i n g in fertilized eggs c o r r e l a t e d well with the storage of m a t e r n a l messenger R N A c o d i n g for F N a n d their t r a n s l a t i o n f r o m fertilization to gastrulation. So far, F N - s y n t h e s i s m a y b e c o n s i d e r e d as a t r a n s c r i p t i o n - i n d e p e n d e n t event in early development.

Discussion T h e i n c o r p o r a t i o n of r a d i o a c t i v e a m i n o acids in p o l y p e p t i d e s has been a n a l y s e d b y t w o - d i m e n sional gel electrophoresis in oogenesis a n d early e m b r y o n i c d e v e l o p m e n t . W e have focused o u r results on the synthesis of F N d u r i n g these two periods. T h e p a t t e r n of F N synthesis has b e e n characterized, a n d it a p p e a r s highly r e p r o d u c i b l e . [ 35S]methionine i n c o r p o r a t i o n i n d i c a t e s that F N synthesis begins d u r i n g oogenesis. U s i n g t w o

exposed for 6 wk. De novo FN synthesis can be seen. (c) Eggs were microinjected with [3~S]methionine after fertilization and proteins extracted at stage 4. 20000 cpm were loaded on the gel, and it was over exposed for 6 wk. These experimental conditions reveal a low rate of FN synthesis during early cleavage. (d) Mid-gastrula eggs (stage 10) were microinjected with [35S]methionine. Labelled proteins were extracted at the end of gastrulation (stage 13), 40000 cpm were loaded orr the first dimension gel, and the second dimension gel was exposed for 2 wk. It appears that FN synthesis occurs during final stages of gastrulation. (e) Actinomycin D and [ 3~S]methionine were microinjected in eggs after fertilization. Radioactive proteins were analysed at 32-cell stage (stage 4) (20000 cpm). The autoradiogram was exposed for 6 wk. FN synthesis can be seen even if transcription is inhibited. (f) Early gastrula (stage 8a) eggs were microinjected with actinomycin and [3SS]methionine. Proteins were extracted at the midgastrula stage (stage 10) and separated on two-dimensional gel (40000 cpm). The autoradiogram was exposed for 2 wk. It indicates that the major spots are still present and in particular the spot corresponding to FN can be seen. The position of FN glycoprotein is indicated by the arrow. M.W: molecular weights of proteins. •

176 m e t h o d s of staining ( C o o m a s s i e blue and silver staining), t w o - d i m e n s i o n a l gels show the spot corr e s p o n d i n g to F N at every stage of e m b r y o n i c d e v e l o p m e n t . F o l l o w i n g these results, we investig a t e d whether this F N spot c o r r e s p o n d e d to F N stored d u r i n g oogenesis or to de n o v o F N synthesis. Consequently, eggs were microinjected with [3H]leucine or [35S]methionine at different stages of embryogenesis. It a p p e a r e d that F N synthesis could be d e t e c t e d d u r i n g early cleavage using [35S]methionine b u t not with [3H]leucine. F N was shown to be synthesized d u r i n g oogenesis. Its synthesis continues at a very low rate d u r i n g first cleavage, b u t the rate increases at the e n d of s e g m e n t a t i o n a n d d u r i n g gastrulation. A similar level of p r o t e i n synthesis d u r i n g embryogenesis was f o u n d for a small n u m b e r of n u c l e a r and p r e d o m i n a n t c y t o p l a s m i c proteins. F o r example, R N A p o l y m e r a s e s a n d D N A polymerases are stored d u r i n g oogenesis a n d early d e v e l o p m e n t ( H o l l i n g e r a n d Smith, 1976; D a v i d son, 1976). M o r e recently W o o d l a n d et al. (1979), using interspecific Xenopus hybrids, showed that histones are stored a n d require m a t e r n a l messenger R N A for synthesis to occur d u r i n g early d e v e l o p m e n t . Brock a n d Reeves (1978) indicate that actin a n d tubulin synthesis is d e t e c t a b l e at the early stages of d e v e l o p m e n t of Xenopus laevis. A c t i n synthesis thus requires stored m a t e r n a l messenger R N A (Sturgess et al., 1980). F N synthesis in oogenesis a n d cleavage could therefore involve newly-synthesized messenger R N A , or equally it m a y have resulted from m a t e r n a l messenger R N A . I n h i b i t i o n of R N A synthesis b y a c t i n o m y c i n D d u r i n g the i n c u b a t i o n of the eggs enables us to d e t e r m i n e which of these two alternatives is correct. O u r results show that F N synthesis is detectable even if a c t i n o m y c i n D is injected before cleavage and d u r i n g gastrulation. Consequently, this suggests that F N synthesis does not rely on transcription b u t d e p e n d s i n s t e a d on stored m a t e r n a l messenger R N A . D u r i n g early cleavage, translation of F N messenger R N A occurs at a low rate a n d increases at the end of cleavage a n d d u r i n g gastrulation. In fact, no functional role is assigned to F N in a m p h i b i a n oocytes or b l a s t o m e r e s during early cleavage. However, at the b l a s t u l a stage a n d dur-

ing gastrulation, an extracellular m a t r i x is revealed b y scanning electron m i c r o s c o p y analysis ( N a k a tsuji and Johnson, 1983) or t h r o u g h the use of lectins ( G u a l a n d r i s et al., 1983). O n e of the c o m p o n e n t s of this extracellular m a t r i x was f o u n d to be F N (Boucaut a n d D a r r i b r r e , 1983). This F N rich extracellular m a t r i x covers the entire surface of the blastocoele r o o f of the P. waltlii blastula. T h e a c c u m u l a t i o n of F N d u r i n g oogenesis a n d cleavage could be e x p l a i n e d b y the need for r a p i d i n c o r p o r a t i o n of F N in the extracellular m a t r i x j u s t prior to gastrulation. In conclusion, as suggested b y Lee et al. (1984) for Xenopus, the presence of F N d u r i n g early stages of d e v e l o p m e n t a n d g a s t r u l a t i o n correlates with the activation of s t o r e d m a t e r n a l messenger RNA.

Acknowledgements W e thank Dr. J.P. T h i e r y for his interest t h r o u g h o u t the course of this work. T h e a u t h o r s are also i n d e b t e d to J.L. D u b a n d , T. Poole a n d G. T u c k e r for r e a d i n g the m a n u s c r i p t , P. G r o u e for technical assistance a n d C. R o n d i n e a u for typing the manuscript. This w o r k was s u p p o r t e d by grants from the M i n i s t e r e de la Recherche et de l'Industrie ( D . G . R . S . T . No. 82 E 1139) a n d the M i n i s t r r e de l ' E d u c a t i o n N a t i o n a l e ( D i r e c t i o n de la Recherche, A R U 83).

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