fibronectin synthesis during oogenesis and early development of the amphibian pleurodeles waltlii

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Cell Differentiation, 14 (1984) 171-177 171 Elsevier Scientific Publishers Ireland, Ltd. 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 electro- phoresis, 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; Kubota 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 move- ments. 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, FN fibrils are present be- tween invaginating mesodermal cells and the over- laying ectodermal cells. Furthermore, microinjec- tion 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 electro- phoresis) to show that FN synthesis in Pleurodeles waltlii can be detected in oocytes, fertilized eggs and cleaving embryos. Our study shows clearly that stored maternal messenger RNA for FN con- tributes to this synthesis. Material and Methods Oocytes Adult females of P. waltfii 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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Page 1: Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

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

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 electro- phoresis, 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; Kubota 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 move- ments. 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, FN fibrils are present be- tween invaginating mesodermal cells and the over- laying ectodermal cells. Furthermore, microinjec- tion 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 electro- phoresis) to show that FN synthesis in P l e u r o d e l e s

wa l t l i i can be detected in oocytes, fertilized eggs and cleaving embryos. Our study shows clearly that stored maternal messenger RNA for FN con- tributes to this synthesis.

Material and Methods

O o c y t e s

Adult females of P. wa 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.

Page 2: Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

172

surgically excised. Free oocytes were obtained with collagenase treatment (1 mg/ml , SIGMA, grade I). Follicular cells were then completely removed by washing with calcium-magnesium-free (CMF- free) Steinberg solution containing EDTA (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 ob- tained 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 (Am- ersham, specific activity > 800 C i / m M , 200 #Ci /ml ) . Then to inhibit protein synthesis, oocytes were washed and incubated at 0 ° C with cyclo- heximide (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 imper- meable 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 /ml , 10 nl) followed by [35S]methionine or [3H]leucine-labelling in the presence of actino- mycin. 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'Far- rell (1975) and run at 300 V for 18 h. Second dimension separation was carried out on exponen- tial 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 (Bou- caut and Darrib6re, 1983). IgG binding to FN was identified with peroxidase conjugated sheep anti- rabbit IgG (Institut Pasteur, 1 /250°) . Specificity was demonstrated by using non-immune rabbit IgG for the first antibody.

Results

Identification of FN in amphibian oocytes and em- bryos

All 2-D electrophoretic separations of proteins corresponding to early stages of development dis-

Page 3: Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

130. 96.

M W x l O -~ 2 2 0

6 9

40.

2 5

4.,5 7 7

2

N~WxlO -3 7

173

4.spH

4 . 5 |

7 4 . 5

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). FN is identified in the region of molecular weight 220 kd by comigration with human and bovine plasma FN. (b) Im- munoblot t ing on nitrocellulose sheet of the two-dimensional gel

Fig. 2. Two-dimensional autoradiograms of [35S]methionine- labelled de novo-synthesized oocyte proteins of P. waltlii ob- tained from various stages. In each case, oocytes were in- cubated 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 molec- ular 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 re- vealed with peroxidase-labelled rabbit IgG (1/250). A spot of 220 kd molecular weight and 5.7 isoelectric point correspond- ing to cellular FN is clearly visible. (c) Two-dimensional silver- stained 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.

Page 4: Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

174

N~,WxlO -3 7.5 2 2 0 - I

,~---

4.3 I

7,5 220- - ~, I

2 5 -

4.apH

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7. 5 4.3 7. 5 4,3 z 2 o . T t . . . . =

I 7.5 4,3

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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

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175

p layed a Coomass ie b lue s ta ined c o m p o n e n t with 220 kd appa ren t molecu la r weight and 5.7 isoelec- tric po in t (Fig. l a ) . As prev ious ly repor ted , this 220 kd c o m p o n e n t comigra ted with ei ther h u m a n or bovine p l a s m a F N ( D a r r i b r r e et al., 1982). Blo t t ing exper iments pe r fo rmed with pur i f ied ant i - F N I g G clear ly ind ica ted the presence of F N in P. waltlii as soon as eggs were fert i l ized (Fig. lb ) . Moreover , the high resolu t ion silver s ta ining pro- cedure showed tha t F N was first de tec tab le in oocytes at s tage II of oogenesis (Fig. lc) .

F N synthesis

Prote in synthesis dur ing oogenesis and ear ly s tages of deve lopmen t was s tudied using [3H]- leucine and (or) [35S]methionine as precursors .

The incorpora t ion of [35S]methionine could be c lear ly seen in oocytes (Fig. 2). M a j o r p ro te ins such as tubul in and f l-act in were heavi ly label led for all s tages ( I - V I ) . The F N spot became rad ioac- t ive at s tage II. Fu r the rmore , compar i son be tween di f ferent stages ind ica t ed that the level of F N synthesis increased dur ing oogenesis (Fig. 2a, b).

The label l ing of fert i l ized eggs and ear ly em- b ryos with [3H]leucine enab led 2 5 - 5 0 rad ioac t ive spots to be ident i f ied (Fig. 3a, b). F r o m fert i l iza- t ion to the 32-cell stage, F N was not de tec tab le (Fig. 3a). However , FN- l abe l l i ng was evident at the late b las tu la s tage (Fig. 3b). In addi t ion , when [3H]leucine was i nco rpo ra t ed in to ear ly and mid- dle gastrulae, F N was d e a r l y label led. Us ing high concen t ra t ions of [35S]methionine as a p recursor (see Mate r ia l and Methods) , FN-syn thes i s could be de tec ted as soon as fer t i l izat ion had occur red (Fig. 3c). A u t o r a d i o g r a m s showed that the ra te of

FN-syn thes i s , as ana lysed with [35S]methionine in- co rpora t ion , increased f rom the ear ly b las tu la stage to the end of gas t ru la t ion (Fig. 3d).

In o rder to ascer ta in the or igin of FN-syn thes i s in fert i l ized eggs, t r anscr ip t ion was inh ib i ted by micro in jec t ing ac t inomyc in D and [35S]methionine pr io r to the first cleavage. Fo l lowing ac t inomyc in t rea tment , embryos d i sp layed no rma l cleavage bu t fa i led to gastrulate , and then were dissociated. Ana lys i s of label l ing using 2-D e lec t rophores is and a u t o r a d i o g r a p h y showed that ma jo r spots were con t inuous ly label led. F o r example , F N synthesis was observed in the presence of ac t inomyc in D. Rad ioac t ive spots co r respond ing to F N were iden- tiffed f rom fert i l ized eggs to gas t ru lae (Fig. 3e, f).

Our results thus show that F N synthesis occurs and increases dur ing oogenesis. The appea rance of F N - l a b e l l i n g in fert i l ized eggs cor re la ted well wi th the s torage of ma te rna l messenger R N A coding for F N and their t rans la t ion f rom fer t i l iza t ion to gas t ru la t ion . So far, FN-syn thes i s m a y be cons id- e red as a t r ansc r ip t ion - independen t event in ear ly deve lopment .

Discussion

The inco rpo ra t ion of rad ioac t ive amino acids in po lypep t ide s has been ana lysed by two-d imen- s ional gel e lec t rophores is in oogenesis and ear ly embryon ic deve lopment . We have focused our re- sults on the synthesis of F N dur ing these two per iods . The pa t t e rn of F N synthesis has been character ized, and it appea r s h ighly reproduc ib le .

[ 35S]methionine inco rpo ra t ion indica tes that F N synthesis begins dur ing oogenesis. Us ing 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. •

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methods of s ta ining (Coomass ie blue and silver staining), two-d imens iona l gels show the spot cor- responding to F N at every stage of embryon ic development . Fol lowing these results, we investi- ga ted whether this F N spot co r responded to F N s tored dur ing oogenesis or to de novo F N synthe- sis. Consequent ly , eggs were microinjec ted with [3H]leucine or [35S]methionine at dif ferent stages of embryogenesis . I t appea red that F N synthesis could be de tec ted dur ing early cleavage using [35S]methionine but not with [3H]leucine.

F N was shown to be synthesized dur ing oogen- esis. Its synthesis cont inues at a very low rate dur ing first cleavage, but the rate increases at the end of segmenta t ion and dur ing gastrulat ion.

A similar level of p ro te in synthesis dur ing em- bryogenesis was found for a small number of nuclear and p r e d o m i n a n t cy top lasmic proteins. F o r example , R N A polymerases and D N A poly- merases are s tored dur ing oogenesis and early deve lopment (Hol l inger and Smith, 1976; David- son, 1976). More recent ly W o o d l a n d et al. (1979), using interspecif ic Xenopus hybr ids , showed that his tones are s tored and require materna l mes- senger R N A for synthesis to occur dur ing ear ly deve lopment . Brock and Reeves (1978) indicate that actin and tubul in synthesis is de tec table at the ear ly stages of deve lopmen t of Xenopus laevis. Act in synthesis thus requires s tored materna l mes- senger R N A (Sturgess et al., 1980). F N synthesis in oogenesis and cleavage could therefore involve newly-synthes ized messenger R N A , or equal ly it m a y have resulted f rom mate rna l messenger R N A . Inhib i t ion of R N A synthesis by ac t inomycin D dur ing the incuba t ion of the eggs enables us to de te rmine which of these two al ternat ives is cor- rect. Our results show that F N synthesis is detecta- ble even if ac t inomycin D is injected before clea- vage and dur ing gast rula t ion. Consequent ly , this suggests that F N synthesis does not rely on t ran- scr ip t ion but depends ins tead on s tored mate rna l messenger R N A . Dur ing early cleavage, t ransla- t ion of F N messenger R N A occurs at a low rate and increases at the end of cleavage and dur ing gast rula t ion.

In fact, no funct ional role is assigned to F N in amph ib i an oocytes or b las tomeres dur ing early cleavage. However , at the b las tu la stage and dur-

ing gas t ru la t ion, an ext racel lu lar mat r ix is revealed by scanning e lect ron mic roscopy analysis (Naka- tsuji and Johnson, 1983) or through the use of lectins (Gua landr i s et al., 1983). One of the com- ponen ts of this ext racel lu lar mat r ix was found to be F N (Boucaut and Dar r ib r re , 1983). This F N - rich ext racel lu lar mat r ix covers the ent ire surface of the b las tocoele roof of the P. waltlii blastula . The accumula t ion of F N dur ing oogenesis and cleavage could be expla ined by the need for rap id incorpora t ion of F N in the ext racel lu lar mat r ix jus t pr ior to gast rula t ion.

In conclusion, as suggested by Lee et al. (1984) for Xenopus, the presence of F N dur ing early stages of deve lopmen t and gas t ru la t ion correlates with the ac t iva t ion of s tored mate rna l messenger R N A .

Acknowledgements

We thank Dr. J.P. Thiery for his interest th roughout the course of this work. The au thors are also indeb ted to J.L. D uba nd , T. Poole and G. Tucker for read ing the manuscr ip t , P. G r o u e for technical ass is tance and C. Rond ineau for typing the manuscr ip t . This work was suppo r t ed by grants f rom the Minis te re de la Recherche et de l ' In- dust r ie (D.G.R.S.T. No. 82 E 1139) and the Min i s t r re de l 'Educa t ion Na t iona l e (Di rec t ion de la Recherche, A R U 83).

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Boucaut, J.C., T. Darribrre, H. Boulekbache and J.P. Thiery: Prevention of gastrulation but not neurulation by antibod- ies to fibronectin in amphibian embryos. Nature (London) 307, 364-367 (1984).

Brock, H.W. and R. Reeves: An investigation of de novo protein synthesis in the South African clawed toad, Xenopus laevis. Dev. Biol. 66, 128-141 (1978).

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