The Ras/Raf Signaling Pathway Is Required for Progression of Mouse ...

MOLECULAR AND CELLULAR BIOLOGY, Oct. 1994, p. 6655-6662

Vol. 14, No. 10

0270-7306/94/$04.00+0 Copyright C 1994, American Society for Microbiology

The Ras/Raf Signaling Pathway Is Required for Progression of Mouse Embryos through the Two-Cell Stage NAOFUMI YAMAUCHI,' ANN A. KIESSLING,2

AND

GEOFFREY M.

COOPER'*

Division of Molecular Genetics, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School,' Department of Surgery, Harvard Medical School, and Faulkner Center for Reproductive Medicine, Faulkner Hospital, 2 Boston, Massachusetts 02115 Received 2 May 1994/Returned for modification 30 June 1994/Accepted 19 July 1994

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. This inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos. The early development of mammalian embryos involves a transition between maternal and embryonic programs of gene expression. In the mouse, maternal mRNAs support meiosis, fertilization, and the first cleavage (4, 40). These maternal messages are then degraded at the two-cell stage, and expression of the embryonic genome, which directs subsequent cleavage divisions, is initiated (14). The two-cell mouse embryo is characterized by a novel cell cycle in which this transition between maternal and embryonic gene expression occurs. Embryonic transcription initiates during the short G1 period (-3 h) of two-cell embryos but is not required for progression to S phase (3, 14, 24, 39). The major transition between maternal and embryonic programs then occurs during the long G2 period (-12 h), and embryonic transcription is required for cleavage to four cells (3, 14, 24). It is noteworthy that these patterns of gene expression in mammalian embryos differ substantially from those of lower organisms, such as Xenopus laevis, in which maternal mRNAs persist and are sufficient to direct development to at least the blastula stage. Thus, the early transition from maternal to embryonic gene expression represents a novel regulatory point in mammalian develop-

signaling in early development (1, 27, 33, 36, 42, 43). For example, both insulin-like growth factor II and its receptor are expressed in early mouse embryos, starting at the two-cell stage, forming an autocrine loop which may play a role in embryonic cell proliferation (43). The ras proto-oncogenes encode small GTP-binding proteins that are activated downstream of protein-tyrosine kinases and play a central role in the intracellular transduction of signals from growth factor receptors (25). Ras function is required for growth factor-induced proliferation or differentiation of a variety of cell types, as indicated by experiments showing that growth factor-induced cellular responses are effectively inhibited by either anti-Ras monoclonal antibody Y13-259 (19, 21, 31, 46) or the dominant negative mutant rasH N-17 (6, 11, 48, 55). The Ras proteins couple growth factor receptors to activation of the Raf protein-serine/threonine kinase (5, 30, 51-54, 56, 58). Raf in turn activates mitogenactivated protein (MAP) kinase via phosphorylation of MAP kinase kinase (10, 20, 22). MAP kinase then phosphorylates a variety of proteins, including transcription factors, leading to growth factor-induced changes in gene expression (16, 28, 41). Since the Ras/Raf pathway is critical to signal transduction from growth factor receptors, one would predict that it would be required for autocrine signaling in eggs or embryos. Consistent with this possibility, we have previously reported that all three members of the ras gene family (rasH, rasK, and rasN) and raf-1 are expressed in early mouse embryos, first as maternal RNAs in oocytes and then as transcripts of the embryonic genome starting at the two-cell stage of embryo development (34). In the present study, we have investigated the potential role of the Ras/Raf signaling pathway in meiosis and early embryo development by using antisense oligonucleotides, anti-Ras monoclonal antibody, and the dominant negative Ras N-17 mutant to inhibit Ras expression and function in mouse oocytes and embryos. Our results indicate that the Ras/Raf pathway is specifically required for development of mouse embryos through the two-cell stage. Since this is the stage at which embryonic transcription is initiated, these

ment.

The potential role of growth factors in the early development of mammalian embryos is unclear. Exogenous growth factors do not appear to be required, since chemically defined media lacking serum or growth factors support the in vitro development of fertilized mouse eggs to blastocysts (7, 12).

Moreover, four-cell embryos developed in media lacking growth factors are capable of giving rise to viable offspring following transfer to surrogate mothers (29). Nonetheless, mouse oocytes and early embryos have been shown to express genes encoding a variety of growth factors and growth factor receptors, suggesting the potential involvement of autocrine

* Corresponding author. Mailing address: Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: (617) 375-8225. Fax: (617) 375-8237.

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results suggest the possible role of the Ras/Raf pathway, and hence of autocrine growth factor signaling, in the regulation of embryonic gene expression. MATERIALS AND METHODS Isolation and culture of oocytes, eggs, and embryos. Cumulus-enclosed oocytes were isolated from ovaries of 18- to 26-day-old B6SJL mice (The Jackson Laboratory) 46 to 50 h after intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin. Oocytes were collected in Dulbecco's phosphate-buffered saline (DPBS; Sigma Chemical Co.) containing 5% fetal bovine serum and 150 ,uM isobutylmethylxanthine to maintain meiotic arrest. Following microinjection, oocytes were cultured in modified Ham's F-10 medium (44) containing 5% fetal bovine serum. For collection of fertilized eggs, 26- to 33-day-old B6SJL or B6D2F1 (Charles River Breeding Laboratories) mice were induced to superovulate with intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin followed 48 h later by 5 IU human chorionic gonadotropin (hCG). Pronuclear-stage eggs were collected 20 to 22 h after hCG injection from oviducts of females that had mated overnight with B6D2F1 males. Eggs were collected in DPBS supplemented with 4 mg of bovine serum albumin (BSA) per ml, treated with 75 IU of hyaluronidase per ml to disperse cumulus and granulosa cells, and transferred to modified Ham's F-10 medium supplemented with 4 mg of BSA per ml. Embryos were cultured in the center well of organ culture dishes (Falcon 3037) containing 1 ml of medium with 3 ml of medium in the outer well. All culture of oocytes and embryos was performed at 370 in 8 to 10% CO2 (pH 7.1 to 7.2). Oligonucleotides and plasmids. Antisense oligonucleotides were synthesized and purified as previously described (32). Two antisense ras oligonucleotides were chosen to hybridize with all three members of the ras gene family. A-ras-1 (5' TCCAACCACCACAAG 3') is complementary to nucleotides 16 to 30 of rasK; it has one mismatch with rasN and two mismatches with rasH. A-ras-2 (5' CACAAAGTGGTTCGT 3') is complementary to nucleotides 73 to 87 of both rasH and rasN; it has one mismatch with rasK. The antisense mos oligonucleotide A-M2mos was previously described (32). The ras expression plasmids pSV-CR and pSV-N17 were constructed by inserting the XbaI-PstI fragments of normal rasH and rasH N-17 genes (48) into XbaI-PstI-digested pSVK3 (Pharmacia), so that the ras genes were expressed from the simian virus 40 early promoter. The activated raf expression plasmid pSV-22W was constructed by insertion of the EcoRI fragment of plasmid raf22W, which contains a c-raf-1 oncogene activated by N-terminal deletion (47), into the EcoRI site of pSVK3. Anti-Ras monoclonal antibody. Rat hybridoma cells producing monoclonal antibody Y13-259 (15) were cultured in RPMI 1640 medium containing Nutridoma-SP (Boehringer Mannheim). Anti-Ras monoclonal antibody was purified from the culture medium by precipitation from 50% ammonium sulfate and protein A-agarose affinity chromatography (Immunopure kit; Pierce), followed by dialysis against phosphate-buffered saline. Microinjection. Oocytes were microinjected in the cytoplasm with 10 pl of antisense oligonucleotides (1 pug/pul) or anti-Ras monoclonal antibody (10 pg/pul) as previously described (32). After microinjection, the oocytes were washed three times with 2.5 ml of DPBS containing 5% fetal bovine serum and transferred to culture medium without isobutylmethylxanthine to initiate meiosis. After 18 to 22 h of culture,

MOL. CELL. BIOL.

the oocytes were denuded and scored for germinal vesicle breakdown, polar body extrusion, and re-formation of a nucleus as an indication of failure to progress to meiosis II or to maintain metaphase II arrest (32). Fertilized eggs and two-cell embryos were microinjected with plasmid DNAs as previously described for microinjection of oocyte nuclei (35). Plasmid DNAs prepared by the alkaline lysis method (26) were reprecipitated three times with sodium acetate and ethanol and rinsed twice with 70% ethanol. The pellet was dried under vacuum and resuspended in injection buffer (48 mM K2HPO4, 14 mM NaH2PO4, 0.25 mM EDTA [pH 7.2]) to a final concentration of 100 to 150 ,ug/ml. The DNA was centrifuged at 15,000 x g in an Eppendorf microcentrifuge for 60 min at 4°C, and the supernatant was loaded into an injection pipette with a diameter of 1 to 2 ,um, using a Picoinjector IM-200 (Medical Systems, Greenvale, N.Y.). Eggs or embryos were visualized by using Hoffman diffractioninterference contrast optics, and nuclei were injected with approximately 5 pl of plasmid DNA solution. Injections were monitored to detect nuclear swelling. Following injection, eggs or embryos were returned to culture medium and monitored for further development. Typically, 65 to 75% of eggs or embryos survived injection. Analysis of oocyte RNAs by RT-PCR. Either single oocytes or groups of five oocytes in 2 to 3 ,ul of DPBS were lysed by addition of 3 ,ul of lysis buffer containing 0.5% Nonidet P-40 and 10 mM dithiothreitol (34). A single round of reverse transcriptase-PCR (RT-PCR), consisting of 40 cycles of amplification, was then performed as previously described (34). PCR products were electrophoresed in a 5% polyacrylamide gel and visualized by staining with ethidium bromide. Embryonic protein synthesis. Following injection, embryos were cultured in 1 ml of modified Earle's balanced salt solution containing 10 ,uM EDTA. In some cases, a-amanitin (100 ,g/ml) was added to inhibit embryonic transcription. At 18 to 22 h after injection (41 to 45 h post-hCG), embryos were transferred to a 50-pld drop of Earle's balanced salt solution containing 10 p,M EDTA and 1 mCi of [35S]methionine[35S]cysteine (Trans35S-label; 1,100 Ci/mmol; ICN) per ml. Embryos were labeled for 3 to 4 h, washed with DPBS containing 40 ,ug of BSA per ml, boiled for 2 min in 2x sodium dodecyl sulfate (SDS) sample buffer (23), and electrophoresed in an SDS-10% polyacrylamide gel. The synthesis of embryonic proteins was quantitated with a Phosphorlmager (Molecular Dynamics).

RESULTS Maternal Ras expression is not required for oocyte meiosis. We initially investigated the possible role of translation of maternal ras mRNAs by microinjection of germinal vesicle stage oocytes with antisense ras oligonucleotides. Two antisense ras oligonucleotides (A-ras-1 and A-ras-2) were chosen to hybridize with all three members of the ras gene family. A-ras-1 is complementary to rasK but has one mismatch with rasN and two mismatches with rasH. A-ras-2 is complementary to rasH and rasN but has one mismatch with rasK. The effectiveness of these antisense oligonucleotides in degrading the maternal ras mRNAs was assessed by RT-PCR analysis of microinjected eggs (Fig. 1). As previously reported (34), maternal transcripts of all three ras genes were detected in uninjected eggs. Transcripts of rasH and rasN were degraded in eggs that were injected with either A-ras-1, A-ras-2, or a mixture of the two antisense oligonucleotides. Injection of A-ras-2 alone was not sufficient to completely degrade rasK mRNA, but this was accomplished by injection with A-ras-1 or

VOL. 14, 1994 C

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REQUIREMENT OF Ras/Raf PATHWAY AT TWO-CELL STAGE 2 1+2

ras N

C

1

2

ras H

C

2 1+2

1

A

B

C

D

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FIG. 1. Degradation of ras mRNAs in antisense oligonucleotideinjected eggs. Uninjected control oocytes (lanes C) or oocytes that had been injected with antisense oligonucleotide A-ras-1 (lanes 1), A-ras-2 (lanes 2), or A-ras-1 plus A-ras-2 (lanes 1+2) were allowed to undergo meiosis. Metaphase II eggs were collected 18 h after injection and analyzed for ras mRNAs by RT-PCR using PCR primers specific for each of the individual ras genes (34). Single eggs were used for detection of rasK and rasN mRNAs, and a pool of five eggs was used for detection of rasH.

A-ras-1 plus A-ras-2. Thus the combination of the two antisense ras oligonucleotides resulted in degradation of the maternal transcripts of all three ras genes. We then determined the effects of such antisense ras oligonucleotide injections on the ability of mouse oocytes to undergo meiosis (Table 1). As a control, we included injections with antisense mos oligonucleotides, which block normal progression of mouse oocytes from meiosis I to meiosis II (32). More than 95% of either uninjected or antisense ras- or mos-injected oocytes underwent germinal vesicle breakdown, and more than 80% of all oocytes completed meiosis I, as indicated by polar body extrusion. As previously reported (32), approximately 70% of the antisense mos-injected oocytes reformed a nucleus within the body of the egg, indicating failure to progress normally to meiosis II. In contrast, the antisense ras-injected oocytes appeared to progress to meiosis II and maintain normal metaphase II arrest, indistinguishable from the uninjected controls. It thus appeared that translation TABLE 1. Effects of antisense ras oligonucleotides and anti-Ras monoclonal antibody on oocyte meiosis Treatment (no. of oocytes)a

Meiotic maturation (% of oocytes)b GVBD

PB

MII

Antisense oligonucleotide Uninjected (378) A-M2mos (39) A-ras-1 (179) A-ras-2 (205) A-ras-1 + A-ras-2 (55)

98 97 97 99 100

84 95 85 88 82

84 31 85

Anti-Ras antibody Uninjected (104) BSA (50) Y13-259 (78)

98 100 100

94 100 99

94 100 99

88 82

a Cumulus-enclosed oocytes from B6SJL mice were microinjected with 10 pl of the indicated oligonucleotides (1 jug/,ul), BSA (10 ,ug/,ul), or anti-Ras monoclonal antibody Y13-259 (15 Rg/pAl). The total number of injected oocytes, from multiple independent experiments, is given in parentheses. b Oocytes were scored 18 to 22 h after initiation of meiosis for germinal vesicle breakdown (GVBD), polar body extrusion (PB), and progression to metaphase II (MII). Failure to progress through meiosis II in A-M2mos-injected oocytes was indicated by re-formation of a nucleus within the body of eggs that had extruded polar bodies (32). Data are presented as the percentage of total injected oocytes that progressed to each of these states.

FIG. 2. Development of preimplantation embryos. Pronuclearstage eggs were microinjected with the control plasmid pSV-CR (day 1) and cultured to the blastocyst stage. (A) Two-cell embryo on day 2; (B) four-cell embryo on day 3; (C) compacted morula on day 4; and (D) blastocyst on day 5.

of ras maternal mRNAs, in contrast to mos, was not required for normal meiotic maturation. To test the possibility that Ras protein synthesized during oocyte growth is required for meiosis, we also microinjected germinal vesicle-stage oocytes with anti-Ras monoclonal antibody Y13-259, which effectively inhibits Ras function in a variety of cell systems (19, 21, 31). However, such anti-Ras antibody microinjection also had no effect on oocyte meiosis (Table 1). Since the same antibody, injected to similar intracellular concentrations, blocks Ras function in other cell types, these results suggest that Ras is not required for normal meiosis of mouse oocytes. The Ras N-17 dominant negative mutant inhibits early embryo development. To investigate the potential role of Ras during subsequent preimplantation development, we microinjected the pronuclei of fertilized eggs with a construct in which the dominant inhibitory rasH N-17 mutant was expressed from the simian virus 40 early promoter (pSV-N17). Controls were either uninjected or injected with a similar construct expressing the normal rasH gene (pSV-CR). Embryos were cultured and scored for development to two cells at 20 to 24 h after injection (day 2), four cells (day 3), compacted morulae (day 4), and

blastocysts (day 5) (Fig. 2). More than 90% of uninjected eggs cleaved to two cells, as did 70 to 80% of eggs injected with either pSV-N17 or pSV-CR (Fig. 3). However, subsequent development of the two-cell embryos to morulae and blastocysts was inhibited by Ras N-17 expression. Thus, 70 to 80% of uninjected or pSV-CR-injected

YAMAUCHI ET AL.

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eggs, but only 30% of pSV-N17 injected eggs, developed to morulae and blastocysts when scored on day 5 (Fig. 3). Ras N-17 expression thus inhibited development by two- to threefold, corresponding to a significant difference between pSV-CR and pSV-N17 injected embryos (P < 0.02 by t test). To determine the stage at which development was inhibited by Ras N-17 expression, we analyzed the distribution of embryos on day 5 of these experiments (Fig. 4). Approximately 50% of the pSV-N17-injected embryos were arrested at the two-cell stage, whereas less than 20% of control embryos were arrested at this point in development. In contrast, only small numbers of either control or pSV-N17-injected embryos were arrested at later developmental stages (three, four, or six cells). These results indicate that the effect of Ras N-17 expression is primarily to inhibit the subsequent development of two-cell

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FIG. 4. Ras N-17 inhibits development past the two-cell stage. Uninjected (=II), pSV-CR-injected ( ), and pSV-N17-injected (_) eggs from experiments summarized in Fig. 3 were scored on day 5 for development to the indicated stages. The data are percentages of embryos at each stage from three independent experiments with 10 to 25 embryos each (total of 45 uninjected, 41 pSV-CR-injected, and 44 pSV-N17-injected eggs). M+B, morulae and blastocytes.

>2 cell

M +B

FIG. 5. Ras N-17 inhibits development of B6D2F1 embryos. Pronuclear-stage eggs of B6D2F1 mice were microinjected as in Fig. 3. Embryos were scored for two cells on day 2, cleavage beyond two cells (>2 cell) on day 3, and morulae plus blastocysts (M+B) on day 5. Data are percentages (±SEM) of development to each stage, derived from 10 to 13 independent experiments with 15 to 35 embryos each. Inhibition of development by injection with pSV-N17 compared with pSV-CR is indicated by * (P < 0.001) and ** (P < 0.005). Symbols are as in Fig. 4.

embryos, since the decreased formation of morulae and blastocysts closely corresponds to an increase of embryos blocked at the two-cell stage. Most of the embryos that cleaved beyond this stage appeared to develop to morulae and blastocysts and may not have expressed sufficient levels of the mutant Ras to effectively inhibit development. The inhibitory effect of Ras N-17 on development through the two-cell stage was confirmed by experiments in a second strain of mice (B6D2F1 instead of B6SJL). In these experiments, embryos were scored on day 2 for formation of two cells, on day 3 for cleavage beyond the two-cell stage, and finally on day 5 for development to morulae and blastocysts (Fig. 5). In the B6D2F1 embryos, injection itself was somewhat toxic, so development of pSV-CR-injected embryos was inhibited relative to the uninjected controls. However, injection of pSV-N17 was significantly inhibitory compared with injection with pSV-CR when embryos were scored either for development beyond the two-cell stage (P < 0.001) or for development to morulae and blastocysts (P < 0.005). Active Raf bypasses the Ras N-17 block to development. The Raf protein-serine/threonine kinase acts downstream of Ras in a variety of differentiated cell types, and expression of activated Raf is able to overcome the inhibitory effects of Ras N-17 on cell proliferation or differentiation (6, 11, 46, 51). To determine whether the effect of Ras N-17 on two-cell embryo development resulted from interference with the Ras/Raf signaling pathway, we therefore coinjected constructs expressing an active raf oncogene (pSV-22W) together with pSV-N17 into pronuclear stage eggs. To avoid nonspecific toxicity in these experiments, each plasmid was injected at a concentration of 75 ,ig/ml (rather than 100 to 150 jig/ml as used for single plasmid injections), so that the total concentration of plasmid DNA did not exceed 150 ,g/ml. The injection of pSV-N17 plus a control plasmid (pSVK3) resulted in inhibition of embryo development comparable to that obtained following injection of higher doses of pSV-N17 alone (compare Fig. 5 and 6). However, coinjection of pSV22W reversed the inhibitory effect of pSV-N17 on embryo development, when scored either for development beyond the two-cell stage on day 3 (Fig. 6A) or for development to

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FIG. 6. Active Raf reverses the Ras N-17 block to embryo development. Pronuclear-stage eggs of B6D2F1 mice were simultaneously microinjected with two plasmid DNAs, each at a concentration of 75 jig/ml, so that the total concentration of plasmid DNA was 150 ,ug/ml. Eggs were injected either with pSV-CR or pSV-N17 plus either a control plasmid (pSVK3; M ) or a plasmid expressing an activated raf oncogene (pSV-22W; _). Embryos were scored for development beyond two cells on day 3 (A) and for morulae and blastocysts (M+B) on day S (B). Data are percentages (±SEM) from five independent experiments with 15 to 30 embryos each except for pSV-CR-plus-pSV-22W-injected eggs (a single experiment). The reversal of Ras N-17 inhibition by Raf is indicated by comparison of the development of pSV-N17-plus-pSV-22W-injected embryos with that of embryos injected with pSV-N17 plus pSVK3; * indicates P < 0.01, and ** indicates P < 0.03.

morulae and blastocysts on day 5 (Fig. 6B). It thus appeared that the inhibitory effect of Ras N-17 on development of two-cell embryos resulted from interference with the Ras/Raf signaling pathway. Although cyclic AMP (cAMP) inhibits Ras/Raf signaling in some cells, such as rat fibroblasts (9, 18, 45, 57), cAMP can instead act downstream of Ras to bypass the inhibitory effect of Ras N-17 in other cell types, such as PC12 cells (49). In addition, cAMP can overcome the inhibition of two-cell mouse embryo development resulting from exposure to purines (13). We therefore tested the possible effect of cAMP on reversing the block to mouse embryo development resulting from Ras N-17 expression. However, treatment with 500 ,uM dibutyryl cAMP failed to reverse the inhibitory effect of pSV-N17 injection (data not shown), although similar treatment effectively reverses purine inhibition of the same stage of embryo development (13). Ras is specifically required at the two-cell stage. The two-cell mouse embryo is the stage at which maternal RNAs are degraded and transcription from the embryonic genome is initiated (14). The requirement for Ras at this stage of embryonic development therefore suggests the possibility that the Ras/Raf signaling pathway is involved in the regulation of embryonic gene expression. It was thus of interest to determine whether Ras function was specifically required during the onset of embryonic transcription in two-cell embryos or was also required for subsequent stages of embryonic development. We therefore microinjected pSV-N17 into the nuclei of blastomeres of late two-cell embryos (46 h post-hCG), in which embryonic transcription was already active (3, 14). In contrast to the results obtained with pronuclear stage eggs, injection of pSV-N17 at this time did not inhibit subsequent cleavage beyond two cells or development to morulae and blastocysts (Table 2). These results therefore indicate that Ras function is specifically required at the two-cell stage but not for subsequent embryonic cleavages. It should be noted, however, that these results do not exclude a requirement for Ras at later stages of embryo development (such as the morula-blastocyst transition), since it is not clear that pSV-N17 injected at the two-cell stage would still be active at this time.

Ras N-17 does not inhibit the onset of embryonic transcription. Since Ras N-17 specifically inhibited development of two-cell embryos, we sought to determine if Ras function was required for the initiation of embryonic transcription. The initial onset of transcription from the embryonic genome is detected during the short G, period of two-cell embryos, followed by a major burst of embryonic transcription during G2 (3, 14). The earliest markers of embryonic genome expression are a group of proteins of approximately 70 kDa, including hsp70, which are detected within 3 h after cleavage to two-cells and whose synthesis is inhibited by ot-amanitin (2, 3, 8, 13, 14, 38). We therefore used synthesis of these 70-kDa proteins to assess the effect of Ras N-17 expression on the initiation of embryonic transcription. As illustrated in Fig. 7A, 70-kDa proteins represented the major products of translation in both control and pSV-N17 injected embryos labeled with [35S]methionine-[35S]cysteine from 41 to 44 h after hCG. As expected, synthesis of these proteins was blocked by ot-amanitin, indicating that they represented products of embryonic transcription. In contrast, the synthesis of other proteins (including a group of proteins of -35 kDa) was not inhibited by cx-amanitin, indicating that they

TABLE 2. Lack of effect of Ras N-17 expression in late two-cell embryos Embryo development"

Treatment'

Uninjected pSV-CR pSV-N17

>2 cells

Morulae + blastocysts

55/71 (78) 99/118 (84) 98/114 (86)

27/42 (64) 52/79 (66) 47/86 (55)

a Late two-cell embryos of B6D2F1 mice (46 h after hCG; day 2) were microinjected in the nucleus of one blastomere with plasmid DNAs (100 or 150 ,ug/ml). Attempts to inject both blastomeres of the same embryo with either pSV-CR or pSV-N17 yielded poor development, presumably because of toxicity. b Data are presented as the fraction of total embryos from two to four independent experiments that developed past two cell on day 3 and to morulae and blastocysts on day 5. The percentages of embryos that developed to each stage are given in parentheses.

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A 1

2

3

B 12

3

98694630FIG. 7. Synthesis of embryonic proteins in pSV-N17- and pSV-CRinjected eggs. Pronuclear-stage eggs of B6D2F1 mice were microinjected with pSV-CR or pSV-N17. Some of the pSV-CR-injected eggs were then cultured in co-amanitin to inhibit embryonic transcription. Two-cell embryos were labeled with [35S]methionine-[35S]cysteine for 3 h starting 41 h after hCG (A) or 4 h starting 44 h after hCG (B), and lysates of 12 embryos were electrophoresed in each lane. The molecular masses of marker proteins are indicated in kilodaltons. The 70-kDa proteins, representing the major products of early embryonic gene expression, are indicated by the arrowheads. Lanes: 1, pSV-CR injected embryos; 2, pSV-N17-injected embryos; 3, pSV-CR-injected embryos cultured in oa-amanitin.

represented translation products of maternal mRNAs (4). The patterns of synthesis of the 70-kDa proteins appeared similar in pSV-CR and pSV-N17-injected embryos (Fig. 7A). To determine if pSV-N17 injection had a partial inhibitory effect, consistent with the observed two- to threefold reduction in subsequent embryo development, the synthesis of these proteins was quantitated by PhosphorImager analysis of pSV-CR and pSV-N17-injected embryos. The ratio (±standard error of the mean [SEM]) of 70-kDa protein synthesis in pSV-CRversus pSV-N17-injected embryos in five similar experiments was 1.1 + 0.1, indicating that Ras is not required for the initial onset of embryonic transcription. A variety of additional proteins were synthesized in embryos labeled from 44 to 48 h after hCG, reflecting the second phase of embryonic transcription (Fig. 7B). The general patterns of protein synthesis were again similar in pSV-CR- and pSV-N17injected embryos (Fig. 7B), and both groups of embryos displayed similar levels of overall protein synthesis, as determined by incorporation of 35S-amino acids into acid-insoluble material (data not shown). It thus appears that Ras function is not required for general activation of the embryonic genome in two-cell embryos, although expression of specific genes needed for further development may be Ras dependent. DISCUSSION The results of this study indicate that the Ras/Raf signaling pathway plays a critical role in the second cell cycle of mouse embryos. Ras function did not appear to be required for oocyte meiosis, since this was unaffected by microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259. Microinjection of pronuclear stage fertilized eggs with constructs expressing the dominant negative Ras N-17 mutant similarly did not affect their cleavage to two-cell embryos but inhibited progression through the two-cell stage. This inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. It is noteworthy that the inhibitory effect of Ras N-17 was manifest at the two-cell stage, since this is the stage at which the transition between maternal and embryonic patterns of gene expression takes place and transcription from the embry-

onic genome is required for further development (4, 14). Perhaps because of this critical reprogramming of gene expression, the two-cell stage appears to represent a particularly sensitive period of mouse embryo development (13, 17). Importantly, microinjection of the Ras N-17 expression construct into late two-cell embryos, which had already initiated embryonic transcription, did not interfere with subsequent cleavages. This specificity suggests that the Ras/Raf pathway may be involved in regulating activation of the embryonic genome. It should be noted, however, that these experiments do not exclude the possibility that Ras/Raf signaling is again required for later stages of development (e.g., the morulablastocyst transition), at which Ras N-17 constructs injected at the two-cell stage may no longer be expressed. The activation of embryonic transcription in two-cell embryos occurs in two stages: the initial onset of transcription is detected within 3 h after cleavage to two cells, while the major burst of embryonic gene expression occurs later, during the long G2 period characteristic of two-cell embryos (3, 14, 24). The earliest markers of the initial onset of embryonic transcription are provided by the synthesis of a group of proteins of approximately 70 kDa, including the heat shock protein hsp70 (2, 3, 8, 14, 38). Synthesis of these proteins was not inhibited by expression of Ras N-17, indicating that Ras is not required for this first phase of embryonic gene expression. Similarly, Ras N-17 expression did not affect the overall pattern of protein synthesis in G2, indicating that Ras function is not required for generalized activation of the embryonic genome. It is of interest that general transcription of the embryonic genome is similarly activated in embryos blocked at the two-cell stage by purines or by suboptimal culture conditions (13, 17). As in the case of Ras N-17-injected embryos, cleavage beyond the two-cell stage is specifically blocked under these conditions, but neither initiation of embryonic transcription nor overall protein synthesis during the second stage of embryonic gene expression is substantially inhibited. It is thus possible that both the sensitivity of two-cell embryos to culture conditions and the requirement for Ras/Raf signaling at this stage of embryogenesis reflect effects on expression of critical regulatory genes required for cleavage and subsequent cell cycles, rather than a general inhibition of transcription in two-cell embryos. Further studies will be needed to assess the effect of Ras N-17 on the transcription of specific genes that are activated in two-cell embryos. Interestingly, these genes include the c-myc proto-oncogene (34), which is induced by growth factor stimulation of a variety of somatic cell types and may also play a role in preimplantation mouse embryo development (37). Alternatively, Ras/Raf signaling may be required for some aspect of the two-cell cycle distinct from regulation of embryonic gene expression. The Ras/Raf pathway of signal transduction is critical to growth factor-induced responses in a variety of cell types, including proliferation of fibroblasts (6, 11, 31), neuronal differentiation of PC12 cells (19, 48), and mesoderm induction in Xenopus embryos (55). Since Ras functions to transmit signals from activated growth factor receptors, the requirement for the Ras/Raf pathway in two-cell embryos suggests that autocrine growth factor signaling is also involved at this stage of early embryo development. One example of an autocrine loop that could play a role is provided by the expression of insulin-like growth factor II and its receptor at the two-cell stage (43). The present results suggest that one important function of such autocrine pathways is to signal critical events in two-cell embryos, most likely involving the changes in gene expression that characterize this stage of mouse embryo development.

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REQUIREMENT OF Ras/Raf PATHWAY AT TWO-CELL STAGE

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