MOLECULAR REPRODUCTION AND DEVELOPMENT 31:1-8 (1992)

Ontogeny of Ha-ras and c-myc mRNA Levels in Rabbit Embryo and Extraembryonic Tissues by Quantitative In Situ Hybridization M.G. MARTINOLI,’ R.D. LAMBERT? F. POTHIER?

AND

G. PELLETIER’

‘MRC Group in Molecular Endocrinology and ‘Ontogeny and Reproduction Group, C.H.U.L. Research Centre, QuCbec, Canada

ABSTRACT A large variety of proto-oncogenes are known t o be of key importance in cellular growth and differentiation during embryonic development. Using quantitative in situ hybridization, we studied in detail the levels of the proto-oncogenes Ha-ras and c-myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prenatal life of rabbit. cDNA probes encoding for Ha-ras (fragment Kpn l-BstE II of 883 bp) and c-myc (fragment Pst l-Pst 1 of 490 bp) were used t o detect specific transcripts in fixed cryostat sections. High levels of Ha-ras and c-myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha-ras and c-myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha-ras and c-myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto-oncogenes, Ha-ras and c-myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto-oncogenes Ha-ras and c-myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prenatal development. Key Words: Oncogenes, Development, Embryo, Placenta, Rabbit, In situ hybridization

INTRODUCTION Proto-oncogenes are a family of genes strongly conserved during animal evolution and nowadays some of them are known to be involved in the production of intracellular growth factors (Cantley et al., 1991). It is therefore not surprising to find them expressed in fast-growing tissues such as the embryo and its related extraembryonic annexes. Indeed, a precise time-dependent pattern of expression of several proto-oncogenes has been described in both embryo and extraembryonic tissues such as the placenta (Szentirmay et al., 1990; Cubits et al., 1988; Tahira et al., 1988; Zhang et al., 1987; Muller et al., 1982). In particular, placental expression of c-myc proto-oncogene is modulated dur-

0 1992 WILEY-LISS, INC.

ing human gestation (Rydnert et al., 1987). The expression of c-ras and c-myc gene families has also been demonstrated in specific stages of development of mouse, rat, and human embryo (Downs e t al., 1989; Ayala et al., 1989; Schmidt e t al., 1989; Mugrauer et al., 1988; Ohlsson and Pfeiffer-Ohlsson, 1986; Mellersh et al., 1986; Pfeifer-Ohlsson et al., 1985). Several data support the role of c-myc and c-ras in the regulation of cellular proliferation and in cellular differentiation of the developing embryo and placenta (Cole, 1986; Ohlsson and Pfeiffer-Ohlsson, 1986; Rydnert et al., 1987; Tahira et al., 1988; Fauquet et al., 1990; Ingraham e t al., 1989). The aim of the present work was to study in detail the level of expression of two proto-oncogenes belonging to two different families: Ha-ras, a GTP-binding protooncogene, and c-myc, a nuclear-binding proto-oncogene, during rabbit prenatal development by using quantitative in situ hybridization.

MATERIALS A N D METHODS Animals Mature New Zealand white rabbits weighing 3-3.5 kg were used. Does in estrus were identified by the presence of oedematiated and purple vulvae and chosen for the experiments. The day of oestrus is mentioned as day “0” in our study. On that day, the does were mated and injected i.v. with 75 I.U. of hCG (human chorionic gonadotropin A.P.L.; Ayerst Laboratories, Montreal, Canada) immediately after to induce pregnancy. The animals were sacrificed at 9, 12, 15, 25, and 30 days of gestation. Tissue Dissection Embryos, embryonic and maternal placentas, and embryonic and interembryonic zones were dissected out (Dugre et al., 1989) and fixed by immersion in 4% paraformaldehyde made in 0.1M phosphate buffer (pH 7.4) for 24 hours before being rinsed in phosphate buffer 0.1M pH 7.4 + 15% sucrose. They were then frozen in isopentane chilled over dry ice and stored a t -80°C

Received June 3, 1991; accepted August 19, 1991. Address reprint requests to Dr. Georges Pelletier, Group in Molecular Endocrinology, C.H.U.L. Research Centre, 2705 boul. Laurier, Quebec, G1V 4G2, Canada.

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Fig. 1. Diagram of the sequences covered by the Ha-ras (A) and c-myc (B) probes. A 883 bp Kpn I-Bst E I1 and a 490 bp Pst I restriction fragment specific for ras and myc respectively were isolated on agarose gel, electroluted, and purified following phenol chloroform extractions. The open boxes represent exons 2, 3, 4, and 5 from ras and exons 1, 2, and 3 from myc.

Fig. 2. Details of the invasive trophoblast a t 9 days of gestation. A c-myc hybridization signal at dark field level. ~ 4 0 0 B: . Higher magnification of two intensely labelled cell bodies. x650.

Ha-ras/c-myc mRNA LEVELS

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Fig. 3. c-myc expression a t 9 days of gestation. A Autoradiograms from X-ray film: a strong labelling can be detected in the decidua (arrows) and in the trophoblast (arrowheads). B RNase pretreatment. x750. C: Dark field microscopy revealing strongly labelled cells in the epithelium of the villi in the decidua. D: RNase pretreatment. X400.

until use. Ten micron longitudinal and coronal sections of embryos, placentas, and zonae were cut with a cryostat and mounted onto gelatine- and poly-l-lysinecoated slides previously baked a t 200°C. For each day of gestation, three animals were used and 24 sections of embryos, placentas, and zonae were cut for each group.

cDNA Probe Labelling Because of the unavailability of rabbit oncogene sequences, cDNA probes were isolated and purified from pEj-ras plasmid (Capon et al., 19831, and from pSVc-myc-1 (Land et al., 1983) (Fig. lA,B). They were then labelled by random primer method (Feinberg and Vogelstein, 1983, 1984) with [35S]dCTP by using a random primer labelling system purchased from Bethesda Research Laboratory.

Briefly, the isolated double-stranded fragments of recombinant cDNAs were labelled with [35S]dCTPand purified on a 0.9 x 15 cm column of Sephadex G-50 fine (Pharmacia) equilibrated with STE buffer pH 8.0 (10 mM Tris-HC1, 50 mM NaC1, and 0.1 mM EDTA) to remove unincorporated nucleotides. The double-stranded products, containing radiolabelled nucleotides (specific activity of approximately lo9 dpm/pg), were taken up in 100 p1 HC1-EDTA buffer and denaturated by heating before use as single-stranded hybridization probes.

In Situ Hybridization In situ hybridization was carried out as previously described (Martinoli and Pelletier, 1989; Watson et al., 1987). Briefly, sections were rinsed in 2x SSC (being 1x SSC 0.15 M NaCl, 0.015M sodium citrate, pH 7.0) for 10 min, permeabilized in the same buffer

M.G. MARTINOLI ET AL.

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Fig. 5. Micrographs illustrating the absence of specific labeling c-myc in both maternal (A) and embryonic (B) placenta at 30 days of gestation. ~ 5 0 0 .

Fig. 4. Histograms showing Ha-ras and c-myc mRNA levels in maternal (A) and embryonic (B) placentas. No differences are observed between the Ha-ras and c-myc mRNA levels in the same gestational stage. In both placentas the expression of the two protooncogenes decreased from midgestation to the end of pregnancy. x 11. Data are expressed as means 5 SEM of relative optical density units after substraction of X-ray film background of 24 sections cut from three animals.

boxes overnight a t 40°C in the dark. The following day, they were washed several times in 2 x SSC, I X SSC, and in 0.5x SSC at room temperature. Finally, slides were air-dried and exposed for autoradiography on X-ray films (Kodak) at 4°C for 4 days. Some slides were dipped in liquid photographic emulsion (Kodak NTB-2) and exposed for 2 weeks. They were then developed with standard procedures, stained containing 0.1% Triton (Sigma) for a further 15 min, with hematoxylin and eosin, and mounted. rinsed twice in 2 x SSC, and finally prehybridized for 1 Densitometric measurement of 24 autoradiographs hour at room temperature in the following buffer: 50% (cut from three specimens) of whole embryo, placenta, deionized formamide, 5 x SSPE (standard saline phos- and zona were obtained with a digitized Amersham’s phate buffer) + EDTA, 0.1% SDS, 5 x Denhardt’s RAS image analysis system. Optical density units, buffer, heterologous nucleic acids (0.05% yeast total after subtraction of X-ray film background, correspond RNA and 0.05% salmon sperm DNA), poly A 2 pg/ml, to the mean O.D. units per pixel (1pixel = 180 nm) of and 4% dextran sulfate. Then 200 pl of the hybridiza- embryo, placenta, trophoblast, and endometrium. Data tion mixture containing the prehybridization buffer are expressed a s means SEM. Statistical signifiand the radioactive cDNA probe a t the saturating cance was determined according to the multiple-range concentration of 7.5 x lo6 cpm/ml was added to each test of Duncan-Kramer (Kramer, 1956). slide. The slides were then incubated in humidified At least two slides per group were treated with 30

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Fig. 6. Histogram showing the level of expression of proto-oncogenes Ha-ras and c-myc in the internal epithelium outside the implantation chamber (1.E.Z.) and in the invasive trophoblast (embryonic zona: E.Z.) at 9 days of gestation. No statistically significative differences in Ha-ras and c-myc mRNA levels are observed between the two tissues. Data are expressed as means ? SEM of relative optical density units after substration of X-ray film background of 24 sections cut from three animals.

0.0 9d

12d

15d

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Days of gestation Fig. 7. Histogram showing Ha-ras and c-myc mRNA levels in whole rabbit embryo. No statistically significant differences were observed between Ha-ras and c-myc mRNA levels. The expression of the two proto-oncogenes increased from day 9 till day 15 of gestation and then dramatically fell to day 25 of pregnancy. Data are expressed as means 2 SEM of relative optical density units after substration of X-ray film background of 24 sections cut from three animals.

mg/ml RNase A (Sigma) for 1 hour at 37°C before hybridization. As additional control some slides were hybridized with [35Sl-labelled probe encoding for r a t GH. RESULTS In Situ Hybridization 35S-labelled Ha-ras and c-myc cDNA probes were used at concentrations of 7.5 x lo6 cpm/ml in all the experiments. This concentration was found to be saturating by comparative optical density measurements (data not shown). All sections of embryo, placenta, trophoblast, and endometrium hybridized with Ha-ras and c-myc cDNA probes show a strong autoradiographic signal after 4 days of exposure, and light microscopy revealed that silver grains were restricted to some specific cells (Fig. 2A,B). RNase treatment of some slides before hybridization partially prevented labelling (Figs. 3B,D, 9B, 10B,D) and a cDNA probe encoding for rat growth hormone (rGH) failed to hybridize with all sections (data not shown). Ha-ras hybridization signal was found to be localized in the same tissue expressing c-myc. Optical density measurements of Ha-ras and c-myc mRNA levels did not reveal statistically significant differences between the two proto-oncogenes among all tissues examined (Figs. 4A,B, 5, 6).

Placenta Large spots of intense radiolabelled signal were observed in the decidua (Fig. 3C) and in the trophoblast a t day 9 of gestation (Figs. 2,3A). In the decidua this signal was exclusively detected in the epithelium of the villi (Fig. 3C). In rabbit maternal placenta Ha-ras and c-myc, mRNA levels were observed to decrease from the ninth to the 25th day of gestation

Fig. 8. Dark field microscopy of rabbit embryo at 9 days of gestation. A weak hybridization signal for Ha-ras can be detected (arrows) over the neural tube. ~ 2 0 0 .

(Fig. 4A). Early embryonic placentas were collected a t day 12 of gestation and they expressed the highest Ha-ras and c-myc mRNA levels through all gestation (Fig. 4B). At 30 days of gestation maternal and embryonic placentas did not reveal any specific labelling (Fig. 5A,B).

Trophoblast and Endometrium Figures 2 and 3 show large labelled spots in the inner layer of the day 9 invasive trophoblast. Because of the

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M.G. MARTINOLI ET AL.

Fig. 9. X-ray autoradiography of a whole embryo a t 12 days of gestation. A: Ha-ras expression is particularly evident in the CNS (arrow),in the liver (Li), and in the lung (L). B: RNase pretreatment of a consecutive section as described in Materials and Methods. x 7.5.

intensity of the reaction, i t was very difficult a t light microscopy level to investigate whether the hybridization signal was localized on giant syncytial cells (Fig. 2B). In the endometrium, Ha-ras and c-myc mRNA levels were detected in some epithelial cells of the villi (data not shown). However, comparative optical density measurements revealed that Ha-ras and c-myc mRNA levels did not appear to be statistically different in the endometrium of the implantation area (E.Z.) as compared to the interimplantation endometrium (I.E.Z.) (Fig. 6) a t day 9 of gestation.

Embryo Autoradiography on X-ray films revealed that Haras and c-myc mRNA levels increased from day 9 till day 15 of gestation (Fig. 7). At 25 days of gestation Ha-ras and c-myc mRNA levels have almost disappeared (Fig. 7) and are undetectable above background levels by 30 days of gestation. At 9 days of pregnancy Ha-ras and c-myc gene expression became detectable in the neural tube (Fig. 8). At 12 days of gestation the autoradiographic signal was particularly located in the central nervous system (CNS) as well as in the liver and in the lung (Fig.gA,B). Later, a t 15 days of gestation, the liver and the lung did not show Ha-ras and c-myc mRNA levels above background; however, the CNS was still intensively labelled (Fig. 10A,B). Very low signal was detected in the 25-day-old embryo (Fig. 10C,D), and a t 30 days of gestation, the specific autoradiographic signal completely disappeared (data not shown). DISCUSSION The present study is the first to show the levels of mRNA encoding for the two proto-oncogenes Ha-ras and c-myc during rabbit prenatal development by using

quantitative in situ hybridization. The expression of Ha-ras and c-myc has been observed in the embryo and extraembryonic tissues a s early as day 9 of gestation and by 30 days of gestation no specific hybridization could be detected, suggesting that both Ha-ras and c-myc gene products might have a role in rabbit cellular growth and differentiation. The present localization of Ha-ras and c-myc mRNA levels in the decidua, the trophoblast, and the endometrium at 9 days of gestation is in good agreement with previous data reporting the expression of several proto-oncogenes in extraembryonic tissues of various animal species by different techniques (Szentirmay e t al., 1990; Rydnert et al., 1987; Ohlsson and Pfeiffer-Ohlsson, 1986; Pfeiffer-Ohlsson et al., 1984). However, attenuation of proto-oncogene expression in the uterine epithelial cells close to the embryo could not be demonstrated in the present study. This suggests that the original hypothesis, indicating that the embryonic environment might attenuate v-scr oncogenesis in chicken embryo (Stoker et al., 19901, cannot be extended to other proto-oncogenes such as Ha-ras and c-myc. Or i t may be the case that in mammals regulation of proto-oncogenes behaves in this respect quite differently from avian species. Rabbits provide a unique model with which to study the developmental regulation of the feto-placental unit because of its invasive implantation (Steer, 1971a). In fact, during implantation the uterine epithelium undergoes a complex series of events including a process of fusion between the symplasma of the endometrial epithelium and the invasive trophoblast of the blastocyst (Enders and Schlafke 1971; Steer, 1970, 1971a,b; Schlafke and Enders, 1975). Thus cells from both the mother and the embryo fuse to give rise to syncytialtrophoblatic tissue (Schlafke and Enders, 1975). At the ultrastructural level these giant cells appear to be

Ha-ras/c-myc mRNA LEVELS

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Fig. 10. Longitudinal section of a rabbit embryo at 12 days of gestation (A and B) and a t 25 days of gestation (C and D). A Ha-ras is still strongly expressed in the CNS (arrows). B: RNase treatment. ~ 5 . 5C: . Ha-ras mRNA levels have dramatically decreased, to become poorly detectable. D: RNase treatment. ~ 3 . 5 .

mammalian species. In particular, c-myc is expressed in human embryonic skin, intestine, brain, kidney, lung, and connective tissue (F'feiffer-Ohlssonet al., 1985), revealing a generalized localization in the developing embryo. c-ras oncoproteins have been detected in the brain and lung of rat (Tanaka et al., 1987) and other members of the ras-gene family are actively involved in the development of the CNS (Ayala et al., 1989). From our results i t clearly appears that the expression of the proto-oncogenes Ha-ras and c-myc is inversely related in rabbit embryo and extraembryonic tissues. Other proto-oncogenes have been described a s following a spatiotemporal pattern of expression during ontogenesis, such a s int-1, which is associated with neural plate formation (Wilkinson et al., 19871, and c-fos, which is associated with late brain development, and c-mos, which is associated with germ cell formation (Gubits e t al., 1988; Mutter and Wolgemuth, 1987). From all these data and our observations, i t seems that some proto-oncogenes might have a role during early stages of embryonic development while others might be active later at the end of gestation or during postnatal life. We detected coexpression of Ha-ras and c-myc mRNA levels in extraembryonic and embryonic tissues but we were not able to determine whether one cell expressed both proto-oncogenes. Other data have reported the colocalization of c-myc and c-sis in the human placenta (Goustin et al., 1985) and recently Suda et al. (1988) have demonstrated that human c-Ha-ras and c-myc introduced into a host 4-day embryo were expressed not only in stem cells but also in many tissues of the developing mouse embryo. The interesting possibility that Ha-ras and c-myc might be expressed in the same cell could be further investigated by performing a technique of double in situ hybridization, which is beyond the scope of the present work. In conclusion, rabbit embryonic development appears to be a n excellent model with which to study the regulation of proto-oncogene expression in vivo. We report colocalization of Ha-ras and c-myc mRNA levels in rabbit embryo and extraembryonic tissues where they exhibit temporal patterns of expression during prenatal development.

electron dense and to contain numerous microtubules (Steer, 1971a). At the light microscopic level, it is, however, difficult to observe where the two tissues have fused. The intensely labelled spots of autoradiographic signal observed in this study are much too large to originate from single normal cells. Since they are located in the region of fusion at the embryo-maternal interface, we propose that these large labelled bodies are the result of cell fusion. Moreover, these data would suggest a particularly strong expression of Ha-ras and c-myc in the fused cells. Our data on the expression of Ha-ras and c-myc in rabbit embryo are consistent with previous work reporting the localization of c-ras and c-myc in other

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