Permissiveness to Moloney Murine Leukemia Virus (MoMuLV) expression was examined during preimplan tation and early postimplantation development of the mouse embryo. Blastocysts and 8th, 9th and 10th day postimplantation embryos were infected in vitro with a MoMuLV-based retroviral vector expressing the lacZ gene driven off an internal rat beta-actin promoter. Beta-galactosidase-positive cells were identified in all embryonic tissues including inner cell mass, epiblast, mesoderm, endoderm and definitive ectoderm. In con trast, embryos infected with a MoMuLV-based vector expressing the lacZ gene driven off the viral LTR showed beta-galactosidase-positive cells only in mesoderm and definitive ectoderm. We conclude that permissiveness to transcriptional activity of the LTR is acquired immedi ately upon differentiation of epiblast during gastrulation of the mouse embryo.

Studies of host-virus relationships during embryogen esis have been stimulated by the hope that the molecu lar control of viral expression may shed light on mechanisms involved in the differentiation of embry onic cells. Early preimplantation mouse embryos are not permissive for replication of the Moloney Murine Leukemia Virus (MoMuLV) (Jaenisch et al. 1975). Integration of the proviral DNA occurs normally but transcription of the genome cannot be detected (Jae nisch et al. 1975; Huszar et al. 1985; Stewart et a/. 1987). In contrast, infection of postimplantation embryos at the onset of organogenesis (9th day of gestation) leads eventually to virus production in every tissues analysed (Jaenisch, 1980; Jaenisch et al. 1981). Similar obser vations have been made with Simian Virus 40 (SV40) and polyoma virus. Totipotent cells of the inner cell mass seem not to be permissive to expression of early functions of SV40 and polyoma virus (Abramczuk et al. 1978). Like murine leukemia virus, this block to ex pression may not be removed before the 9th or possibly 10th day of development (Kelly and Condamine, 1982). It is possible that this transition from nonpermissiveness in preimplantation mouse embryos to productive viral infection in postimplantation embryos may reflect some more fundamental change in endogenous gene ex pression occurring in the early postimplantation phase of development.

The block to productive infection has been further studied using murine embryonal carcinoma (EC) cells. EC cells are derived from malignant stem cells of teratocarcinomas and share many properties with nor mal embryonic stem cells (Kleinsmith and Pierce, 1964; Martin, 1980). In particular, EC cells are similar to preimplantation mouse embryos in that they are non-permissive for expression of several viruses including MoMuLV (Teich et al. 1977; Gautsch, 1980; Speers et al. 1980). It has been shown that transcription is blocked by two independent mechanisms: (1) enhancer elements in the MoMuLV long terminal repeat (LTR) are not functional in undifferentiated F9 EC cells (Linney et al. 1984; Gorman et al. 1985; Weiher et al. 1987; Loh et al. 1987; Feuer et al. 1989); (2) a negative regulatory factor in F9 EC cells interacts with a region of the MoMuLV genome in the vicinity of the tRNA primer-binding site (Barklis et al. 1986; Weiher et al. 1987; Loh et al. 1987,1988; Feuer et al. 1989). Finally, it appears that, superimposed on this early block to expression, is an extensive methylation of the provirus, which occurs within a few days after integration of the proviral DNA (Gautsch and Wilson, 1983). This meth-ylation maintains the block to MoMuLV expression even after differentiation of EC cells (Niwa et al. 1983). It is most likely that the same mechanisms are respon sible for the block to MoMuLV expression in the preimplantation mouse embryo. It has been shown that the provirus becomes heavily methylated following infection of preimplantation embryos (Jâhner et al. 1982; Jaenisch and Jahner, 1984). As with EC cells that are allowed to differentiate, the block to MoMuLV expression is maintained during embryonic development (Jä hner et al. 1982).

In this work, we investigated the pattern and timing of MoMuLV activation between the time of blastocyst formation and the onset of organogenesis. We asked at which stage of development does transcription first occur and in which tissue(s) of the embryo is this activity first detected. These questions were addressed using a replication-defective helper-free MuMoLV-based retroviral vector carrying the E. coli lacZ gene driven by the LTR. Expression of lacZ in this vector was compared with one in which lacZ was expressed from an internal rat beta-actin promoter. Mouse embryos were infected at various stages of development and cultured in vitro. Subsequently, the frequency and distribution of beta-galactosidase-positive cells in the different embryonic and extraembryonic tissues were analysed.

(1) Recovery and culture of embryos

Outbred PO (Pathology, Oxford) mice were used in all experiments. The recovery and manipulation of all embryos was carried out in PB1 medium (Whittingham and Wales, 1969) containing 10% fetal calf serum (FCS). Blastocyst outgrowths were cultured in LabTek chamber/slides (4-5 embryos per 1.5 cm2 * well) in alpha-medium+10 % FCS (Gardner, 1985) at 37°C in an atmosphere of 5% CO2. Infection was carried out in a mixture of alpha-medium+ 10% FCS (50%) and medium containing the virus (50%) + 10μgml−1 polybrene. Postimplantation embryos were cultured in rotating (30 revs min−1) 30 ml universal tubes (Sterilin) containing 3 ml of culture medium made up of DMEM (Flow Laboratories) (50%) and rat serum (50%) (New et al. 1976; Tam and Snow, 1980). The culture medium was sterilized by filtration (Sartorius, pore size 0.45 μ m) and equilibrated with 5% CO2 in air overnight. For embryos infected by culturing them in medium containing the virus, culture supernatants from virus producer cells (DMEM+5 % FCS+10μgml−1 polybrene) were used instead of normal DMEM. 8th day embryos were cultured in medium gassed with 5% CO2, 5% O2 and 90% N2. 9th day embryos were cultured in medium gassed with 5 % CO2, 20 % O2 and 75 % N2. 10th day embryos were cultured in medium gassed with 5% CO2, 40% O2 and 55% N2. Cultures were regassed 20 and 30 h after the start of the culture with the appropriate gas mixtures (Beddington, 1987).

The structure of the recombinant lacZ transducing vector BAG is described in Price et al. (1987). The lacZ transducing genome pIRV is carried by the plasmid pIRV-neo-act-lacZ described in Beddington et al. (1989). Both retroviral vectors are produced by the Psi-2 packaging cell line (Mann et al. 1983). Virus was concentrated essentially as described by Sanes et al. (1986). Virus was harvested in DMEM+5 % foetal calf serum (106lacZ colony forming units (CFU) ml−1). Harvesting medium was filtered (Sartorius, pore size 0.45 μm) to remove cells and debris. Virus was then concentrated by centrifugation (14 000 revs min−1, 12h, 2°C) and resuspended in l/200th of foetal calf serum to yield about 108lacZ CFU ml−1 and stored at —20°C for 2-3 months. Virus preparations were titrated by infecting NIH3T3 cells plated at a density of 4x 104 * cells cm−2 with diluted samples of the virus stocks in the presence of 10μgml−1 polybrene. Cells were grown to confluency (2-3 days) and stained for lacZ activity. pIRV and BAG producer cells were periodically checked for production of replication-competent virus by infecting fresh fibroblasts with culture supernatants from BAG-or plRV-infected fibroblast cultures and by staining the recipient cells for lacZ activity (see ‘Histochemistry’ for staining procedure).

(3) Infection of embryos

Blastocysts were isolated on the 4th day of gestation essen tially as described by Hogan et al. (1986). The zona pellucida was removed in acidified Tyrode’s solution (Nicolson et al. 1975). They were cultured for three days in medium contain ing the virus (titre of virus in the culture medium: 5×105lacZ CFU ml−1) until the inner cell mass formed an outgrowth with the trophoblast expanding laterally. The medium was re placed every 12 h to provide fresh virus during this period. Embryos were subsequently cultured in virus-free medium for a further 24 h and stained to reveal lacZ expression.

9th day and 10th day postimplantation embryos were infected by culturing them in medium containing the virus (titre of virus in the culture medium: 5x10slacZ CFU ml−1) for 8h with visceral yolk sac and amnion reflected. Then embryos were cultured in virus-free medium for a further 36 h. 8th day postimplantation embryos were infected by injecting concentrated virus (108lacZ CFU ml−1) either inside the amniotic cavity or inside the exocoelom or between epiblast and endoderm. Embryos were injected in a drop of PB1 medium+10% FCS covered with paraffin oil (Boots, UK, Ltd) in the lid of a culture dish (Sterilin) placed on the fixed stage of a dissecting binocular microscope. Injecting and handling pipettes (diameter of the opening: 10 and 140 /un respectively) were attached to tubing filled with paraffin oil and controlled by de Fonbrune suction and force pump and Aglar syringe respectively. Prior to injection, polybrene was added to the virus at a final concentration of 100μg ml−1.

(4) Histochemistry

After culture grossly abnormal embryos were discarded. The remainder were processed for E. coli beta-galactosidase activity. Embryos were washed in 0.1M phosphate buffer (pH 7.4) and fixed for 5–10 min in 0.2% glutaraldehyde (Gurr) in the same buffer containing 2mM MgCl2 and 5mM EGTÁ. They were washed for 2h in three changes of 0.1M phosphate buffer (pH 7.4) containing 2niM MgCl2, 0.01% (W/V) sodium desoxycholate and 0.02% (W/V) Nonidet P-40. Staining was carried out at 37°C in a solution of the above buffer containing 5ITIM K3Fe(CN)g, 5mM K4Fe(CN)6.6H2O, ImM spermidine hydrochloride, 0.001% sodium chloride and Imgml−1 4-chloro-5-bromo-3-indolyl-beta-galactosidase (X-gal). X-gal was dissolved in dimethyl formamide at a concentration of 40 mg ml−1. Embryos and cells were left to stain between 24 and 48h. All reagents, unless specified, were obtained from Sigma.

After staining, most embryos were postfixed in Carnoys’ fluid (30 min), dehydrated with absolute ethanol and toluene and embedded in paraffin wax. They were serially sectioned at 7μm. Some embryos were postfixed in 0.2% glutaraldehyde (15 min), dehydrated, incubated in propylene oxide (10 min), propylene oxide/Araldite CY212 (30min), then embedded in Araldite and serially sectioned at 5μm. All sections were counterstained with eosin before mounting in DPX and viewed in a light microscope.

(1) Experimental strategy

Embryos were isolated at various stages of develop ment (blastocysts, postimplantation embryos during the 8th, the 9th and the 10th day of gestation). They were infected in vitro with the lacZ transducing vectors either by culturing them in medium containing the virus (blastocysts, 9th and 10th day postimplantation em bryos) or by injecting the vectors directly into the conceptus (8th and 9th day postimplantation embryos). All embryos were cultured in vitro for a further 24-36 h period in virus-free medium to allow infected cells to divide, a prerequisite to viral integration and ex pression. Beta-galactosidase-positive cells were ident ified by X-gal staining.

Two replication-defective vectors were used. Both are MoMuLV-based vectors carrying the E. coll lacZ gene as reporter gene as illustrated in Fig. 1. (1) BAG expresses the lacZ gene driven off the LTR (Price et al. 1987). Therefore, production of beta-galactosidase in BAG-infected cells was used to monitor transcriptional activity of the LTR. It must be pointed out that the BAG vector also contains the MoMuLV 5’ leader segment including the primer binding site. Therefore, it contains all the civ-acting elements known to be in volved in repression of viral transcription in undifferen tiated EC cells. (2) pIRV expresses the lacZ gene driven by the rat beta-actin promoter (Beddington et al. 1989) and was used as a control to monitor viral infection. It was assumed that every infected cell would express lacZ. Therefore, if no beta-galactosidase-posi tive cell is detected following infection with BAG, the control with pIRV ensures that it actually results from nonpermissiveness of the target cells to transcriptional activity of the LTR and is not simply due to the failure of virus to infect postimplantation tissues.

Fig. 1.

Diagram of BAG and pIRV lac Z-tranducing vectors. BAG has been described in Price et al. (1987). pIRV is the viral genome inserted in the plRV-Neo-Act-LacZ plasmid described in Beddington et al. (1989).

Fig. 1.

Diagram of BAG and pIRV lac Z-tranducing vectors. BAG has been described in Price et al. (1987). pIRV is the viral genome inserted in the plRV-Neo-Act-LacZ plasmid described in Beddington et al. (1989).

(2) Development of embryos in vitro

Development of 8th day embryos was not radically affected by the injection procedure although 10 nl of virus suspension, as much as the volume of amniotic fluid in the amniotic cavity at this stage (Burgoyne et al. 1983), was injected into the embryos. Over 90% of the embryos developed beating hearts and initiated somite formation during the 48 h of culture (Fig. 2G). Most embryos showed signs of a yolk sac circulation. How ever, only a few embryos showed complete closure of the neural tube and axis rotation. About 10% of embryos were severely retarded and abnormal and were discarded before analysis of lacZ expression.

Fig. 2.

Embryos following infection with pIRV or BAG vectors, in vitro culture and X-gal staining. (A) Blastocyst outgrowth infected with pIRV with beta-galactosidase positive cell in the inner cell mass. Bar=20; μm. (B) Transverse section through an embryo infected with the pIRV vector injected into the amniotic cavity during the 8th day of development and showing positive cells in the amnion (mesoderm). Bar=200m. (C) Transverse section through an embryo infected with pIRV injected into the amniotic cavity during the 8th day of development and showing positive cells among red blood cells and extraembryonic mesoderm. Bar=10μm. (D) Transverse section through an embryo infected with pIRV injected into the amniotic cavity during the 8th day of development and showing positive cells in the gut endoderm. Bar=20μm. (E) Transverse section through an embryo infected with BAG injected into the exocoelom during the 8th day of development and showing positive red blood cells. Bar=20Jum. (F) Transverse section through an embryo infected with pIRV during the 9th day of development and showing positive cells in mesoderm of a limb bud. Bar=20μm. (G) Whole mount of an embryo infected with BAG injected into the exocoelom during the 8th day of development and showing positive cells in the extraembryonic mesoderm of the visceral yolk sac (VYS). Bar=200μ m. (H) Whole mount of an embryo infected with pIRV injected into the amniotic cavity during the 9th day of development and showing positive cells in the left anterior limb bud. Bar=200μm. (I) Transverse section through an embryo infected with BAG during the 10th day of development and showing positive cells in surface ectoderm. Bar=10; μ m.

Fig. 2.

Embryos following infection with pIRV or BAG vectors, in vitro culture and X-gal staining. (A) Blastocyst outgrowth infected with pIRV with beta-galactosidase positive cell in the inner cell mass. Bar=20; μm. (B) Transverse section through an embryo infected with the pIRV vector injected into the amniotic cavity during the 8th day of development and showing positive cells in the amnion (mesoderm). Bar=200m. (C) Transverse section through an embryo infected with pIRV injected into the amniotic cavity during the 8th day of development and showing positive cells among red blood cells and extraembryonic mesoderm. Bar=10μm. (D) Transverse section through an embryo infected with pIRV injected into the amniotic cavity during the 8th day of development and showing positive cells in the gut endoderm. Bar=20μm. (E) Transverse section through an embryo infected with BAG injected into the exocoelom during the 8th day of development and showing positive red blood cells. Bar=20Jum. (F) Transverse section through an embryo infected with pIRV during the 9th day of development and showing positive cells in mesoderm of a limb bud. Bar=20μm. (G) Whole mount of an embryo infected with BAG injected into the exocoelom during the 8th day of development and showing positive cells in the extraembryonic mesoderm of the visceral yolk sac (VYS). Bar=200μ m. (H) Whole mount of an embryo infected with pIRV injected into the amniotic cavity during the 9th day of development and showing positive cells in the left anterior limb bud. Bar=200μm. (I) Transverse section through an embryo infected with BAG during the 10th day of development and showing positive cells in surface ectoderm. Bar=10; μ m.

In contrast, development of 9th day embryos was impaired by the infection procedure. They were cul tured with visceral yolk sac and amnion opened, which resulted in abnormal morphogenesis of the posterior part of the embryos and limited growth and differen tiation of the visceral yolk sac. However, the heart was beating, the anterior neural tube was completely closed and a large number of somites (>15) were generally clearly visible. Again, about 10% of growth-arrested embryos were discarded. A few 9th day embryos were also infected by injecting the virus inside the amniotic cavity. After 36 h of culture, most of them showed no significant difference from unoperated embryos of the same age developed in vitro (Fig. 2H).

10th day embryos were also cultured with visceral yolk sac and amnion reflected. Despite the absence of yolk sac circulation, considerable growth occurred dur ing the culture period.

(3) Infection of blastocyst outgrowths

77 blastocysts were infected with pIRV by culturing them for three days in medium containing the virus (5×105lacZ CFU ml−1). During this time, they formed trophoblast outgrowth and the inner cell mass differen tiated into epiblast and primitive endoderm. They were cultured for a further 24 h in virus-free medium. 37 embryos contained beta-galactosidase producing cells in the inner cell mass after staining (Fig. 2A) and only three contained positive cells in the trophoblast. 66 embryos were infected under identical conditions with BAG (5×105lacZ CFUml−1). No embryo contained beta-galactosidase-positive cells either in the inner cell mass or in the trophoblast (Table 1). Therefore, the rat beta-actin promoter driving the lacZ gene in the pIRV vector works very efficiently in early embryonic cells, particularly the ICM and its derivatives. In contrast, no expression is detected when the reporter gene is driven by the LTR.

Table 1:

Distribution and frequencies of positive cells In blastocyst outgrowths

Distribution and frequencies of positive cells In blastocyst outgrowths
Distribution and frequencies of positive cells In blastocyst outgrowths

(4) Infection of 8th day postimplantation embryos (mid-gastrulation)

Late-primitive-streak-stage embryos (8th day of ges tation) were infected by injecting the virus inside the amniotic cavity in order to ensure access to the undiffer-entiated epiblast. Embryos were then cultured in vitro for 48 h. 64 embryos were infected with the pIRV vector (1000 lacZ CFU/embryo). Of these, 40 (62%) proved positive after X-gal staining (Table 2) and 36 of these were serially sectioned and the distribution of positive cells was recorded. 22 embryos had positive cells in definitive ectoderm (neurectoderm, surface ectoderm and ectoderm of the amnion). 34 embryos had positive cells in mesoderm including the heart, head and trunk mesoderm, red blood cells, mesoderm of the visceral yolk sac, amnion and allantois (Fig. 2B,C). 14 embryos had positive cells in gut or visceral embryonic endo derm (Fig. 2D). Nine embryos had positive cells in derivatives of all three germ layers. Therefore, the rat beta-actin promoter seems to be active in every differ entiated derivative of the epiblast.

TABLE 2:

Distribution of positive cells In embryos infected during the 8th day of development (Injection Inside the amniotic cavity).

Distribution of positive cells In embryos infected during the 8th day of development (Injection Inside the amniotic cavity).
Distribution of positive cells In embryos infected during the 8th day of development (Injection Inside the amniotic cavity).

115 embryos were infected by injecting the BAG vector (1000 lacZ CFU/embryo) into the amniotic cavity. Only six (5%) displayed positive cells after staining (Table 2). All were located in the extraembry-onic mesoderm of the yolk sac. No embryo had positive cells in embryonic tissues. 36 negative embryos were serially sectioned to confirm the absence of positive cells in the inner tissues of the embryos.

The frequencies of positive cells following infection of epiblast with pIRV and BAG are significantly different (X2=70.8; P<0.001). Expression of the reporter gene driven off the LTR is observed only at a very low frequency and exclusively in the extraembry onic mesoderm of the visceral yolk sac. These data suggest that epiblast is not permissive to transcriptional activity of the LTR.

A second experiment was carried out where virus was injected inside the exocoelom in order to infect directly cells already differentiated into extraembryonic meso derm. Seven embryos were infected with the pIRV vector (1000 lacZ CFU/embryo) and cultured in vitro for 48 h as in the previous experiment. All contained positive cells after X-gal staining (Table 3). They were serially sectioned. Positive cells were found mostly in the extraembryonic mesoderm of the yolk sac and among red blood cells in blood islands. Similarly, 22 embryos were infected with the BAG vector by inject ing the virus (1000 lacZ CFU/embryo) inside the exocoelom. 10 embryos (45 %) contained positive cells after staining (Table 3). Of these, six were serially sectioned and showed positive cells in extraembryonic mesoderm of the yolk sac and/or among red blood cells in blood islands (Fig. 2E,G). Therefore, neither the frequency of positive embryos nor the distribution of positive cells following infection of the extraembryonic mesoderm with the pIRV and the BAG vectors appear radically different. This suggests that extraembryonic mesoderm of the 8th day postimplantation embryo is permissive to transcriptional activity of the LTR.

TABLE 3:

Distribution of positive cells In embryos infected during the 8th day of development (Injection Inside the amniotic exocasiuon).

Distribution of positive cells In embryos infected during the 8th day of development (Injection Inside the amniotic exocasiuon).
Distribution of positive cells In embryos infected during the 8th day of development (Injection Inside the amniotic exocasiuon).

In a third experiment, we asked if embryonic meso derm of the 8th day postimplantation embryo is also permissive to transcriptional activity of the LTR. LacZ transducing vectors were injected between the epiblast and the endoderm in order to hit directly cells already differentiated into embryonic mesoderm. 17 embryos were infected with the pIRV vector (100 lacZ CFU/ embryo). Of these, ten (59%) displayed positive cells (Table 4). Seven were serially sectioned and showed positive cells in mesoderm and gut endoderm. No embryo had positive cells in either neurectoderm or surface ectoderm. Similarly, 39 embryos were infected with, the BAG vector (100 lacZ CFU/embryo). 12 embryos (31%) were found to contain positive cells after staining (Table 4). Six were serially sectioned. All had positive cells, located exclusively in embryonic mesoderm. These results suggest that embryonic meso derm of the 8th day postimplantation embryo is also permissive to LTR activity. In contrast to pIRV infec tions, no positive cells were detected in gut endoderm with the BAG vector. Absence of positive cells in definitive ectoderm with pIRV is also noteworthy. A possible explanation is that viral particles cannot cross the basal lamina underlying the epiblast (Wartiovaara et al. 1979; Leivo et al. 1980) or that viral receptors are located only on the amniotic surface.

TABLE 4:

Distribution of positive cells In embryos infected during the 8th day of development (Injection between epbiast and endoderm).

Distribution of positive cells In embryos infected during the 8th day of development (Injection between epbiast and endoderm).
Distribution of positive cells In embryos infected during the 8th day of development (Injection between epbiast and endoderm).

Results obtained from these experiments suggest that transcriptional activity of the LTR is observed only when infected cells have differentiated into mesoderm or extraembryonic mesoderm prior to viral infection. The frequencies of positive cells following infection of these tissues with pIRV or BAG are almost identical. In contrast, when cells are infected before differentiating into mesoderm (i.e. infection of epiblast) they are not permissive to transcriptional activity of the LTR even if they later differentiate into mesoderm. The few positive cells identified in extraembryonic mesoderm after injection of BAG into the amniotic cavity most likely result from leakage of virus out of the amniotic cavity into the exocoelom.

(5) Infection of 9th day postimplantation embryos ( early-somite-stage)

If permissiveness to transcriptional activity of the LTR is acquired when epiblast differentiates into mesoderm, is it also acquired when epiblast differentiates into definitive ectoderm? Early-somite-stage embryos (9th day of gestation) were infected with the pIRV or BAG vectors by culturing embryos whose viscera) yolk sac and amnion had been reflected in medium containing virus. The neural folds form during the 9th day of gestation. Therefore, it is possible to infect cells which have already differentiated into neurectoderm and surface ectoderm.

64 embryos were infected with the pIRV vector by culturing them in medium containing the virus (final concentration=5× 105lacZ CFU ml1) for 8h. They were subsequently cultured for a further 40 h in virus-free medium. All contained positive cells after X-gal staining (Table 5). Ten were serially sectioned and the distribution and frequencies of positive cells recorded. In contrast to embryos infected during the 8th day of development, positive cells always appeared in clusters, which presumably corresponded to clones resulting from division of the primary infected cells. All the embryos had positive cells in surface ectoderm. Six embryos had positive cells in neurectoderm and seven embryos had positive cells in mesoderm (Fig. 2F). No embryo had positive cells in either vis ceral endoderm or gut endoderm. Similar results were obtained from embryos infected by injecting the pIRV vector into the amniotic cavity (Fig. 2H). Similarly, 76 embryos were infected under the same conditions with the BAG vector (final concentration=5 × 105lacZ CFUml−1). 37 (49%) contained positive cells after staining (Table 5). Ten were serially sectioned. Positive cells were identified in neurectoderm and mesoderm. As for embryos infected with the pIRV vector, no embryo had positive cells in either visceral or gut endoderm. However, as opposed to embryos infected with pIRV, no embryo infected with BAG had positive cells in surface ectoderm.

TABLE 5:

’ Distribution ot positive cells in embryos infected during the 9lh day of development

’ Distribution ot positive cells in embryos infected during the 9lh day of development
’ Distribution ot positive cells in embryos infected during the 9lh day of development

Therefore, the frequencies ofclusters of positive cells in neurectoderm with pIRV and BAG are not signifi cantly different. This suggests that early neurectoderm cells of the 9th day postimplantation embryo are permissive to transcriptional activity of the LTR. In contrast, no expression of the lacZ gene driven off the LTR could be detected in surface ectoderm. On the other hand, no positive cells were observed with pIRV in either gut or visceral endoderm despite the endo derm being directly accessible to viral particles during infection.

(6) Infection of 10th day postimplantation embryos

In order to ascertain when surface ectoderm becomes permissive to LTR transcriptional activity, 10th day embryos were infected with the pIRV and BAG vec tors. After reflection of the visceral yolk sac and amnion, embryos were cultured in medium containing the virus.

Five embryos were infected with the pIRV vector (final concentration=5 × 105lacZ CFU ml−1) for eight hours, then cultured for a further 40 h in virus-free medium. All displayed positive cells after X-gal staining (Table 6) and were serially sectioned. Clusters of posi tive cells were observed in surface ectoderm (10.6 clusters/embryo). Positive cells were also identified in the pericardium. However, in several cases, it was not possible to determine whether these positive cells were in the ectoderm or mesoderm of the pericardium. Similarly, seven embryos were infected with the BAG vector under the same conditions. All displayed posi tive cells after staining (Table 6) and were serially sectioned. 31 clusters of positive cells were identified in surface ectoderm (4.4 clusters/embryo) distributed on the head and trunk (Fig. 21). Four clones were also identified in the pericardium.

TABLE 6:

Distribution of positive cells in embryos infected during the 10th day of development.

Distribution of positive cells in embryos infected during the 10th day of development.
Distribution of positive cells in embryos infected during the 10th day of development.

These results show that surface ectoderm is permiss ive to transcriptional activity of the LTR during the 10th day of gestation although the frequency of positive cells with BAG appeared slightly lower than that obtained with pIRV (T=3.14; F=0.01). This difference may reflect a transition state where surface ectoderm is becoming permissive to LTR expression. Again, despite endoderm being directly accessible to viral particles when embryos are cultured in medium containing the virus, no positive cells were identified in the gut endoderm of embryos infected with either the pIRV or the BAG vector.

Justification of the experimental system

The objective of this work was to determine the developmental stage at which transcription of the MoMuLV provirus is no longer repressed. It was carried out on embryos developing in vitro in order to monitor very precisely the site of injection of the virus and the time of infection. Helper-free replication defective vectors were used in order to prevent horizontal spreading of virus during the culture period.

There are three main requirements for any embry onic cell to be infected by a helper-free replication defective retroviral vector: (i) they have to be directly accessible to viral particles because there can be no horizontal spread of virus; (ii) they have to be able to bind viral particles and to translocate the requisite viral protein and the viral RNAs; (iii) they have to divide because integration of proviral DNA into the host genome, which is a prerequisite to expression, occurs only during DNA replication (Varmus et al. 1977). Therefore, a retroviral vector, which should allow ubiquitous expression of the lacZ gene in every infected cell, was used as control of viral infection. The rat beta actin promoter was chosen because it is thought to be constitutively active in all cells, regardless of tissue type. It has been shown to produce ubiquitous ex pression of lacZ in midgestation embryonic tissues (Beddington et al. 1989). In the experiments reported here, pIRV infection of blastocyst outgrowths resulted in expression of lacZ in epiblast, primitive endoderm and trophectoderm. pIRV infection of early-cleavage-stage embryos led to a similar conclusion (data not shown). Similarly, pIRV infection of epiblast at mid-gastrulation resulted in expression of lacZ, albeit at different frequencies, in all its differentiated deriva tives. No obvious difference in the intensity of the staining was seen between cells of different tissues. Therefore, we concluded that the rat beta-actin pro moter is indeed an ubiquitous promoter in our exper imental system. Differences in the frequency of positive cells between the various epiblast derivatives following pIRV infection of epiblast at midgastrulation (Table 2) may simply reflect relative differences in the size of each prospective tissue area during gastrulation.

In these experiments, transcriptional activity of the LTR was determined from the frequency of positive embryos infected with BAG versus the frequency of positive embryos infected with pIRV. When no positive embryos were identified following infection with BAG, it was concluded that the LTR was inactive only if the frequency of positive embryos infected with pIRV was statistically significant (P<0.001). We could not provide direct evidence that a silent provirus was indeed present in BAG-infected tissues. However, it must be pointed out that: (i) the same number of BAG and pIRV infectious particles (titrated on NIH3T3) were used to infect embryos; (ii) both vectors have the same LTRs and were produced with the same packaging cell line. Therefore, infection and integration efficiencies of BAG and pIRV are expected to be identical.

ICM and epiblast are not permissive to transcriptional activity of the LTR

Early-cleavage-stage embryos and blastocysts are not permissive to MoMuLV expression (Jaenisch et al. 1975, 1981). Absence of transcriptional activity of the LTR following infection of early-cleavage-stage em bryos was confirmed in transgenic mice by using repli cation-defective vectors carrying exogenous genes under the control of the LTR (Rubenstein et al. 1984; Huszar et al. 1985; Stewart et al. 1987). Absence of positive cells in the inner cell mass following infection of blastocyst outgrowths with BAG provides further evi dence for this (Fig. 3). With respect to trophectoderm, the frequency of positive cells in this tissue was very low following pIRV infection. Therefore, although no beta galactosidase activity was detected in trophectoderm using the BAG vector, it is not possible to draw any conclusions about LTR activity in this tissue.

Fig. 3.

Diagram illustrating the status of the differentiated derivatives of the ICM regarding permissiveness to transcriptional activity of the LTR. 4th, 5th, 8th, 9th, and 10th indicate the day of gestation. +, permissiveness to LTR activity; —, non permissiveness to LTR activity; ?, permissiveness not determined.

Fig. 3.

Diagram illustrating the status of the differentiated derivatives of the ICM regarding permissiveness to transcriptional activity of the LTR. 4th, 5th, 8th, 9th, and 10th indicate the day of gestation. +, permissiveness to LTR activity; —, non permissiveness to LTR activity; ?, permissiveness not determined.

Our data provide strong evidence that the epiblast is not yet permissive to transcriptional activity of the LTR at the late primitive streak stage (8th day of gestation) (Fig. 3). Permissiveness seems to be acquired only when epiblast differentiates into mesoderm and defini tive ectoderm during gastrulation. Interestingly, epi blast of the 8th day embryo is still relatively undifferen tiated. No obvious differences in developmental potential can be detected between epiblast located in different parts of the embryo (Beddington, 1981; 1982; 1983) and it can also give rise to teratocarcinomas, indicating the presence of pluripotent stem cell progenitors (Solter et al. 1980). It is tempting then to speculate that LTR inactivity may be closely associated with the undifferentiated state of the inner cell mass and of the epiblast. The presence of negative regulatory factors or the absence of positive factors in undifferentiated cells might be directly or indirectly responsible for re pression of LTR activity.

Permissiveness to transcriptional activity of the LTR is acquired in the differentiated derivatives of the epiblast

Resistance of preimplantation embryos to productive infection is known to disappear by the 9th day of gestation (early somite stage) when MoMuLV injected into embryos in vivo leads to productive infection in many different cell types (Jaenisch, 1980; Jaenisch et al. 1981). However, as replication-competent virus was used in these experiments, it was not possible to determine if all the tissues of the 9th day embryo were permissive to retroviral expression. It may be restricted to one or few tissues, which subsequently generate infectious viruses spreading into neighbouring tissues as they become permissive to productive infection. Our data show that permissiveness to transcriptional activity of the LTR seems to be acquired as early as the 8th day of gestation, when epiblast differentiates into meso derm and extraembryonic mesoderm. LTR activity is also acquired when epiblast differentiates into neurec-toderm during the 9th day of development. Surface ectoderm does not become permissive to LTR activity until the 10th day (Fig. 3). Thus, it is clear that: (i) permissiveness is acquired only in differentiated deriva tives of the epiblast (embryonic and extraembryonic mesoderm, neurectoderm and surface ectoderm); (ii) not all the differentiated derivatives of the epiblast become permissive simultaneously at gastrulation. Sur face ectoderm requires an extra day of gestation, with respect to neurectoderm, for the LTR to become an active promoter.

Positive cells in embryos infected with the BAG vector were observed in the mesoderm following injec tion of virus inside the amniotic cavity on the 9th day of gestation. Since mesoderm is not directly exposed to the amniotic cavity, this observation was unexpected. One possible explanation is that the invaginating epi blast of the 9th day embryo, located in the primitive streak at the posterior end of the embryo, is no longer made of undifferentiated cells and, as such, has become permissive to virus expression. Certainly, 9th day em bryos have lost their capability of giving rise to terato carcinomas following transplantation to ectopic sites (Damjanov et al. 1971) suggesting that few or no pluripotent stem cell progenitors remain at this stage (Robertson and Bradley, 1986) despite the persistence of a primitive streak. A more likely explanation is that the positive cells identified in mesoderm following infection with the BAG vector may be migrating neural crest cells. This second hypothesis is largely supported by the observation that most lac Z-positive cells found were located in the head mesenchyme which is almost entirely neural crest in origin (Noden, 1988). Some positive cells were also identified in trunk mesoderm located in the expected neural crest cell migration pathways (Erickson et al. 1989).

It must be pointed out that no obvious difference in the intensity of staining could be detected between pIRV- and BAG-expressing cells. Therefore, it appears that transcriptional activity of the LTR in mesoderm and neurectoderm reaches a level comparable to the transcriptional activity of the beta-actin promoter very quickly following differentiation of the epiblast.

Injection of helper-free replication-defective vectors into 9th day embryos in vivo has recently been reported (Compere et al. 1989). These vectors express the ras and/or myc oncogene(s) under the transcriptional con trol of the LTR. They produce tumors in a great variety of tissues including surface epithelium. This implies that surface ectoderm was permissive to transcriptional activity of the LTR at the time of infection (9th day of gestation). Our data do not support this conclusion. It is possible that a very low level of transcriptional activity takes place in cells of surface ectoderm on the 9th day of development but it is not high enough to produce a detectable level of beta-galactosidase. By contrast, low levels of expression of the ras oncogene are known to be sufficient to induce a transformed phenotype (Compere et al. 1989). In addition, stimulation of the MoMuLV enhancer by the product of the ras oncogene has been reported (Wasylyk et al. 1987).

No beta-galactosidase-positive cells were identified in gut endoderm following infection of 9th and 10th day embryos in medium containing the pIRV vector (Table 5 and 6). The same result was obtained with 8th day embryos (data not shown). For this reason, we could not determine the status of definitive endoderm regarding permissiveness to transcriptional activity of the LTR. This observation cannot be due simply to the lack of activity of the rat beta-actin promoter in endoderm because positive cells were identified in gut endoderm following injection of virus inside the amni otic cavity of 8th day embryos. Also, in transgenic embryos containing a 4.3 kb beta-actin-ZacZ fragment of the pIRV vector, expression is found in gut endo derm (Beddington et al. 1989). Therefore, it is possible to infect endoderm cells only via integration into the epiblast and its subsequent differentiation into endo derm. Once differentiated, the endoderm cells appear to be resistant to viral infection, perhaps due to high protease activity, which may interrupt the normal retrovirus integration pathway.

Mechanisms that restrict retroviral expression in epiblast

Recent investigations on restriction of MoMuLV ex pression in undifferentiated F9 EC cells have provided compelling evidence that two independent mechanisms are probably involved in the block to expression, (1) inactivity of the MoMuLV enhancer located in the LTR and (2) interaction of negative regulatory factors in the vicinity of the primer-binding site (Loh et al. 1987; Weiher et al. 1987; Feuer et al. 1989). Both deletion and competition experiments have now shown that non function of the MoMuLV enhancer in EC cells most probably results from the absence of positive regulatory factors although the binding of a putative repressor has recently been reported (Tsukiyama et al. 1989). Assuming that the same mechanisms are also involved in repressing provirus transcription in the mouse em bryo, activation of the enhancer may reflect the pres ence of new ubiquitous transcription factors in the differentiation derivatives of the epiblast. As far as the binding of negative regulatory factor(s) in the vicinity of the primer-binding site is concerned, the function in embryonic cells of such repressors remains obscure. It has been postulated that such repressors would block transcription elongation (Loh et al. 1988; Feuer et al. 1989).

However, it remains to be determined whether both mechanisms are down-regulated simultaneously at gas trulation. Myeloproliferative Sarcoma Virus (MPSV) has been shown to be expressed more efficiently in EC cells than MoMuLV (Franz et al. 1986; Seliger et al. 1986). Analysis of hybrid retroviral vectors between MPSV and MoMuLV have demonstrated that struc tural differences both within the U3 portion of the LTR and within the region encoding the 5’ leader segment of MPSV are necessary for MPSV expression in EC cells. Each of the two inhibitory elements (MoMuLV LTR and MoMuLV 5’ leader segment) can prevent high-level transcription in EC cells. With the proviso that the same mechanisms are also involved in repressing pro virus transcription in the mouse embryo, it is possible that activity of the MoMuLV enhancer may be acquired before gastrulation in the epiblast and then, as revealed by our data, the negative regulatory factors binding to the 5’ leader segment would maintain repression until gastrulation. However, the converse may also be true.

Our data provide evidence that nonpermissiveness to transcriptional activity of MoMuLV is a feature of the epiblast of the early postimplantation embryo. Differ entiation of the epiblast into mesoderm and neurecto derm triggers activation of the provirus. No terminal differentiation is required. This suggests that differen tiation of the epiblast may involve a major change in the production of new ubiquitous transcription activators and/or repressors.

We would like to thank Jack Price for providing us with the BAG vector, and Hartmut Land for helpful discussion. This work was supported by the Imperial Cancer Research Fund.

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