Morphology of Mouse Egg Cylinder Development In Vitro: A Light and Electron Microscopic Study LYNN M.WILEY AND ROGER A. PEDERSEN Laboratory of Radiobiology, Universityof California,Sun Francisco 94143
ABSTRACT Phase contrast, light, and electron microscopy showed that mouse egg cylinder development from the blastocyst stage in vitro closely resembled the egg cylinder stages of the fourth to seventh days of gestation in vivo. Primary endoderm and ectoderm formed on day 2 of culture, and on day 3 visceral and parietal endoderm could be distinguished. Mesoderm formed on days 6.5-7 by delaminating from a thickened region of embryonic ectoderm. Extraembryonic ectoderm developed as a single layer of cuboidal epithelial cells that appeared to originate from polar trophoblast cells. By day 8 of culture egg cylinders had an ectoplacental cavity, a n exocoelom, a proamnionic cavity, a chorion, and an allantois and resembled egg cylinders in vivo after seven days' gestation. However, cultured blastocysts took longer to develop to these egg cylinder stages than embryos in vivo (8 days in vitro vs. 4 days in vivo) and were shorter (600p m in vitro vs. 700 p m in vivo). The development of mural trophoblast, parietal endoderm, Reichert's membrane, and ectoplacental cone also differed from that of their counterparts in vivo. Nevertheless, because the egg cylinder itself, which produces the embryo proper, develops similarly in vitro as in vivo, we suggest that cultured mouse blastocysts provide an excellent model for studies on early postimplantation embryogenesis. Early postimplantation development has been largely inaccessible to study in vitro. Recently, however, mouse preimplantation embryos have been successfully cultured to egg cylinder stages (Hsu, '71; Pienkowski et al., '74), which occasionally produce somites and heart-like structures (Hsu, '72, '73; Hsu et al., '74). Histological (Hsu, '72) and fine structural (Solter et al., '74; Hsu et al., '74) studies of cultured embryos at the egg cylinder stage show that their primary germ layers and extraembryonic structures resemble those of equivalent-stage embryos in vivo (Reinius, '65; Solter et al., '70). However, none of these studies compare the sequential development of cultured egg cylinders with early postimplantation development in vivo. Such a comparison is essential before the cultured egg cylinder is to serve as an in vitro model for early postimplantation development. To this end, we compared the development of mouse egg cylinders from the blastocyst stage in vitro with the reported anatomical, histological, fine structural and temporal features of early postimplantation development J. EXP. ZOOL., 200: 389-402.
in vivo. We based this comparison on the development of the three anatomic regions of the blastocyst: the inner cell mass (ICM); the polar trophoblast, which overlies the ICM; and the mural trophoblast, which encloses the blastocoel. In vivo, the ICM is believed to produce the visceral and parietal endoderm, the embryonic ectoderm and mesoderm, and the structures that arise from these germ layers. Classically, the extraembryonic ectoderm was also thought to be an ICM derivative (Jenkinson, '02) but recent experimental evidence indicates that i t originates from the polar trophoblast (Gardner and Johnson, '75). Other polar trophoblast derivatives are the ectoplacental cone (Gardner et al., '73) and the secondary trophoblast cells. The mural trophoblast produces the primary trophoblast cells, which later become giant cells (Snell and Stevens, '66).The ICM and the polar trophoblast contribute to the formation of the egg cylinder, which ultimately produces the fetus and the fetal membranes. At seven to seven and one-half days, the mouse egg cylinder in vivo consists of two segments, composed of visceral endoderm, embryonic ectoderm, and
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mesoderm and contains three cavities: the amnionic cavity, the exocoelom, and the ectoplacental cavity. In addition, it has three extraembryonic structures: the amnion, the chorion, and the allantois (Snell and Stevens, '66).Other extraembryonic structures - the ectoplacental cone, mural trophoblast giant cells, parietal endoderm, and Reichert's membrane - are not a part of the egg cylinder in vivo (Snell and Stevens, '66). We show that mouse egg cylinder development from the blastocyst stage in vitro resembles that during four to seven days of gestation with respect to the formation of endoderm, ectoderm, mesoderm, and extraembryonic ectoderm and the morphology of the egg cylinder structures but it differs with respect to the fate of those structures that are not a part of the egg cylinder in vivo.
resembling egg cylinders after eight days of culture. Consequently, only the more advanced embryos in each age group were analyzed in detail by sectioning. Selected embryos were photographed by phase-contrast microscopy and then were fixed with 2.5%glutaraldehyde in 0.1 M phosphate buffer followed by 2.0%osmium tetroxide in the same buffer. Fixed embryos were dehydrated in an ethanol series during which they were stained en bloc with uranyl acetate (three parts of aqueous saturated uranyl acetate to seven parts of absolute ethanol for 20 minutes). After being passed through propylene oxide, the embryos were embedded in Epon (Luft, '61). Plastic coverslips were peeled away from the polymerized Epon blocks, which were trimmed so that midsaggittal serial sections of the embryos could be prepared (fig. 1). MATERIALS AND METHODS For light microscopy, thick sections (0.5 Embryos were obtained from randomly bred pm) were cut with glass knives, dried onto Dub: (ICR) mice (Flow Research Animals, slides, and stained with Toluidine Blue 0. SecDublin, Virginia) after induction of su- tions were covered with immersion oil and perovulation with 5 IU of pregnant mares' then observed and photographed with a serum gonadotropin (Sigma) followed 48 bright-field, 63 X Zeiss planapochromatic hours later with 5 IU of human chorionic objective and a Wratten No. 22 orange filter. gonadotropin (HCG) (Ayerst Labs) and mat- Montages of the photographs were coning. Cultures were started with early blas- structed to provide complete images of the tocysts, taken from uteri 92 to 96 hours (4 sections. days) after HCG injection; this was desigFor electron microscopy, thin sections (600nated as day 1 of culture. 700 A) were cut with a diamond knife on a All embryos were grown in Eagle's basal Reichert ultratome and picked up on copper medium (BME) containing 102 mM NaCl and grids containing support films of carbonoptimal concentrations of essential amino coated Parlodion. Sections were treated seacids (Spindle and Pedersen, '73),5%fetal calf quentially with saturated uranyl acetate (30 serum, and 5%newborn calf serum (GIBCO). minutes) and lead citrate (Reynolds, '63) to Embryos were flushed from uteri with bicar- enhance contrast. Sections were observed and bonate-free Hanks' balanced salt solution (pH photographed with a n Hitachi HS-8 electron 7.0) modified to contain 129 mM NaC1, 1.71 microscope. mM calcium lactate, 0.25 mM sodium pyruRESULTS vate, lo5 IUAiter of penicillin, 50 mg/liter of During the 8-day culture period mouse blasstreptomycin, and vitamins, amino acids, and serum as in modified BME. So that the em- tocysts developed into segmented egg cylinbryos would remain attached to the substrata ders supported by a trophoblast sheet; they while being processed for light and electron averaged 600 p m in length. Serial sections microscopy, groups of 15 blastocysts were revealed that each segment of these egg cylinallowed to attach and grow out on solvent-re- ders (proximal, middle, and distal) contained sistant plastic coverslips (Sykes and Basrur, a cavity (the ectoplacental cavity, the exo'71) (Thermanox Lux, Thousand Oaks, Cali- coelom, and the proamniotic cavity, respecfornia). The coverslips were placed in 35-mm tively). The egg cylinders were attached to tissue culture dishes containing 3 ml of me- the trophoblast layer by their proximal segdium, which was not changed during the cul- ment. These embryos consisted of three priture period. mary germ layers (visceral endoderm, embryOf the several hundred cultured blastocysts onic ectoderm, and mesoderm) and had that we examined, 20-25%formed structures extraembryonic structures (ectoplacental
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I
a
3
Fig. 1 Schematic drawing of the embryo-fixation process. Embryos are flat-embedded in Epon (l), which is then trimmed (2) so that the embryos can he sectioned midsaattally (3)
closing the egg cylinder appeared thinnest around the end of the distal segment. Occasionally there were a few small, spherical cells scattered over the free surface of the trophoPhase contrast microscopy of blast sheet, most often around the base of the whole embryos proximal segment (figs. 2d-f). In order to identify these cell layers and Blastocysts, which were unhatched on day 1 of culture, hatched out of the zona pellucida structures and to correlate them with their and began adhering to the coverslips on day 2 counterparts in vivo, we fixed and sectioned (fig. 2a). On the morning of day 2, the inner the embryos for light and electron microscell mass (ICM) consisted of two distinct COPY. layers of cells (fig. 2b). Later, on day 3, the Light microscopy of thick sections blastocysts gradually flattened, collapsing the On the first day of culture, cells flattened blastocoel, as the mural trophoblast spread out on the coverslips and became giant cells over the free surface of the ICM (fig. 3a) as the first sign of endoderm formation. Early on (fig. 2c). On day 4, the mural trophoblast, which had day 2, while some blastocysts were still enclosed the ICM, formed a sheet of giant unhatched, primitive endoderm was apparent cells on the coverslip beneath a group of as a single layer of cuboidal cells (fig. 3b), smaller cells. This (proximal) group in turn which on day 3 (figs. 3c-f) resembled visceral supported another mass of cells (the distal endoderm of embryos in vivo (Reinius, '65). In group), which consisted of a solid core of cells addition, single cells or strands of cells surrounded by a conspicuous layer of dome- extended from the margins of the ICM along the inner surface of the mural trophoblast shaped cells (fig. 2d). During day 4, both groups enlarged con- (figs. 3c-e) and resembled parietal endoderm siderably, and late on day 4 the proximal cells developing in vivo a t four and one-half to group also became enclosed by a layer of cells, five days of gestation (Snell and Stevens, '66). so that the proximal and distal groups became Embryonic ectoderm on day 3 consisted of the proximal and distal segments of a n elon- lightly stained cells with large, round nuclei gated structure that was enclosed by a con- and prominent nucleoli (figs. 3c-f) and retinuous layer of cells and rested on a sheet of sembled the embryonic ectoderm in vivo on mural trophoblast (fig. 2e). At this time a days 4-5 of gestation (Snell and Stevens, '66; small cavity appeared in the distal segment Reinius, '65). The polar trophoblast remained next to the embryonic ectoderm instead of (fig. 2e). During the next four days, the region be- spreading out on the coverslip like the mural tween the proximal and distal segments elon- trophoblast (figs. 3f,g). Early on day 4, polar trophoblast cells corregated, so that by the end of the &day culture period embryos had three segments (fig. 2f). sponded in position to and presumably formed There was no conspicuous cavity in the prox- the proximal group of cells observed in whole imal segment, but the middle segment was embryos (figs. 3g, 4a). At the time that the hollow and contained a sac-like structure. A proximal group of cells became covered by viscrescent-shaped structure spanned the junc- ceral endoderm to become the proximal segtion of the middle and the proximal segments ment (fig. 4b), the cells in the proximal seg(fig. 2f). The small cavity in the distal seg- ment that were adjacent to the embryonic ment had enlarged. The layer of cells en- ectoderm became epithelial-like. Their posicone, chorion, and allantois) characteristic of 7-day-old egg-cylinder-stage embryos developing in vivo (Snell and Stevens, '66).
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tion corresponded with that of the extraembryonic ectoderm in vivo at five to six days of gestation (Snell and Stevens, '66). The remaining cells of the proximal segment were continuous with the mural trophoblast sheet and corresponded in position with the ectoplacental cone of embryos in vivo (Snell and Stevens, '66). Late on day 4, in the distal segment a proamnionic cavity appeared within the embryonic ectoderm (fig. 4b), which by now was a pseudostratified columnar epithelium typical of embryonic ectoderm in vivo (Solter et al., '70; Reinius, '65). After the blastocoel disappeared, there was no obvious layer of parietal endoderm cells like that seen on day 3 lining the mural trophoblast (fig. 3f). Only rarely did we see cells in sectioned day-4 embryos (fig. 3g) that resembled the small, spherical cells scattered over the mural trophoblast sheet of whole embryos (figs. 2d-f). However, there was evidence of parietal endoderm cell activity a t the base of the proximal segment in the form of a n amorphous, lightly staining extracellular material that resembled Reichert's membrane (figs. 3g, 4a,b), which is secreted by parietal endoderm in vivo (Pierce et al., '62). This material was present in various amounts a t the base of the egg cylinders throughout the rest of the 8-day culture period. On day 5, the epithelial region of the proximal segment became a new cell layer, the extraembryonic ectoderm, and elongated to produce the middle segment (fig. 5). This segment was hollow and was lined by this new layer of cuboidal cells. The embryonic ectoderm fused with this layer of cuboidal cells, which caused the proamniotic cavity to become continuous with the cavity of the middle segment. A similar fusion occurs in vivo after five and one-half to six days (Snell and Stevens, '66). This inner layer of small, cuboidal cells in the middle segment had the same position and histological appearance as the extraembryonic ectoderm in embryos in vivo a t five to five and onehalf days of gestation. In the distal segment, the visceral endoderm and the embryonic ectoderm cells retained their characteristic morphologies. Days 6-7 (fig.6) were characterized by the appearance of mesoderm cells. These appeared to originate from a thickened portion of embryonic ectoderm near the junction of the distal and middle segments and to occupy the space between the ectoderm and visceral
endoderm as a mass of loosely arranged, small cells. The origin of these cells corresponded with the primitive streak region at seven days of gestation in vivo (Solter et al., '70; Snell and Stevens, '66; Reinius, '65). On day 8 (fig. 7), each of the three segments contained a cavity. The proximal segment had a crescent-shaped cavity bound on one end by the mass of ectoplacental cone cells and on the other by extraembryonic ectoderm. This cavity appears to have been formed by the extraembryonic ectoderm and a layer of mesoderm cells receding from the middle segment, a phenomenon similar to the formation of the ectoplacental cavity and chorion of embryos in vivo a t seven to eight days of gestation (Razek, '72; Snell and Stevens, '66). The cavity of the middle segment, the exocoelom, was lined by mesoderm and often contained a sac-like structure, presumably the allantois, which appeared to consist of mesoderm cells and which was attached to the wall of the exocoelom by a narrow stalk. The distal segment contained a vesicle of embryonic ectoderm whose lumen was the proamniotic cavity. The visceral endoderm surrounding the egg cylinder was highly vacuolated and columnar over the proximal and middle segments but was thin and squamous over the distal segment, particularly at its tip. The embryonic ectoderm remained pseudostratified, and a loosely arranged group of mesoderm cells had extended completely around the tip of the distal segment between the embryonic ectoderm and the squamous visceral endoderm. At this Fig. 2 Phase contrast microscopy of whole embryos. (a) Day-2 hatched blastocyst showing inner cell mass (ICM), mural trophoblast (m-TB), polar trophoblast (p-TB). (b) Day-2 blastocyst whose ICM consists of two distinct cell layers farrows 1 and 2). (c) Day-3 embryo whose mural trophoblast (m-TB1 is spreading out onto the coverslip and undergoinggiant cell transformation.Arrows 1 and 2 point to the two cell layers of the ICM. (d) Day-4 embryo, which consists of a proximal (PRO) and a distal (DIS) group of cells. The distal group of cells consists of a solid core (arrow 1) surrounded by a layer of dome-shaped cells (arrow 2). Small spherical cells (ssc) are scattered over the mural trophoblast sheet. (e) Late on day 4 the proximal group of cells now also consists of an inner core of cells surrounded by an outer layer, like the distal group (DIS). The asterisk indicates a small cavity within the distal segment. (f)Day8 egg cylinder consisting of three segments: a proximal (PRO), a middle (MID), and a distal (DIS) segment. The shadow (S) within the hollow middle segment is a sac-like structure. A crescent-shaped structure (C) spans the junction of the proximal and middle segments. The asterisk indicates a cavity within the distal segment. M a g nifications: a-e, X 240; f, X 120.
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Fig. 3 Thick sections: days 1-4. (a) Unhatched blastocyst on day 1of culture. The flattened cells over the free surface of the ICM represent the first sign of endoderm differentiation. (b) A fully expanded unhatched blastocyst with cuboidal endoderm (END1 cells and ectoderm (ECT) cells. (c1 Day-3 embryo after hatching and attachment. Both visceral endoderm (v-END)and parietal endoderm (p-END1 are present. The mural trophoblast (m-TB) is spreading out on the coverslip as the blastocyst flattens. (d,e1 Slightly more advanced day-3 embryo. (f) A day-3 embryo with a completely flattened blastocoel. (gf A day-4 embryo shortly after the mural trophoblast (m-TB) has completely moved off of the ICM derivatives. There are two small spherical cells (sschear the proximal group of cells (pTB) that may correspond to those hypothesized as being parietal endoderm in whole day4 embryos. Magnification: X 296.
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Fig. 4 Thick sections: day 4. (a) A day-4 embryo after the proximal group of cells and the distal group of cells (ECT and V-END1have enlarged. (b) A day-4 embryo after the visceral endoderm (FEND) has covered the proximal group of cells and the extraembryonic ectoderm (x-ECT) has appeared. Magnification: X 296.
stage, then, embryos consisted of a t least three embryonic cell types-endoderm, ectoderm, and mesoderm-and two extra-embryonic cell types-extraembryonic ectoderm and ectoplacental cone. Mitotic figures were frequently observed in all cell types, particularly in embryonic ectoderm and in the ectoplacental cone.
Electron microscopy of thin sections Electron microscopy was done to verify the identity and determine the fate of presumed parietal endoderm cells in early embryos and to confirm that cells which appeared to originate from the embryonic ectoderm were mesoderm cells. To verify the identity of parietal endoderm cells, we characterized presumed parietal endoderm cells at day 3 of culture; we then looked for similar cells in older embryos. We found that on day 3 these cells were cuboidal and had a high nucleocytoplasmic ratio (fig. 8). The most characteristic fine structural feature was the well-developed rough endoplasmic reticulum (RER), which was filled with an amorphous material whose electron density matched that of the prominent basal lamina separating these cells from the trophoblast cells (fig. 8). These fine structural characteristics are similar to those of parietal endoderm cells in vivo (Pierce et al., '62). When the blastocoel collapsed completely, mural trophoblast cells appeared to sandwich
the parietal endoderm cells (fig. 8). After the mural trophoblast moved off the ICM, the only remaining identifiable parietal endoderm cells were those occasionally observed at the base of the proximal segment in association with a n amorphous, lightly stained extracellular material that resembled Reichert's membrane (Pierce et al., '62). To determine whether cells that appeared to originate from the embryonic ectoderm resembled mesoderm, we made thin sections of embryos on day 7 of culture and studied the area of the presumed primitive streak (fig. 6 ) . The small, oblong, loosely arranged cells that appeared between the embryonic ectoderm and the visceral endoderm were oriented with their long axis perpendicular to that of the embryonic ectodermal cells. These small cells lacked cell junctions, had randomly distributed cytoplasmic organelles, and had large nuclei with smooth contours and two or three prominent nucleoli (fig. 9). There were a few profiles of rough endoplasmic reticulum, but most of the ribosomes were arranged into polysomes; there were well-developed Golgi and numerous small mitochondria. Although several of these fine structural features are common also to embryonic ectodermal cells, the small size and lack of cell junctions, in particular, distinguish mesodermal cells, and are characteristic of mesodermal cells of egg cylinders after six and one-half to seven days of gestation in vivo (Solter et al., '70).
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Fig. 5 Thick sections: day 5. Day-5 egg cylinder after the embryonic ectoderm (e-ECT) and extraembryonic ectoderm (x-ECT)have fused to enclose a common cavity. These are semi-serial sections that run lateral (a) to medial, (d). Magnification: X 296.
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Fig. 6 Thick sections: day 7. A day7 egg cylinder that illustrates mesoderm delamination. A portion of the embryonic ectoderm fe-EL”I’) is thicker (arrow), and loosely arranged cefls, probably corresponding to mesoderm (MES)cells on day 7 in vivo, appear to delaminate from it to occupy the apace between the embryonic ectoderm (e-ECT)and visceral endoderm b-END). A bend in the middle of this egg cylinder produces the apparent discontinuity between the x-ECT and ectoplacental cone (EPC); other sections showed that they are connected as in figure 5. The hole a t the tip of the distal segment is probably an artifact. Magnification: X 236.
Fig. 7 Thick sections: day 8. Egg cylinder a t the end o f the %day culture period. Reichert’s membrane (RM), proamnionic cavity (I), exocoelom 12), ectoplacental cavity (3). Magnification: X 236.
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Fig. 8 Thin sections: parietal endoderm in a day-3 embryo. A day-3 embryo (left side of embryo in fig. 30. in which the mural trophoblast cells (m-TB)together with the adjacent parietal endoderm (pEND) are migrating away from the ICM. The cytoplasm in the parietal endoderm cells contains many profiles of rough endoplasmic reticulum (*) filled with an amorphous material of the same electron density as the basal lamina (arrows) that separates the parietal endoderm from the mural trophoblast (m-TB). Magnification: X 4,105.
Fig. 9 Thin sections:mesoderm cells in a day-7 egg cylinder. A day-7 embryo (same embryo as in fig. 6) undergoing mesoderm (MES) delamination. The small, fusiform mesoderm cells have large nuclei, a few profiles of rough endoplasmic reticulum, small mitochondria, prominent Golgi (G), and numerous polysomes. In spite of the lengthy expanses of mutual contact, very few, if any, cell junctions are evident between mesoderm cells, in contrast to the well-developedjunctions between visceral endoderm cells and in the embryonic ectoderm. Magnification: X 4,105.
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From comparisons of the development of egg cylinders cultured from blastocysts with published observations of early mouse embryo development in vivo we conclude that the morphology of egg cylinders developing from blastocysts in vitro resembles that of mouse embryos in vivo during the fourth to seventh days of gestation. Cultured blastocysts, however, developed more slowly to equivalent egg cylinder stages (8 days in vitro vs. 4 days in vivo) and formed shorter egg cylinders (600 p m in vitro vs. 700 p m in vivo). Both of these features may have resulted from our inability to supply the embryos with their optimal culture conditions, since these conditions are still being elucidated (flsu, '72; Pedersen and Spindle, '76). The major differences between post-blastocyst development in vitro and in vivo involved the fate of four structures that are not a part of the egg cylinder itself in vivo; these are the mural trophoblast, the parietal endoderm, Reichert's membrane, and the ectoplacental cone. In vivo, the mural trophoblast forms the wall of the blastocoel, which becomes the yolk cavity and is lined by Reichert's membrane and parietal endoderm cells (Snell and Stevens, '66). In vitro, however, the mural trophoblast does not enclose a three-dimensional cavity but instead spreads as a two-dimensional sheet of giant cells on the coverslips (Cole and Paul, '65). Because parietal endoderm cells, which were initially present during attachment and outgrowth, were later observed only a t the base of the proximal segment of the egg cylinder, our observations are consistent with those of Hsu et al. ('74) but differ with the observation of Solter et al. ('74) that parietal endoderm cells were interspersed with the visceral endoderm cells of cultured mouse embryos. However, phase contrast microscopy of whole embryos a t days 4-8 of culture showed that small, round cells were scattered over the mural trophoblast sheet, particularly around the base of the proximal segment (figs. 2d,f). These could have been parietal endoderm cells like those reported by Sherman and Salomon ('75). Because such cells were rarely attached to the mural trophoblast in sectioned embryos, they may have adhered poorly and been lost during fixation. In the thick sections of cultured embryos we saw no extracellular material resembling
Reichert's membrane over the surface of the mural trophoblast sheet after the blastocoel had disappeared. Glutaraldehyde fixation preserves Reichert's membrane (Reinius, ,651, so we conclude that it probably was not present over the surface of the trophoblast sheet. The only material that resembled Reichert's membrane in cultured embryos was at the base of the proximal segment where parietal endoderm cells were seen. The ectoplacental cone originates from proliferating polar trophoblast cells in vivo (Gardner et al., '731, projects into the uterine epithelium, and is not covered by visceral endoderm (Snell and Stevens, '66). Consequently, in vivo, the egg cylinder consists of two segments enclosed by visceral endoderm instead of the three segments we describe for cultured egg cylinders. We suggest that in vitro the proliferating polar trophoblast cells, encountering the unyielding coverslip instead of uterine epithelium, protrude inward, become covered by visceral endoderm, and thus become the proximal segment. It was not possible to determine whether the visceral endoderm covering the proximal segment originated de nouo from the ectoplacental cone or migrated from the visceral endoderm covering the adjacent (distal) segment of late day4 embryos (fig. 4b). The time that primitive endoderm and ectoderm form in vitro (day 2 of culture) corresponds to the fifth day of gestation, when Reinius ('65) observed endoderm formation in vivo in mouse embryos of the CBA strain, and was about one day later than the time of endoderm formation in the hybrid embryos studied by Snell and Stevens ('66). Both in vivo and in vitro, parietal and visceral endoderm can be distinguished from each other during the 24 hours after endoderm differentiation. During the same period, the morphology of ectoderm cells changes very little. During morphological differentiation of primitive endoderm and ectoderm, the ICM is sensitive to drugs that interfere with gene expression, such as 5-bromodeoxyuridine (Sherman and Atienza, '75; Pedersen and Spindle, '76) and actinomycin D (Rowinski et al., '75; Glass et al., '76). Consequently, morphological differentiation of endoderm and ectoderm in mouse embryos may require concurrent gene expression. Mesoderm differentiation occurs in cultured embryos on day 7, when the embryos' total age is ten days (3 days in vivo plus 7 days in cul-
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plantation mouse and rabbit embryos, and cell strains ture), compared with six and one-half (Snell derived from them. In: Preimplantation Stages of Pregand Stevens, '66) or seven and one-half nancy. Ciba Foundation Symposium. G. E. W. Wol(Reinius, '65) days total age in vivo. This stenholme and M. OConnor, eds. J. & A. Churchill, Londifference in timing probably represents a don, pp. 82-122. cumulative delay of embryos developing Gardner, R. L. 1972 An investigation of inner cell mass and trophoblast tissues following their isolation from beyond the blastocyst stage in vitro. In culthe mouse blastocyst. J. Embryol. Exp. Morphol.,28: 279tured embryos mesoderm always appeared to 312. originate in the distal segment from the 1975 Analysis of determination and differentiation in the early mammalian embryo using intrathickened embryonic ectoderm near the juncand interspecific chimaeras. In: The Developmental Bioltion of the distal and middle segment, as ogy of Reproduction. C. L. Markert and J. Papaconstanobserved by Snell and Stevens ('661, and did tinou, eds. Academic Press, New York, pp. 207-236. not originate in the middle segment as re- Gardner, R. L., and M. H. Johnson 1973 Investigation of early mammalian development using interspecific ported by Razek ('72). chimaeras between rat and mouse. Nature New Biology, Extraembryonic ectoderm, which was first 246: 86-89. noticed late on day 4 of culture, appeared to 1975 Investigation of cellular interaction and originate in the proximal segment and was deployment in the early mammalian embryo using interspecific chimaeras between the rat and mouse. In: always attached to the polar trophoblast or to Cell Patterning. Vol. 29. Ciba Foundation Symposium. the ectoplacental cone cells, and i t was only Associated Scientific Publishers, New York, pp. 183-200. transiently attached to the (ICM-derived)em- Gardner, R. L., V. E. Papaioannou and S. C. Barton 1973 bryonic ectoderm on day 5. Extraembryonic Origin of the ectoplacental cone and secondary giant cells in mouse blastocysts reconstituted from isolated ectoderm, which consisted of a simple layer of trophoblast and inner cell mass. J. Embryol. Exp. Morcuboidal cells, contrasted sharply with embryphol., 30: 561-572. onic ectoderm, which consisted of a layer of Glass, R. H., A. I. Spindle and R. A. Pedersen 1976 pseudostratified columnar cells. These obserDifferential inhibition of trophoblast outgrowth and inner cell mass growth by actinomycin D in cultured vations are consistent with a polar trophomouse embryos. J. Reprod. Fert., 48: 443-445. blast origin for extraembryonic ectoderm, and Hsu, Y.-C. 1971 Post-blastocyst differentiation in vitro. ectoplacental cone cells, as found by Gardner Nature, 231: 100-102. and co-workers (Gardner et al., '73; Gardner, 1972 Differentiation in vitro of mouse embryos beyond the implantation stage. Nature, 239: 200-202. '75; Gardner and Johnson, "73, '75). 1973 Differentiation in vitro of mouse embryos Because the egg cylinder itself, which proto the stage of early somite. Devel. Biol., 33: 403-411. duces the embryo proper, develops so similarly Hsu, Y.-C., J. Baskar, L. C. Stevens and J. E. Rash 1974 Dein vitro and in vivo, we conclude that cultured velopment in vitro of mouse embryos from the two-cell egg stage to the early somite stage. J. Embryol. Exp. embryos provide an excellent experimental Morphol., 31: 235-245. system to study differentiation, development J. W. 1902 Observations on the histology and morphogenetic movements of the primary Jenkinson, and physiology of the placenta of the mouse. Tijdschr. germ layers and their inductive interactions ned. dierk. Vereen., 2: 124-198. during early mammalian development. Fur- Luft, J. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. thermore, this histological analysis provides a guide for isolating and studying the proper- Pederson, R. A., and A. I. Spindle 1976 Genetic effects on mammalian development during and after implantation. ties of particular cell populations from the In: Embryogenesis in Mammals. Ciba Foundation Symdifferent stages of egg cylinder development posium 40. Elsevier, Amsterdam and New York, pp. 133154. in vitro. ACKNOWLEDGMENTS
The authors would like to acknowledge the assistance of Doctor Akiko Spindle and of Ms. Kitty Wu in culturing the embryos and to thank Doctor Patricia G. Calarco for reading the manuscript and Ms. Mimi Zeiger for editorial assistance. This work was performed under the auspices of the United States Energy Research and Development Administration. LITERATURE CITED Cole, R. J.,and J. Paul 1965 Properties of cultured preim-
Pienkowski, M., D. Solter and H. Koprowski 1974 Early mouse embryos: growth and differentiation in vitro. Exp. cell Res., 85: 424-428. Pierce, G. B., Jr., A. R. Midgley Jr., J. S. Ram and J. D. Feldman 1962 Parietal yolk sac carcinoma: clue to the histogenesis of Reichert's membrane of the mouse embryo. Amer. J. Pathol., 41: 549-566. Razek, H. A. 1972 New aspects of the differentiation of the presomite mouse embryo. Z. Anat. Entwick1.-Gesch., 135: 265-278. Reinius, S. 1965 Morphology of the mouse embryo, from the time of implantation to mesoderm formation. 2. Zellforsch., 68: 711-723. Reynolds, E. S. 1963 The use of lead citrate a t high pH as an electron opaque stain in electron microscopy. J. Cell Biol.. 17: 208-212. Rowinski, J., D. Solter and H. Koprowski 1975 Mouse em-
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bryo development in vitro: effects of inhibitors of RNA and protein synthesis on blastocyst and post-blastocyst embryos. J. Exp. Zool., 192: 133-142. Sherman, M. I., and S. I. Atienza 1975 Effects of bromodeoxyuridine, cytosine arabinoside and colcemid upon in vitro development of mouse blastocysts. J. Embryol. Exp. Morphol., 34: 467-484. Sherman, M. I., and D. S. Salomon 1975 The relationships between the early mouse embryo and its environment. In: The Developmental Biology of Reproduction. C. L. Markert and J. Papconstantinou, eds. Academic Press, New York, pp. 277-309. Snell, G. D., and L. C. Stevens 1966 Early Embryology. In: Biology of the Laboratory Mouse. Seconded. E. L. Green, ed. McGraw-Hill, New York, pp. 205-245.
Solter, D., W. Biczysko, M. Pienkowski and H. Koprowski 1974 Ultrastructure of mouse egg cylinders developed in vitro. Anat. Rec., 180: 263-280. Solter, D., I. Damjanov and N. Skreb 1970 Ultrastructure of mouse egg-cylinder. 2. Anat. Entwick1.-Gesch., 132: 291-298. Solter, D., and B. B. Knowles 1975 Immunosurgery of mouse blastocyst. Proc. Nat. Acad. Sci. (U.S.A.), 72: 5099-5102. Spindle, A. I., and R. A. Pedersen 1973 Hatching, attachment, and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements. J. Exp. Zool., 186: 305-318. Sykes, A. K., and P. K. Basrur 1971 A melinex coverslip method for ultrastructural studies of monolayer cultures. In Vitro, 7: 68-73.
ABSTRACT Phase contrast, light, and electron microscopy showed that mouse egg cylinder development from the blastocyst stage in vitro closely resembled the egg cylinder stages of the fourth to seventh days of gestation in vivo. Primary endoderm and ectoderm formed on day 2 of culture, and on day 3 visceral and parietal endoderm could be distinguished. Mesoderm formed on days 6.5-7 by delaminating from a thickened region of embryonic ectoderm. Extraembryonic ectoderm developed as a single layer of cuboidal epithelial cells that appeared to originate from polar trophoblast cells. By day 8 of culture egg cylinders had an ectoplacental cavity, a n exocoelom, a proamnionic cavity, a chorion, and an allantois and resembled egg cylinders in vivo after seven days' gestation. However, cultured blastocysts took longer to develop to these egg cylinder stages than embryos in vivo (8 days in vitro vs. 4 days in vivo) and were shorter (600p m in vitro vs. 700 p m in vivo). The development of mural trophoblast, parietal endoderm, Reichert's membrane, and ectoplacental cone also differed from that of their counterparts in vivo. Nevertheless, because the egg cylinder itself, which produces the embryo proper, develops similarly in vitro as in vivo, we suggest that cultured mouse blastocysts provide an excellent model for studies on early postimplantation embryogenesis. Early postimplantation development has been largely inaccessible to study in vitro. Recently, however, mouse preimplantation embryos have been successfully cultured to egg cylinder stages (Hsu, '71; Pienkowski et al., '74), which occasionally produce somites and heart-like structures (Hsu, '72, '73; Hsu et al., '74). Histological (Hsu, '72) and fine structural (Solter et al., '74; Hsu et al., '74) studies of cultured embryos at the egg cylinder stage show that their primary germ layers and extraembryonic structures resemble those of equivalent-stage embryos in vivo (Reinius, '65; Solter et al., '70). However, none of these studies compare the sequential development of cultured egg cylinders with early postimplantation development in vivo. Such a comparison is essential before the cultured egg cylinder is to serve as an in vitro model for early postimplantation development. To this end, we compared the development of mouse egg cylinders from the blastocyst stage in vitro with the reported anatomical, histological, fine structural and temporal features of early postimplantation development J. EXP. ZOOL., 200: 389-402.
in vivo. We based this comparison on the development of the three anatomic regions of the blastocyst: the inner cell mass (ICM); the polar trophoblast, which overlies the ICM; and the mural trophoblast, which encloses the blastocoel. In vivo, the ICM is believed to produce the visceral and parietal endoderm, the embryonic ectoderm and mesoderm, and the structures that arise from these germ layers. Classically, the extraembryonic ectoderm was also thought to be an ICM derivative (Jenkinson, '02) but recent experimental evidence indicates that i t originates from the polar trophoblast (Gardner and Johnson, '75). Other polar trophoblast derivatives are the ectoplacental cone (Gardner et al., '73) and the secondary trophoblast cells. The mural trophoblast produces the primary trophoblast cells, which later become giant cells (Snell and Stevens, '66).The ICM and the polar trophoblast contribute to the formation of the egg cylinder, which ultimately produces the fetus and the fetal membranes. At seven to seven and one-half days, the mouse egg cylinder in vivo consists of two segments, composed of visceral endoderm, embryonic ectoderm, and
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mesoderm and contains three cavities: the amnionic cavity, the exocoelom, and the ectoplacental cavity. In addition, it has three extraembryonic structures: the amnion, the chorion, and the allantois (Snell and Stevens, '66).Other extraembryonic structures - the ectoplacental cone, mural trophoblast giant cells, parietal endoderm, and Reichert's membrane - are not a part of the egg cylinder in vivo (Snell and Stevens, '66). We show that mouse egg cylinder development from the blastocyst stage in vitro resembles that during four to seven days of gestation with respect to the formation of endoderm, ectoderm, mesoderm, and extraembryonic ectoderm and the morphology of the egg cylinder structures but it differs with respect to the fate of those structures that are not a part of the egg cylinder in vivo.
resembling egg cylinders after eight days of culture. Consequently, only the more advanced embryos in each age group were analyzed in detail by sectioning. Selected embryos were photographed by phase-contrast microscopy and then were fixed with 2.5%glutaraldehyde in 0.1 M phosphate buffer followed by 2.0%osmium tetroxide in the same buffer. Fixed embryos were dehydrated in an ethanol series during which they were stained en bloc with uranyl acetate (three parts of aqueous saturated uranyl acetate to seven parts of absolute ethanol for 20 minutes). After being passed through propylene oxide, the embryos were embedded in Epon (Luft, '61). Plastic coverslips were peeled away from the polymerized Epon blocks, which were trimmed so that midsaggittal serial sections of the embryos could be prepared (fig. 1). MATERIALS AND METHODS For light microscopy, thick sections (0.5 Embryos were obtained from randomly bred pm) were cut with glass knives, dried onto Dub: (ICR) mice (Flow Research Animals, slides, and stained with Toluidine Blue 0. SecDublin, Virginia) after induction of su- tions were covered with immersion oil and perovulation with 5 IU of pregnant mares' then observed and photographed with a serum gonadotropin (Sigma) followed 48 bright-field, 63 X Zeiss planapochromatic hours later with 5 IU of human chorionic objective and a Wratten No. 22 orange filter. gonadotropin (HCG) (Ayerst Labs) and mat- Montages of the photographs were coning. Cultures were started with early blas- structed to provide complete images of the tocysts, taken from uteri 92 to 96 hours (4 sections. days) after HCG injection; this was desigFor electron microscopy, thin sections (600nated as day 1 of culture. 700 A) were cut with a diamond knife on a All embryos were grown in Eagle's basal Reichert ultratome and picked up on copper medium (BME) containing 102 mM NaCl and grids containing support films of carbonoptimal concentrations of essential amino coated Parlodion. Sections were treated seacids (Spindle and Pedersen, '73),5%fetal calf quentially with saturated uranyl acetate (30 serum, and 5%newborn calf serum (GIBCO). minutes) and lead citrate (Reynolds, '63) to Embryos were flushed from uteri with bicar- enhance contrast. Sections were observed and bonate-free Hanks' balanced salt solution (pH photographed with a n Hitachi HS-8 electron 7.0) modified to contain 129 mM NaC1, 1.71 microscope. mM calcium lactate, 0.25 mM sodium pyruRESULTS vate, lo5 IUAiter of penicillin, 50 mg/liter of During the 8-day culture period mouse blasstreptomycin, and vitamins, amino acids, and serum as in modified BME. So that the em- tocysts developed into segmented egg cylinbryos would remain attached to the substrata ders supported by a trophoblast sheet; they while being processed for light and electron averaged 600 p m in length. Serial sections microscopy, groups of 15 blastocysts were revealed that each segment of these egg cylinallowed to attach and grow out on solvent-re- ders (proximal, middle, and distal) contained sistant plastic coverslips (Sykes and Basrur, a cavity (the ectoplacental cavity, the exo'71) (Thermanox Lux, Thousand Oaks, Cali- coelom, and the proamniotic cavity, respecfornia). The coverslips were placed in 35-mm tively). The egg cylinders were attached to tissue culture dishes containing 3 ml of me- the trophoblast layer by their proximal segdium, which was not changed during the cul- ment. These embryos consisted of three priture period. mary germ layers (visceral endoderm, embryOf the several hundred cultured blastocysts onic ectoderm, and mesoderm) and had that we examined, 20-25%formed structures extraembryonic structures (ectoplacental
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I
a
3
Fig. 1 Schematic drawing of the embryo-fixation process. Embryos are flat-embedded in Epon (l), which is then trimmed (2) so that the embryos can he sectioned midsaattally (3)
closing the egg cylinder appeared thinnest around the end of the distal segment. Occasionally there were a few small, spherical cells scattered over the free surface of the trophoPhase contrast microscopy of blast sheet, most often around the base of the whole embryos proximal segment (figs. 2d-f). In order to identify these cell layers and Blastocysts, which were unhatched on day 1 of culture, hatched out of the zona pellucida structures and to correlate them with their and began adhering to the coverslips on day 2 counterparts in vivo, we fixed and sectioned (fig. 2a). On the morning of day 2, the inner the embryos for light and electron microscell mass (ICM) consisted of two distinct COPY. layers of cells (fig. 2b). Later, on day 3, the Light microscopy of thick sections blastocysts gradually flattened, collapsing the On the first day of culture, cells flattened blastocoel, as the mural trophoblast spread out on the coverslips and became giant cells over the free surface of the ICM (fig. 3a) as the first sign of endoderm formation. Early on (fig. 2c). On day 4, the mural trophoblast, which had day 2, while some blastocysts were still enclosed the ICM, formed a sheet of giant unhatched, primitive endoderm was apparent cells on the coverslip beneath a group of as a single layer of cuboidal cells (fig. 3b), smaller cells. This (proximal) group in turn which on day 3 (figs. 3c-f) resembled visceral supported another mass of cells (the distal endoderm of embryos in vivo (Reinius, '65). In group), which consisted of a solid core of cells addition, single cells or strands of cells surrounded by a conspicuous layer of dome- extended from the margins of the ICM along the inner surface of the mural trophoblast shaped cells (fig. 2d). During day 4, both groups enlarged con- (figs. 3c-e) and resembled parietal endoderm siderably, and late on day 4 the proximal cells developing in vivo a t four and one-half to group also became enclosed by a layer of cells, five days of gestation (Snell and Stevens, '66). so that the proximal and distal groups became Embryonic ectoderm on day 3 consisted of the proximal and distal segments of a n elon- lightly stained cells with large, round nuclei gated structure that was enclosed by a con- and prominent nucleoli (figs. 3c-f) and retinuous layer of cells and rested on a sheet of sembled the embryonic ectoderm in vivo on mural trophoblast (fig. 2e). At this time a days 4-5 of gestation (Snell and Stevens, '66; small cavity appeared in the distal segment Reinius, '65). The polar trophoblast remained next to the embryonic ectoderm instead of (fig. 2e). During the next four days, the region be- spreading out on the coverslip like the mural tween the proximal and distal segments elon- trophoblast (figs. 3f,g). Early on day 4, polar trophoblast cells corregated, so that by the end of the &day culture period embryos had three segments (fig. 2f). sponded in position to and presumably formed There was no conspicuous cavity in the prox- the proximal group of cells observed in whole imal segment, but the middle segment was embryos (figs. 3g, 4a). At the time that the hollow and contained a sac-like structure. A proximal group of cells became covered by viscrescent-shaped structure spanned the junc- ceral endoderm to become the proximal segtion of the middle and the proximal segments ment (fig. 4b), the cells in the proximal seg(fig. 2f). The small cavity in the distal seg- ment that were adjacent to the embryonic ment had enlarged. The layer of cells en- ectoderm became epithelial-like. Their posicone, chorion, and allantois) characteristic of 7-day-old egg-cylinder-stage embryos developing in vivo (Snell and Stevens, '66).
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tion corresponded with that of the extraembryonic ectoderm in vivo at five to six days of gestation (Snell and Stevens, '66). The remaining cells of the proximal segment were continuous with the mural trophoblast sheet and corresponded in position with the ectoplacental cone of embryos in vivo (Snell and Stevens, '66). Late on day 4, in the distal segment a proamnionic cavity appeared within the embryonic ectoderm (fig. 4b), which by now was a pseudostratified columnar epithelium typical of embryonic ectoderm in vivo (Solter et al., '70; Reinius, '65). After the blastocoel disappeared, there was no obvious layer of parietal endoderm cells like that seen on day 3 lining the mural trophoblast (fig. 3f). Only rarely did we see cells in sectioned day-4 embryos (fig. 3g) that resembled the small, spherical cells scattered over the mural trophoblast sheet of whole embryos (figs. 2d-f). However, there was evidence of parietal endoderm cell activity a t the base of the proximal segment in the form of a n amorphous, lightly staining extracellular material that resembled Reichert's membrane (figs. 3g, 4a,b), which is secreted by parietal endoderm in vivo (Pierce et al., '62). This material was present in various amounts a t the base of the egg cylinders throughout the rest of the 8-day culture period. On day 5, the epithelial region of the proximal segment became a new cell layer, the extraembryonic ectoderm, and elongated to produce the middle segment (fig. 5). This segment was hollow and was lined by this new layer of cuboidal cells. The embryonic ectoderm fused with this layer of cuboidal cells, which caused the proamniotic cavity to become continuous with the cavity of the middle segment. A similar fusion occurs in vivo after five and one-half to six days (Snell and Stevens, '66). This inner layer of small, cuboidal cells in the middle segment had the same position and histological appearance as the extraembryonic ectoderm in embryos in vivo a t five to five and onehalf days of gestation. In the distal segment, the visceral endoderm and the embryonic ectoderm cells retained their characteristic morphologies. Days 6-7 (fig.6) were characterized by the appearance of mesoderm cells. These appeared to originate from a thickened portion of embryonic ectoderm near the junction of the distal and middle segments and to occupy the space between the ectoderm and visceral
endoderm as a mass of loosely arranged, small cells. The origin of these cells corresponded with the primitive streak region at seven days of gestation in vivo (Solter et al., '70; Snell and Stevens, '66; Reinius, '65). On day 8 (fig. 7), each of the three segments contained a cavity. The proximal segment had a crescent-shaped cavity bound on one end by the mass of ectoplacental cone cells and on the other by extraembryonic ectoderm. This cavity appears to have been formed by the extraembryonic ectoderm and a layer of mesoderm cells receding from the middle segment, a phenomenon similar to the formation of the ectoplacental cavity and chorion of embryos in vivo a t seven to eight days of gestation (Razek, '72; Snell and Stevens, '66). The cavity of the middle segment, the exocoelom, was lined by mesoderm and often contained a sac-like structure, presumably the allantois, which appeared to consist of mesoderm cells and which was attached to the wall of the exocoelom by a narrow stalk. The distal segment contained a vesicle of embryonic ectoderm whose lumen was the proamniotic cavity. The visceral endoderm surrounding the egg cylinder was highly vacuolated and columnar over the proximal and middle segments but was thin and squamous over the distal segment, particularly at its tip. The embryonic ectoderm remained pseudostratified, and a loosely arranged group of mesoderm cells had extended completely around the tip of the distal segment between the embryonic ectoderm and the squamous visceral endoderm. At this Fig. 2 Phase contrast microscopy of whole embryos. (a) Day-2 hatched blastocyst showing inner cell mass (ICM), mural trophoblast (m-TB), polar trophoblast (p-TB). (b) Day-2 blastocyst whose ICM consists of two distinct cell layers farrows 1 and 2). (c) Day-3 embryo whose mural trophoblast (m-TB1 is spreading out onto the coverslip and undergoinggiant cell transformation.Arrows 1 and 2 point to the two cell layers of the ICM. (d) Day-4 embryo, which consists of a proximal (PRO) and a distal (DIS) group of cells. The distal group of cells consists of a solid core (arrow 1) surrounded by a layer of dome-shaped cells (arrow 2). Small spherical cells (ssc) are scattered over the mural trophoblast sheet. (e) Late on day 4 the proximal group of cells now also consists of an inner core of cells surrounded by an outer layer, like the distal group (DIS). The asterisk indicates a small cavity within the distal segment. (f)Day8 egg cylinder consisting of three segments: a proximal (PRO), a middle (MID), and a distal (DIS) segment. The shadow (S) within the hollow middle segment is a sac-like structure. A crescent-shaped structure (C) spans the junction of the proximal and middle segments. The asterisk indicates a cavity within the distal segment. M a g nifications: a-e, X 240; f, X 120.
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Figure 2
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Fig. 3 Thick sections: days 1-4. (a) Unhatched blastocyst on day 1of culture. The flattened cells over the free surface of the ICM represent the first sign of endoderm differentiation. (b) A fully expanded unhatched blastocyst with cuboidal endoderm (END1 cells and ectoderm (ECT) cells. (c1 Day-3 embryo after hatching and attachment. Both visceral endoderm (v-END)and parietal endoderm (p-END1 are present. The mural trophoblast (m-TB) is spreading out on the coverslip as the blastocyst flattens. (d,e1 Slightly more advanced day-3 embryo. (f) A day-3 embryo with a completely flattened blastocoel. (gf A day-4 embryo shortly after the mural trophoblast (m-TB) has completely moved off of the ICM derivatives. There are two small spherical cells (sschear the proximal group of cells (pTB) that may correspond to those hypothesized as being parietal endoderm in whole day4 embryos. Magnification: X 296.
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Fig. 4 Thick sections: day 4. (a) A day-4 embryo after the proximal group of cells and the distal group of cells (ECT and V-END1have enlarged. (b) A day-4 embryo after the visceral endoderm (FEND) has covered the proximal group of cells and the extraembryonic ectoderm (x-ECT) has appeared. Magnification: X 296.
stage, then, embryos consisted of a t least three embryonic cell types-endoderm, ectoderm, and mesoderm-and two extra-embryonic cell types-extraembryonic ectoderm and ectoplacental cone. Mitotic figures were frequently observed in all cell types, particularly in embryonic ectoderm and in the ectoplacental cone.
Electron microscopy of thin sections Electron microscopy was done to verify the identity and determine the fate of presumed parietal endoderm cells in early embryos and to confirm that cells which appeared to originate from the embryonic ectoderm were mesoderm cells. To verify the identity of parietal endoderm cells, we characterized presumed parietal endoderm cells at day 3 of culture; we then looked for similar cells in older embryos. We found that on day 3 these cells were cuboidal and had a high nucleocytoplasmic ratio (fig. 8). The most characteristic fine structural feature was the well-developed rough endoplasmic reticulum (RER), which was filled with an amorphous material whose electron density matched that of the prominent basal lamina separating these cells from the trophoblast cells (fig. 8). These fine structural characteristics are similar to those of parietal endoderm cells in vivo (Pierce et al., '62). When the blastocoel collapsed completely, mural trophoblast cells appeared to sandwich
the parietal endoderm cells (fig. 8). After the mural trophoblast moved off the ICM, the only remaining identifiable parietal endoderm cells were those occasionally observed at the base of the proximal segment in association with a n amorphous, lightly stained extracellular material that resembled Reichert's membrane (Pierce et al., '62). To determine whether cells that appeared to originate from the embryonic ectoderm resembled mesoderm, we made thin sections of embryos on day 7 of culture and studied the area of the presumed primitive streak (fig. 6 ) . The small, oblong, loosely arranged cells that appeared between the embryonic ectoderm and the visceral endoderm were oriented with their long axis perpendicular to that of the embryonic ectodermal cells. These small cells lacked cell junctions, had randomly distributed cytoplasmic organelles, and had large nuclei with smooth contours and two or three prominent nucleoli (fig. 9). There were a few profiles of rough endoplasmic reticulum, but most of the ribosomes were arranged into polysomes; there were well-developed Golgi and numerous small mitochondria. Although several of these fine structural features are common also to embryonic ectodermal cells, the small size and lack of cell junctions, in particular, distinguish mesodermal cells, and are characteristic of mesodermal cells of egg cylinders after six and one-half to seven days of gestation in vivo (Solter et al., '70).
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Fig. 5 Thick sections: day 5. Day-5 egg cylinder after the embryonic ectoderm (e-ECT) and extraembryonic ectoderm (x-ECT)have fused to enclose a common cavity. These are semi-serial sections that run lateral (a) to medial, (d). Magnification: X 296.
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Fig. 6 Thick sections: day 7. A day7 egg cylinder that illustrates mesoderm delamination. A portion of the embryonic ectoderm fe-EL”I’) is thicker (arrow), and loosely arranged cefls, probably corresponding to mesoderm (MES)cells on day 7 in vivo, appear to delaminate from it to occupy the apace between the embryonic ectoderm (e-ECT)and visceral endoderm b-END). A bend in the middle of this egg cylinder produces the apparent discontinuity between the x-ECT and ectoplacental cone (EPC); other sections showed that they are connected as in figure 5. The hole a t the tip of the distal segment is probably an artifact. Magnification: X 236.
Fig. 7 Thick sections: day 8. Egg cylinder a t the end o f the %day culture period. Reichert’s membrane (RM), proamnionic cavity (I), exocoelom 12), ectoplacental cavity (3). Magnification: X 236.
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Fig. 8 Thin sections: parietal endoderm in a day-3 embryo. A day-3 embryo (left side of embryo in fig. 30. in which the mural trophoblast cells (m-TB)together with the adjacent parietal endoderm (pEND) are migrating away from the ICM. The cytoplasm in the parietal endoderm cells contains many profiles of rough endoplasmic reticulum (*) filled with an amorphous material of the same electron density as the basal lamina (arrows) that separates the parietal endoderm from the mural trophoblast (m-TB). Magnification: X 4,105.
Fig. 9 Thin sections:mesoderm cells in a day-7 egg cylinder. A day-7 embryo (same embryo as in fig. 6) undergoing mesoderm (MES) delamination. The small, fusiform mesoderm cells have large nuclei, a few profiles of rough endoplasmic reticulum, small mitochondria, prominent Golgi (G), and numerous polysomes. In spite of the lengthy expanses of mutual contact, very few, if any, cell junctions are evident between mesoderm cells, in contrast to the well-developedjunctions between visceral endoderm cells and in the embryonic ectoderm. Magnification: X 4,105.
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From comparisons of the development of egg cylinders cultured from blastocysts with published observations of early mouse embryo development in vivo we conclude that the morphology of egg cylinders developing from blastocysts in vitro resembles that of mouse embryos in vivo during the fourth to seventh days of gestation. Cultured blastocysts, however, developed more slowly to equivalent egg cylinder stages (8 days in vitro vs. 4 days in vivo) and formed shorter egg cylinders (600 p m in vitro vs. 700 p m in vivo). Both of these features may have resulted from our inability to supply the embryos with their optimal culture conditions, since these conditions are still being elucidated (flsu, '72; Pedersen and Spindle, '76). The major differences between post-blastocyst development in vitro and in vivo involved the fate of four structures that are not a part of the egg cylinder itself in vivo; these are the mural trophoblast, the parietal endoderm, Reichert's membrane, and the ectoplacental cone. In vivo, the mural trophoblast forms the wall of the blastocoel, which becomes the yolk cavity and is lined by Reichert's membrane and parietal endoderm cells (Snell and Stevens, '66). In vitro, however, the mural trophoblast does not enclose a three-dimensional cavity but instead spreads as a two-dimensional sheet of giant cells on the coverslips (Cole and Paul, '65). Because parietal endoderm cells, which were initially present during attachment and outgrowth, were later observed only a t the base of the proximal segment of the egg cylinder, our observations are consistent with those of Hsu et al. ('74) but differ with the observation of Solter et al. ('74) that parietal endoderm cells were interspersed with the visceral endoderm cells of cultured mouse embryos. However, phase contrast microscopy of whole embryos a t days 4-8 of culture showed that small, round cells were scattered over the mural trophoblast sheet, particularly around the base of the proximal segment (figs. 2d,f). These could have been parietal endoderm cells like those reported by Sherman and Salomon ('75). Because such cells were rarely attached to the mural trophoblast in sectioned embryos, they may have adhered poorly and been lost during fixation. In the thick sections of cultured embryos we saw no extracellular material resembling
Reichert's membrane over the surface of the mural trophoblast sheet after the blastocoel had disappeared. Glutaraldehyde fixation preserves Reichert's membrane (Reinius, ,651, so we conclude that it probably was not present over the surface of the trophoblast sheet. The only material that resembled Reichert's membrane in cultured embryos was at the base of the proximal segment where parietal endoderm cells were seen. The ectoplacental cone originates from proliferating polar trophoblast cells in vivo (Gardner et al., '731, projects into the uterine epithelium, and is not covered by visceral endoderm (Snell and Stevens, '66). Consequently, in vivo, the egg cylinder consists of two segments enclosed by visceral endoderm instead of the three segments we describe for cultured egg cylinders. We suggest that in vitro the proliferating polar trophoblast cells, encountering the unyielding coverslip instead of uterine epithelium, protrude inward, become covered by visceral endoderm, and thus become the proximal segment. It was not possible to determine whether the visceral endoderm covering the proximal segment originated de nouo from the ectoplacental cone or migrated from the visceral endoderm covering the adjacent (distal) segment of late day4 embryos (fig. 4b). The time that primitive endoderm and ectoderm form in vitro (day 2 of culture) corresponds to the fifth day of gestation, when Reinius ('65) observed endoderm formation in vivo in mouse embryos of the CBA strain, and was about one day later than the time of endoderm formation in the hybrid embryos studied by Snell and Stevens ('66). Both in vivo and in vitro, parietal and visceral endoderm can be distinguished from each other during the 24 hours after endoderm differentiation. During the same period, the morphology of ectoderm cells changes very little. During morphological differentiation of primitive endoderm and ectoderm, the ICM is sensitive to drugs that interfere with gene expression, such as 5-bromodeoxyuridine (Sherman and Atienza, '75; Pedersen and Spindle, '76) and actinomycin D (Rowinski et al., '75; Glass et al., '76). Consequently, morphological differentiation of endoderm and ectoderm in mouse embryos may require concurrent gene expression. Mesoderm differentiation occurs in cultured embryos on day 7, when the embryos' total age is ten days (3 days in vivo plus 7 days in cul-
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plantation mouse and rabbit embryos, and cell strains ture), compared with six and one-half (Snell derived from them. In: Preimplantation Stages of Pregand Stevens, '66) or seven and one-half nancy. Ciba Foundation Symposium. G. E. W. Wol(Reinius, '65) days total age in vivo. This stenholme and M. OConnor, eds. J. & A. Churchill, Londifference in timing probably represents a don, pp. 82-122. cumulative delay of embryos developing Gardner, R. L. 1972 An investigation of inner cell mass and trophoblast tissues following their isolation from beyond the blastocyst stage in vitro. In culthe mouse blastocyst. J. Embryol. Exp. Morphol.,28: 279tured embryos mesoderm always appeared to 312. originate in the distal segment from the 1975 Analysis of determination and differentiation in the early mammalian embryo using intrathickened embryonic ectoderm near the juncand interspecific chimaeras. In: The Developmental Bioltion of the distal and middle segment, as ogy of Reproduction. C. L. Markert and J. Papaconstanobserved by Snell and Stevens ('661, and did tinou, eds. Academic Press, New York, pp. 207-236. not originate in the middle segment as re- Gardner, R. L., and M. H. Johnson 1973 Investigation of early mammalian development using interspecific ported by Razek ('72). chimaeras between rat and mouse. Nature New Biology, Extraembryonic ectoderm, which was first 246: 86-89. noticed late on day 4 of culture, appeared to 1975 Investigation of cellular interaction and originate in the proximal segment and was deployment in the early mammalian embryo using interspecific chimaeras between the rat and mouse. In: always attached to the polar trophoblast or to Cell Patterning. Vol. 29. Ciba Foundation Symposium. the ectoplacental cone cells, and i t was only Associated Scientific Publishers, New York, pp. 183-200. transiently attached to the (ICM-derived)em- Gardner, R. L., V. E. Papaioannou and S. C. Barton 1973 bryonic ectoderm on day 5. Extraembryonic Origin of the ectoplacental cone and secondary giant cells in mouse blastocysts reconstituted from isolated ectoderm, which consisted of a simple layer of trophoblast and inner cell mass. J. Embryol. Exp. Morcuboidal cells, contrasted sharply with embryphol., 30: 561-572. onic ectoderm, which consisted of a layer of Glass, R. H., A. I. Spindle and R. A. Pedersen 1976 pseudostratified columnar cells. These obserDifferential inhibition of trophoblast outgrowth and inner cell mass growth by actinomycin D in cultured vations are consistent with a polar trophomouse embryos. J. Reprod. Fert., 48: 443-445. blast origin for extraembryonic ectoderm, and Hsu, Y.-C. 1971 Post-blastocyst differentiation in vitro. ectoplacental cone cells, as found by Gardner Nature, 231: 100-102. and co-workers (Gardner et al., '73; Gardner, 1972 Differentiation in vitro of mouse embryos beyond the implantation stage. Nature, 239: 200-202. '75; Gardner and Johnson, "73, '75). 1973 Differentiation in vitro of mouse embryos Because the egg cylinder itself, which proto the stage of early somite. Devel. Biol., 33: 403-411. duces the embryo proper, develops so similarly Hsu, Y.-C., J. Baskar, L. C. Stevens and J. E. Rash 1974 Dein vitro and in vivo, we conclude that cultured velopment in vitro of mouse embryos from the two-cell egg stage to the early somite stage. J. Embryol. Exp. embryos provide an excellent experimental Morphol., 31: 235-245. system to study differentiation, development J. W. 1902 Observations on the histology and morphogenetic movements of the primary Jenkinson, and physiology of the placenta of the mouse. Tijdschr. germ layers and their inductive interactions ned. dierk. Vereen., 2: 124-198. during early mammalian development. Fur- Luft, J. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. thermore, this histological analysis provides a guide for isolating and studying the proper- Pederson, R. A., and A. I. Spindle 1976 Genetic effects on mammalian development during and after implantation. ties of particular cell populations from the In: Embryogenesis in Mammals. Ciba Foundation Symdifferent stages of egg cylinder development posium 40. Elsevier, Amsterdam and New York, pp. 133154. in vitro. ACKNOWLEDGMENTS
The authors would like to acknowledge the assistance of Doctor Akiko Spindle and of Ms. Kitty Wu in culturing the embryos and to thank Doctor Patricia G. Calarco for reading the manuscript and Ms. Mimi Zeiger for editorial assistance. This work was performed under the auspices of the United States Energy Research and Development Administration. LITERATURE CITED Cole, R. J.,and J. Paul 1965 Properties of cultured preim-
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