Cell, Vol. 18. 41 l-422.

October

1979,

Copyright

0 1979 by MIT

Gap Junctional Communication in the Post-implantation Mouse Embryo

Cecilia W. Lo* and Norton 6. Gilula The Rockefeller University New York, New York 10021

Summary We studied the extent of cell-to-cell communication via junctional channels in in vitro-implanted mouse blastocysts by monitoring ionic coupling and the spread of two injected low molecular weight dyes, fluorescein and Lucifer yellow. In the early attached embryos, both trophoblasts and cells of the inner cell mass (ICM) were ionically coupled to one another. Dye injections in either trophoblasts or ICM cells resulted in spread to the entire embryo. As older and more developed embryos were examined, the spread of injected dye was progressively more limited. In the most developed embryos examined, dye injected into a cell in the ICM region resulted in spread throughout the ICM but not into the surrounding trophoblast cells, while dye injected into a trophoblast cell did not spread to any other cell in the embryo. Simultaneous monitoring of ionic coupling and dye injections in embryos of intermediate stages in this transition revealed that the trophoblast and ICM cells were ionically coupled, even across the apparent boundary where no dye was observed to pass. In the latest stage embryos examined in which no injected dye was observed to move out of the ICM, ionic coupling was still observed between the cells of the ICM and the trophoblasts. Furthermore, in the more developed embryos, dye injected into the ICM region frequently was not transferred to all the cells of the ICM, thus suggesting a further compartmentalization of dye spread within the ICM. Our observations that ionic coupling is more extensive than the detectable spread of injected dyes may perhaps reflect a reduced number of junctional channels. With fewer channels less dye would pass between cells, so that, together with continuous quenching, the transfer of injected dye would not be detectable. This partial segregation of cell-to-cell communication as indicated by the limited dye spread may parallel specific differentiation processes, in particular that of giant trophoblast, embryonic ectoderm and extraembryonic endoderm differentiation. Introduction Cells of the early mouse embryo are not linked by junctional channels until the onset of compaction at the 8-cell stage (Lo and Gilula, 1979). At that time, * Present address: Department of Biological Chemistry, Medical School, Boston, Massachusetts 021 15.

Harvard

both ionic coupling and the ability to pass injected fluorescein are detected simultaneously. As the embryo develops into a blastocyst (on day 4) all the cells remain linked to one another by junctional channels. The blastocyst then begins implantation in utero on day 5. During implantation the embryo establishes intimate contacts with the uterine epithelium, culminating in the formation of the placenta. At some regions of cell-to-cell contact, specialized junctions (including gap junctions) link the maternal uterine cells with the trophoblast ceils of the embryo (Tachi, Tachi and Linder, 1970). In considering the possibility that gap junctional channels may mediate the transmission of morphogenetic gradients during embryogenesis (Wolpert, 1978) it would be interesting and important to know whether there is junctional communication between the maternal uterine cells and the embryo proper in organisms whose fetus depends upon the placenta for continued growth and development. The existence of gap junctions between trophoblast cells and uterine epithelial cells early during implantation indicates that, at least during that time, the maternal and embryonic cells of the placenta are probably coupled to each other. At present, however, it is not known whether the cells of the embryo proper are linked via junctional pathways to the cells of the forming placenta. When mouse embryos are maintained in vitro they undergo a process resembling implantation through which the embryos become firmly attached to the culture vessel. Furthermore, after attaching and spreading on the culture dish, the mouse embryo will continue its development in vitro very much as it would in vivo, but at a slower rate. If culture conditions are carefully monitored, embryos up to the 12-somite stage (with beating hearts) can develop in vitro from embryos of the 2-cell stage (Hsu, 1979). This system is ideal for exploring the extent of cell-to-cell communication in the developing embryo of a placental mammal. It is particularly useful for studying whether the portion of the embryo which gives rise to the fetus proper, the inner cell mass (KM), is linked via junctional channels to the trophoblasts after implantation. In this study we examined the extent of gap junctionmediated cell-to-cell communication in the in vitroimplanted mouse embryo at various stages of development by monitoring for the presence of ionic coupling and the spread of injected fluorescent dyes. The presence of specialized membrane junctions in these embryos was also analyzed in freeze-fracture replicas and correlated with the observations obtained from the electrophysiological studies. Results Development To correlate

of Mouse Embryos in Vitro the pattern of intercellular communication

Cl?11 412

with the development of the mouse embryo, we examined the morphological organization of the mouse embryo at various times after attachment in vitro. Blastocysts were harvested from pregnant mice on day 4 and immediately placed in culture. On day 6, they hatch spontaneously from the zona pellucida and shortly thereafter become attached to the petri dish. As the trophoblast cells migrate and spread onto the dish the blastocyst cavity collapses, and eventually the hollow sphere of cells is transformed into a sheet or cell monolayer in which cells of the inner cell mass remain at the center, surrounded by trophoblasts. This process of hatching, attachment and spreading occurs rather rapidly and is probably completed within 24 hr (by late day 6). The trophoblast cells are fibroblastic and characteristically vacuolated for the first 024-36 hr after attachment (Figure 1). In contrast, the cells of the ICM are small and epithelial-like, each cell having a large nucleus with a prominent nucleolus (Figure 1). The ICM initially consists of only 1-2 cell layers; within 24 hr after attachment, however, rapid cell proliferation accompanies the transformation of the ICM into a multi-layered structure. By late day 7 or day 8 (or 24-48 hr after attachment), the trophoblast cells are significantly increased in size while remaining as a monolayer; these cells are referred to as giant cells (Figure 2). The numerous vacuoles, which were previously so prominent, are no longer present. At this late stage further proliferation has taken place in the ICM, resulting in the formation of a large three-dimensional mass of cells (Figure 2). The inner cell mass of such late attached embryos is comprised of two readily distinguishable cell types, embryonic ectoderm and extraembryonic endoderm (Figures 2c and 2d). The extraembryonic endodermal cells are easily identified by the large number of granules within their cytoplasm, while the presumptive embryonic ectodermal cells are small and morphologically similar to the undifferentiated stem cells of the

ICM of the early embryo. The ICM frequently appears to be organized into two distinct cell groups, corresponding to a segregation of the extraembryonic endoderm from the embryonic ectoderm. Such attached embryos continue to grow and develop for a week or longer, resulting in the formation of massive structures resembling the egg cylinder of the in vivo mouse embryo. To take advantage of the simplicity of the earlier embryos, which have only three major cell types and a relatively small number of cells, only attached mouse embryos of days 6, 7 and 8 were studied. For convenience, these embryos have been grouped into four stages, corresponding to distinct progressions in the development of the mouse embryo as described above: stage 1 -embryos within 24-36 hr after attachment or day 6 embryos; stage 2 and 3-embryos 36-48 hr following attachment or late day 6 to day 7 embryos; and stage 4-embryos 36-72 hr after attachment or late day 7 or day 8 embryos. The stage 2 and 3 embryos are at an intermediate stage in the transformation of the primitive trophoblast cells to giant cells and the progression of the undifferentiated ICM toward a large three-dimensional mass composed mainly of embryonic ectoderm and extraembryonic endoderm. Due to the natural asynchrony of embryonic development, the time scale outlined here is only a rough approximation, and even among embryos within one litter there is frequent overlap between stages. The distinction between stage 2 and stage 3 is based upon the observed changes in the pattern of intercellular communication; at present this distinction can not be made cytologically. Electrophysiology Ionic coupling between cells of the early mouse embryos (stage 1) was examined with microelectrode impalements of cells in the ICM and in the surrounding trophoblast monolayer. Coupling was detected

b Figure

1. Early Attached

Mouse

Embryo

(Stage

1)

(a) Phase-light micrograph of an early attached mouse embryo in culture. The central group of small cells which contain large nuclei with prominent nuclei are the cells of the inner cell mass. The surrounding cells are trophoblasts and at this stage their cytoplasm is filled with refractile vacuoles. (b) Thick section from a perpendicular plane of a mouse embryo at a similar stage in culture. The large central mass is the inner cell mass (icm) and the flat cells in the periphery are the trophoblasts (1). Bar = 45.6 pm in (a) and 29 pm in (b).

Communication 413

in the Post-implantation

Mouse

Embryo

(Stage

4)

02 Figure

2. Late Attached

Mouse

Embryo

(a) Phase micrograph of a late attached mouse embryo. The three-dimensional mass in the center is the inner cell mass (icm). The large flat cells in the periphery are trophoblasts (t). (b) Thick section through the horizontal plane of such an embryo. The cells in the center with small nuclei are cells of the inner cell mass. The surrounding cells with large nuclei are the trophoblast (t) or giant cells. (c) and (d) Two thick sections cut through a perpendicular plane of such an embryo. The large cell mass is the ICM; it consists of two morphologically distinct cell types, the embryonic ectoderm (ec) and the extra embryonic endoderm (en). The cytoplasm of endoderm cells is filled with large vacuoles. The flat cells are the trophoblasts (t). Bar = 45.6 pm in (a) and 36.6 pm in (b-d).

throughout the entire embryo, so that the ICM and trophoblast cells were coupled among themselves and also to each other (Figure 3). Fluorescein injected into a cell in either the ICM region or the trophoblast region was passed extensively to all or most of the cells of the embryo (Figure 4). In some instances ionic coupling was monitored simultaneously with fluorescein dye injections, and in all cases coupling and fluorescein transfer were observed at the same time. These observations demonstrate that, at this point, all the cells of the embryo are directly linked to one another via low resistance or gap junctional channels. Examination of the more developed embryo (stage 2) for ionic coupling revealed that the entire embryo was ionically coupled, as before. Although fluorescein injected into a cell of the ICM was extensively passed to the surrounding ICM cells, however, much less dye was observed to spread into the trophoblast cells (Figure 5). This pattern of limited dye spread to trophoblasts was observed even when the injection was maintained for an extended period (up to 90 min) with the use of large current pulses (up to 15 x 1 O-’ A). With simultaneous impalements of a fluorescein-filled electrode into the ICM region and a KCI-filled electrode into a trophoblast cell, it was found that tropho-

blast cells located beyond the boundary of the dye spread were always ionically coupled to cells within the ICM which were heavily filled with dye (Figures 5g and 5h). These observations indicated that ionic coupling in the embryo at this stage was more extensive than the spread of injected fluorescein. The stage 3 mouse embryo was observed to undergo further changes in the extent of fluorescein dye transfer. At this time of development in vitro, injection of dye into the ICM region resulted in the spread of dye to all the surrounding ICM cells, but only a faint trace of dye was detected in the adjacent peripheral trophoblasts, even with prolonged injections using large current pulses as before (data not shown). Simultaneous monitoring of ionic coupling with fluorescein injection revealed the continued existence of ionic coupling across the boundary of dye spread, as observed with embryos of the previous stage (data not shown). By stage 4, fluorescein injected into the ICM region spread throughout the ICM as before but no dye was detected in the trophoblast cells, even with prolonged injections with large current pulses (Figures 6d-6f). In addition, fluorescein injected into any trophoblast cell did not pass into any other cell (Figures 6a-6c

Cell 414

Figure

3. Ionic Coupling

in the Stage

1 Mouse

Embryo

The extent of ionic coupling between cells in various regions of the early attached mouse embryo (stage 1) was monitored with microelectrode Impalements. (a-f) are phase micrographs which demonstrate the regions impaled, and (a’-f’) are the corresponding electrical recordings obtained from those impaled regions. As a current pulse (top trace) was injected into one call, a voltage deflection was detected in that cell (a-d’, bottom trace: e’-f’, middle trace) and also in the other impaled cell (al-d’. middle trace; e’-f’. bottom trace). Bar in (a) = 45.6 pm in (a-f). In (a’f’), the horizontal calibration bar = 100 msec and the vertical calibration bar = 1 nA and 10 mV. Figure 4. Fluorescein Dye Spread in the Stage 1 Mouse Embryo

Communication 415

Figure

in the Post-implantation

4. Fluorescein

Dye Spread

Mouse

in the Stage

Embryo

1 Mouse

Embryo

(a) A trophoblast cell of an early attached mouse embryo was impaled with a microelectrode. cell and the fluorescence images were recorded at various times after the start of injection: min. Bar = 45.6 pm in (a-f).

and 6g-60. Because the giant trophoblasts are extremely thin, it was technically impossible to simultaneously monitor ionic coupling and dye injections. Ionic coupling measurements alone were also very difficult, but ionic coupling was observed in two cases of successful simultaneous impalements of an ICM cell and a giant trophoblast cell (Figure 7). Examination of cells in the ICM at this stage often revealed a further compartmentalization of dye transfer within the ICM itself. Injection of dye into the ICM resulted in a limited spread to some of the surrounding cells with a sharp boundary delineating the region of the ICM beyond which the dye did not enter (Figure 8). Even injections with large current pulses for long time periods (l-l .5 hr) did not alter this pattern of

(b-f) Fluorescein was injected into the trophoblast (b) 4 min, (c) 6 min. (d) 16 min. (e) 20 min and (f) 31

limited dye spread. Sometimes the region of the ICM ascribed by the boundary of dye spread coincided with the grouping of the cells into distinct aggregates or clumps which were visible in the light microscope. Thick sections revealed that the extraembryonic endodermal cells of the late attached embryos were often associated with one another in large aggregates. Sometimes they completely surrounded an inner aggregate of embryonic ectodermal cells, while at other times they were only partially associated with the embryonic ectodermal cells, the rest of the aggregate being spread on the trophoblast monolayer (Figures 2c and 2d). It is possible that this further compartmentalization of the dye spread in the ICM may correspond to the additional segregation and differentia-

Cell 416

Figure

5. Ionic Coupling

and Fluorescein

Dye Spread

in the Stage 2 Mouse

Embryo

(a) A stage 2 embryo was impaled with two microelectrodes, one in a trophoblast cell and the other in a cell bordering on the ICM region. (b-f) Fluorescein was injected into a cell in the ICM region and the fluorescence images were recorded at various times after the start of injection: (b) 4 min. (c) Q min. (d) 12 min. (e) 19 min and (f) 31 min. (Q-h) Lower magnifications of (a) and (f). Note that the impaled trophoblast cell is beyond the region filled by the dye. Bar = 45.6 pm in (a-f) and 72.5 pm in (g-h). (Inset) As a current pulse (top trace) was passed during the fluorescein infection from the microelectrode on the right, a voltage deflection was detected in that cell (middle trace) and also in the trophoblast cell (bottom trace) on the left. The horizontal calibration bar = 100 msec and the vertical calibration bar = 5 nA and 10 mV.

Communication 417

Figure

in the Post-implantation

6. Fluorescein

Dye Transfer

Mouse

Embryo

in the Stage 4 Mouse

Embryo

(a. d. e) A late-attached embryo was successively impaled with a microelectrode in three different regions: (a) a giant trophoblast, (d) inner cell mass and (e) another giant trophoblast. (b-c, e-f, h-i) Fluorescein was injected into the impaled cells and the fluorescence images were recorded at various times after the start of injection: (b) 4 min. (c) 22 min. (e) 4 min. (f) 26 min. (h) 4 min and (i) 19 min. Bar = 67.4 pm in (a-i).

Figure

7. Ionic Coupling

in the Stage 4 Mouse

Embryo

(a) Two cells, a trophoblast and a cell in the ICM, were impaled with microelectrodes. Bar = 45.6 Km. (b) As a current pulse (top trace) injected into the cell in the ICM, a voltage deflection was detected in that cell (middle trace) and also in the trophoblast cell (bottom trace). horizontal calibration bar = 100 msec and the vertical calibration bar = 1 nA and 10 mV.

tion of two cell types in the ICM (the embryonic derm and the extraembryonic endoderm).

ecto-

Lucifer Yellow In light of the recent report that the fluorescent dye Lucifer yellbw may give a different result from fluorescein in the extent of dye spread observed in dye injection experiments (Stewart, 1978; Bennett, Spira

was The

and Spray, 1978) the stage 4 embryos were injected with Lucifer yellow. The extent of dye spread upon injection of Lucifer yellow into the ICM or the trophoblast giant cell was indistinguishable from injections of fluorescein into similar embryos (Figures 9 and 10). Examination of earlier stages revealed more extensive dye spread patterns which were identical to those obtained with fluorescein injections (data not shown).

Cell 418

Figure

8. Segregation

of Fluorescein

Dye Transfer

in the ICM of tk be Stage 4 Mouse

Embryo

(a) A late attached mouse embryo was impaled at the ICM region vvith a microelectrode. (b-d) Fluorescein was injected into the impaled I cell and fluorescence images were recorded at various times after the start of dye injection: (b) 4 min, (c) 13 min and (d) 31 min. Bar = 45.6 pm in (z 1-d).

Freeze-Fracture Freeze-fracture replicas of blastocyst embryos that had just begun attaching to the substratum and whose blastocoelic cavities still remained expanded (before stage 1) revealed a complex network of tight junctional ridges and grooves on the outer trophoblast cells (Figure 1 la). Gap junctional particles and pits were intercalated at some regions within this complex array of tight junctions (Figure 11 a, inset). These observations are consistent with both the presence of a permeability seal in the blastocyst and the ability of the cells of the blastocyst to communicate (Lo and Gilula, 1979). Freeze-fracture replicas of embryos (stage 1) that have completely spread out on the substratum revealed that the tight junctional ridges and grooves were no longer present in such extensive networks (Figure 11 b). In many regions, few if any tight junctional elements were present. When tight junctions were still present, they were found as one or two strands with few interconnecting elements. The surface of the trophoblast cells was also characterized by the presence of many pinocytosis-associated membrane fracture face interruptions (Figure 11 b). At the light microscope level, the cytoplasm of the trophoblasts of this stage contained large refractile vacuoles (Figure 1) and these vacuoles were also detected in freeze-fracture replicas. Gap junctions were rarely

found between the trophoblast cells at this stage, but they were found in large numbers in the ICM region. The ICM regions were easily distinguishable from the trophoblast regions by the difference in their overall three-dimensional morphology, which was also reflected in the shadowing angle [The ICM regions are slightly elevated from the level of the extremely flat trophoblast cells, and they consist of a group of cells arranged in a hemisphere (Figure 1 b)]. Discussion The studies reported here demonstrate that the cells of the in vitro implanted mouse embryo remain ionitally coupled throughout the stages examined, but that the spread of injected dye is confined within sharp boundaries in the later stage embryos. Simultaneous monitoring of dye injections and coupling demonstrated that ionic coupling clearly occurs across the boundary beyond which no dye was transferred. One possible explanation for the occurrence of coupling without dye transfer is that the pore size of the junctional channels is reduced so that although ions can pass, fluorescein or Lucifer yellow cannot. At present there is no evidence for the existence of different types of mammalian junctional channels with varying pore sizes. In addition, the ultrastructure of the gap junctions of these embryos, as revealed in

Communication 419

Figure

in the Post-implantation

9. Lucifer

Yellow Dye Transfer

Mouse

Embryo

in the ICM of a Stage

4 Mouse

Embryo

(a) A cell in the ICM was impaled with a microelectrode. Lucifer yellow was injected into the impaled cell and fluorescence images were recorded at various times after the start of injection: (b) 4 min. (c) 19 min and (d) 49 min. (e-f) Lower magnifications of (a) and (d); (f) is 54 min. Bar = 45.6 pm in (a-d) and 72.5 pm in (e-f).

freeze-fracture replicas, is identical to that of the typical gap junctions observed in many other cell types of both embryonic and adult origin (Sheridan, 1976). The gap junctions themselves therefore do not appear to be ultrastructurally altered during development. Furthermore, the observed ionic coupling without dye transfer probably cannot be explained by the mediation of ion movement by a nonjunctional/extracellular pathway created by the presence of tight junctions. Freeze-fracture replicas have revealed that most of the tight junctions are no longer present in the early attached mouse embryos. The detection of ionic coupling without dye transfer

may perhaps reflect differences in flux (rate of transfer per cm* junctional membrane) observed as a consequence of the inherent different sensitivities of the assay methods used-that is, ionic coupling is probably more sensitive then the detection of dye transfer. Such differences in flux may simply result from a difference in the number of junctional channels linking cells. Thus the apparent lack of dye transfer may not necessarily indicate the inability of dye to pass, but only that the amount passed is below the limit of detection. Thus, in the late embryos, fluorescein injetted into the ICM may be transferred to the trophoblasts but at such a slow rate of flux that the rate of

Cell 420

Figure

10.

Absence

of Lucifer

Yellow Dye Transfer

(a) A trophoblast cell of a stage 4 embryo other cell even after 48 min of continuous

in a Trophoblast

Cell of a Stage 4 Mouse

was impaled with a microelectrode. (b) Lucifer injection. Bar = 45.6 pm in (a) and (b).

dye quenching exceeds the rate at which the dye accumulates; dye transfer would not be visible in such cases. The lack of dye transfer between the giant trophoblasts of stage 4 embryos and the further segregation or compartmentalization of dye spread observed within the ICM may reflect similar changes in flux. Cell-to-cell communication perhaps should not be considered as a simple all-or-none phenomenon, and quantitative differences or the variation in flux of communication across defined boundaries may in fact permit the segregation of semi-independent communication compartments. In such compartments, separate morphogenetic gradients may be generated independently of other surrounding compartments, and perhaps without this partial isolation appropriate thresholds of components of presumptive morphogenetic gradients may not be able to form. The low level of junctional communication which continues to interlink different communication compartments (as detected by the presence of ionic coupling) may allow the coordination of differentiation between several different compartments without affecting the formation of separate gradients within each compartment. The breakoff in dye spread between the ICM and the trophoblasts during implantation in vitro is consistent with such a hypothesis. That is, if morphogenetic gradients of the ICM are forming via junctional channels, then it would be critical to isolate this communication compartment from the mother when the embryonic trophoblasts are making intimate contacts, including the formation of gap junctions with the maFigure

11.

Freeze-Fracture

Replicas

of Early Attached

Mouse

yellow

Embryo injected

into the impaled

cell did not move into any

ternal uterine epithelium. In addition, the further segregation of dye spread within the ICM may reflect the formation of semi-independent communication compartments necessary for the generation of morphogenetic gradients involved in the differentiation of the embryonic ectoderm. In a previous study of cell-to-cell communication in the insect cuticle, ionic coupling was detected between the epidermal cells of two adjacent body segments (Warner and Lawrence, 1973; Caveny, 1974). Since each body segment is thought to contain an independent morphogenetic gradient, the detection of ionic coupling across the segmental boundary seemed incompatible with a hypothesis of gap junction-mediated formation of gradients. Based on the observations and proposed hypothesis of this study, however, ionic coupling between independent developmental compartments is not unexpected, and it remains to be seen whether injected dyes such as fluorescein will move across the segmental boundary. The hypothesis proposed in this paper would also explain the observation of polyclones (Garcia-Bellido, 1975; Crick and Lawrence, 1975) in insect development, in which the cell lineage of any adult structures can only be traced back to a group of cells (never to just one cell) in the larvae. This can be explained by the supposition that only when a critical number of cells are present and have formed a communication compartment can the necessary morphogenetic gradients be generated to elicit the appropriate differentiation and development. This hypothesis for gap junction-mediated regula-

Embryos

(a) Freeze-fracture replica of an embryo shortly after attachment (before stage 1) and before the collapse of the blastocoelic cavity. The cells are linked by complex networks of tight junctional ridges (black and white arrow) on the P face and grooves on the E face (black arrow). Magnification 68.000X. (Inset) Gap junctional particles (black and white arrow) and pits (black arrow) are frequently intercalated within the tight junctional e!ements. Magnification 84.000X. (b) Freeze-fracture replica of a stage 1 embryo. The tight junctional grooves (black and white arrow) exist primarily as single strands. Circular indentation (black arrow) on the cell surface may represent sites of ongoing formation of pinocytic vesicles. Magnification 30,000X. (Inset) A gap junction in the ICM region of a stage 1 embryo. The junctional plaque is comprised of particles (black arrow) and complementary pits (black and white arrow). Magnification 88.000X.

Communication 421

in the Post-implantation

Mouse

Embryo

Cell 422

tion of embryonic differentiation is not intended to explain all regulatory events in embryogenesis, and certainly other mechanisms are operative either simultaneously or at different times in the same organism. It is hoped that a thorough examination of cellto-cell communication properties at the developmental compartment boundaries in other systems will provide further insight into the possible role of gap junctions in development.

discussions concerning electrophysiology, Dr. Sidney Strickland and Mary Jean Sawey for help and advice in the mating of mice and the handling of mouse embryos, Asneth Kloesman and Kathy Wall for help in the preparation of figures and Madeleine Naylor for secretarial assistance. This research was supported by grants from the USPHS and The Rockefeller Foundation. N. B. G. is a recipient of a Research Career Development Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Experimental

Received

Procedures

The mice and mating protocols used were the same as those reported in the accompanying paper (Lo and Gilula. 1979). Pregnant mice were sacrificed on day 4 of pregnancy by cervical dislocation, and their uteri were removed. The embryos were flushed from each uterine horn and maintained in Dulbecco’s modified Eagle’s medium (Grand Island Biological) with 10% fetal bovine serum (Flow Laboratories) and 50 U-50 pg/ml of penicillin-streptomycin (Grand Island Biological) at 37°C in a 5% CO, incubator in 35 mm petri dishes (BioQuest. BBL and Falcon Products). Shortly before the start of electrophysiological recordings, the medium was aspirated and replaced with Leibovitz’s medium (Grand Island Biological) with 10% fetal bovine serum. The phosphate buffering in Leibovitz’s medium allowed the maintenance of a pH of 7.3 during the electrophysiological recordings. Electrophysiology The equipment and methods used have been reported elsewhere (Gilula. Epstein and Beers, 1978; Lo and Gilula. 1979). and will only be briefly described here. Microelectrodes for ionic coupling measurements were filled with 3 M KCI; the tip resistances were 40-80 MD. Microelectrodes for dye injections were filled with either 5% sodium fluorescein (w/v) or 3% Lucifer yellow CH (w/v) (dilithium salt); these had tip resistances of 15-20 MO as measured when filled with 3 M KCI. (Lucifer yellow CH was provided by W. Stewart of the NIH). Photographs of fluorescein injections were recorded as before, but for Lucifer yellow the exposure time was increased to 4.5 min (Lucifer yellow has a lower quantum yield than fluorescein; W. Stewart, personal communication). Thick-Section Light Microscopy The embryos were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) for 20 min at room temperature, post-fixed in 1% osmium tetroxide in veronal acetate buffer for 1 hr and then stained en bloc with uranyl acetate in veronal acetate buffer for 1 hr. The samples were dehydrated in a graded series of ethanol and embedded in Epon 812. Thick sections (l-2 pm) were cut with glass knives, mounted on glass slides and stained with toluidine,blue. Photographs were taken on Kodak Plus-X film with a Zeiss photomicroscope II. Freeze-Fracture For obtaining freeze-fracture replicas, embryos were allowed to attach on collagen-coated Balzers gold carriers for the double replica device (Balzers High Vacuum Corp.; Santa Ana. California) (Kalderon, Epstein and Gilula, 1977) instead of on petri dishes. They were fixed with 2.5% glutaraldehyde (Polysciences; Warrington. Pennsylvania) in Dulbecco’s phosphate-buffered saline (pH 7.3; Grand Island Biological) and then treated with 25% glycerol in 0.1 M cacodylate buffer (pH 7.3) at room temperature for 1 hr prior to freezing. The glycerinated samples were sandwiched with an identical blank carrier and rapidly frozen in liquid nitrogen-cooled Freon 22. The frozen sandwiched carriers were mounted in the Balzers (Freeze Etch Unit BA 360M). then forced open at - 115°C and immediately shadowed with platinum and carbon. The replicas were cleaned with sodium hypochlorite and examined in a Siemens 101 electron microscope. Acknowledgments We would like to thank Theodore

Lawrence

for assistance

and helpful

May 2. 1979

References Bennett, M. V. L.. Spira, M. E. and Spray, D. C. (1978). Permeability of gap junctions between embryonic cells of Fundulus: a reevaluation. Dev. Biol. 65, 114-I 25. Caveney. movement 31 l-322.

S. (1974). Intercellular of small ions between

communication insect epidermal

Crick, F. H. C. and Lawrence, polyclones in insect development.

in a positional field: cells. Dev. Biol. 40,

P. A. (1975). Compartments Science 789, 340-347.

and

Garcia-Bellido, A. (1975). Genetic control of wing disc development in Drosophila. In Cell Patterning, Ciba Foundation Symposium, 29 (Amsterdam:Elsevier), pp. 161-I 82. Gilula, N. B., Epstein, M. L. and Beers, W. H. (1978). Cell-to

Gap junctional communication in the post-implantation mouse embryo.

Cell, Vol. 18. 41 l-422. October 1979, Copyright 0 1979 by MIT Gap Junctional Communication in the Post-implantation Mouse Embryo Cecilia W. Lo*...
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