Flow of cells from polar to mural trophectoderm is polarized in the mouse blastocyst

R.L. Gardner

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During growth of the blastocyst there is a net flow of cells from the polar to the mural trophectoderm which is presumed to be radially symmetrical. However, such a pattern of cell movement is inconsistent with findings from a recent clonal analysis. To visualize the overall flow of cells directly, the polar trophectoderm of expanding blastocysts was labelled globally with fluorescent microspheres. Following further growth, the great majority of blastocysts that remained labelled throughout the polar trophectoderm exhibited a polarized rather than radial spread of label into the mural region. This was the case regardless of the labelling technique, whether the blastocysts were grown in utero or in vitro, or had the zona pellucida removed or left on. Intriguingly, where there were two foci of spread of label into the mural trophectoderm rather than one, these were diametrically opposite each other. In further experiments, fluorescent lineage labels were used to distinguish junctional trophectoderm cells with and without an extension onto the blastocoelic surface of the inner cell mass. The location of clones formed following further blastocyst growth provided no evidence that egress of cells from the polar trophectoderm is restricted circumferentially by the presence of junctional cells having an extension.

Key words: mouse blastocyst/mural trophectoderm/polarized growth/polar trophectoderm/trophectoderm


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell proliferation occurs throughout the trophectoderm in the early mouse blastocyst. Later, it declines progressively in the mural region of the tissue surrounding the blastocoele (Copp, 1978Go), so that by the onset of implantation mitoses are restricted to the polar region overlying the inner cell mass (ICM). Following cessation of mitotic activity in the mural trophectoderm, its cells begin to form primary trophoblast giant cells, a process that is signalled by onset of endoreduplication of the genome (Barlow et al., 1972Go). Although the polar trophectoderm retains its mitotic activity through interaction with the ICM (reviewed in Gardner, 1985; Gardner and Beddington, 1988), its cell number increases very little as the blastocyst grows (Copp, 1978Go). This is because there is a net flow of cells from polar to mural trophectoderm once blastocyst expansion begins (Copp, 1979Go; Cruz and Pedersen, 1985Go; Dyce et al., 1987Go; Gardner, 1996Go). The outflow of surplus polar trophectoderm cells is assumed to be radially symmetrical so that the further from the ICM trophectoderm cells lie, the longer they are presumed to have resided in the mural region. Such a simple relationship between the age and position of cells was invoked to explain why morphological giant transformation of mural trophectoderm cells (Dickson, 1963Go; McRae and Church, 1990Go) invariably spreads radially from its site of initiation at the abembryonic pole of the late blastocyst (Gardner and Papaioannou, 1975Go). However, DNA measurements cast doubt on whether morphological transformation of mural cells relates to their age and hence to the switch to an endoreduplicative mode of growth (Gardner and Davies, 1993Go).

There is no direct evidence that the flow of cells from polar to mural trophectoderm is radial and, indeed, two recent findings raise the possibility that it is not. One is that the mouse conceptus is already bilaterally rather than radially symmetrical at the early blastocyst stage (Gardner, 1997Go), as was reported much earlier for the rat (Huber, 1915Go). However, particularly since bilateral symmetry becomes temporarily obscured during blastocyst expansion (Gardner, 1997Go), polar to mural flow of cells might nonetheless conform to a radial pattern. The other finding, which is more difficult to reconcile with a radially symmetrical flow of cells, emerged from a recent clonal analysis of growth of the polar trophectoderm (Gardner, 1996Go). Here, ionophoretic injection of horseradish peroxidase was used to label either the central polar trophectoderm cell or a single peripheral one in 3.5 days post-coitum (d.p.c.) blastocysts which were then cultured for ~ 1 or 2 days thereafter. Clones formed by labelled central polar cells were typically displaced towards the abembryonic pole of the blastocyst, in accordance with the results of earlier studies (Cruz and Pedersen, 1985Go; Dyce et al., 1987Go). In contrast, only about one-quarter of the clones formed by labelled peripheral polar trophectoderm cells behaved thus, half retaining their ancestral location, and the remainder actually shifting towards, rather than away from, the centre of the polar trophectoderm during the first day of culture (Gardner, 1996Go). Such variable deployment of peripheral clones suggests that the spread of cells from polar to mural trophectoderm is polarized rather than radially symmetrical.

In the present study the entire polar trophectoderm was labelled selectively with fluorescent latex microspheres (Fleming and George, 1987Go) so as to enable the overall pattern of flow of its cells into the mural region to be visualized. Regardless of whether they were cultured with or without the zona pellucida, or returned to the uterus for further development, labelled blastocysts almost invariably showed polarized rather than radially symmetrical spread of label into the mural trophectoderm thereafter. In additional blastocysts, individual junctional trophectoderm cells were labelled with a fluorescent tracer in order to determine whether they extended a process onto the blastocoelic surface of the ICM. Cells with such an extension showed no greater tendency to retain a junctional location than those without. Hence, the hypothesis that cells with an extension might thereby be held at the polar–mural junction and thus circumferentially restrict egress of cells from the polar trophectoderm (Gardner, 1996Go) appears untenable.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Production, recovery and culture of blastocysts
All mice were of the PO (Pathology, Oxford) closed-bred strain which were kept on either of two lighting regimes. For most, the daily dark period was from 13:00 to 23:00 h (altered lighting); for the remainder it was from 19:00 to 07:00 h (standard lighting). Females were selected for oestrus by external inspection (Champlin et al., 1973Go), paired with fertile or vasectomized males before onset of the dark period, and checked for the presence of a vaginal plug the following morning. The day of the plug was recorded as the first day of pregnancy or pseudopregnancy.

All blastocyst donor females on altered lighting were killed and their uterine horns flushed between 10:00 and 12:00 h on the fourth d.p.c., while those on standard lighting were killed and flushed between 14:00 and 15:00 h. MTF-HEPES (Gardner and Sakkas, 1993Go) was used as the medium for recovery, storage and manipulation of blastocysts, and for their transplantation to recipient uteri. Concepti that had not reached the early or expanding blastocyst stage (for staging, see Gardner, 1997) at the time of recovery were cultured until they did so. For growth in vitro, blastocysts were placed in pre-equilibrated drops of alpha medium (Stanners et al., 1971Go) plus 10% inactivated fetal calf serum ({alpha}+IFCS) overlaid with light paraffin oil (BDH, Poole, UK) in 30 mm disposable tissue culture dishes, and incubated at 37°C in 5% CO2 in air.

Blastocyst manipulation
All manipulations were done using a Leitz micromanipulator assembly with the blastocysts immobilized by gentle suction on the tip of a holding pipette (Gardner, 1978Go; Gardner and Davies, 1998Go). Global labelling of the polar trophectoderm was achieved by exposing it to fluorescent latex microspheres (Fluoresbrite YG, carboxylate; Polysciences, Warrington, PA, USA) of a size which its cells could readily endocytose. Several batches of microspheres whose mean diameter ranged from ~0.20 to 1.66 µm were used in different experiments. Three to four drops of a stock suspension in water were added to 2–5 ml of phosphate-buffered saline (PBS), MTF-HEPES, or {alpha}+IFCS which, depending on the size of the microspheres, was then passed through a sterile 0.22 or 0.45 µm filter. For batches of microspheres that were 0.5 µm or more in diameter, those that were retained were then resuspended by back-flushing up to 1.0 ml of medium through the filter from its distal end. For batches of ~ 0.20 µm, microspheres that passed through a 0.45 µm filter but were subsequently retained in a 0.22 µm filter were used. Immediately before being dispensed into culture or manipulation chamber drops, the final suspensions were agitated on a whirlimixer to disperse aggregates and then microfuged at ~2250 g for up to 30 s to sediment residual clumps.

Two different ways of exposing the polar trophectoderm selectively to the microspheres were adopted. One was to slit the zona pellucida over the polar region in order to obtain externalization of the ICM and overlying polar trophectoderm during subsequent culture so that only the latter would be exposed to the microspheres. The other was to inject a suspension of microspheres under the zona at the centre of the polar region.

Slitting of the zona was carried out as described previously (Tsunoda et al., 1986Go), taking care to ensure that the slit was both wide and well-centred on the polar trophectoderm. In later experiments, a pair of slits set approximately at right-angles to each other was made in the zona, rather than a single one. In most experiments, the polar trophectoderm was exposed briefly to a suspension of microspheres in Dulbecco A PBS once it had completed herniation in culture. Providing the blastocysts were first rinsed in PBS on removal from culture, the microspheres adhered rapidly to the exposed trophectoderm. In some later experiments, blastocysts were exposed to the microspheres during herniation by including them in culture medium. Following exposure of the polar trophectoderm to the microspheres, blastocysts were rinsed extensively before being examined briefly by fluorescence microscopy using standard fluorescein filters to check the quality of labelling (Figure 1Go). They were then returned to culture directly or exposed briefly to acidified Tyrode's (AT) saline to remove the zona pellucida before further culture or transfer to the uteri of day 3 pseudopregnant females (Gardner and Davies, 1998Go). No adverse effects on development have been discerned after single, or even repeated, exposure of preimplantation mouse concepti to 450–490 nm light (Fleming and George, 1987Go; Zernicka-Goetz et al., 1997Go).



View larger version (68K):
[in this window]
[in a new window]
 
Figure 1. Brightfield (A) and fluorescent (B) images of four blastocysts photographed immediately after global labelling of the herniated polar trophectoderm with fluorescent microspheres. Scale bar = 100 µm.

 
Microspheres were injected subzonally with micropipettes pulled from microelectrode capillaries (Clark Electromedical, Pangbourne, UK) and connected to a De Fonbrune suction-and-force pump via a continuous column of mineral oil. The micropipettes had a smooth, straight, heat-polished tip of 3–4 µm outer diameter. Because it was difficult to push such pipettes through the zona without damaging the underlying trophectoderm, in initial experiments a small slit was first made in the zona with a fine needle. However, a slit that was small enough to avoid herniation of the polar trophectoderm was very hard to relocate for inserting the pipette. Later it was found that the microsphere injection pipette could be inserted without damaging cells if its tip was repeatedly moved from side to side slightly once it had firmly indented the zona so as to erode the latter's more resistant inner layer. Limiting spread of the microspheres required careful ejection of the suspension so that transient opening up of the perivitelline space was restricted to the polar region. After rinsing, blastocysts were examined briefly by fluorescence microscopy to check the distribution of the microspheres before they were transferred to culture or to the uterus with the zona left on.

To determine whether junctional trophectoderm cells had an extension onto the blastocoelic surface of the ICM they were labelled either ionophoretically with 10 kDa tetramethylrhodamine dextran-lysine-biotin (TMRDLB; Molecular Probes Inc., Eugene, OR, USA) in 0.1 mol/l KCl (Gardner and Cockroft, 1998Go), or by pressure injection with DiI (also from Molecular Probes) in mineral oil (Gardner, 1997Go). Some blastocysts were examined in detail by fluorescence microscopy following injection: the remainder were examined just long enough to ascertain whether or not the labelled cell had an unequivocal extension before being cultured separately in vitro overnight for recording the distribution of the resulting clones during the following morning.

Scoring of labelled blastocysts
On recovery from culture or the uterus, blastocysts were returned to hanging drops in Puliv manipulation chambers for detailed examination by fluorescence microscopy and brightfield microscopy. A solid, fine-tipped siliconized glass needle was used to orient them appropriately, and a holding pipette to immobilize them for photography. Blastocysts that had been labelled with the strongly fluorescent microspheres were usually fixed first because they deteriorated very rapidly when examined fresh. For fixation, they were immersed for at least 40 min in 1% glutaraldehyde in PBS that included 10 kDa polyvinylpyrrolidone (PVP) at 4 mg/ml. Thereafter, the blastocysts were rinsed and examined either in deionized water containing PVP at 4 mg/ml or in MTF-HEPES diluted ~ 4-fold in deionized water so they remained expanded. The presence of the PVP prevented adhesion of the blastocysts to surfaces and their consequent damage.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Labelling of the polar trophectoderm
Blastocysts for labelling were selected at an expanding stage before the zona pellucida began to thin obviously. Neither exposure of the herniated trophectoderm to the microspheres nor their subzonal injection proved ideal labelling methods. Growth of the polar trophectoderm could clearly be perturbed during herniation, even when a cross-wise pair of wide slits rather than a single slit was made in the zona. This was evident from the partial or complete separation of the polar trophectoderm from the ICM that occurred in a minority of herniated blastocysts. Occasionally, such separation led to relocation of ICM so that the entire patch of labelled polar trophectoderm was consigned to the mural region shortly after labelling. Hence, blastocysts with obvious cavitation between the polar trophectoderm and ICM following herniation were discarded. A further problem with herniation was the susceptibility of the exposed polar trophectoderm cells to lysis during both labelling with microspheres and their subsequent exposure to AT saline.

With subzonal injection of the microspheres it was harder to control the distribution of labelling, so that a higher proportion of blastocysts had to be rejected. Commonly, the microspheres either became disseminated well beyond the limit of the polar trophectoderm or were confined to only part of it. This was because the perivitelline space often failed to open up uniformly as the suspension was ejected from the pipette, presumably because of local differences in the rigidity of trophectoderm cells or in the strength of their attachment to the zona.

The results of microsphere labelling of the polar trophectoderm by both methods are summarized in Table IGo. Not all recovered blastocysts were classified as scorable, for reasons that are given in the footnote to the table. Importantly, to guard against artefactual relocation of the ICM, all blastocysts which did not retain labelling throughout the polar trophectoderm were rejected. The highest proportion of unscorables, mostly due to damage to the polar trophectoderm, was in the herniation series where the zona was removed with AT saline prior to culture. Regardless of the method of labelling, or whether the zona was left on or removed thereafter, the great majority of scorable blastocysts in all series exhibited a single, circumferentially restricted, coherent patch of fluorescent cells in the mural trophectoderm (Table IGo; Figure 2Go). Outside this patch, the mural trophectoderm was either completely unlabelled or had the label confined to cells at its immediate junction with the polar trophectoderm. When blastocysts were viewed from the embryonic pole, the proportion of their circumference that the patch occupied was commonly around 50%, but ranged from ~ 25% to 70%. The distal limit of spread of the label also varied considerably, from proximal to the equator of the blastocyst to close to the abembryonic pole. Patches which extended further distally generally appeared narrower than those that penetrated the mural trophectoderm more modestly. More significantly, in the overwhelming majority of cases, the distal extremity of the patch lay diametrically opposite the centre of the region where the mural trophectoderm remained essentially unlabelled. Typically, spread of the label from the polar to the mural trophectoderm took the form depicted in Figure 3Go. Most of the scorable blastocysts that did not exhibit this pattern showed a limited more or less radially symmetrical spread of label. That cases where spread was not obviously polarized were encountered, most commonly following subzonal injection of the beads, may be attributable to the greater difficulty in obtaining uniform labelling of the polar trophectoderm by this method. The remaining scorable blastocysts were particularly interesting in exhibiting two circumferentially limited patches of labelling in the mural trophectoderm that were diametrically opposite each other (Figure 4Go). In one of these blastocysts the two patches extended about equally into the mural trophectoderm, while in all the others they did so unequally.


View this table:
[in this window]
[in a new window]
 
Table I. Distribution of fluorescence in blastocysts developing in vitro or in vivo after global microsphere labelling of the polar trophectoderm
 


View larger version (81K):
[in this window]
[in a new window]
 
Figure 2. (AH) Photographs of blastocysts cultured overnight following selective labelling of the polar trophectoderm with fluorescent microspheres. (A) Brightfield and (B) fluorescence image of the same blastocyst in side view. (C, D) Combined brightfield/fluorescence images of another blastocyst viewed from the embryonic pole with polar (C) versus equatorial focus (D). (EH) Brightfield (E, G) versus fluorescence (F, H) images of two further blastocysts viewed from the embryonic pole with equatorial focus. Scale bars = 50 µm.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 3. Diagrammatic embryonic polar (A), lateral (B) and frontal (C) views of a blastocyst with respect to microsphere labelling to show typical overall pattern of spread of the labelled polar trophectoderm cells. The dots represent bead labelling in the trophectoderm, and the shading delimits the inner cell mass.

 


View larger version (172K):
[in this window]
[in a new window]
 
Figure 4. (A, B) Brightfield and fluorescence embryonic polar views of same blastocyst with equatorial focus. In (B), note that a narrow and a broad focus of egress of microsphere-labelled cells lie opposite each other. (C, D) Brightfield and fluorescent lateral views of a blastocyst with two diametrically opposing foci of spread of label from the polar to the mural trophectoderm. Scale bar = 50 µm.

 
Lineage-labelling of individual junctional trophectoderm cells
Since labelled cells were clearly damaged rapidly by exposure to fluorescence microscopy, the incidence of junctional trophectoderm cells with an extension, and the form of such extensions, was assessed initially in expanding fourth d.p.c. blastocysts that were not cultured thereafter. This meant that cells could be labelled strongly with TMRDLB or DiI and then examined very thoroughly by fluorescence microscopy so as to minimize the risk of overlooking thinner or smaller extensions. Junctional trophectoderm cells could usually be identified readily because they spanned the boundary between the polar and mural region. However, where mural and polar cells abutted at the junction it was often difficult to decide which was better placed to extend onto the blastocoelic surface of the ICM. While both candidate cells were labelled in blastocysts which were not required to develop further, only one junctional cell was injected per blastocyst destined for culture.

In the non-cultured series, between one and eight individual junctional cells or cell pairs were labelled in each of 21 blastocysts, and a complete circumferential ring of eight such cells in a further blastocyst. Altogether, a little more than half the injected junctional cells or cell pairs (49/92) exhibited an unequivocal extension onto the blastocoelic surface of the ICM. While some extensions reached the centre, others obviously fell short of it. Regardless of length, the extensions varied in shape from broad triangles to much narrower, cable-like projections. In the blastocyst in which the complete circumferential ring of eight junctional trophectoderm cells was labelled successfully, three cells (an adjacent pair and a separate one) each had a long extension and a fourth a much shorter one, while the remaining four cells altogether lacked an extension. Examples of labelled junctional cells with an extension are shown in Figure 5Go.



View larger version (90K):
[in this window]
[in a new window]
 
Figure 5. (AD) Lateral brightfield (A, C) and corresponding fluorescence images (B, D) of two blastocysts with a labelled junctional trophectoderm cell that has an extension onto the blastocoelic surface of the inner cell mass (ICM). (E, F) Brightfield (E) and fluorescence abembryonic polar view (F) of a third blastocyst with a broad triangular extension onto the blastocoelic surface of the ICM. Scale bar = 50 µm.

 
To compare the fate of cells with and without an extension, one junctional cell was labelled with DiI in each of a further series of expanding fourth d.p.c. blastocysts. The blastocysts were then exposed briefly to fluorescence microscopy to enable them to be sorted for subsequent overnight culture according to whether their labelled cell had or lacked an extension. As shown in Table IIGo, the proximal boundary of most of the resulting fluorescent clones was displaced murally, regardless of whether their labelled junctional precursor cell had or lacked an extension at the time of labelling.


View this table:
[in this window]
[in a new window]
 
Table II. Location of clones formed by junctional trophectoderm cells with or without an extension onto the inner cell mass
 
Finally, live expanding blastocysts were examined closely by light microscopy to see whether the region of spread of cells from polar to mural trophectoderm could be identified without recourse to labelling. No consistent regional differences in cell density or shape were seen. Nevertheless, in occasional blastocysts a cell lying distal to the mural side of the polar–mural junction exhibited a thinnish process that extended onto the blastocoelic surface of the ICM near its edge. Initially, when viewed from directly above by brightfield or differential interface contrast (DIC) optics, such cells often appeared to be binucleate. However, on examination at higher magnification the more proximal of the two nuclei was found to be at a slightly higher focal plane. In lateral view, the image was consistent with the cell bearing an extension being partially overlapped by its immediately proximal neighbour (see Figure 6Go for interpretative diagrams).



View larger version (23K):
[in this window]
[in a new window]
 
Figure 6. Diagrams illustrating displacement murally of junctional cells with an extension, viewed from the side on the left, and from above on the right. Note the substantial overlap of the cell with an extension by its immediately proximal neighbour. The thick lines between adjacent trophectoderm cells depict tight junctions.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two techniques with different limitations were used to obtain global labelling of the polar trophectoderm with fluorescent microspheres so as to enable the overall pattern of movement of cells into the mural trophectoderm during growth of the blastocyst to be determined. The findings were essentially the same, regardless of the technique and of whether the blastocysts developed subsequently in vitro or in utero. Thus, the great majority of scorable blastocysts exhibited a coherent patch of labelling in the mural trophectoderm which varied considerably in how far it extended both into and around the tissue. The centre of the region that was either completely devoid of label or in which labelling was present in only the most proximal mural cells, typically lay diametrically opposite the most distal boundary of the labelled patch. Furthermore, acquisition of label by the mural trophectoderm was not accompanied by its disappearance from the polar region. Hence, mural labelling must have resulted from the efflux of surplus cells from the polar trophectoderm rather than relocation of the ICM (Kirby et al., 1967Go). Parenthetically, such persistence of labelling throughout the polar trophectoderm is obviously in conflict with the hypothesis that growth of the tissue depends on recruitment of cells from the ICM (Winkel and Pedersen, 1988; see also Dyce et al., 1987; Gardner and Nichols, 1991).

Collectively, the above findings confirm the conclusion suggested by an earlier clonal analysis (Gardner, 1996Go) that the polar to mural flow of cells in the trophectoderm which accompanies growth of the blastocyst is normally polarized rather than, as had been assumed hitherto, radially symmetrical. That the flow was not discernibly polarized in all cases may be because, in order to assess the quality of labelling, all blastocysts had to be exposed to fluorescence microscopy before culture or transfer. Although both the duration and level of excitation were kept to a minimum, and blastocysts with obviously dead or excluded cells were discounted, the possibility remains that the polar trophectoderm occasionally suffered more subtle damage that perturbed its subsequent growth. The incidence of non-polarized spread was higher in blastocysts that were labelled by subzonal injection which also tended to give denser labelling. That radiation damage might also account for the several cases where spread of the label was bi- rather than uni-polar also cannot be excluded. It would, nevertheless, be intriguing to know whether cells egressed from opposite sides of the polar trophectoderm simultaneously or sequentially in these specimens.

The fact that the cells which leave the polar trophectoderm typically form a single coherent patch in the mural region argues that they must have emigrated in essentially the same direction throughout the 15–20 h between labelling and scoring. What is not clear is whether they leave the polar region on a front that is as broad as the patch they eventually form in the mural trophectoderm, or whether they spread circumferentially after emerging more focally. Limited observations on blastocysts cultured for just a few hours after labelling suggest that spread of the label is not confined initially to a narrow segment of the polar–mural junction (R.L. Gardner, unpublished data).

The question of how the spread of cells from polar to mural trophectoderm is restricted radially during blastocyst growth has been discussed elsewhere (Gardner, 1996Go). In particular, the possibility was considered that trophectoderm cells which extend a process over the blastocoelic surface of the ICM (Ducibella et al., 1975Go; Fleming et al., 1984Go) might thereby be anchored enduringly at the polar–mural junction (Gardner, 1996Go). It was argued that prevention of the net distal movement of junctional clones could restrict egress of cells from the polar region, provided that part of the junction was occupied by cells which were not so anchored. The results of lineage-labelling junctional cells reported here offer no support for this hypothesis. Thus, while not all junctional cells extend onto the ICM, those that do not tend to be interspersed among those that do. More significantly, clones formed by junctional cells with a extension showed net displacement murally during subsequent culture as often as those without one. Hence, extensions are clearly transient rather than enduring features of junctional cells and therefore cannot be responsible for the circumferential restriction in polar to mural flow during blastocyst growth. The incidence of junctional cells with an extension was found to be much lower than was expected from earlier findings (Fleming et al., 1984Go). This may reflect better growth of blastocysts in vitro in the present study, since the proportion of junctional cells with an extension would be expected to vary inversely with the rate of polar to mural cell flow. Previous observations (Soltynska, 1985Go) on freshly recovered blastocysts are consistent with this possibility. Whereas extensions were found to cover the surface of the ICM almost entirely in nascent blastocysts, in expanded blastocysts much of this surface was uncovered. Morphological observations consistent with withdrawal of extensions as cells are displaced murally are summarized diagrammatically in Figure 6Go, which also indicates how such displacement might be achieved without disrupting the tight junctional permeability seal between adjacent trophectodermal cells through their partially overlapping each other.

One implication of the present findings is that distance from the ICM cannot be used as a measure of the length of time for which cells have resided in the mural trophectoderm. In view of the uniform way in which morphological giant transformation of mural cells spreads from the abembryonic pole, the present findings make it even less likely that this process bears any relationship to commitment to endoreduplication of the genome.

Finally, it is intriguing that whenever the label spread into the mural trophectoderm at two discrete sites rather than just one, these were always more or less diametrically opposite each other. It suggests that the orientation of flow of cells from the polar to the mural trophectoderm may be related to the axis of bilateral symmetry of the early blastocyst, a possibility that is now being explored.


    Acknowledgments
 
I thank Tim Davies and Ann Yates for their help in preparing the manuscript and the Royal Society and the Wellcome Trust for support.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Barlow, P., Owen, D.A.J. and Graham, C. (1972) DNA synthesis in the preimplantation mouse embryo. J. Embryol. Exp. Morphol., 27, 431–445.[ISI][Medline]

Champlin, A.K., Dorr, D.L. and Gates, A.H. (1973) Determining the stage of the estrous cycle in the mouse by the appearance of the vagina. Biol. Reprod., 8, 491–494.[ISI][Medline]

Copp, A.J. (1978) Interaction between inner cell mass and trophectoderm of the mouse blastocyst. I. A study of cellular proliferation. J. Embryol. Exp. Morphol., 48, 109–125.[ISI][Medline]

Copp, A.J. (1979) Interaction between inner cell mass and trophectoderm of the mouse blastocyst. II. The fate of the polar trophectoderm. J. Embryol. Exp. Morphol., 51, 109–120.[ISI][Medline]

Cruz, Y.P. and Pedersen, R.A. (1985) Cell fate in the polar trophectoderm of the mouse blastocyst as studied by microinjection of lineage tracers. Dev. Biol., 112, 73–83.[ISI][Medline]

Dickson, A.D. (1963) Trophoblast giant cell transformation of mouse blastocysts. J. Reprod. Fertil., 6, 465–466.[ISI]

Ducibella, T., Albertini, D.F., Anderson, E. et al. (1975) The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development. Dev. Biol., 45, 231–250.[ISI][Medline]

Dyce, G., George, M., Goodall, H. et al. (1987) Do trophectoderm and inner cell mass cells in the mouse blastocyst maintain discrete lineages. Development, 100, 685–698.[Abstract]

Fleming, T.P. and George, M.A. (1987) Fluorescent latex microparticles: a non-invasive short-term cell lineage marker suitable for use in the mouse early embryo. Roux's Arch. Dev. Biol., 196, 1–11.

Fleming, T.P., Warren, P.D., Chisholm, T.C. et al. (1984) Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst. J. Embryol. Exp. Morphol., 84, 63–90.[ISI][Medline]

Gardner, D.K. and Sakkas, D. (1993) Mouse embryo cleavage, metabolism and viability: role of medium composition. Hum. Reprod., 8, 288–295.[Abstract]

Gardner, R.L. (1978) Production of chimeras by injecting cells or tissues into the blastocyst. In Daniel, J.C., Jr (ed.), Methods in Mammalian Reproduction. Academic Press, New York, pp. 137–165.

Gardner, R.L. (1985) Origin and development of the trophectoderm and inner cell mass. In Edwards, R.G., Purdy, J.M. and Steptoe, P.C. (eds), Implantation of the Human Embryo. Academic Press, London, pp. 155–178.

Gardner, R.L. (1996) Clonal analysis of growth of the polar trophectoderm in the mouse. Hum. Reprod., 11, 1979–1984.[Abstract]

Gardner, R.L. (1997) The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse. Development, 124, 289–301.[Abstract/Free Full Text]

Gardner, R.L. and Beddington, R.S.P. (1988) Multi-lineage `stem' cells in the mammalian embryo. J. Cell Sci., 10 (Suppl.), 11–27.

Gardner, R.L. and Cockroft, D.L. (1998) Complete dissipation of coherent clonal growth occurs before gastrulation in mouse epiblast. Development, 125, 2397–2402.[Abstract/Free Full Text]

Gardner, R.L. and Davies, T.J. (1993) Lack of coupling between onset of giant transformation and genome endoreduplication in the mural trophectoderm of the mouse blastocyst. J. Exp. Zool., 265, 54–60.[ISI][Medline]

Gardner, R.L. and Davies, T.J. (1998) Mouse chimeras and the analysis of development. In Tuan, R. and Lo, C. (eds), Developmental Biology Protocols. Humana Press, Totowa, USA.

Gardner, R.L. and Nichols, J. (1991) An investigation of the fate of cells transplanted orthotopically between morulae/nascent blastocysts in the mouse. Hum. Reprod., 6, 25–35.[Abstract]

Gardner, R.L. and Papaioannou, V.E. (1975) Differentiation in the trophectoderm and inner cell mass. In Balls, M and Wild, A.E. (eds), The Early Development of Mammals: British Society for Developmental Biology Symposium 2. Cambridge University Press, Cambridge, pp. 107–132.

Huber, C.G. (1915) The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of the mesoderm anlage: end of the first to end of the 9th day. J. Morphol., 31, 235–245.

Kirby, D.R.S., Potts, D.M. and Wilson, I.B. (1967) On the orientation of the implanting blastocyst. J. Embryol. Exp. Morphol., 17, 527–532.[ISI][Medline]

McRae, A.C. and Church, R.B. (1990) Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocyst. J. Reprod. Fertil., 88, 31–40.[Abstract]

Soltynska, M.S. (1985) Ultrastructure of mouse blastocysts during blastocoele expansion. Roux's Arch. Dev. Biol., 194, 425–428.

Stanners, C.P., Eliceiri, G.L. and Green, H. (1971) Two types of ribosome in mouse-hamster hybrid cells. Nature New Biol., 230, 52–54.[ISI][Medline]

Tsunoda, Y., Yasui, T., Nakamura, K. et al. (1986) Effect of cutting the zona on the pronuclear transplantation in the mouse. J. Exp. Zool., 240, 119–125.[ISI][Medline]

Winkel, G.K. and Pedersen, R.A. (1988) Fate of the inner cell mass in mouse embryos as studied by microinjection of lineage tracers. Dev. Biol., 127, 143–156.[ISI][Medline]

Zernicka-Goetz, G., Pines, J., McLean-Hunter, S. et al. (1997) Following cell fate in the living mouse embryo. Development, 124, 1133–1137.[Abstract/Free Full Text]

Submitted on June 8, 1999; accepted on November 16, 1999.