Mammalian Development Laboratory, University of Oxford, Department of Zoology, South Parks Road, Oxford OX1 3PS, UK
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Abstract |
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Key words: bilateral symmetry/mouse blastocyst/mural trophectoderm/polarized growth/polar trophectoderm
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Introduction |
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Among the questions raised by the early acquisition of bilateral symmetry is its significance for subsequent morphogenesis of the conceptus and, consequently, of the fetus itself (Gardner et al., 1992). Of interest in this context are findings from an earlier clonal analysis of growth of the trophectoderm which suggested that the flow of surplus polar cells into the mural region (Copp, 1978a
; Cruz and Pedersen, 1985
) was polarized rather than radially symmetrical (Gardner, 1996
). This was confirmed by more recent experiments in which the entire polar trophectoderm was labelled selectively through endocytosis of fluorescent microspheres. Polar to mural movement of cells was found thus to be restricted circumferentially during the subsequent growth of blastocysts, typically yielding a single coherent patch of label that extended well into the mural region (Gardner, 2000
). Moreover, in the few cases where there were two patches rather than one, these were diametrically opposite each other. This suggested that the direction of flow of polar trophectoderm cells might be related to the blastocyst's axis of bilateral symmetry. If the outflow of polar cells were aligned with the bilateral axis, it would argue that this axis was conserved during blastocyst expansion. It might also explain how the bilateral axis acquires polarity. To account for the latter it has been proposed that trophectoderm cells become less deformable once they have resided in the mural region for some time, possibly through deposition of extracellular matrix components on their blastocoelic surface (Biggers et al., 2000
; Summers et al., 2000
). Hence, the hydrostatic pressure of the fluid in the blastocoele (Watson, 1992
) should cause relatively greater stretching and attenuation of cells that have entered the mural trophectoderm most recently. The consequent deformation of a coherent patch of cells in the proximal part of the mural region could account for the observed tilting of one side of the inner cell mass (ICM)/polar trophectoderm complex with respect to the blastocyst's EmAb axis (Gardner, 1998
). Tilting of this complex is the most obvious manifestation of polarity of the bilateral axis, and Smith has defined the point where it is furthest from the abembryonic pole as the anterior end of the bilateral axis, and the diametrically opposite point as its posterior end (Smith, 1980
).
A further question is the extent to which growth of the mural trophectoderm depends on proliferation of cells residing within it as opposed to recruitment from the mitotically more active polar region. This is of interest in view of evidence that fibroblast growth factor (FGF) signalling is required to sustain the cycling of all cells beyond 5th cleavage in the mouse (Chai et al., 1998), and that mural trophectoderm cells become postmitotic by the late blastocyst stage (Gardner and Davies, 1993
). Although mitoses occur within the mural trophectoderm during blastocyst growth (Copp, 1978b
), these might represent the completion of cycles to which cells were committed whilst they were still in the polar region.
The direction of movement of polar trophectoderm cells with respect to the bilateral axis of the early blastocyst was examined both clonally and by labelling the tissue globally. It was found by global labelling to be non-random and, within the limits of the resolution that was attainable, closer to parallel than orthogonal to the bilateral axis. The results of clonal analysis were consistent with this conclusion, and also revealed that new cycles could be initiated in mural cells that were remote from the polar region.
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Materials and methods |
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Marking the bilateral axis
Early blastocysts were immobilized on a standard holding pipette (Gardner and Davies, 2000) with their EmAb axis vertical and embryonic pole uppermost. Small drops of soya oil were injected into the substance of the zona pellucida (ZP) via a very fine-tipped pipette pulled from a microelectrode capillary (GC100F-15; Harvard, Edenbridge, UK) and connected to a microinjector system (IM-6 Narishige, Japan) filled with heavy paraffin oil (Gardner, 2001
). The drops were sited either at both ends of the bilateral axis, which was taken as their greater diameter (GD) or, as a control for the resolution of marking, at the both ends of the lesser diameter (LD).
Polar trophectoderm labelling
The entire polar trophectoderm was labelled selectively by injecting 1 µm fluorescent latex microspheres (Fluoresbrite; YG Carboxylate, Polysciences, Warrington, PA, USA) that are readily endocytosed by trophectoderm cells locally under the ZP, as described elsewhere (Gardner, 2000
). Individual trophectoderm cells were labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes Inc., Eugene, OR, USA), horseradish peroxidase (HRP; Boehringer-Ingelheim UJK, Bracknell, UK), 40 kDa tetramethylrhodamine dextran lysine (TMRDL) or fluorescein dextran lysine (FDL; Molecular Probes, Inc.) or, in some cases, a mixture of both HRP and TMRDL or FDL. DiI was prepared as a saturated stock in ethanol, with undissolved material being sedimented by centrifugation, before aliquots were diluted 1:5 with soya oil. Drops of the DiI in oil were injected against or into the cell to be labelled using the type of pipette and injector system described earlier for marking the ZP. Labelling of cells with HRP, either alone or together with TRMDL or FDL, was done ionophoretically (Gardner, 1996
, 1997
). All three labels were prepared at a final concentration of 68% (w/v) in 0.1 mol/l KCl. Staining for HRP activity was carried out essentially as described elsewhere (Beddington and Lawson, 1990
). Where a fluorescent label was injected alone or together with HRP, the targeted cell was inspected briefly by fluorescence microscopy shortly thereafter to confirm labelling and to determine whether the label had spread to another cell. Such spreading is not uncommon in early mouse development because intercellular bridges between sister cells often remain patent for many hours following cytokinesis (Goodall and Johnson, 1984
).
Preparation of labelled blastocysts for scoring following culture
Blastocysts whose polar trophectoderm had been labelled globally with fluorescent microspheres were fixed before being examined following culture (Gardner, 2000), as were those in which HRP was used as a cell label (Gardner, 1996
). For examination, all blastocysts were placed in hanging drops in Puliv chambers (Gardner and Davies, 2000
) so that they could be oriented optimally for immobilization on a holding pipette during photography. For assessing the relationship of polar trophectoderm cell movement to the early bilateral axis, blastocysts were immobilized with their embryonic pole uppermost and, depending on which had been marked, either their GD or LD parallel to the long axis of the holding pipette. Brightfield and epifluorescence images were then taken of each specimen, and the resulting prints from both the GD and LD series were coded by one author for scoring by the other.
Scoring
Analysis of clones was based on the expectation that cells lying at the site of outflow from the polar trophectoderm should be the first, and those diametrically opposite this site among the last, to leave this region (Gardner, 1996). The distribution of clones should therefore differ in two related respects, depending on whether or not their progenitors were aligned with the direction of outflow. Aligned progenitors should yield a higher proportion of clones that are retained within the polar trophectoderm (Figure 1
). Moreover, clones from opposite pairs of aligned progenitors should more often lie at different levels along the EmAb axis than those from non-aligned progenitors (Figure 1
). Clones were recorded as polar if none of their cells lay entirely within the mural region. Pairs of clones were recorded as being disparate in level along the EmAb axis if they differed by
2 cell diameters in the position of their proximal or distal boundary.
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Results |
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Five out of a total of 97 clones were discounted in the GD series because they included binucleate cells or debris. A single scorable clone was present in 16 blastocysts and pairs of clones in a further 38. Three clones were discounted in the LD series in which 13 blastocysts with single and 38 blastocysts with pairs of clones were scored. The results of analysis of the distribution of all the scorable clones is summarized in Table II, from which it is evident that both polar retention and axial disparity were significantly more common among GD than LD clones. The mean size of clones was similar for the two series (see footnote to Table II
).
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Global labelling of polar trophectoderm in blastocysts with the GD or LD marked with oil drops in ZP
Early oval blastocysts had a small oil drop injected into the ZP at each end of the GD or LD before their entire polar trophectoderm was labelled with fluorescent microspheres. The blastocysts were then cultured overnight, and recovered before they hatched so that the direction of polar to mural flow relative to the axes defined by opposing oil drops in the ZP could be determined (Figures 2 and 3). Flow was scored as `on axis' if its centre was within 45° of the axis defined by the oil drops, and as `off axis' if it was outside this angle. From the results summarized in Figure 2
[on axis/off axis: GD 29/17 (63%), LD 12/33 (27%)] it is clear that the GD and LD series differed significantly in the frequency with which the flow was `on axis' versus `off axis' (
2 = 12.15 for 1 d.f.; P < 0.001). However, while there were significantly more `off' than `on axis' cases in the LD series, in the GD series the `on axis' did not outnumber the `off axis' to the same extent (Figure 2
). Nonetheless, for the combined data there was a significant bias in the direction of flow towards the GD rather than the LD (
2 = 6.18 for 1 d.f ; P < 0. 02). Among 14 blastocysts in the GD series retaining an intact 2nd polar body at analysis, this persisting marker of the animal pole of the zygote (Gardner, 1997
) lay towards the side of the polar to mural cell flow in eight cases and away from it in the remaining six.
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The results of experiments in which blastocysts were cultured for 1 or 2 days following lineage-labelling of single abembryonic mural versus polar trophectoderm cells are summarized in Table IV. Mural cells yielded clones composed of up to five cells after
1 day in culture, and up to eight cells after 2 days (Figure 4
). Both the maximum and mean sizes of polar clones were greater than mural at both intervals. Because single cell clones occurred only after direct labelling (Table IV
), this may have occasionally resulted in damage that was sufficient to prevent or delay further proliferation (e.g. Cruz and Pedersen, 1985
). However, the great majority of directly labelled cells could not have been affected adversely since their mean clone size was not consistently smaller than that of indirectly labelled cells.
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Discussion |
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The relationship of polar to mural flow to the bilateral axis was first investigated by examining the distribution of clones resulting from labelling of peripheral polar cells that were either aligned with, or orthogonal to, this axis. This approach was based on the expectation that clonal descendants of a peripheral polar cell lying at the site of egress should be the first, and those of a cell lying diametrically opposite this site the last, to enter the mural region. Consequently, polar retention should be more frequent among aligned than non-aligned clones, as also should a disparity between members of pairs of clones in their location along the EmAb axis (Figure 1). Both expectations were fulfilled and position-dependent differences in growth could be discounted as a confounding factor. The extent to which `on' and `off' axis clones differ in location should first increase and then decrease according to the amount of polar growth that intervenes between labelling and scoring. There is no reason to suppose the difference should be greatest after the 1618 h of culture employed in the present study. However, in view of the considerable asynchrony in cell cycles both within and between conceptuses (Barlow et al., 1972
; Chisholm et al., 1985
), a more optimal interval might prove hard to attain. The resolution achievable by lineage-labelling single cells obviously depends on the proportion of the blastocyst's circumference orthogonal to the EmAb axis that is occupied by the resulting clone. This is clearly limited by the proportion of the circumference of the blastocyst orthogonal to its EmAb axis occupied by clonal descendants of the labelled cell. According to the cell counts on living blastocysts optically sectioned by DIC, this would not be <10% even where clones were oriented strictly proximo-distally, and possibly up to 25% where they were not.
The second approach was to determine the direction of bulk movement into the mural region of surplus polar cells relative to pairs of oil drops in the ZP that marked either the GD (bilateral axis) or the LD at the early blastocyst stage. Within the limits of resolution noted above, the centre of the outflow of surplus polar cells was found to be `off axis' significantly more often for the LD than for the GD. However, because blastocysts had to be scored whilst they still retained the ZP, the bilateral axis was seldom discernibly polarized. This meant that it was possible to record only the size of the angles by which the centre of flow departed from the bilateral axis and not to which side of this axis they lay. Hence, the possibility that the mean direction of flow is at some angle of <45° to the bilateral axis rather than coincident with it cannot be discounted. Nonetheless, when considered in conjunction with those from the clonal analysis, these findings are consistent with the notion that the polar to mural flow of cells in the trophectoderm corresponds with the axis of bilateral symmetry of the blastocyst and hence with the AV axis of the zygote. However, because the flow could be either towards or away from a persisting 2nd polar body, it clearly cannot depend on information in the zygote for its polarity as opposed to orientation.
Finally, the mural trophectoderm cell furthest from the ICM was lineage-labelled in early blastocysts to determine how many times it could divide during their subsequent growth in vitro. Since macromolecular labels can pass from an injected cell to its sister, not all labelled cells are necessarily descendants of the injected one. Sufficient transfer of label for an uninjected cell to stain as strongly as its injected sister can only occur if there is extensive cytoplasmic continuity between them as, for example, before the intercellular bridge formed during cytokinesis becomes attenuated. In such circumstances, full spread of a co-injected fluorochrome should be discernible almost immediately after labelling. While partial spread of label may go undetected at the time of labelling, it can be identified at scoring as differential staining among positive cells. Thus, in one blastocyst, four cells were uniformly well stained and a further four uniformly less well stained for HRP activity. These were therefore classified as a pair of sister clones. In all other cases where more than two labelled cells were present they were similarly stained, and there was no reason to doubt that they were the clonal descendants of a single labelled cell that had divided after the start of culture. Hence, notwithstanding evidence that FGF signalling is needed for all cells of the mouse conceptus to complete 5th cleavage (Chai et al., 1998), trophectoderm cells can initiate at least two new cycles after they have become remote from the ICM. This might be because such obligatory signalling can license more than one cell cycle or that it initially occurs throughout the blastocyst, and only later becomes confined to the ICM.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 15, 2002; accepted on March 6, 2002.