1 Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Genetics,
Tennis Court Road, Cambridge CB2 1QR, UK
2 Department of Experimental Embryology, Polish Academy of Science, Jastrzebiec
05-552, Poland
3 Développement des Vertébrés, Institut Jacques Monod,
UMR7592 CNRS Universités Paris 6, 7. 2, place Jussieu, 75005 Paris,
France
4 Department of Microbiology, Shiga University of Medical Science, Otsu, Shiga
520-2192, Japan
* Author for correspondence (e-mail: mzg{at}mole.bio.cam.ac.uk)
Accepted 29 November 2004
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SUMMARY |
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Key words: Cleavage pattern, Cell fate, Chimaeras, Mouse embryo
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Introduction |
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Since in a major group of embryos (ME embryos) the progeny of individual
four-cell blastomeres tends to follow different fates, the question arises as
to whether they are equivalent to each other. This could happen, for example,
as a consequence of their spatial relationship per se or because they inherit
different properties when they divide. Indeed it has been proposed that an
equatorial division of the two-cell blastomere might partition `animal' and
`vegetal' components between daughter cells, whereas a meridional division
would not (Gardner and Davies,
2003). Irrespective of whether such partitioning takes place, can
cells have different developmental properties that reflect their positions in
the embryo as early as the four-cell stage?
In the mouse, the only individual blastomeres recorded to undergo normal
development to term have been from the two-cell stage embryo
(Tarkowski, 1959;
Tsunoda and McLaren, 1983
;
Papaioannou et al., 1989
).
Thus far, it has not been possible to demonstrate whether all individual
blastomeres at the four-cell stage have this capability. Individual four-cell
blastomeres will form miniature blastocysts
(Tarkowski and Wroblewska,
1967
; Rossant,
1976
), but there is only one reported case of such a blastocyst
ever giving rise to a postimplantation embryo
(Rossant, 1976
). This can be
attributed to the difficulty of a four-cell blastomere to generate sufficient
cells as the blastocyst forms to allow some to be enclosed and develop as
inner cell mass (ICM) precursors. Thus, to determine whether single
blastomeres have a full developmental capacity, they have been aggregated with
other, `carrier', blastomeres. Such studies provided evidence that four-cell
blastomeres can retain the ability to form ICM and trophectoderm lineages
(Hillman et al., 1972
;
Kelly, 1977
). They also showed
that individual four-cell blastomeres, when aggregated with carrier cells,
were in some cases able to contribute exclusively to the resulting animals
(Kelly, 1977
). These
experiments indicate the totipotency of at least some of the blastomeres at
these early developmental stages. However, despite numerous efforts,
quadruplet mice have never been produced from a single embryo
(Tarkowski et al., 2001
).
In all previous experiments to analyse the developmental potency of blastomeres, chimaeras were constructed without reference to the origins of the donor cells. Knowledge of the ways in which individual four-cell blastomeres become arranged, on the one hand, and the blastocyst becomes populated with their progeny, on the other, has now allowed us to examine whether four-cell blastomeres are equivalent in their developmental abilities. To this end, we have generated chimaeras comprising four-cell blastomeres of single types that have not only defined spatial origins, but also `preferred' developmental fate. Here, we report that such chimaeras differ in their developmental properties. We show that specific cells in the four-cell stage embryo have a reduced ability to develop successfully when aggregated with cells of a similar type. We describe the developmental defects shown by such embryos and discuss how these might arise.
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Materials and methods |
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Non-invasive labelling of individual blastomeres at the two- and four-cell stage
All embryos were micromanipulated with Leica micromanipulators using a De
Fonbrune suction-force pump and observed using an inverted (Leica) microscope
with DIC optics. To mark blastomeres, DiD or DiI (Molecular Probes) was
applied using a blunt-ended micromanipulation micropipette as previously
(Piotrowska et al., 2001). The
first step was to label one of the two-cell stage blastomeres with a red dye
(between 45.5 and 47 hours after hCG). Ten two-cell stage embryos were placed
in the manipulation chamber at the same time and
80 two-cell embryos
(recovered from four mice) that had a visible polar body between the
blastomeres were labelled in each experiment. Approximately 95% of embryos
survived the labelling procedure. As soon as the dye-labelling procedure was
completed (up to 1.5 hours for 80 embryos), one or both two-cell stage
blastomeres were labelled with beads (a step taking up to 2 hours for 80
embryos). Labelling of the vegetal pole of two-cell blastomeres with
fluorescent beads was carried out as previously
(Piotrowska and Zernicka-Goetz,
2001
). In brief, green fluorescent beads (
2-3 µm in size)
were first treated with 300 µg/ml phytohaemagglutin for 30 minutes. Only
these beads, which settled down on the bottom of the dish were subsequently
picked by `sticking' them to the micromanipulation micropipette. They were
subsequently inserted through a previous `cut' in the zona, made by inserting
and removing a micropipette, and deposited on the blastomere membrane.
Labelled embryos were cultured in vitro in drops of KSOM medium and 4 mg/ml
BSA, under paraffin oil in an atmosphere of 5% CO2 in air at
37.5°C.
To monitor the order and orientation of the second cleavage divisions, and
then at the four-cell stage to examine the arrangements of blastomeres,
labelled embryos were cultured in the 5% CO2 incubator at 37°C
and after 1 hour of uninterrupted culture checked by fluorescent microscopy at
intervals of 20-40 minutes over a period of 5 hours. If within this time both
blastomeres had divided to the four-cell stage, the embryos were discarded.
Those embryos in which the red labelled blastomere had divided first were
collected into one drop of medium and those in which the unlabelled two-cell
blastomere was first to divide into another. In some experiments (as specified
in the Results), four-cell blastomeres located furthest away from the polar
body were labelled with a blue dye. Embryos were finally analysed to reveal
the positions of the progeny of the labelled cells at the blastocyst stage. In
lineage tracing experiments blastocysts were observed when still alive using a
BioRad confocal microscope taking optical sections every 7 µm. By examining
all sections in each series, it was possible to determine the distribution of
cells labelled by specific dyes in the embryonic (polar trophectoderm and
deeper ICM cells) and abembryonic (mural trophectoderm) parts of the
blastocyst. The boundary zone between these two parts was defined as a cell
layer, approximately one cell deep and parallel to the `roof' of the
blastocoel cavity as previously
(Piotrowska et al., 2001).
After confocal sectioning, the zona pellucida was removed by a short treatment
with Acid Tyrode's reagent and next each of the embryos was disassociated by
treatment with 1% trypsin for 5 minutes dispersing them by thorough pipetting
to count the total number of cells in each embryo.
Making chimaeras from specific four-cell stage blastomeres
One blastomere was labelled at the late two-cell stage by fluorescent DiD
as described above. Following the division from the two- to the 4-cell stage,
all marked embryos were classified into groups according to whether the
labelled or unlabelled blastomere first underwent the two- to four-cell stage
division. Embryos were examined at the four-cell stage and those with
tetrahedral morphology (three blastomeres gathered around the attached polar
body and the fourth one more distal) were scored from the position of labelled
and unlabeled blastomeres as being either ME or EM (see Results). The products
of the meridional division (M-division) were not distinguished from each other
and are referred to as m-blastomeres, but products of the oblique or rather
more equatorial division (E-division) differed in their position, with one of
the blastomeres always being more distal from the second polar body, a marker
of the animal pole (Gardner,
1997). The blastomere most proximal to the polar body was termed
e1 and the one most distal, e2. Control groups of chimeric embryos were
generated from m-blastomeres. Our discovery that marked membrane in the
vegetal position of the two-cell blastomere could be displaced in the course
of the E-division, but not in the M-division, required labelling of the
vegetal pole of two-cell stage blastomeres by attaching beads as described
above. Thus, in the second set of experiments, we used only `vegetally marked'
or unmarked `animal sisters' to make chimaeras. We do not use these terms to
infer that all cellular components are partitioned respecting their true
animal-vegetal origins. We refer to four-cell stage blastomeres carrying a
bead arising from an E-division as e1+ or e2+, depending upon the position of
the cell. Cells not marked with beads are termed e1- or e2-. Meridional
chimaeras comprised completely separated individual m-blastomeres that were
then associated. To control for any effect of beads being attached to the
blastomere surface, we also generated chimaeras with meridionally dividing
blastomeres that carried beads. These developed normally.
Before the individual blastomeres were isolated to generate chimaeras, the zona pellucida was digested with 0.5% pronase in Ringer's solution at 37.5°C for 15 minutes. When the zona pellucida started to become `thin', we gently rinsed the embryos and transferred them to a drop of M2 medium in a glass chamber on the stage of an inverted Leica microscope. This step had to be carried out with great precision in order to avoid any change in the specific configuration of four-cell stage blastomeres. Individual blastomeres of known origins were delicately aspirated using a biopsy pipette and removed from embryo (see also Fig. 4). Four (or three as specified in the text) such blastomeres of a single cell-type from different embryos were placed together into a small depression made in the bottom of culture dish and cultured in KSOM alongside three `helper' embryos enclosed in their zones of albino MF1 strain. We routinely added helper embryos from this different strain to our individual experimental chimaeras as we found that the embryos developed better when cultured in a group.
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In a third additional series of experiments, we generated chimaeras of individual e2+ four-cell blastomeres by surrounding them with four other four-cell blastomeres from random positions. We thus selected individual e2+ cells from a line expressing GFP-H2B and aggregated these with four randomly selected non-labelled four-cell blastomeres of a wild-type strain. The chimaeras were cultured in vitro to the advanced morula/blastocyst stage, transferred to foster mothers and then recovered at E5.5 for examination by confocal microscopy.
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Results |
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We found that the developmental success of the group of chimaeras that were
generated from the e2 blastomeres was significantly different. The two groups
of control chimaeras, of m-blastomeres that had divided meridionally to
four-cell stage, had a high probability of survival. Of these groups, between
85% (22/26) and 69% (18/26) developed to term, depending whether m-blastomere
was a progeny of the earlier or later dividing two-cell blastomere. By
contrast, the proportion of surviving progeny of the chimaeras of e2
blastomeres was reduced. This was most dramatic in chimaeras comprising e2
blastomeres taken from the later dividing two-cell blastomeres of ME embryos.
The development of such chimaeras into viable pups was reduced to 30% (8/27).
This was a statistically significant difference compared either with control
chimaeras generated from later dividing m-blastomeres (P<0.1,
2 test 1 d.f.
2 is 2,846) or with chimaeras
generated from earlier dividing m-blastomeres (P<0.05,
2 test 1 d.f. -
2 is 4,726). The survival to
term of chimaeras comprising e2- blastomeres of EM embryos was also lower than
the m-controls; however, as many as 46% (11/24) of embryos of this group gave
rise to pups. This indicates that e2 blastomeres from ME embryos had
significant differences from the other cells of the four-cell embryo.
Progeny of blastomeres derived from later E-divisions of ME embryos are allocated to specific blastocyst regions
Our previous lineage tracing experiments showed that e2 blastomeres from ME
embryos contributed most of their progeny to the abembryonic rather than the
embryonic part of the blastocyst (23 out of 26 embryos)
(Piotrowska-Nitsche and Zernicka-Goetz,
2005). This could either be as a clone of cells that comprised the
mural trophectoderm or a clone contributing mainly to the boundary zone
between the embryonic and abembryonic parts. We wondered whether these
different fates might reflect alternative positioning of blastomeres at the
four-cell stage.
One way that such a situation could arise would be if there had been displacement of all or part of the two-cell blastomere that undertakes an equatorial or oblique division. To test whether this could be the case, we labelled one two-cell stage blastomere with red dye and its sister with a fluorescent bead at a point opposite the second polar body (the `vegetal' pole; Fig. 2A). We then selected those embryos in which the red cell divided first through the meridional plane (Fig. 2B) and then scored the position of the fluorescent bead when the other two-cell blastomere had undergone its E-division (Fig. 2C). We found that there were approximately equal proportions of embryos in which the e2 cell arising was labelled with the bead (68 blastomeres) or not (64 blastomeres) (Fig. 2C,E). Time-lapse observations indicated that following labelling, membrane marked by the bead could indeed either remain at the vegetal position or be displaced towards and even beyond the position of cleavage (see Movies 1 and 2 in the supplementary material). From this point onwards, we will continue to refer to the four-cell blastomeres arising from the later second cleavage in ME embryos as e1 and e2, reflecting whether they are located proximal or distal to the second polar body. However, we will adopt an additional + or - symbol to reflect whether or not they inherit a bead that was placed at a `vegetal' position in their parental two-cell stage blastomere.
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In multiple experiments that transfer groups of chimaeras to foster
mothers, we found that of a total of 22 chimaeras derived from e2+ four-cell
blastomeres, none was able to develop to term
(Table 2). Similarly, none of
the 22 chimaeras constructed from e1+ blastomeres could develop to term. By
contrast, 27% (6/22) and 18% (4/22) of chimaeras derived from their respective
e1- or e2- sister four-cell stage blastomeres from the same embryos were able
to do so. The differences in survival between e2+ and e1- chimaeras was
statistically significant (P<0.05, 2 test 1 d.f. -
2 is 4.726). Thus, it appears that chimaeras generated from
blastomeres that inherit at the very least some components of the `vegetal'
membrane from the egg have dramatically reduced developmental survival in
comparison with both their sisters and m-cell cousins. Both e1- and e2-
chimaeras also appeared to be compromised in their developmental abilities in
comparison to chimaeras generated from the m-blastomeres that are the products
of the early meridional division (Table
2).
Chimaeras of like-blastomeres arising from the later E-division of ME embryos show defects during pre- and postimplantation development
We noted that a significant proportion of the chimaeras derived from the
four categories of blastomere arising from the later E-division developed to
an advanced morulae but did not reach the blastocyst stage at the same time as
their m-cell-derived counterparts (Table
2). Some of such e1 and e2 chimaeras did not form blastocysts even
when permitted an additional 10 hours of development. This suggested that
developmental defects in at least some of these embryos may be already arising
prior to implantation. Thus, we carried out an additional series of
experiments to examine further their development prior to and
postimplantation.
To this end, we again generated the chimaeras from four-cell blastomeres arising from the later E-division of ME embryos, noting first whether this division had resulted in the `vegetal' membrane being displaced. We found that irrespective of such displacement, three blastomere chimaeras constructed from both e1+ or e2+ and e1- or e2- blastomeres from the equatorial division developed into morulae but showed a variety of abnormalities at subsequent stages, including apparent failure of development to the blastocyst (Fig. 5). We observed two types of abnormalities: apparently arrested morulae (14/33 of combined e1+ and e2+ chimaeras; 9/32 of e1- and e2- chimaeras); and formation of trophoblastic vesicles (3/33 of combined e1+ and e2+ chimaeras; 6/32 of e1- and e2- chimaeras). However, as many as 53% (17/32) of the combined groups of e1- and e2-, and 42% (14/33) of e1+ and e2+ chimaeras developed to apparently normal blastocysts, although often with reduced ICM. Such reduced size of ICM was especially apparent in e1+ and e2+ chimaeras (Fig. 5C,E,F). Optical sectioning indicated that such e1/e2- or e1/e2+ chimeric blastocysts had a reduced mean number of cells [mean of 33, range 23 to 43 (n=22); and mean of 29, range 20 to 37 (n=23), respectively] in comparison with control chimaeras derived from the earlier, meridionally dividing blastomere (mean of 49; range 30 to 56, n=20). A small series (n=10) of in situ experiments indicated that when blastocysts developed from either the e1 or e2 chimaeras, they expressed Oct4 in the ICM (Fig. 5M-P).
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To examine the molecular patterning of some of the above chimaeras at E6.5
we performed a small series in situ hybridisation to detect Fgf8, Cer1 or
Bmp4. Fgf8 is first expressed in the posterior epiblast and in the
anterior visceral endoderm (AVE) at pre-streak stages and is maintained in the
primitive streak after the onset of gastrulation (Crossley et al., 1995).
Cer1 is expressed in the distal and then AVE cells from E5.5 to E6.5
(Belo et al., 1997;
Stanley et al., 2000
). BMP4 is
expressed in a ring of extra-embryonic ectoderm abutting the proximal epiblast
from pre-streak stages onwards (Winnier et
al., 1995
; Lawson et al.,
1999
). In agreement with their normal morphology control m
chimaeras show normal expression of Fgf8 and Cer1 in the
posterior epiblast and the AVE respectively
(Fig. 5X). An example of an e2+
chimaeric embryo with delayed development is shown in
Fig. 5X'. This embryo
showed a thick Cer1-positive AVE that is reminiscent of wild-type
E5.75 embryos, when distal visceral endoderm cells have just reached an
anterior position. However, although the embryo is small, the expression of
Bmp4 appears normal. This is also the case in another larger e2+
chimaera (Fig. 5Y). Other small
e2+ chimaeras (Fig. 5Y')
showed no expression of Fgf8 and Cer1, indicating their
patterning was severely affected. Nevertheless, three germ layers could be
distinguished morphologically.
An example of an e1- chimaera with a visceral endoderm layer as well as an inner epithelial layer, but with an abnormally placed ectoplacental cone (on one side of the embryo) is shown in Fig. 5Z. Bmp4 expression is relatively normal in a ring of cells in the inner epithelium, presumably marking the boundary between the epiblast and the extra-embryonic ectoderm. Cer1 is expressed in a group of visceral endoderm cells, presumably marking the distal tip of the embryo. Another example of an e1- chimaera shows normal expression of Cer1 and Fgf8, as observed in control m chimaeras (Fig. 5Z' compare with 5X). Thus, it appears that chimaeras of like-blastomeres arising from the later E-division show variety of defects during their pre- and postimplantation development.
Development of vegetally marked four-cell stage blastomeres when surrounded in chimaeras by blastomeres from random positions
The preceding experiments indicate that when four-cell blastomeres of
equivalent type are aggregated into chimaeras their development can be
compromised to an extent that depends upon their spatial location and history.
As the greatest defects were seen in chimaeras derived from either e1+ or e2+
blastomeres, we wished to see the extent to which a single such cell placed
into a chimaera surrounded by other four-cell blastomeres from random
positions would contribute to the development of different lineages. We thus
selected individual e1+ or e2+ cells from a line expressing GFP-H2B and
aggregated these with four randomly selected non-labelled four-cell
blastomeres of a wild-type strain (Fig.
6C). The chimaeras were cultured in vitro to the advanced
morula/blastocyst stage, transferred to foster mothers and then recovered for
examination at E5.5. The experiments showed that, for nine such embryos
recovered, the labelled e1+ or e2+ derived cells could contribute to all
tissue types (Fig. 6). Similar
results were found for m-blastomere controls (n=20). Thus, when
surrounded by randomly selected blastomeres, it would appear that the e1+ and
e2+ blastomeres have full developmental potential.
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Discussion |
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These results demonstrate that when surrounded by like cells, not all the
four-cell blastomeres of the mouse embryo are equally able to achieve their
full developmental capability. How does this outcome relate to previous
findings about developmental capacities of individual mouse blastomeres?
Earlier studies testing whether individual four-cell blastomeres were able to
develop into mice, selected cells without referring to their site of origin or
fate before they were aggregated into chimaeras with `carrier' blastomeres
(Kelly, 1977;
Tarkowski et al., 2001
). To
our knowledge, the only previous attempt to generate chimaeras from
blastomeres with spatially defined origins was from our own laboratory, in
which we selected and isolated one four-cell blastomere with an attached polar
body and let it divide to give one one-eighth of a blastomere with an attached
polar body (by definition an `animal blastomere') and another that we presumed
to be a `vegetal blastomere' (Ciemerych et
al., 2000
). When we made chimaeras of five of such one-eighth
`animal blastomeres' or five presumptive `vegetal blastomeres', we found that
26-31% of them developed to term. However, our lack of understanding of the
exact early cleavage pattern at that time meant that we could not have
excluded with certainty `animal blastomeres' or others with greater
developmental potency from the `vegetal blastomere' chimaeras.
The present findings focus on the development of particular four-cell
blastomeres from one specific pattern of cleavage that allow us to predict not
only the origin of individual blastomeres but also their developmental fate up
to the blastocyst stage. However, we cannot exclude the possibility that
blastomeres that inherit `vegetal' egg components in some other cleavage
patterns may also differ in their developmental success. Indeed, in
experiments in which we monitored the developmental success of the two
minority groups of embryos in which the second cleavage divisions appear to be
either successively meridional (MM) or successively equatorial/oblique (EE)
using a polar body as a marker
(Piotrowska-Nitsche and Zernicka-Goetz,
2005), the EE embryos showed significantly poorer development.
Whether this reflects `animal-vegetal' partitioning in all of the cells of
such four-cell embryos would require further study.
However, the reduced ability of the products of the later equatorial cleavage in ME embryos to develop as single-cell type chimaeras could also in part reflect their parentage from the later dividing blastomere. We note that, in general, chimaeras generated from later dividing blastomeres survived less well than those made from the earlier dividing cells. This can be seen by the better survival of chimaeras made from four-cell blastomeres descended from the earlier dividing blastomere of EM embryos (Table 1). In chimaeras generated from three cells from different regions of the four-cell ME embryo this may be accentuated (Table 2). Thus, chimaeras of the earlier dividing m-cells attained a mean size of 49 cells at the blastocyst stage and achieved 88% developmental success to term. This developmental success correlates with the ability of specific cells to proliferate when compared with chimaeras generated from the e- progeny of the later second cleavage. These e- chimaeras together achieved 23% developmental success. The mean cell number of e- chimaeras at the blastocyst stage was 33. However, although blastocysts from e+ cell chimaeras (with marked vegetal membrane) attained a similar mean cell number (29), none of these developed to term. Thus, a combination of at least two `factors' could affect blastomeres achieving their full developmental ability. First of all, it would appear that cells arising in the later division have a reduced proliferative ability. Second, the products of the equatorial division appear to have inherent differences. Further study will be required to resolve the relative contribution of these factors. Thus far, there is no evidence that would allow us to conclude that there are any specific components in mouse egg that are spatially distributed along the oocyte animal-vegetal axis. We therefore wish to stress that in interpreting these results it is important to bear in mind that differences might arise between blastomeres, at least in part, owing to their specific arrangements and interactions with each other.
We cannot fully account for the mechanism whereby marked `vegetal' membrane
becomes displaced so that it lies more proximal to the polar body in some
equatorially dividing cells at the four-cell stage. It has been reported that
in rabbit embryos the cross-wise arrangement of blastomeres at the four-cell
stage was the consequence of two meridional divisions in which one group of
cells underwent a 90° rotation (Gulyas,
1975). It has also been suggested that this might be a possibility
in the mouse embryo. However, a cell labelling study with beads by Gardner
(Gardner, 2002
) concluded that
the most common tetrahedral form of the mouse embryo arose from the meridional
division of one two-cell blastomere and the approximately equatorial division
of the other. Thus, although the possibility of predominantly sequential
meridional divisions associated with 90° rotation of cells as suggested by
Gulyas (Gulyas, 1975
) was
dismissed, our observations of the displacement of the marked `vegetal'
membrane make it of interest to re-examine this question once again in future.
Indeed it is not clear why the study of Gardner
(Gardner, 2002
) did not
apparently detect the movement of `vegetally' marked membrane. One possibility
is that by labelling a larger area of `vegetal' membrane with more diffusely
distributed microspheres, the movement of a smaller restricted region was not
detectable. An alternative possibility is that because Gardner's study used a
smaller number of embryos, this membrane behaviour was not seen as we detected
it only in about half of the ME embryos. In this light, we also note that
beads placed at the `vegetal' pole of meridionally dividing cells showed
little movement in relation to the polar body position. Our study indicates
that some of the equatorial/oblique divisions may be difficult to classify as
parts of the membrane may behave independently of cytoplasmic components, let
alone the spindle itself. Thus, it seems that these divisions may be
equatorial in some aspects and not others. Consequently, it appears that at
least some potential `animal' and `vegetal' egg components do not retain their
initial spatial positioning in the second equatorial division.
Importantly, irrespective of whether the `vegetal' membrane marked in our experiments is displaced, the four-cell blastomere that carries it tends to have specific blastocyst fate and developmental properties. We stress that this does not mean the fate of this cell is absolutely fixed. Indeed, when surrounded by cells of random origins it can contribute to a variety of embryonic lineages. However, when surrounded by cells of similar origins, as in the e1+ and e2+ chimaeras, it seems less able to do so. Thus, the e1- and e2- blastomeres are pluripotent in their ability to respond to developmental signals. However, they may be in the process of loosing their own ability to generate such signals as a result of their placement in the embryo. Alternatively, they may have other deficiencies in comparison with their neighbouring blastomeres, a component of which can relate to slower rate of division as discussed above. Although we observed that a significant number of e1+and e2+ chimaeras already had defects at the preimplantation stages, the ICM of some was able to develop into epiblast, indicating that they were able to make not only extra-embryonic but also embryonic tissues. Interestingly, the extra-embryonic tissues in these chimeric embryos were not always properly organised. This was also evident in e1- and e2- chimaeras of their sister cell type. Although understanding the precise reasons behind the demise of such chimaeras will require much further study, our results indicate that developmental abnormalities appear to be not only due to the effects of a delay in the formation of the ICM and subsequently the epiblast, even though this could be a contributing factor.
In summary, a better understanding of the spatial and temporal cleavage patterns of the mouse embryos has allowed us to undertake studies of the developmental properties of individual four-cell stage blastomeres. This indicates that blastomeres differ in realising their developmental abilities from as early as the four-cell stage and that blastomeres inheriting at least some `vegetal' component(s), partitioned in the later second cleavage, are significantly compromised in their development when aggregated with cells of the same origin. Alternatively, such four-cell blastomeres could be lacking critical components from the animal part of the egg. It has long been known that it is difficult to obtain identical quadruplets in mice from individual four-cell stage blastomeres. Even when combined together to increase total cell numbers at the time of blastocyst formation, not all combinations of individual four-cell blastomeres have similar chances for successful development to term. Our findings that not all four-cell stage blastomeres have equal ability to achieve their full development when surrounded by like cells could account in significant part for this difficulty.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/3/479/DC1
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ACKNOWLEDGMENTS |
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