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RESEARCH |
Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK
Correspondence should be addressed to M H Johnson; Email: mhj{at}mole.bio.cam.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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In this paper, we derive quantitative data on cleavage for the pre-implantation period of development. We describe the direct measurement of total conceptus volume and nucleo-cytoplasmic ratio and examine whether the emergent cell lineages differ in cell volume and nucleo-cytoplasmic ratio.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Imaging concepti
Nuclei were stained by incubating concepti in M2 + 4% BSA containing 10 µg/ml Hoechst 33258 (Sigma; 1 mg/ml stock) for 2–4 h. Cytoplasm was stained by a 15 min incubation in the calcium dye Calcein AM (0.5 µg/ml in M2 + 4% BSA; Molecular Probes, Eugene, ON, USA). Membranes were stained with the styryl dye FM4-64 (3.025 µg/ml; Molecular Probes), which was added to the viewing chamber. In the majority of experiments, triple staining was used. Hoechst-stained concepti were rinsed thoroughly in protein-free PBI (phosphate buffered medium 1; supplied courtesy of Sheila Barton, Department of Physiology, University of Cambridge, Downing Street, Cambridge, UK) and placed in 1 ml of PBI in the centre of a 30 mm petri dish to the base of which had been fixed a No. 1 coverslip using Sylgard (Dow Corning). The cover-slips were pre-coated with either poly-L-lysine (1% in PBS) or (mostly) phytohaemagglutinin (0.1–0.2 mg/ml; Sigma) followed by washing with PBI. After concepti had firmly adhered to the coverslip, 1 ml M2 + 4% BSA was added. Whole concepti were imaged with their zonae intact at 37 °C using a Leica TCS-SP-MP two-photon excitation and confocal microscope. Visualization was by sequential two-photon excitation and confocal microscopy, using a picosecond pulsed tunable Tsunami laser (Spectra Physics, Mountain View, CA, USA), tuned to 775 nm to excite Hoechst and Calcein and the 568 nm line of a Krypton laser to excite FM4-64. A water immersion x 63 lens with a numerical aperture of 1.2 and a cover-glass correction collar were used. Emitted fluorescent light was captured in discrete windows (Hoechst 400–480 nm, FM4-64 600–700 nm, and Calcein 505–545 nm). The confocal pinhole was set to 1 Airy disk equivalent for the objective lens in both confocal and two-photon imaging. A Z-series of images was collected from each subject, capturing an image every 1 µm. Every image was used for three-dimensional (3D) reconstruction, while every second image was used for stereology.
Analytical procedures
The Cavalieri principle (Gunderson & Jenson 1987) was used to estimate volumes of concepti, ICMs, single cells and their nucleo-cytoplasmic components. Images on screen (521 x 521 mm) were overlain with quadratic grids with a spacing of 20 mm for cytoplasm and 10 mm for nuclei. The number of intersections overlying nuclei and cytoplasm was counted. The near-spherical shape of the concepti meant that less than the accepted maximum of 200 points per object from 10–15 sections could be counted, giving an error coefficient of 5–10% (Roberts et al. 1993, 1994). The volume of the conceptus (excluding blastocoelic volume) or its constituent cytoplasm or nuclei could then be calculated. For single-cell analysis, Z-stacks were captured through the conceptus and individual cells within these images were chosen for analysis. Where cell subpopulations were analysed, random selection of cells was attempted. However, not all cells within the total image were delineated clearly enough to obtain an accurate volume. Data from these cells are not included, and so a degree of non-random selection cannot be excluded. The nucleo-cytoplasmic ratio was also calculated for isolated single cells, although these are not comparable directly to those obtained through whole conceptus analysis due to problems with different levels of nuclear staining and filter corrections. Independent estimates of total conceptus volume were also made on 3D reconstructions (Fig. 1
) made from sectional data using Imaris v.3 software (BitPlane AG, Zurich, Switzerland). Gamma correction and a median filter were applied to sectional images to achieve a satisfactory 3D image. Volume estimates of the same concepti made using the Cavalieri method and 3D reconstructions did not differ significantly (P < 0.0001). 3D reconstruction using Imaris proved impossible to use reliably for calculation of the nucleo-cytoplasmic ratio, for technical reasons. Thus, the degree of variability in staining between individual nuclei prevented reconstruction adequate for measurement purposes (see Fig. 1c).
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and the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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. The total volume of concepti (excluding blastocoelic volume) increases slightly during the pre-implantation period, and can be accounted for entirely by the increasing total nuclear volume with each successive cell cycle (Fig. 3a), while whole embryo cytoplasmic volume remains constant. Mean cytoplasmic volume per cell was calculated by division from whole conceptus data and found to decrease exponentially throughout the pre-implantation period (Fig. 3b and c), consistent with cleavage divisions continuing throughout this period. The data from Fig. 3a were also used to calculate the changing mean nucleo-cytoplasmic ratio, which was found to increase during pre-implantation development (Fig 3d ). Together with data showing that cytoplasmic volume remains constant (Fig 3a), these results imply that within the conceptus as a whole, interphase growth has not occurred and that cleavage divisions have not terminated by the time of attachment.
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e; P = 0.0015). No difference was seen between mural and polar trophoblast subpopulations (P = 0.122).
Volumes and nucleo-cytoplasmic ratios from individual cells over the first seven developmental cell cycles
The averaged data from whole volume analysis suggest continuance of cleavage throughout pre-implantation development. However, the data from the late blastocyst stage indicate that nucleo-cytoplasmic ratios of subpopulations differ. To explore when such differences might emerge, direct measurement of individual blastomere volume and nucleo-cytoplasmic ratio at different stages of development was undertaken. Whole conceptus optical images were taken and individual blastomeres within these selected for cell-by-cell volumetric analysis. Concepti with total numbers approximating a serial doubling of blastomere numbers were used to reduce problems of blastomere asynchrony. Data were collected during the first seven developmental cell cycles and, from the 16-cell stage onwards, cells were categorized according to their inner and outer positions within the conceptus (Table 1). For early cell cycles (first to fourth), distribution plots of cell volumes calculated in this way were found to be normally distributed and unimodal, whereas subsequent cell cycles (fifth to seventh) were spread in an increasingly bimodal distribution. Cell volume was again found to decline by approximately half with each cell cycle (Fig. 4a), and was not significantly different to values calculated from whole conceptus analysis. Converting these data to a linear plot, it is clear that from the fifth (16-cell stage) developmental cell cycle onwards that the volume of outer cells was significantly greater than that of inner cells (P = 0.0131). A significant difference was also found in the nucleo-cytoplasmic ratio of inner and outer cells at the late blastocyst stage (P < 0.0001; Fig. 4b); the nucleo-cytoplasmic ratio of the trophoblastic population appearing to stabilize. ES cell volume (Fig. 4a) was also measured and found to be higher than that of either inner or outer cells of 3.5-day blastocysts. Within the pluriblast cell population, no difference in volume or nucleo-cytoplasmic ratio of juxtacoelic putative hypoblast precursors and deeper putative epiblast precursors was found (Fig. 4a and b). ES cell nucleo-cytoplasmic ratio (Fig. 4b) was found to be similar to that of trophoblast cells from 3.5-day blastocysts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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However, the use of averaged whole conceptus data masks variation in emergent subpopulations. The most evident subpopulations to emerge over the pre-implantation period are the trophoblast and pluriblast cell populations that make up the outer transporting epithelium and the ICM of the blastocyst respectively (Johnson & Selwood 1996, Johnson & McConnell 2004). Most cells in these two subpopulations are allocated in two waves at the 8- to 16-cell and 16- to 32-cell stages to outer and inner locations respectively (Fleming et al. 1986, Fleming 1987, Johnson & McConnell 2004). Qualitative observations on 16-cell concepti have shown that, as a population, the inner (pluriblast) cells are smaller than the outer (trophoblast) cells (Handyside 1980, Johnson & Ziomek 1981). Our quantitative analysis of individual cell volumes has confirmed these earlier observations. Thus, between the one-cell and eight-cell stages, volume distributions are unimodal, and suggest an approximate halving of cell volume with each division. At the 16-cell stage and later, there is a significant difference between the volume of inner and outer cells. By the expanding blastocyst stage, ICM cells continue to reduce in size and so become smaller than those from trophoblastic populations. These volume differences are also reflected in diverging nucleo-cytoplasmic ratios for trophoblast and pluriblast. Thus, the trophoblast nucleo-cytoplasmic ratio appears to be plateauing (Figs 3e, 4a and b). There was no significant volume difference between cells from mural and polar trophoblast subpopulations, nor between ICM cells adjacent to the blastocoele (potential hypoblast?) and those deep within the ICM (potential epiblast?; Fig. 4; Chisholm et al. 1985, Rossant 1986, Johnson & McConnell 2004).
The size differential between inner and outer cell sub-populations could arise in a number of ways. The differential could reflect an eccentricity of the cleavage planes at the 8- to 16-cell and 16- to 32-cell divisions. Qualitative observations have already suggested that this is the case (Johnson & Ziomek 1981, Ziomek et al. 1982, Reeve & Kelly 1983), and it is known that these divisions do generate cell subpopulations that differ in phenotype and potential (reviewed in Johnson & McConnell 2004). Thus, differential division is likely to provide at least part of the explanation. An alternative explanation might be that a growth increase in cytoplasmic volume occurs during the cell cycle in outer cells, indicating that cleavage ends earlier in this subpopulation. This seems to us unlikely given the overall average stability of cytoplasmic volume and the absence of net total growth, since it would require inner cells to shrink during the cell cycle. A third possibility is that at later stages some cells in each of the two subpopulations are in different developmental cell cycles and so of different sizes (assuming that non-growth cleavage divisions are indeed continuing). This is a possible explanation, given that there is an increasing asynchrony of blastomere divisions with each successive developmental cell cycle, such that cycles might overlap (Chisholm et al. 1985). In addition, there is evidence of differences in cell cycle length between inner and outer cells and of mitotic arrest at later expanded blastocyst stages (Barlow et al. 1972, Johnson & Ziomek 1981, MacQueen & Johnson 1983, Surani & Barton 1984). We have attempted to minimize the problem of overlapping developmental cell cycles by identifying for single-cell analysis concepti with exactly 16, 32 or 64 cells. However, it is possible that trophoblast cells as a population are delayed compared with pluriblast cells and so this may account for part of the size difference between them. A fourth explanation of volume differences between cell subpopulations at blastocyst stages is that from the mid-32-cell stage onwards, blastocoele expansion occurs (Smith & McLaren 1977, Chisholm et al. 1985). Fluid transport across the mural trophoblast cells (Borland et al. 1977, Wiley 1984) might lead to increases in their volume, although the presence of membrane ion channels, which regulate trophoblastic cell volume and prevent swelling or shrinkage, make this unlikely (Kolajova et al. 2001), as does the observation that there is no significant difference in volume between mural and polar trophoblast (Fig. 3e). Finally, it is possible that the difference in sizes between the two subpopulations might be an artifact of the non-random selection of cells for measurement within each subpopulation in the second part of the study. Whereas this cannot be excluded formally, it seems unlikely, since similar differentials were observed when averaged estimates from whole ICM and total trophoblast were made in the first part of the study.
Overall, the most likely interpretation of the subpopulation differentials, given the data on volumetric constancy of the whole conceptus, is that they arise initially by asymmetric division, but developmental cell cycle overlap may account for some of the later divergence. If this conclusion is correct, then cleavage has not ended for either cell sub-population by the time of attachment. It is reasonable that net growth should not occur until after a secure nutritional contact with the maternal uterine epithelium has been achieved. Interestingly, the volume of the ES cells was measured as being of the same order as day 4–5 trophoblast or pluriblast. ES cells are thought to be equivalent to the late pluriblastic population, so the observation suggests that cleavage may have neared completion by attachment.
Do data in the literature support a nucleo-cytoplasmic ratio of the order seen at the 64-cell stage? Surprisingly, there are few published studies on nucleo-cytoplasmic volumetric ratios in ‘mature’ cell types. Data derived using a fluorescence partitioning method suggest that a percentage nucleo-cytoplasmic ratio of between 18 and 32% is found in cultured cells as diverse as endothelial cells, macrophages, fibroblasts, and CV-1 and PtK2 cell lines. Moreover, this ratio range was allometric, being observed in cells of different sizes and with different cDNA contents (Swanson et al. 1991). These values approximate to the values observed around the 40-cell stage and in trophoblast cells and ES cells in culture reported here, but are lower than we observed for pluriblast cells, in which the nucleus comes to occupy a large proportion of the cell. Different measurement techniques may account for part of this difference, but without further work it cannot be excluded that multi-potential stem cells in vivo may pass through a phase of greatly reduced cytoplasmic volume.
There are two implications of our study to which we wish to draw attention. First, a role for nucleo-cytoplasmic ratio as a form of developmental timer has been proposed for a number of developmental systems (reviewed in Johnson & Day 2000) including the mouse (evidence reviewed in Day et al. 2001). The usual mechanistic model proposed is the titration of a cytoplasmic factor in the oocyte against increasing amounts of DNA (Prioleau et al. 1994). Our observations suggest that such a timing mechanism could (i) operate until attachment and possibly beyond in some or all cells of the conceptus, and (ii) operate differentially in inner and outer cells prior to implantation to activate developmental programmes at different times in the two emergent lineages, perhaps contributing to the initiation of different transcriptional patterns.
Second, the notion of what constitutes cleavage may need to be clarified. We have operated a strict definition based on absence of net growth and the parcelling out of cytoplasm into cells of decreasing size and increasing nucleo-cytoplasmic ratio. A series of other developmental characteristics has been identified in association with cleavage in a range of organisms and this association has sometimes lead to their elision into the definition of cleavage (O’Farrell 2004). For example, in Xenopus, the mid-blastula transition (MBT) occurs during a specific developmental cell cycle and is associated with characteristic changes to cell cycle length, the appearance of G1 and G2 phases, cell division asynchrony and motility, changes in protein turnover, and the initiation of transcriptional and apoptotic activity (Newport & Kirschner 1982a, 1982b, Newport & Kirschner 1984, Sible et al. 1997, Stack & Newport 1997). The MBT is often implicitly taken to mark the end of cleavage, even though further reductions in cell volume and nucleo-cytoplasmic ratio may occur. The developmental properties newly expressed at the MBT have also been taken as the mark of a non-cleavage phenotype, even though this is clearly not a straightforward case even in Xenopus (Sible et al. 1997). It is certainly not the case in the mouse since transcriptional activation, long and asynchronous cell cycles with checkpoints and G phases, cell motility and cyto-differentiation are all occurring during, and not after, the period of cleavage. Analogies between different organisms on the basis of cleavage similarities and differences may therefore require some qualification (O’Farrell 2004).
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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; R 2 = 0.2526), cytoplasmic compartment (
; R 2 = 0.0012) and nuclear compartment (grey triangles; R 2 = 0.7951) plotted against total number of nuclei per conceptus. Each point represents a single mouse conceptus (n = 86 in panels a–d). (b) Calculated mean cell volume, generated by division of each conceptus volume by the number of nuclei within it. Each point represents a single conceptus. (c) Data from (b) shown as a logarithmic plot; R 2 = 0.9651. (d) Calculated mean nucleo-cytoplasmic ratio (shown as a percentage) throughout the pre-implantation period in relation to total cell number. Each point represents a single conceptus and was generated by dividing the summed volume of nuclei by the total cytoplasmic volume; R 2 = 0.8494. (e) Nucleo-cytoplasmic ratio (shown as a percentage) from whole subpopulations within 3.5-day blastocysts. ICM data are from isolated ICMs (n = 15); trophoblast data are from intact conceptus images (n = 10 concepti for polar trophoblast and n = 15 concepti for mural trophoblast). The total cytoplasmic volume of each subpopulation from each embryo was divided by its subpopulation total nuclear volume.
; R 2 = 0.9822). Values for ES cells (
; n = 40), epiblast (
; n = 16) are also shown. (b) Mean values of nucleo-cytoplasmic ratio of individual blastomeres; n values for individual blastomeres are shown in Table 1