Abstract
Setting aside pluripotent cells that give rise to the future body is a central cell fate decision in mammalian development. It requires that some blastomeres divide asymmetrically to direct cells to the inside of the embryo. Despite its importance, it is unknown whether the decision to divide symmetrically versus asymmetrically shows any spatial or temporal pattern, whether it is lineage-dependent or occurs at random, or whether it influences the orientation of the embryonic-abembryonic axis. To address these questions, we developed time-lapse microscopy to enable a complete 3D analysis of the origins, fates and divisions of all cells from the 2- to 32-cell blastocyst stage. This showed how in the majority of embryos, individual blastomeres give rise to distinct blastocyst regions. Tracking the division orientation of all cells revealed a spatial and temporal relationship between symmetric and asymmetric divisions and how this contributes to the generation of inside and outside cells and thus embryo patterning. We found that the blastocyst cavity, defining the abembryonic pole, forms where symmetric divisions predominate. Tracking cell ancestry indicated that the pattern of symmetric/asymmetric divisions of a blastomere can be influenced by its origin in relation to the animal-vegetal axis of the zygote. Thus, it appears that the orientation of the embryonic-abembryonic axis is anticipated by earlier cell division patterns. Together, our results suggest that two steps influence the allocation of cells to the blastocyst. The first step, involving orientation of 2- to 4-cell divisions along the animal-vegetal axis, can affect the second step, the establishment of inside and outside cell populations by asymmetric 8- to 32-cell divisions.
🔬 Techniques
🔭 Microscopes
💻 Software
✨ Fluorophores
🧪 Sample Preparation
🏭 Microscope Brands
💻 Software Details
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📋 Methods
Generation of 4D movies F1
(C57BL/6xCBA) females were mated with males expressing EGFP-H2B ( Hadjantonakis and Papaioannou, 2004 ). 2-cell embryos were collected in M2 medium and then cultured in KSOM ( Piotrowska-Nitsche et al., 2005 ). Time-lapse imaging was performed using a Zeiss Axiovert microscope, Hamamatsu camera and Kinetic-imaging software.
Fluorescence and DIC
Z-stacks were collected every 15 min, on 15 different planes for each time point, from 2-cell to blastocyst stage. Obtained PICT files were converted to TIF using VisBio (LOCI, Wisconsin) and ImageJ (NIH, Bethesda).
Analysis of 4D movies
All cells were followed manually using SIMI Biocell software ( Schnabel et al., 1997 ). 3D coordinates of nuclei were saved on average every two to three frames and analysed as described in Results. All cell tracing was carried out blindly, before assigning embryos to sub-groups, and cross-checked by two researchers. Cell divisions were classified as symmetric or asymmetric for all 8- and 16-cell blastomeres by scoring the position of daughter cells relative to the embryo surface one frame after and one before the next division in both DIC and fluorescence (see also Results). The timing of development was assessed as the period between successive 2 nd to 5 th cleavages. To describe the relative position of blastomeres in the 2- to 4-cell cleavage we measured the angle between their apposing planes 15 min after division of the second blastomere using SIMI Biocell. We rotated the 3D representation of the embryo to look laterally at the axis defined by the daughters of the first 2- to 4-cell division and read the angle between this axis and that of the second 2- to 4-cell division ( Fig. 4A ). We calculated the distance between polar body (PB) and the centre of the two daughter cells during division in pixels using SIMI Biocell ( Fig. 4B ). Descendants of meridionally dividing blastomeres (M) were positioned equidistantly from the PB. After equatorial/oblique division (E), only one of the two daughter cells touched the PB and the distances between daughters and the PB differed by approximately one cell diameter (≈25-35 pixel). From the 80 cavitated embryos, we analysed 66. Embryos were excluded because M and E divisions occurred synchronously, or the PB did not stay attached before the 2 nd cleavage, or the movie ended before cavitation.
Show full methods section
Generation of 4D movies F1
(C57BL/6xCBA) females were mated with males expressing EGFP-H2B ( Hadjantonakis and Papaioannou, 2004 ). 2-cell embryos were collected in M2 medium and then cultured in KSOM ( Piotrowska-Nitsche et al., 2005 ). Time-lapse imaging was performed using a Zeiss Axiovert microscope, Hamamatsu camera and Kinetic-imaging software.
Fluorescence and DIC
Z-stacks were collected every 15 min, on 15 different planes for each time point, from 2-cell to blastocyst stage. Obtained PICT files were converted to TIF using VisBio (LOCI, Wisconsin) and ImageJ (NIH, Bethesda).
Analysis of 4D movies
All cells were followed manually using SIMI Biocell software ( Schnabel et al., 1997 ). 3D coordinates of nuclei were saved on average every two to three frames and analysed as described in Results. All cell tracing was carried out blindly, before assigning embryos to sub-groups, and cross-checked by two researchers. Cell divisions were classified as symmetric or asymmetric for all 8- and 16-cell blastomeres by scoring the position of daughter cells relative to the embryo surface one frame after and one before the next division in both DIC and fluorescence (see also Results). The timing of development was assessed as the period between successive 2 nd to 5 th cleavages. To describe the relative position of blastomeres in the 2- to 4-cell cleavage we measured the angle between their apposing planes 15 min after division of the second blastomere using SIMI Biocell. We rotated the 3D representation of the embryo to look laterally at the axis defined by the daughters of the first 2- to 4-cell division and read the angle between this axis and that of the second 2- to 4-cell division ( Fig. 4A ). We calculated the distance between polar body (PB) and the centre of the two daughter cells during division in pixels using SIMI Biocell ( Fig. 4B ). Descendants of meridionally dividing blastomeres (M) were positioned equidistantly from the PB. After equatorial/oblique division (E), only one of the two daughter cells touched the PB and the distances between daughters and the PB differed by approximately one cell diameter (≈25-35 pixel). From the 80 cavitated embryos, we analysed 66. Embryos were excluded because M and E divisions occurred synchronously, or the PB did not stay attached before the 2 nd cleavage, or the movie ended before cavitation.
Supplementary Material Movie 1 Movie 2 Supplementary Material
📊 Figures
Fig. 1
4D analysis of early mouse development
Lineage generated with SIMI Biocell. Merges of 3D representations and DIC images from 2-cell-stage to blastocyst are shown (2-cell-stage descendants are coloured red or blue).
Fig. 2
Blastocysts show distinctive clonal patterns
( A-D ) Embryos were analysed using the centres of gravity of the clones made up of the descendants of the 8-cell-stage blastomeres. ( A ) Merge of DIC and 3D representation of a blastocyst (Colouring...
Fig. 3
Model for the generation of blastocyst pattern
The 32-cell embryo consists of two clones derived from 2-cell blastomeres, which show an arrangement reminiscent of a u201cbaseballu201d. Based on the arrangement of 2-cell-stage clones there are thre...
Fig. 4
The influence of the animal-vegetal axis on the generation of different blastocyst patterns
( A )-( C ) Classification of embryos according to sequence and orientation of second cleavage divisions. ( A ) To measure the angle (u03b1) between the division planes of the 2-cell blastomeres (whit...
Fig. 5
Analysis of division orientation
Analysis of division orientation at the 4 th and 5 th cleavage divisions. ( A ) Percentage of asymmetric and symmetric divisions in 4 th and 5 th cleavage (average u00b1 SEM). ( B ) Analysis of cell d...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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