2011 Jul 15
Lineage Mapping the Pre-Implantation Mouse Embryo by Two-Photon Microscopy, New Insights Into the Segregation of Cell Fates
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Lineage Mapping the Pre-Implantation Mouse Embryo by Two-Photon Microscopy, New Insights Into the Segregation of Cell Fates
The first lineage segregation in the pre-implantation mouse embryo gives rise to cells of the inner cell mass and the trophectoderm. Segregation into these two lineages during the 8-cell to 32-cell stages is accompanied by a significant amount of cell displacement, and as such it has been difficult to accurately track cellular behavior using conventional imaging techniques. Consequently, how cellular behaviors correlate with cell fate choices is still not fully understood. To achieve the high spatial and temporal resolution necessary for tracking individual cell lineages, we utilized two-photon light-scanning microscopy (TPLSM) to visualize and follow every cell in the embryo using fluorescent markers. We found that cells undergoing asymmetric cell fate divisions originate from a unique population of cells that have been previously classified as either outer or inner cells. This imaging technique coupled with a tracking algorithm we developed allows us to show that these cells, which we refer to as intermediate cells, share features of inner cells but exhibit different dynamic behaviors and a tendency to expose their cell surface in the mouse embryo between the fourth and fifth cleavages. We provide an accurate description of the correlation between cell division order and cell fate, and demonstrate that cell cleavage angle is a more accurate indicator of cellular polarity than cell fate. Our studies demonstrate the utility of two-photon imaging in answering questions in the pre-implantation field that have previously been difficult or impossible to address. Our studies provide a framework for the future use of specific markers to track cell fate molecularly and with high accuracy.
Copyright © 2011 Elsevier Inc. All rights reserved.
Two-photon live imaging of pre-implantation development. (A) 3D reconstruction of mouse embryos expressing H2B-GFP (green) imaged by TPLSM in IMARIS software at different time-points during the 8, 16, 32, and 32-cell blastocyst stages. Z resolution 2 μm, 820 nm wavelength. Each 8-cell nucleus is marked by a ball of a distinct color, which is also used to mark all of that 8-cell’s progeny. (B) P12 mouse pups born from embryo transfer after being imaged by TPLSM for 48 hours from an 8 to 32/64-cell stage under the same conditions as in (A).
Three cell populations in 16-cell embryos identified by lineage tracing (A) Numbers and percentages for each of the three 16-cell stage cell types. Outer 16-cell parents account for 72.3% of all 16-cell stage cells, while intermediate parents constitute 21.3% and inner parents 6.3%. 16-cell stage outer cells divide symmetrically to produce two TE 32-cell daughters 81.9% of the time, and the remaining 18.1% give rise to one TE and one transient-outer, putative PE daughter cell. Intermediate cells found at the 16-cell stage divide to give rise to asymmetric outer and inner 32-cell daughters 68.18% or entirely inner progeny 31.82% of the time. By contrast, inner 16-cell stage cells only give rise to inner daughters at the 32-cell stage (100% of the time).. (B) Example of a lineage graph depicting the position of a cell’s nuclei from the center of the embryo. Radial distance index (RDI) plotted for each time-point over the course of the entire time-lapse (see Materials and Methods). RDI 0 represents the center of the embryo. The lineage in this example follows a single 8-cell parent (red line) into two 16-cell daughters (blue lines) and 4 32-cell granddaughters (pink and green lines). This particular lineage gave rise to two outer TE cells (pink) and two inner ICM cells (green). (C) The radial distance index (RDI) was calculated for each cell at each time point during the 16-cell stage from the first frame in which the 16-cell appeared after the 4
th cleavage to the first frame when the cell undergoes the 5 th cleavage. The RDI is calculated as the distance of each nucleus from the center of the embryo, 0 (see Materials and Methods). The RDI for each time point for each cell during the 16-cell stage was then grouped according to that cell’s lineage: outer, transient-outer, asymmetric and symmetric intermediate cells, and inner cells as determined by lineage tracing and visual analysis, resulting in a dot-plot of all cell positions for each time point during the 16-cell stage for each category. There is a significant difference in RDI between outer cells and the intermediate cells, as well as inner and intermediate cells (p-value <0.0001). (D) Lineage trees for the 8–32 cell stage. Four different lineages starting from the 8-cell stage are described in the text, giving rise to both inner and outer progeny by the 32-cell blastocyst stage. n refers to the number of cells analyzed in each category.
Intermediate cells in 16-cell stage embryos have outer surface exposure and low levels of Cdx2 expression. (A) Cell surface exposure versus RDI. Cells with outer membrane exposure based on phalloidin staining of actin are grouped according to their RDIs. Cells with an RDI between 2.3 and 1.56 have 100% exposure, RDIs between 1.55 and 1.0 are exposed 72.19% of the time, and RDIs of less than 1.0 are exposed 22.46% of the time. n refers to the number of embryos analyzed. Error bars represent standard error of the mean (SEM). Significantly higher percentages of cells with RDI between 1.55-1.0 are exposed than those with RDI of less than 1.0, p-value <0.0001. (B) Examples of intermediate cells with surface exposure. Four examples of individual 16-cell stage embryos are shown with Alexa Fluor 568 Phalloidin in red and DAPI staining in blue. Spots represent nuclear positions and embryos are cut with orthogonal slices in the IMARIS program for easier visualization. Yellow spots denote nuclei in intermediate positions whose cell-surface is highlighted using a transparent green layover. The RDI of each yellow-marked cell nucleus is as follows 1) 1.37, 2) 1.25, 3) 1.19, and 4) 1.38. Scale bar = 10 μm. (C) Cdx2 expression versus nuclear position. The relative Cdx2 expression of a cell’s closest, outer-most neighbor is compared between cells with an RDI of 2.3-1.56; mean of 0.985, 1.55-1.0; mean 0.413, and RDI values less than 1.0; mean 0.384. n refers to the number of cells analyzed in each category. Error bars represent SEM. Expression levels between RDIs of 2.3-1.56 and 1.55-1.0 are significantly different, p-value <0.0001. n refers to the number of cells analyzed.
Cell cycle length and cell lineage. (A) A graph of average cell-cycle length as grouped by cell lineage. For each 16-cell the cell cycle time was calculated as the time between the start frame in which the 16-cell first split from its 8-cell parent during the 4
th cleavage and the end frame in which the 16-cell split into two 32-cell daughters during the 5 th cleavage. 16-cell stage cells were grouped according to their lineage; Int. to O/I refers to 16-cell intermediate cells that give rise to outer and inner 32-cell progenies. Int. to I/I refers to 16-cell intermediate cells that give rise to two inner 32-cell progenies. I to I/I refers to 16-cell inner cells that give rise to two inner 32-cell progenies. O to O/O refers to 16-cell outer cells that give rise to two outer cell progenies. O to O/T-O refers to 16-cell outer cells that give rise to an outer and a transient outer cell progenies. Lineages for each bar are shown to the right of the graph. n refers to the number of cells analyzed in each category. Error bars represent SEM. p-value = 0.0147. (B) Cell division order as grouped by lineage type at the 5 th cell division. 16-cell parents that divided to give rise to two outer 32-cell daughters (O/O) divided early in the 5 th cleavage, while divisions from 16-cell parents that resulted in two ICM (I/I) progeny occurred later. 16-cell outer cells that divide into an outer TE cell and a transient-outer cell (O/T-O) that occupies the ICM at the 32-cell stage again behave like outer cells and divide earlier than inner or bi-potent intermediate cells (O/I). (C) Cell division order as grouped by lineage type at the 4 th cell division. The cleavage order was determined by following the order in which cells during the 4 th and 5 th cleavages divided and grouping them according to lineage. During the 4 th cleavage 8-cell parents which gave rise to two outer 16-cell daughters (O/O) preferentially divided before other cells in the embryo, while the cleavage order of 8-cell parents which divided to give rise to outer and a bi-potent intermediate daughters (O/Int.) was more evenly distributed. 8-cell parents that would give rise to inner and outer 16-cell daughters (O/I) tended to divide later than other cells in the embryo.
Cell division angle correlates with cell polarity but not cell fates. (A) The cell division angle was determined as described in the Materials and Methods. Briefly, the more symmetric a division (tangential to the surface of the embryo) the closer the cell division angle will be to 0º. An asymmetric division, with one cell moving away from the center of the embryo and one toward, will have a cell division angle closer to 180º. (B) The division angle as grouped by the cell lineage for the 5
th cleavage. Degrees in-between 0° and 180° represent all the angles taken by cells dividing during the 16- to 32-cell stage. The division angle of each dividing cell is then grouped based on its lineage. During the 5 th cleavage the division angles are shown for 16-cell parents that are outer, outer 16-cells that will give rise to transient-outer, and intermediate and inner 16-cell parents. The outer cells have a tendency of dividing symmetrically as compared to the other three16-cell populations, p-value <0.0001. (C) The division angle as grouped by the cell lineage for the 4 th cleavage. During the 4 th cleavage 8-cell parents that divide to produce outer and inner (O/Inner), outer and intermediate (O/Int) or two outer 16-cell daughters (O/O). The 8 cells that divide to give two outer cells (O/O) have a tendency to divide symmetrically as compared to the two other types of divisions. p-value < 0.0001. Red dashed lines denote the mean for each group, while SEM bars are in blue. (D) Evidence of cell sorting after asymmetric cell division. Lineage graph of an entirely outer-lineage based on the radial distance index (RDI) of cell nuclei over time from the 8-cell parent to 32-cell progeny. At the 8–16 cell division (open arrow) there is a clear asymmetric division of the 16-cell daughters as indicated by the large difference in RDI between the starting frames of the 16-cell daughters. During the 16-32 cell division, the 32-cell daughters indicated by the pink line again divide asymmetrically (solid arrow). In this case, one 32-cell daughter falls inward close to the center of the embryo (dotted arrow) for nearly 40 frames (~4.5 hours), but then sorts back to the surface of the embryo and occupies a position in the TE post-cavitation. n refers to the number of cells analyzed in each category.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Cell Differentiation / physiology
Cell Lineage / physiology
Embryonic Development / physiology
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Image Processing, Computer-Assisted
Microscopy, Fluorescence / methods
Green Fluorescent Proteins