Oct4 kinetics predict cell lineage patterning in the early mammalian embryo

Journal name:
Nature Cell Biology
Volume:
13,
Pages:
117–123
Year published:
DOI:
doi:10.1038/ncb2154
Received
Accepted
Published online
Corrected online

Abstract

Transcription factors are central to sustaining pluripotency, yet little is known about transcription factor dynamics in defining pluripotency in the early mammalian embryo. Here, we establish a fluorescence decay after photoactivation (FDAP) assay to quantitatively study the kinetic behaviour of Oct4, a key transcription factor controlling pre-implantation development in the mouse embryo. FDAP measurements reveal that each cell in a developing embryo shows one of two distinct Oct4 kinetics, before there are any morphologically distinguishable differences or outward signs of lineage patterning. The differences revealed by FDAP are due to differences in the accessibility of Oct4 to its DNA binding sites in the nucleus. Lineage tracing of the cells in the two distinct sub-populations demonstrates that the Oct4 kinetics predict lineages of the early embryo. Cells with slower Oct4 kinetics are more likely to give rise to the pluripotent cell lineage that contributes to the inner cell mass. Those with faster Oct4 kinetics contribute mostly to the extra-embryonic lineage. Our findings identify Oct4 kinetics, rather than differences in total transcription factor expression levels, as a predictive measure of developmental cell lineage patterning in the early mouse embryo.

At a glance

Figures

  1. Selective photoactivation in live mouse embryos allows imaging of Oct4-paGFP kinetic behaviours.
    Figure 1: Selective photoactivation in live mouse embryos allows imaging of Oct4–paGFP kinetic behaviours.

    (a) Schematic diagram of photoactivation of paGFP (green) within a defined volume (grey sphere) using 820 nm light. (b) Photoactivation of cytoplasmic (cytopl) paGFP within a small ROI (red circle) localized to one cell of a live 8-cell stage embryo. Following photoactivation paGFP fluorescence spreads throughout the cytoplasm of the photoactivated cell. (c) Photoactivation of Oct4–paGFP within a ROI localized to a single cell nucleus of the embryo. (d) Schematic representation of the FDAP assay. Following photoactivation within a single cell nucleus, Oct4–paGFP fluorescence (green) is tracked in four dimensions with confocal time-lapse imaging. (e) FDAP analysis. The photoactivated Oct4–paGFP (green in simplified model, left) is initially in the nucleus and then moves from the nucleus to the cytoplasm. The parameters kin, kout and kdeg are the rates of Oct4–paGFP import into the nucleus from the cytoplasm, export from the nucleus to the cytoplasm, and the rate of overall degradation within the entire cell, respectively. 1−μ denotes the immobile fraction of nuclear Oct4-paGFP. Theoretical time development of the average Oct4-paGFP fluorescence profile (I/Io; normalized to the initial level of nuclear Oct4–paGFP fluorescence) in a FDAP experiment. (f) Representative FDAP time-lapse images showing Oct4–paGFP fluorescence over time in the cell nucleus photoactivated in c. Scale bar, 10 μm.

  2. Oct4-paGFP kinetic behaviours identify two cell populations in the mouse embryo.
    Figure 2: Oct4–paGFP kinetic behaviours identify two cell populations in the mouse embryo.

    (a) Examples of a pre-compaction embryo showing two photoactivated cell nuclei. Scale bar corresponds to 15 μm. (b, c) FDAP curves show average fluorescence intensity of Oct4–paGFP within the cell nuclei photoactivated in a over time. Green lines are exponential fits to fluorescence data. (d) FDAP curves for Oct4–paGFP obtained from several cell nuclei of pre-compaction embryos combining 4-cell stage and 8-cell stage data. Cells show two distinct kinetic behaviours, divided into clusters 1 and 2. (e) FDAP curves for Oct4ΔHD–paGFP at pre-compaction stages. (f) FDAP curves for Oct4–paGFP in cells that overexpress Sox17 at pre-compaction stages. (g) Quantification of Oct4–paGFP nuclear export, import and immobile fraction. Cl 1, cluster 1; Cl 2, cluster 2; ΔHD, Oct4ΔHD–paGFP; Sox17, Sox17 overexpressed. In d–g, Cl 1, n = 11; Cl 2, n = 5; ΔHD, n = 6; Sox 17, n = 15. Asterisks show statistically significant differences (export, P < 0.015; import, P < 0.006, immobile P < 1 × 10−5). Error bars show standard deviations.

  3. Oct4-paGFP kinetics are uncorrelated to total fluorescence in the cell nucleus.
    Figure 3: Oct4–paGFP kinetics are uncorrelated to total fluorescence in the cell nucleus.

    Relation between the initial level of Oct4–paGFP fluorescence within the cell nucleus and the export rate, import rate and immobile fraction values obtained using FDAP analysis for single cells from Fig. 2d. The initial values of Oct4–paGFP fluorescence are unrelated to any of the kinetic parameters.

  4. Oct4-paGFP kinetics predict patterning of inside and outside cells.
    Figure 4: Oct4–paGFP kinetics predict patterning of inside and outside cells.

    (a) Schematic representation of the experimental strategy used. Following FDAP analysis and photoactivation of a membrane-targeted paGFP, embryos undergo compaction and the pattern of cell division is determined. (b) Examples of two pre-compaction embryos (left panels) show Oct4–paGFP fluorescence following photoactivation of single cell nuclei, and mpaGFP fluorescence following photoactivation of part of the membrane. The FDAP curves show the distinct Oct4–paGFP kinetic behaviours in each cell. Following compaction and division (right panels), cell 1 (top row) divided asymmetrically, generating one inside and one outside cell. Cell 2 (lower row) divided symmetrically, generating two outside cells. Surface rendered three-dimensional views confirm that the inside cell is buried within the embryo whereas outside cells have part of their surface exposed to the outer region. A membrane-targeted RFP (pink) marks half of the cells in these embryos. The FDAP curves on the right show the distinct Oct4–paGFP kinetic behaviours in each daughter cell of the original photoactivated cell. Scale bar, 15 μm. (c) Quantification of symmetric and asymmetric divisions undertaken following compaction by cells that showed the FDAP profiles of clusters 1 and 2 at pre-compaction stages. (d) FDAP curves show Oct4–paGFP kinetic behaviours in several inside and outside cells at post-compaction stages. (e) Quantification of Oct4–paGFP nuclear export, import and immobile fraction. Asterisks show statistical significant differences (export, P = 0.0012; import, P = 1.4737 × 10−4, immobile P = 1.4154 × 10−5). Error bars show standard deviations. In d, e, inside cells, n = 9; outside cells, n = 6.

  5. Schematic illustration of Oct4-paGFP kinetics and cell lineage allocation in the early mouse embryo.
    Figure 5: Schematic illustration of Oct4–paGFP kinetics and cell lineage allocation in the early mouse embryo.

    Different accessibility of Oct4 DNA binding sites among cells, possibly due to a differential chromatin structure, the presence of an excess of another factor that blocks access or the absence of a cofactor required for high-affinity binding, results in segregation of Oct4–paGFP kinetic properties before lineage allocation. Cells with slower kinetics and a large immobile fraction divide more frequently in an asymmetric manner during the 8- to 16-cell transition, contributing more cells to the pluripotent cell lineage, whereas cells with faster kinetics and a small immobile fraction contribute more cells to the extra-embryonic lineage through symmetric divisions.

Change history

Corrected online 28 January 2011
In the version of this article initially published online and in print, the values for kout and kin in table 1 were incorrect. This error has been corrected in both the HTML and PDF versions of the article.

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Affiliations

  1. Division of Biology, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125, USA.

    • Nicolas Plachta,
    • Shirley Pease,
    • Scott E. Fraser &
    • Periklis Pantazis
  2. Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

    • Tobias Bollenbach
  3. Present address: Institute of Science and Technology Austria, Am Campus 1, A-3400 Klosterneuburg, Austria.

    • Tobias Bollenbach

Contributions

T.B. performed the quantitative analysis. S.P. performed microinjections into mouse embryos. N.P., S.E.F. and P.P. designed and N.P. and P.P. carried out all other experiments.

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The authors declare no competing financial interests.

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