Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy

Abstract

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotencyassociated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERGassociated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems.

Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo

“…While the constricting daughter becomes more cuboid, the daughter cell that remains outside adopts a wedge-like shape ( Figure 1F). Our segmented embryos contain 2.9 ± 0.3 inner cells on average by the 16-cell stage (n = 33 embryos), in line with previous work (Kaur et al, 2013;Morris et al, 2010;Plachta et al, 2011). A similar proportion of cells is seen to undergo apical constriction using different membrane labeling approaches ( Figures S2A-S2E).…”

Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis

“…The obtained intensity values correspond to the average number of fluorescent particles diffusing over time in the defined volume. A recent study investigated the dynamics of individual Oct4-paGFP as well as other pluripotency-related TFs in vivo using FCS (Kaur et al, 2013). Interestingly, the results obtained from the sub-space observation of a limited amount of TF diffusion rates were consistent with the previously performed population analysis of TF kinetics using FDAP (Kaur et al, 2013).…”

Lineage specification in the mouse preimplantation embryo

“…Thus, outer cells at the 16-cell stage have higher OCT4 motility than inner cells. The low OCT4 motility is likely to be due to OCT4 binding to DNA; a truncated mutant form of OCT4 that lacks the DNA-binding homeodomain shows only high motility (Plachta et al, 2011;Kaur et al, 2013). Surprisingly, high and low OCT4 motility can be already distinguished in the blastomeres of 4-and 8-cell embryos (Plachta et al, 2011).…”