Fig. 2

Oct4 and Nanog bind in mesendoderm of zebrafish blastula embryos.

(a) Schematic location of the presumptive mesendoderm (ME) and ectoderm (EC) in an embryo at oblong stage [3.5 hr post-fertilization (hpf)]. Scale bar: 200 µm. (b) Schematic diagram of FLIM-FRET (Fluorescence Lifetime Microscopy-Forster Resonance Energy Transfer). GFP lifetime (t1) of the donor is reduced if an acceptor (mCherry) is in close proximity (1-10 nm); this is the reduced lifetime (t2). If the acceptor is not in close proximity (>10 nm) to the donor, donor lifetime remains unchanged (t2 similar to t1). Lifetimes are measured with time-correlated single-photon counting. (c) Lifetime values and FLIM images of GFP-Oct4 alone and in the presence of a linked mCherry protein in single cells. Scale bar: 10 µm. In the same graph, lifetime values and FLIM images of GFP-Oct4ΔHD alone and co-expressing mCherry-Nanog in single nuclei. Scale bar: 5 µm. (d) Lifetime values and FLIM images of GFP-Oct4 lifetime alone and in the presence of mCherry-Nanog in the nucleus of individual cells from mesendoderm or ectoderm. Scale bar: 5 µm. The percentage of binding is indicated at the top right corner of the FLIM images. Values represent the median and quartile ranges of data from three to five independent experiments (n = 20-40 cell nuclei from 10 to 15 embryos; ****p<0.0001). n.s. over bars indicates non-significant differences. See also Figure 2 - figure supplements 1-4, Figure 2 - source data 1 and 2.