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. 2016 Sep 29;5:e11475. doi: 10.7554/eLife.11475

Figure 1. Oct4 DNA-bound active fraction controls zebrafish gastrulation.

(a) Schematic diagram of fluorescence correlation spectroscopy (FCS). GFP-tagged nuclear protein is localized in the nucleus of embryonic cells. Fluorescence molecules diffuse through a confocal volume (<1 μm3) within a single-cell nucleus and generate fluctuating fluorescence intensity. The autocorrelation function (ACF) of the fluctuation is fit to obtain the absolute protein concentration (C) and the diffusion coefficient (D), where N is the number of molecules. Scale bar: 10 μm. (b) Lateral view of pou5f3 morphant embryos expressing GFP-Oct4 rescued by GFP-Oct4 mRNA at the blastula [3.5 hr post-fertilization (hpf)] and gastrula (7 hpf) stages and non-rescued embryos (arrested) at the blastula stage. The non-rescued embryos also express GFP-Oct4 but remain at the blastula stage and do not develop further. Scale bar: 200 μm. (c) ACF of the intensity traces of GFP-Oct4 and GFP-Oct4ΔHD in rescued and non-rescued embryos at the blastula stage. The ACF were fit by two-component anomalous diffusion model. Curves are normalized to compare differences in protein activity (indicated by arrows). (d) Raw data of residuals from fit curves shown in c. (e) Concentration and DNA-bound fraction levels derived from the FCS measurements in c. Values represent the mean ± SEM of data from three to five independent experiments (n = 39–125 cell nuclei from 10 to 15 embryos ****p<0.0001; **p<0.01). n.s. over bars indicates non-significant differences. See also Figure 1—figure supplements 1–3, Figure 1—source data 1 and Materials and methods.

DOI: http://dx.doi.org/10.7554/eLife.11475.003

Figure 1—source data 1. Quantification of GFP-Oct4 concentration and activity in zebrafish rescued and non-rescued embryos.
Values for concentration and diffusion parameters were derived from the analysis of FCS data with the ACFs fit by two-component anomalous diffusion model. D1, D2: Diffusion coefficients of the fast and slow diffusion components, respectively. F2: Slow component fraction. α1, α2: anomalous parameters of the fast and slow diffusion components, respectively. Values represent mean ± SEM of data from three to five independent experiments (n represents the number of cell nuclei from 10 to 15 embryos; ****p<0.0001; **p<0.01; *p<0.05). Details of the rescue and the FCS analyses are shown in Figure 1—figure supplements 1–3 and Material and methods.
DOI: http://dx.doi.org/10.7554/eLife.11475.004
elife-11475-fig1-data1.docx (85KB, docx)
DOI: 10.7554/eLife.11475.004

Figure 1.

Figure 1—figure supplement 1. GFP-Oct4 rescues zebrafish Pou5f3 function.

Figure 1—figure supplement 1.

(a) Phenotypes of pou5f3 knockdown using morpholino (MO) antisense. Arrows show a constriction in the interface between the yolk and the blastoderm, prohibiting gastrulation in embryos arrested at the blastula stage after MO injection. Embryos not arrested show zygotic phenotype: they do not develop the mid–hindbrain boundary (*MHB) at the 24-hpf stage. (b) Relative percentages of different phenotypes according to the dose of pou5f3 MO injected. Phenotypes are expressed as a percent of whole (n = 150–400 embryos per condition). As a control, a mismatch MO sequence was injected resulting in 100% of embryos with wt phenotype. 100% of embryos were arrested at the blastula stage using 8 ng of MO. Lower-dosed 4-ng and 2-ng embryos continued through gastrulation but did not develop the MHB, according to the percentages shown. (c) Phenotypes of rescued embryos. Rescue of maternal pou5f3 function, as shown by restoring normal gastrulation. Rescue of maternal-zygotic pou5f3 function, as shown by MHB formation at 24 hpf. (d and e) Relative percentages of (d) maternal and (e) maternal-zygotic rescue according to different amounts of injected mRNA. Phenotypes of the rescued embryos are expressed as a percent of whole (n = 170–400 embryos per condition).
DOI: http://dx.doi.org/10.7554/eLife.11475.005
Figure 1—figure supplement 2. One- and two-component anomalous diffusion model for GFP and GFP-Oct4.

Figure 1—figure supplement 2.

(a) Autocorrelation function (ACF) of free GFP fit by two- and one-component anomalous diffusion models. Two-component anomalous diffusion model converged to effective one-component anomalous diffusion for a real one-component system, such as free GFP. (b) Raw data of residuals from fit curves shown in a. (c) Quantitative parameters derived from the ACFs. F1: Fast component fraction. D1, D2: Diffusion coefficients of the fast and slow diffusion components, respectively. α1, α2: anomalous parameters of the fast and slow diffusion components, respectively. I/N: Ratio of mean intensity extracted directly from the time traces and the mean number of molecules in the focus extracted from the fit, referred to as molecular brightness. n: number of cells. (d) The GFP-Oct4-free diffusion coefficient (D1) determined by global fitting and averaging individual fits yields provides similar diffusion time, tauD1. Values represent mean ± SEM of data from three independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.11475.006
Figure 1—figure supplement 3. Oct4 concentration and DNA-bound active fraction in embryos rescued with different amount of GFP-Oct4 mRNA.

Figure 1—figure supplement 3.

(a) Autocorrelation function (ACF) of the intensity traces of GFP-Oct4 by the two-component fit anomalous diffusion model in rescued embryos with different amounts of GFP-Oct4 mRNA at blastula (oblong; 3.5 hpf) and gastrula (60% epiboly; 7 hpf) stages. Double-headed arrow in the graph indicates the difference in the amplitude of the curves, which corresponds to the difference in protein concentrations. (b) Raw data of residuals from fit curves shown in a. (c, d) Concentration (c) and DNA-bound fraction (d) derived from the FCS measurements in a. Changes in the GFP-Oct4 concentration at oblong stage in rescued embryos were concordant with the proportion of mRNA injected. However, the Oct4 DNA-bound fraction did not significantly change with varying concentrations. Concentration and Oct4 DNA-bound fraction remained similar in rescued embryos at 60% epiboly. Values represent mean ± SEM of data from three independent experiments (n > 35 cell nuclei from 10 to 15 embryos; **p<0.01). n.s. over bars indicates non-significant differences. See also Figure 1—source data 1.
DOI: http://dx.doi.org/10.7554/eLife.11475.007