Nuclear F-actin detected by phalloidin in fixed zebrafish early embryos. Zebrafish embryos were fixed at the 32-, 128-, 1000-cell, and sphere stage, and stained with Acti-stain™ 555 phalloidin and Hoechst. Low (A) and high (B) power views of single confocal sections are shown. (C) Relative intensity profile plots of lines indicated in B. The intensity is normalized using the average cytoplasmic intensity. Phalloidin signals were clearly observed in nuclei of the 32-, 128-, and 1000-cell stages. Scale bars: 50 μm (A), 10 μm (B).

Nuclear F-actin detected by specific probes in living zebrafish early embryos. (A) Schematic of visualizing nuclear F-actin in living zebrafish embryos. Zebrafish embryos were injected with fluorescent probes, including those for actin and chromatin, and mounted on a glass-bottomed dish in agarose gel, facing the animal pole down. Time-lapse z-stack (typically up to 100 μm deep) images were collected using an inverted confocal microscope with the indicated objective lenses. In the later stages, nuclei within ∼70 μm (sphere) and ∼50 μm (dome) from the bottom (mostly the surface and second layers) are applied for quantitative analysis. (B) F-actin in living zebrafish embryos. Zebrafish embryos were injected with UtrCH-sfGFP as an F-actin probe and Cy5-labeled Fab for H3K9ac as a chromatin marker. Every 90 s, 25 z-stack fluorescence images with 4 μm intervals were acquired using a confocal microscope. Single z-sections are displayed. Accumulation of UtrCH-sfGFP in nuclei is clearly observed in the eight- to the 1000-cell stage embryos (arrowheads). Insets show the zoomed images of nuclei indicated by red arrowheads. See also Movie 1. Scale bar: 100 μm.

Nuclear F-actin levels increased in the late interphase. Zebrafish embryos were injected with UtrCH-sfGFP and H3K9ac Fab-Cy5, and images were acquired using the same procedure as described in Fig. 2B. (A) Single confocal sections of a nucleus at different cell stages are depicted with the time (min) after the onset of the interphase. (B) The nucleus to cytoplasmic (N/C) intensity ratios of UtrCH-sfGFP were measured from three independent experiments. One embryo was analyzed in each experiment. The mean values of UtrCH-sfGFP N/C ratios with the standard deviations (s.d.) and the number of nuclei, as identified by H3K9ac Fab-Cy5, are plotted. N/C ratios of UtrCH-sfGFP are above 1.0 (dashed line) up to the 1000-cell stage. (C) The N/C intensity ratios of UtrCH-sfGFP in the enveloping layer (dashed line) and deeper layer (solid line) cells of the 512-cell (orange) and sphere-(blue) stage embryos. Mean values with the standard deviations (s.d.) from five nuclei of each are shown. The N/C ratios of UtrCH-sfGFP at the peak in the 512-cell stage embryos were not significant (P=0.25). The right panel shows the max intensity projection image from four z-slices with 4 μm intervals of the 512-cell stage embryo just before NEBD. Some nuclei in the enveloping and deeper layer cell are shown by arrowheads and arrows, respectively. Scale bars: 20 μm (A), 100 μm (C).

LifeAct-sfGFP accumulates in nuclei in early-stage embryos. Zebrafish embryos were injected with LifeAct-sfGFP as an actin probe, H3S28ph Fab-Cy3 as a mitotic chromosome marker, and H3K9ac Fab-Cy5 as a chromatin marker. Every 90 s, 25 z-stack fluorescence images with 4 μm intervals were acquired using a confocal microscope. (A) Single confocal sections of a nucleus at the 512-cell stage (2.75 hpf) are shown. LifeAct-sfGFP accumulated in the nucleus up to 12 min and disappeared at 13.5 min, when H3S28ph was observed on condensed chromosomes. Scale bar: 20 μm. (B) Interphase nuclei were selected based on a low H3S28ph Fab signal (N/C ratio <2.2). The numbers of nuclei with different ranges of LifeAct-sfGFP N/C ratio in every time point are plotted as cumulative bars. After the 1000-cell stage, the number of nuclei with LifeAct-sfGFP N/C ratio <1.0 increased. A representative result from three independent experiments/embryos is shown.

F-actin that accumulated in the nucleus remained there after nuclear envelope breakdown (NEBD). Zebrafish embryos were injected with UtrCH-sfGFP as an F-actin probe, TMR-labeled 155-kDa dextran to monitor NEBD, and JF646-LANA as a chromatin marker. Every 12.5 s, confocal sections were acquired during the 1000-cell (A) and the high (B) stages. UtrCH-sfGFP accumulated in nuclei (A and B, −37.5 s to −12.5 s) and remained in the vicinity of chromosomes even after NEBD (A and B, 0 s to 37.5 s). Representative images of one of the two embryos analyzed are shown. (C) N/C intensity ratio of UtrCH-sfGFP and TMR-Dextran were measured (n=10 cells) at the 512-cell, 1000-cell, and high stages. The mean values were shown with the s.d. Nuclear actin levels increased at the late interphase to prophase (from −50 s to 0 s) with a peak at around NEBD (0 s). After NEBD, UtrCH-sfGFP gradually decreased from chromatin (from 0 s to 50 s) during the prometaphase. Scale bars: 20 μm.

Actin patches remained in the vicinity of chromosomes during prometaphase and disappeared at the metaphase in the 1000-cell stage embryos. Zebrafish embryos were injected with UtrCH-sfGFP, TMR-tubulin, and JF646-LANA. Every 15 s, confocal sections were acquired. A representative nucleus from three independent experiments/embryos is shown. (A) Single sections of UtrCH-sfGFP, TMR-tubulin, and JF646-LANA at the 1000-cell stage embryo, and their merged and magnified images are shown. Relative intensity profile plots of lines are indicated at the bottom. The intensity is normalized using maximum and minimum intensity. UtrCH-sfGFP signals are located by chromosomes. (B) The surface rendering three-dimensional images are shown (green: UtrCH-sfGFP, magenta: JF646-LANA, cyan: TMR-Tubulin). UtrCH-sfGFP accumulated in the nucleus (−15 s) remained in the vicinity of condensing chromosomes after NEBD (from 0 s to 15 s) and disappeared before the chromosomes were aligned at the metaphase (60 s). Just after NEBD (0 s and 15 s), TMR-Tubulin does not appear to reach chromosomes when UtrCH-sfGFP patches are around. When UtrCH-sfGFP patches partially disappear (15 s and 30 s), TMR-Tubulin appears to capture condensing chromosomes (15 s and 30 s). See also Movie 4 for a large area view and Movie 5 for a magnified view. Scale bars: 20 μm. See Figs. S3, S4, and S5 for the high, sphere, and dome stages.

Transcription inhibition delayed the nuclear actin decrease after the 1000-cell stage. (A) Zebrafish embryos were injected with α-amanitin to inhibit RNA-polymerase II-mediated transcription, or the vehicle (PBS), with Cy3-labeled morpholino antisense oligonucleotide for miR-430 transcripts as a transcription marker, and JF646-LANA as a chromatin marker. Every 90 s, 25 z-stack images with 4 μm intervals were acquired using a confocal microscope. Seven slices at each time point from the 128-cell to dome stage were depicted and the max intensity projection images are shown. (B) Embryos were grown to the 1000-cell stage and oblong stage before fixation and staining with Hoechst and Acti-stain™ 555 phalloidin. Single confocal sections are shown with N/C ratios of phalloidin intensity (N=10 cells from three embryos). In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles;×indicates the means; and data points are plotted as closed circles (PBS, blue; α-amanitin, orange). P-values obtained with a Student's t-test (unpaired, two-tailed) are also shown (*P<0.05). At the 1000-cell stage, clear phalloidin signals are observed in the nuclei of both embryos treated without or with α-amanitin. At the oblong stage, phalloidin signals are not concentrated in the nucleus of embryos without α-amanitin but are still observed in the nucleus of α-amanitin-treated embryos. (C) Zebrafish embryos were injected with α-amanitin, or PBS, and then with UtrCH-sfGFP and H3K9ac Fab-Cy5. Every 90 s, 25 z-stack fluorescence images with 4 μm intervals were acquired. The mean values of N/C ratios and the number of nuclei (control: blue, +α-amanitin: orange) are plotted with the s.d. from the 128-cell to dome stage for two independent experiments/embryos. α-amanitin-injected embryos showed the higher N/C ratio of UtrCH-sfGFP value in later stages (sphere and dome). Single confocal sections at the sphere stage are shown on the right. UtrCH-sfGFP remained accumulated in some nuclei during the sphere stage (orange arrowheads). Scale bars: 10 μm (A) and 20 μm (B and C).