Journal: Biology Open

Article Title: Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo

doi: 10.1242/bio.059783

Figure Lengend Snippet: 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).

Article Snippet: After fixation, embryos were washed three times with PBS and stained with 2 μg/ml Hoechst 33342 (Nacalai Tesque, 04929-82) and 20 nM Acti-stain™ 555 phalloidin (Cytoskeleton, PHDH1-A) in PBS containing 0.05% Triton X-100 at 4°C overnight in the dark.

Techniques: Staining

Journal: Biology Open

Article Title: Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo

doi: 10.1242/bio.059783

Figure Lengend Snippet: 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).

Article Snippet: After fixation, embryos were washed three times with PBS and stained with 2 μg/ml Hoechst 33342 (Nacalai Tesque, 04929-82) and 20 nM Acti-stain™ 555 phalloidin (Cytoskeleton, PHDH1-A) in PBS containing 0.05% Triton X-100 at 4°C overnight in the dark.

Techniques: Inhibition, Injection, Labeling, Marker, Microscopy, Staining, Two Tailed Test, Fluorescence