Histone modifications. Stacks of optical sections were collected with a ZEISS LSM700 or LSM880 Confocal Microscope and analyzed using Volocity software. Signal intensity for histone modifications was normalized to signal intensity for unmodified histones for each nucleus. Normalized values were averaged at given embryonic stages and plotted (Figs. 1D, 2, D, E, and J, 3B, and 4E, and figs. S1D and S3, C and D).

Quantitation of histone modifications by Volocity. Nuclei were identified in three dimensions using the DAPI channel, and sum signal intensity of histone modification was calculated for each nucleus in interphase. Mitotic nuclei were excluded from the analysis manually based on DAPI morphology. Mean cytoplasmic background was measured at a random point for each embryo and was used to extrapolate the sum background signal for each nucleus, which depends on nuclear volume. To calculate the total amount of background signal in the nucleus, mean cytosolic signal was multiplied by the voxel counts for each nucleus. Sum background intensity was subtracted from the sum signal intensity for that nucleus. For each nucleus, signal intensity of the histone modification was normalized to signal intensity of histones. Unmodified histones were similar between different genotypes and served as a staining control. Note that sum signal intensity for any mark positively correlates with the volume of the nucleus (36), and normalizing the sum intensity of marks to histone H3 controls for changes in nuclear volume at different stages of embryogenesis.

Transmission electron microscopy (TEM). Adult hermaphrodites were treated by high-pressure freeze fixation and freeze substitution into 2% osmium tetroxide and 0.1% uranyl acetate in acetone, rinsed in 4% distilled H2O in acetone, and then rinsed in propylene oxide before embedding in plastic resin. The adults were thin-sectioned, lengthwise onto Pioloform-coated slot grids on a diamond knife. The thin sections were counterstained with uranyl acetate and then with lead citrate (4) before examination in a Philips CM10 electron microscope. Fertilized early embryos could be found in a row within the uterus of each adult, and sample images of nuclei from each embryo were collected at high resolution with an Olympus Morada digital camera. Images were collected at intervals across serial sections of each embryo to sample many different nuclei. The digital camera offers a much greater range of contrast levels compared to EM film images, and it becomes possible to detect weakly staining objects with minimal poststaining, just by digital contrast enhancement. However, we applied similar amounts of counterstain to each sample to avoid artifacts, spurious results, or undue contrast enhancement by digital manipulations (37).

TEM image processing. Raw images were processed with Photoshop as 8-bit grayscale images. Images from different preparations were standardized for accurate comparison. The cytoplasm was used to adjust signal intensity range for each image, but image contrast was not altered. Adjusted images were then saved and quantified with ImageJ by line-scan analysis.

TEM/H3K9me3 line-scan analysis. Random lines were drawn across the center of different nuclei, and the intensity was measured. SD was calculated for each individual line. The SD of 30 lines was averaged for each strain and listed on the plot. A randomly selected line profile for a nucleus is shown as an example (Fig. 2B for TEM and Fig. 4D for H3K9me3). The SD describes the morphology of the nucleus; that is, a higher SD stems from a more punctate staining pattern that alternates between high and low values (that is, electron-dense heterochromatin and electron-lucent nucleoplasm or H3K9me3-positive and -negative regions in the nucleus).

TEM percentage of EDR. A black-and-white image was created for each nucleus in ImageJ using the thresholding function. The threshold was manually chosen to best reflect EDRs in >200-cell WT embryos and was used to quantify all nuclei (threshold, 39 to 150). The inner nuclear membrane was manually traced to define an outermost ring, and the number of black pixels versus total pixels within the ring was used to quantify %EDRs.

MET-2/ARLE-14/LIN-65 line-scan analysis. Lines that go through the center of the nucleus were drawn across the cell, and the intensity was measured. Each line had 100 bins. The intensity in each bin was averaged for 30 lines, and the average line was plotted. Error bars denote the SEM at each bin (Figs. 3G, 5, B and D, 6B, and fig. S5, C and F).

Definition and quantitation of hubs. Nuclear hubs were defined by intensity thresholding in ImageJ. The threshold was selected manually in WT nuclei at the 51- to 100-cell stage, and the same threshold was applied to all the images in a given data set. The intensity measurements for each defined hub were averaged to yield the intensity of “hubs” at given embryonic stages. “Nonhub” was defined as nuclear areas that were below the intensity threshold. The mean intensity in nonhub areas was measured for each nucleus and averaged across 30 nuclei. Noninterphase nuclei were discarded manually. For intensity measurements in the cytosol, at least four random areas in the cytosol were chosen for every cell that was in interphase. The measured intensities were averaged.

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