Picture from Weeks JC et al. (2016) MicroPubl Biol "Microfluidic EPG Recordings Show Striking Pharyngeal Pumping Phenotype in a C. ...."

Reviewed by: Chris Link; Nicole Liachko.

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure 6. Immunolocalization of DAO-5 during mitosis. A) DAO-5 is not detected in mitotic cells of wildtype embryos (asterisk). B) In ncl-1 mutants, weak and diffuse nucleoplasmic labeling is observed from prophase (asterisk) to telophase (arrows). Putative remnants of DAO-5-positive nucleoli can be detected in prophase. C) DAO-5 reassociates with forming nucleoli in late telophase/early G1. Bars: 5 um.

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

A) DAO-5 is not detected in mitotic cells of wildtype embryos (asterisk).

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure 2. Co-localization of DAO-5 with rDNA or fibrillarin. Immunolabeling and DNA FISH were performed sequentially on the same sample. Single optical section of part of a 30-cell embryo showing the rDNA signal (A), the DAO-5 signal (B) and the merged image (C). Shown in D is a 3D reconstruction of part of the sample. Note the presence of occasional DAO-5-positive bodies in the nucleoplasm of embryonic nuclei (arrow). E-H) Immunostaining was performed for DAO-5 and FIB-1 in early embryos. Single optical section (thickness of 800 nm) showing the DAO-5 signal (in red, E) in the nucleus of a 2-cell embryo. FIB-1 signal (in green, F) on the same optical section. Overlay of the DAO-5 and FIB-1 signals (G, scale bar: 5 mm). Intensity profiles (H) along the lines shown in E (DAO-5, red) and in F (FIB-1, green).

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure S5 Co-localization of DAO-5 and FIB-1. A, D) Single optical sections (thickness of 800 nm) of the DAO-5 signal (red) in nuclei of 2-cell embryos. B, E) FIB-1 signal (green) on the same section. C, F) Overlay of the DAO-5 and FIB-1 signals. The vast majority of signals co-localize, but occasional foci are labeled by only one of the nucleolar markers (arrows in D and E). G) DAO-5 staining (red) in a 3 mm slice through part of a 28-cell embryo. H) FIB-1 staining in the same slice. I) Overlay of the DAO-5 and FIB-1 signals, which co-localize completely. Note that not all nuclei are complete in this rendering of part of a 28-cell embryo and that, as a consequence, not every nucleus displays 2 nucleoli. Bars: 5 um.

Picture from Gandhi, Jay et al. (2021) MicroPubl Biol

Figure 1. In dhc-1; mel-28 double mutants, proximal oocytes often fail to mature: (A) Immunolocalizations of phosphorylated histone H3 and DAO-5 in proximal gonads captured using structured illumination. The dotted line outlines the proximal gonad, and the -1 oocyte is the right-most oocyte in all panels. In the merged images, blue indicates DAPI staining, red indicates phosphorylated histone H3, and green represents DAO-5. Arrowheads indicate DAO-5-positive nuclei in the merged images. Scale bar = 10 μM. (B) Frequency of DAO-5-positive nuclei in the four most proximal oocytes in each strain. (C) Frequency of nuclei with phosphorylated histone H3 in the four most proximal oocytes in each strain. We used a Fisher's Exact test to compare each mutant with the wild-type at the equivalent oocyte position. *=p<0.05, ** =p<0.01, ***=p<0.001. (D) The number of oocytes scored at each position for each strain.

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure 1. Immunolocalization of nucleolar markers in embryonic and adult nuclei. A, B) In the adult gonad, prominent nucleoli are easily detected in all nuclei (arrows), except in those of the most mature oocytes. Accordingly, only a faint nucleoplasmic DAO-5 signal (in red) can be detected in these late oocytes (arrowhead). C, D) At the 2-cell stage, the DAO-5 and FIB-1 antibodies label numerous dots of varying size and intensity throughout the nucleoplasm. E,F) The first appearance of distinct nucleoli (arrows) occurs at the 6- to 8-cell stage. Shown here are projections of parts of 8-cell embryos. G) At later stages, all somatic cells display one, or in most cases two DAO-5-labeled nucleoli. Shown here is a 2.5 mm slice from a 45- cell embryo. H) On this single optical section of adult intestinal nuclei, DAO-5 is clearly found in ring-like structures in a DNA-poor region. On a projection of all optical sections, numerous dot-like DAO-5-positive structures are seen throughout the nucleoplasm (inset). In this and other figures, the contour of the labeled embryos is dotted and DNA is counterstained with DAPI (in blue). Bars: 5 um.

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure S4 A substantial pool of DAO-5 is found in the nucleoplasm of embryonic nuclei. The DAO-5 signal on a single representative optical section (here from a 45-cell embryo, thickness of 2 um) was segmented and the pixel number and mean pixel intensity was measured for each segment. The source 16-bit image is shown in A. The mean background pixel intensity was also measured in an area of similar size outside of the nucleus (light blue in the segmented image shown in B). The mean background value is 1306. The signal-to-noise ratio is 2.2 in the nucleoplasm (green) and 13.2 and 14.9 in the two nucleoli (red). The sum total intensity of the DAO-5 signal in the nucleoplasm represents 48% of the total signal intensity measured in the nucleus.

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure 8. Ultrastructural immunolocalization of DAO-5 in high-pressure frozen and cryo-substituted embryos. A) In early embryos (~10-20-cell stage), the nucleolus (arrow) appears as an electron-dense structure with no obvious internal organization. B) Post-embedding immunolabeling of DAO-5 confirms that the protein is associated with the nucleolus. C) In slightly later embryos (~30-40-cell stage), initial signs of granular structures (arrowheads) are found at the periphery of the nucleolus. The DAO-5 signal appears to be excluded from this region. D) A section showing two granular nucleoli in the nucleus of a ,30-40-cell stage. E) Higher magnification of the region boxed in D. The arrow points to the nucleolus. F) In yet older embryos (~60-80-cell stage), the segregation of granular structures (arrowheads) at the periphery of the nucleolus is more pronounced. li, lipid droplet; mi, mitochondria; nm, nuclear membrane; ri, ribosomes. Bars: A, D and F, 500 nm; B, C and E, 200 nm.

Picture from Korekova D et al. (2012) PLoS One "Nucleologenesis in the Caenorhabditis elegans embryo."

Figure S3 Immunolocalization of nucleolar markers in whole embryos at or shortly before gastrulation. A) DAO-5 (in red) in a 26-cell embryo. B) FIB-1 (in red) in a 24-cell embryo. DNA is counterstained with DAPI (blue). The entire embryos were scanned by laser scanning confocal microscopy. Shown for each marker are representative maximal projections of top and bottom parts of the embryo (approximately 10 mm in thickness). Mitotic cells are not labeled (asterisks). One nucleus (arrows, that of the germ cell precursor, see text) fails to show the typical nucleolar staining observed in surrounding cells. Images were scanned at the same magnification (pixel size of 95 nm695 nm). Nuclei appear somewhat smaller after immunostaining for FIB-1 due to slight shrinking during acetone fixation (for DAO-5, samples were fixed with formaldehyde in 1X PBS). Bar: 5 um.