Nucleologenesis in the Caenorhabditis elegans Embryo

In the Caenorhabditis elegans nematode, the oocyte nucleolus disappears prior to fertilization. We have now investigated the re-formation of the nucleolus in the early embryo of this model organism by immunostaining for fibrillarin and DAO-5, a putative NOLC1/Nopp140 homolog involved in ribosome assembly. We find that labeled nucleoli first appear in somatic cells at around the 8-cell stage, at a time when transcription of the embryonic genome begins. Quantitative analysis of radial positioning showed the nucleolus to be localized at the nuclear periphery in a majority of early embryonic nuclei. At the ultrastructural level, the embryonic nucleolus appears to be composed of a relatively homogenous core surrounded by a crescent-shaped granular structure. Prior to embryonic genome activation, fibrillarin and DAO-5 staining is seen in numerous small nucleoplasmic foci. This staining pattern persists in the germline up to the ∼100-cell stage, until the P4 germ cell divides to give rise to the Z2/Z3 primordial germ cells and embryonic transcription is activated in this lineage. In the ncl-1 mutant, which is characterized by increased transcription of rDNA, DAO-5-labeled nucleoli are already present at the 2-cell stage. Our results suggest a link between the activation of transcription and the initial formation of nucleoli in the C. elegans embryo

Immunolocalization of DAO-5 in <i>ncl-1</i> mutants.

<p>In <i>ncl-1</i> mutants, well-formed DAO-5-positive nucleoli can be detected at the 4-cell stage (B and D arrows), which is never the case in wildtype embryos (A and C). Note that DAO-5-positive structures persist into prophase in early <i>ncl-1</i> mutants (asterisks in D). At later stages (here 14-cell), nucleoli are bigger in <i>ncl-1</i> mutants (F) than in wildtype (E). DAO-5 labeling describes a ring-like structure in the mutant strain. Bars: 5 µm.</p

Co-localization of DAO-5 with rDNA or fibrillarin.

<p>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 µm). Intensity profiles (H) along the lines shown in E (DAO-5, red) and in F (FIB-1, green).</p

Immunolocalization of nucleolar markers in embryonic and adult nuclei.

<p>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 µm 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 µm.</p

Immunolocalization of DAO-5 during mitosis.

<p>A) DAO-5 is not detected in mitotic cells of wildtype embryos (asterisk). B) In <i>ncl-1</i> 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 µm.</p

Distinct localization of nucleolar markers in the early germline.

<p>In each image, DAO-5 (A–C) or FIB-1 (D–F) is in red; PGL-1, a marker of the germline, is in green; DNA is in blue. A, D) In the 4-cell embryo, the distribution of nucleolar markers is dotted in both somatic and germ cells. B, E) At later stages (here around 25-cell), the distribution of nucleolar markers remains dotted only in the unique germline precursor. C, F) In yet older embryos, which contain two germ cell precurors (Z2 and Z3), nucleolar markers are found in distinct nucleoli in each of these cells. Note that all images are maximal projections centered on the germ cell nuclei and that, as a consequence, not all somatic nuclei are complete. Bars: 5 µm.</p

Radial positioning of DAO-5 in embryonic nuclei.

<p>The distance between each DAO-5 positive voxel and the nearest nuclear border was measured for 37 embryonic nuclei. Three patterns were observed. Representative nuclei are shown. A) Both nucleoli in the interior of the nucleus (n = 5, 13%, nucleoli pseudo-colored in green). B) One nucleolus at the nuclear periphery and the other in the interior (n = 7, 19%, nucleoli pseudo-colored in yellow). C) Both nucleoli at the periphery (n = 25, 68%, nucleoli pseudo-colored in red). D) For each pattern, mean distances (± standard deviation of the mean) were plotted in bins of 0.3 µm using the color scheme described above. The nuclear periphery is arbitrarily set on the right-hand side of the graph.</p

Detection of pre-rRNA molecules in early embryos.

<p>RNA FISH was performed using a probe against ITS1 (signal in green). DNA is counterstained with DAPI (blue). Asterisks indicate the remnants of the polar bodies. A dotted-like distribution of RNA FISH signals was never observed. Images A, C and D were overexposed to reveal weak signals (also revealed upon overexposure are probe aggregates non specifically bound to the eggshell of the embryo). A) A 2-cell embryo with both cells in S-phase shows no signal, even upon overexposure. B, C) Two-cell embryos with cells at different stages of mitosis display weak signals only upon overexposure (panel C). Note the absence of signal in anaphase (arrow in C). D) The difference between nuclei in S-phase (negative) and in prophase (arrows, positive) is clearly seen in this 4-cell embryo. E) At later timepoints (here a ∼80-cell embryo), robust signals are detected in the nucleoli (compare B and E, same exposition). F) No signal is detected after RNAse treatment.</p

Ultrastructural immunolocalization of DAO-5 in high-pressure frozen and cryo-substituted embryos.

<p>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.</p