CLS-2 first appeared on the centrosomes at prophase in some embryos. By prometaphase, centrosome staining was evident in most embryos and the signal also entered the nucleus. CLS-2 localized to the kinetochore interfaces and on the spindle at metaphase in one-cell embryos, in addition to the centrosomes. Spindle staining persisted into anaphase, especially in the mid spindle region; anaphase spindle staining was not reported in C. elegans mitotic embryos previously, but was shown for meiotic spindles. Centrosome staining also persisted through anaphase and into telophase. All embryos also showed cytoplasmic staining which was absent after RNAi depletion. In multicellular embryos, interphase cells showed strong cytoplasmic staining that appeared enriched at cell contacts but did not appear to colocalize with regions of the cells showing the densest tubulin staining. Mitotic cells in multicellular embryos exhibited centrosome, kinetochore and spindle staining through at least the 28-cell stage.
Asymmetric expression of imb-2 in AWCON was also observed in our initial assessment of the imb-2 expression pattern using imb-2 fosmid::GFP in which we fused GFP with the C terminus of IMB-2 by homologous recombination, that rescued imb-2(vy10) mutants.
NOS-2 protein is expressed sequentially during embryogenesis. First detected expression of NOS-2 in the 1-cell stage; at this stage, NOS-2 was present uniformly throughout the embryo. No NOS-2 expression was detected in 2- to 20-cell embryos. NOS-2 expression reappeared in the 28-cell stage in the cytoplasm of the germline blastomere P4. In some embryos, NOS-2 staining appeared to be concentrated in a few perinuclear foci. NOS-2 expression continued in P4 and its two daughters Z2 and Z3 until approximately the 200-cell stage. NOS-2 levels decreased sharply in later stages and were undetectable by the 550-cell stage.