UBA-1 in C. elegans Development Figure 5. In situ hybridization of gonads. Panels on the left show the uba-1 expression pattern in dissected gonads. Panels on the right show nuclear morphology by DAPI staining. From top to bottom, gonads are from adult hermaphrodites during oogenesis, adult males during spermatogenesis, fem-1(hc17) adult hermaphrodites that produce only oocytes, and fem-3(q23) adult hermaphrodites that make only sperm. At least 20 gonads were examined for each genotype or sex. DT, distal tip of gonad; P, pachytene region. doi:10.1371/journal.pgen.1000131.g005 multicellular embryonic lethality resulting from reduced APC function. This phenotype is produced by hypomorphic mutations in APC components or by synthetic interactions between some pairs of temperature-sensitive alleles (i.e., each single mutation has no effect at the permissive temperature, whereas the combination of both mutations causes maternal embryonic lethality) [29]. Since UBA-1 and APC function in the same enzymatic cascade, mutations in both might likewise exhibit a synthetic interaction. Therefore, we tested the uba-1(it129) allele in combination with APC components. Double mutants of uba-1(it129) with either the APC subunit mat-3(or180) [19] or the APC activator fzy-1(h1983) [53] resulted in maternal embryonic lethality at the permissive temperature (Table 3). Early embryogenesis was examined in uba-1(it129) adult hermaphrodites shifted to 25u for defects in meiotic progression or A-P polarity in the first cell division. An oma-1::GFP transgene was used to allow visualization of embryonic polarity [54]. In wild- type hermaphrodites, OMA-1::GFP protein is evenly distributed throughout the cytosol and excluded from the intact pronuclei of the one-celled embryo. Our observations at 25uC indicate that the protein is also concentrated on the sperm centrioles and mitotic spindle. Ubiquitin-mediated proteolysis at the first cell division degrades the bulk of OMA-1::GFP. The protein is absent in the anterior (A) cell of the two-celled embryo, and the remaining OMA-1::GFP becomes associated with P granules in the posterior (P) cell. In uba-1(it129) animals, the pattern of OMA-1::GFP in one-celled embryos is indistinguishable from wild-type. OMA- 1::GFP degradation during the first cell division is likewise identical, and the protein persists only in the P cell. However, progression of the zygote through the first division is slower than normal for uba-1(it129) embryos. The delayed progression leads to an increase in the number of one-celled embryos within the uterus, which is easily visualized by the presence of OMA-1::GFP (Figure 7A). Wild-type hermaphrodites typically contain a single one-celled embryo in each arm of the gonad; in contrast, uba- 1(it129) mutants possess an average of three one-celled embryos per gonad arm (Figure 7B). In addition, 35% of the uba-1(it129) hermaphrodites contained a crushed zygote within the uterus. Formation of the rigid eggshell is completed late in meiosis, so these crushed zygotes might be either an indirect consequence of the observed meiotic delay or indicate a structural requirement for ubiquitination in the embryo immediately following fertilization. The delay in progression through the first embryonic division was examined in greater detail. Upon fertilization, the oocyte nucleus completes the first and second meiotic divisions. The oocyte and sperm pronuclei meet and fuse, then undergo the first mitotic division. The percentage of embryos observed at each of these stages (meiosis, pronuclear migration and fusion, and mitosis) was determined for wild-type and uba-1(it129) animals. The fraction of one-celled embryos in the meiotic and pronuclear stages was equivalent in wild type, but approximately two-fold higher in the meiotic stage for uba-1(it129) embryos (Figure 7C), suggesting that meiosis is acutely sensitive to UBA-1. Despite this significantly skewed distribution (p,0.001 by Pearson’s chi-square PLoS Genetics | www.plosgenetics.org 8 July 2008 | Volume 4 | Issue 7 | e1000131