Author Summary Proteins that control an organism’s development must first be turned on at the proper time and place, and then turned off when they are no longer needed. One of the ‘‘off’’ signals occurs through the attachment of a small protein, known as ubiquitin, to the target protein, which typically leads to the destruction of the target. Attachment of ubiquitin is controlled by a series of enzymes, the first of which is known as E1. Most organisms have a single gene for the E1 enzyme, and its activity is crucial for the degradation of a wide range of target proteins throughout development. We have identified a temperature-sensitive mutation in the E1 enzyme of the nematode Caenorhabdi- tis elegans. By manipulating the growth temperature, we have determined the various functions of E1 at different stages of development. We find that this enzyme controls embryonic and larval development, sperm fertility, and body size. We also characterized sex-specific roles for E1; males exhibit progressive paralysis and defects in the tail, which is used for mating. In addition to the knowledge gained, this mutation provides a means of identifying both the functions of other ubiquitin enzymes during develop- ment as well as the target proteins that are marked for destruction. developmental defects such as changes in cell fate specification and embryonic lethality. Formation of the A-P axis and progression of the embryonic cell cycle requires the activities of a class of E3 complexes known as Cullin-RING ligases [21–27]. Mutations in components of the APC also affect A-P polarity, possibly as a consequence of defects in meiosis [28,29]. The E1 ubiquitin-activating enzyme lies at the apex of the enzymatic cascade, and manipulation of its activity might provide a crucial entry point for identifying the myriad roles performed by ubiquitin during development. Temperature-sensitive alleles of E1 have been identified in mammalian cell lines as cell cycle mutations that exhibit reduced ubiquitination and degradation of substrate proteins [30,31]. Similarly, a temperature-sensitive allele of E1 in yeast dramatically reduces ubiquitin conjugation and also leads to cell cycle arrest [32]. Conditional alleles have also been isolated in Drosophila in a screen for suppressors of hid- induced apoptosis during eye development [33]. Detailed characterization demonstrated the complexity of ubiquitin regu- lation in this system. Whereas weak alleles of the E1-encoding Uba1 gene block apoptosis, strong alleles promote cell cycle arrest and death. Furthermore, these pro-apototic alleles promote non- autonomous proliferation in adjacent cells via elevated levels of Notch signaling. We report here the isolation of a temperature-sensitive mutation in the C. elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Prior results for RNAi of uba-1 reported maternal sterility and embryonic lethality, with defects in meiotic progres- sion [34–36]. The uba-1(it129) mutation recapitulates these phenotypes and also reveals several novel functions, including roles in sperm fertility, body size, and sex-specific development. The uba-1(it129) mutation reduces in vivo levels of ubiquitin conjugates and causes a delay in meiotic progression in the early embryo, consistent with a reduction in E1 activity. The uba- 1(it129) mutation also demonstrates synthetic lethal interactions with known components of the anaphase-promoting complex and, as such, provides a sensitized genetic background for identifying new in vivo functions for other components of the ubiquitin cascade. UBA-1 in C. elegans Development Results Phenotypic Characterization The temperature-sensitive it129 allele was isolated by Diane Shakes and, on the basis of sperm sterility and larval lethality, was provisionally designated as spe-32 (S. Ward, pers. comm.). We have determined that spe-32 is allelic to uba-1 (see below), the sole E1 ubiquitin-activating enzyme in C. elegans, and have adopted the latter gene name for the sake of clarity. Our detailed character- ization of uba-1(it129) demonstrates a number of phenotypes, some of which are sex-specific, in addition to those mentioned above. Different phenotypes are manifested at different developmental stages (summarized in Table 1). To facilitate characterization, temperature-shift experiments were performed with age-synchro- nized populations of uba-1(it129) hermaphrodites. Adults shifted to the restrictive temperature produce dead embryos, and the number is equal to the number of progeny produced by wild- type animals at this temperature (Figure 1A). Embryonic arrest is heterogeneous, based on the variable morphology of the embryos and the broad range in the number of nuclei observed with DAPI staining (Figure 1B). Temperature shift at any stage of embryo- genesis leads to normal hatching, but 100% of the resulting larvae die at the L2 stage (data not shown). Thus, the uba-1 gene product is essential for both embryonic and larval development. Larvae that are shifted to the restrictive temperature at the L3 stage exhibit normal somatic development. However, reproduc- tion is adversely affected in the adult hermaphrodite. These sterile animals lay only unfertilized oocytes instead of embryos, but produce viable progeny when mated to wild type males, indicating that the sterility is sperm-specific (Figure 1C). Viability of these outcross progeny is high (96%), suggesting that oocyte develop- ment (which occurs subsequent to sperm production in the hermaphrodite) is largely unaffected by the mutation. Detailed characterization of the spermatogenesis defect (described below) indicates that these hermaphrodites produce appropriate numbers of morphologically normal sperm, but that the sperm are incapable of fertilization. All of the above phenotypes are fully recessive, as heterozygous hermaphrodites are indistinguishable from wild type. These phenotypes are largely though not completely rescued in uba- 1(it129) homozygous animals at the permissive temperature. There is an increase in embryonic lethality as well as a decrease in the number of embryos produced (Figure 1A), which indicates that the uba-1(it129) gene product is not fully functional at 15uC. In addition, temperature causes a small but significant (p,0.001 by Student’s t-test) difference in body size between wild-type and uba-1(it129) adults (Figure 1D). When reared at 15uC, uba-1(it129) hermaphrodites are 16% longer than wild type. The opposite Table 1. Summary of uba-1(it129) phenotypes. Stage shifted Adult Embryo L2/L3 larva doi:10.1371/journal.pgen.1000131.t001 Phenotype(s) F1 embryonic lethality Paralysis (male only) Larval lethality Sperm-specific sterility Change in body size Tail defect (male only) Paralysis (male only) PLoS Genetics | www.plosgenetics.org 2 July 2008 | Volume 4 | Issue 7 | e1000131