We have identified a homolog of the mammalian p53 tumor suppressor protein in the nematode Caenorhabditis elegans that is expressed ubiquitously in embryos. The gene encoding this protein, cep-1, promotes DNA damage-induced apoptosis and is required for normal meiotic chromosome segregation in the germ line. Moreover, although somatic apoptosis is unaffected, cep-1 mutants show hypersensitivity to hypoxia-induced lethality and decreased longevity in response to starvation-induced stress. Overexpression of CEP-1 promotes widespread caspase-independent cell death, demonstrating the critical importance of regulating p53 function at appropriate levels. These findings show that C. elegans p53 mediates multiple stress responses in the soma, and mediates apoptosis and meiotic chromosome segregation in the germ line.

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. We report that the MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. We find that SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. Our results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network.

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Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele ofzen-4, an MKLP1 homologue in the nematodeCaenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

The molecular pathways that link nutritional cues to developmental programs are poorly understood. Caenorhabditis elegans hatchlings arrest in a dormant state termed "L1 diapause" until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. We report that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation.

. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities are most striking between the precursors of proteinase A and human lysosomal cathepsin D; these similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

Specification of the endoderm precursor, the E cell, in Caenorhabditis elegans requires a genomic region called the Endoderm Determining Region (EDR). We showed previously that end-1, a gene within the EDR encoding a GATA-type transcription factor, restores endoderm specification to embryos deleted for the EDR and obtained evidence for genetic redundancy in this process. Here, we report molecular identification of end-3, a nearby paralog of end-1 in the EDR, and show that end-1 and end-3 together define the endoderm-specifying properties of the EDR. Both genes are expressed in the early E lineage and each is individually sufficient to specify endodermal fate in the E cell and in non-endodermal precursors when ectopically expressed. The loss of function of both end genes, but not either one alone, eliminates endoderm in nearly all embryos and results in conversion of E into a C-like mesectodermal precursor, similar to deletions of the EDR. While two putative end-1 null mutants display no overt phenotype, a missense mutation that alters a residue in the zinc finger domain of END-3 results in misspecification of E in approximately 9% of mutant embryos. We report that the EDR in C. briggsae, which is estimated to have diverged from C. elegans approximately 50--120 myr ago, contains three end-like genes, resulting from both the ancient duplication that produced end-1 and end-3 in C. elegans, and a more recent duplication of end-3 in the lineage specific to C. briggsae. Transgenes containing the C. briggsae end homologs show E lineage-specific expression and function in C. elegans, demonstrating their functional conservation. Moreover, RNAi experiments indicate that the C. briggsae end genes also function redundantly to specify endoderm. We propose that duplicated end genes have been maintained over long periods of evolution, owing in part to their synergistic function.

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