Calreticulin, a calcium-binding molecular chaperone, is required for stress response and fertility in Caenorhabditis elegans.

Calreticulin (CRT), a Ca(2+)-binding protein known to have many cellular functions, including regulation of Ca(2+) homoeostasis and chaperone activity, is essential for heart and brain development during embryogenesis in mice. Here, we report the functional characterization of Caenorhabditis elegans calreticulin (crt-1). A crt-1 null mutant does not result in embryonic lethality but shows temperature-dependent reproduction defects. In C. elegans CRT-1 is expressed in the intestine, pharynx, body-wall muscles, head neurons, coelomocytes, and in sperm. crt-1 males exhibit reduced mating efficiency and defects late in sperm development in addition to defects in oocyte development and/or somatic gonad function in hermaphrodites. Furthermore, crt-1 and itr-1 (inositol triphosphate receptor) together are required for normal behavioral rhythms. crt-1 transcript level is elevated under stress conditions, suggesting that CRT-1 may be important for stress-induced chaperoning function in C. elegans.

cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans.

A high frequency of apoptosis is a conserved hallmark of oocyte development. In C. elegans, about half of all developing oocytes are normally killed by a physiological germline-specific apoptosis pathway, apparently so that they donate cytoplasm to the survivors. We have investigated the functions of CGH-1, the C. elegans ortholog of the predicted RNA helicase ste13/ME31B/RCK/p54, which is germline-associated in metazoans and required for sexual reproduction in yeast. We show that CGH-1 is expressed specifically in the germline and early embryo, and is localized to P granules and other possible mRNA-protein particles. cgh-1 is required for oocyte and sperm function. It is also needed to prevent the physiological germline apoptosis mechanism killing essentially all developing oocytes, making lack of cgh-1 function the first stimulus identified that can trigger this mechanism. We conclude that cgh-1 and its orthologs may perform conserved functions during gametogenesis, that in C. elegans certain aspects of oocyte development are monitored by the physiological germline apoptosis pathway, and that similar surveillance mechanisms may contribute to germline apoptosis in other species.

Every sperm is sacred: fertilization in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is an attractive model system for the study of fertilization. C. elegans exists as a self-fertilizing hermaphrodite or as a male. This unusual situation provides an excellent opportunity to identify and maintain sterile mutants that affect sperm and no other cells. Analysis of these mutants can identify genes that encode proteins required for gamete recognition, adhesion, signaling, fusion, and/or activation at fertilization. These genes can also provide a starting point for the identification of additional molecules required for fertility. This review describes progress in the genetic and molecular dissection of fertilization in C. elegans and related studies on sperm competition.

Sperm competition in the absence of fertilization in Caenorhabditis elegans.

Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal that sperm competition within a hermaphrodite does not require successful fertilization. However, sperm competition does require normal sperm motility. Additionally, sperm competition is not an absolute process because oocytes not fertilized by male-derived sperm can sometimes be fertilized by hermaphrodite-derived sperm. These results indicate that outcrossed progeny result from a wild-type cross because male-derived sperm are competitively superior and hermaphrodite-derived sperm become unavailable to oocytes. The sperm competition assays described in this study will be useful in further classifying the large number of currently identified mutations that alter sperm function and development in C. elegans.

The C. elegans spe-9 gene encodes a sperm transmembrane protein that contains EGF-like repeats and is required for fertilization.

In the nematode worm C. elegans, individuals with mutations in the spe-9 gene produce spermatozoa with wild-type morphology and motility that cannot fertilize oocytes even after contact between gametes. Therefore, disruption of spe-9 function affects either gamete recognition, adhesion, signaling, and/or fusion. The spe-9 gene encodes a sperm transmembrane protein with an extracellular domain that contains ten epidermal growth factor-like repeats. A common feature of proteins that include epidermal growth factor-like motifs is their involvement in extracellular functions such as adhesive and ligand-receptor interactions. Additionally, the overall structure of the predicted SPE-9 protein is similar to that of ligands for the Notch/LIN-12/GLP-1 family of transmembrane receptors. These results suggest that SPE-9 functions in the specialized cell-cell interactions required for fertilization.

Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling.

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Here, we report the identification of several genes that are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate.

Dpbx, a new homeobox gene closely related to the human proto-oncogene pbx1 molecular structure and developmental expression.

Recently, a new class of homeodomain containing proteins, pbx1, pbx2, and pbx3 has been described. pbx proteins are most closely related to two yeast regulatory proteins, a1 and alpha 2. Here, we identify and characterize the pbx homolog in Drosophila, designated Dpbx. Dpbx is 95% identical to the pbx proteins within the homeodomain and, more remarkably, is 85% to 88% identical within a 201 amino acid region adjacent to the homeodomain. Cytologically, the Dpbx gene is located on the X chromosome at 14A. mRNA expression is both maternal and zygotic and occurs throughout the life cycle. Prior to full germband retraction, Dpbx is rather ubiquitously present and variations are minor. The most notable feature of Dpbx expression is that after germband retraction, high levels of Dpbx are observed in the anterior portion of the ventral nerve cord.

Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonized by extramacrochaetae.

The spatially restricted activities of achaete (ac) and scute (sc) are thought to define proneural clusters of potential sensory organ precursor cells in the imaginal discs of Drosophila. These genes encode transcriptional regulators of the basic helix-loop-helix (bHLH) class. We have found that direct, positive transcriptional autoregulation by the ac protein and cross-regulation by sc are essential for high-level expression of the ac promoter in the proneural cluster pattern and that autoactivation is important for the bristle-promoting function of the ac gene. These auto- and cross-regulatory activities are antagonized in a dose-dependent manner by the inhibitory HLH protein encoded by the extramacrochaetae (emc) gene. We have found that emc is expressed in the wing imaginal disc in a pattern strongly complementary to that of the proneural clusters. Our results indicate that emc plays an essential early role in defining territories of bristle-forming potential.

A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents.

The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents. Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pI of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg++ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50-100 mM. Nuclease 3 exhibits a temperature optimum of 42 degrees C and is insensitive to N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)