NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans.

BACKGROUND: The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins. By binding regulatory elements in the 3' untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions. In C. elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis. RESULTS: We report here that FBF and NANOS-3 (NOS-3), one of three C. elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays. We have delineated the portions of each protein required for this interaction. Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3). The roles of the three nos genes overlap during germ-line development. In certain nos-deficient animals, the hermaphrodite sperm-oocyte switch was defective, leading to the production of excess sperm and no oocytes. In other nos-deficient animals, the entire germ line died during larval development. This germ-line death did not require CED-3, a protease required for apoptosis. CONCLUSIONS: The data suggest that NOS-3 participates in the sperm-oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA. NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF. The three C. elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival. We propose that this may have been the primitive function of nanos genes.

Repression by the 3' UTR of fem-3, a sex-determining gene, relies on a ubiquitous mog-dependent control in Caenorhabditis elegans.

The fem-3 sex-determining gene is repressed post-transcriptionally via a regulatory element in its 3' untranslated region (UTR) to achieve the switch from spermatogenesis to oogenesis in the Caenorhabditis elegans hermaphrodite germ line. In this paper, we investigate the fem-3 3' UTR control in somatic tissues using transgenic reporter assays, and we also identify six genes essential for this control. First, we find that a reporter transgene bearing a wild-type fem-3 3' UTR is repressed in somatic tissues, whereas one bearing a mutant fem-3 3' UTR is derepressed. Moreover, control by mutant 3' UTRs is temperature sensitive as predicted from the temperature sensitivity of the fem-3 gain-of-function (gf) mutations. Secondly, we find a fem-3 3' UTR RNA-binding activity in somatic tissues, in addition to the previously reported germ-line-specific binding by FBF. Thirdly, we find that each of six genes, mog-1-mog-6, is required for repression by the fem-3 3' UTR. Therefore, the mog genes not only affect the sperm/oocyte switch in the germ line, but also function in somatic tissues. We suggest that the mog genes may encode components of a ubiquitous machinery that is used for fem-3 3' UTR-mediated repression and the sperm/oocyte switch.

Notch/LIN-12 signaling: transduction by regulated protein slicing.

Intercellular signaling through the Notch/LIN-12 transmembrane receptors regulates growth and differentiation during animal development. Moreover, defects in the conserved Notch/LIN-12 pathway are linked to human diseases. Here, we review models for two key steps in Notch/LIN-12 signaling: ligand-mediated activation of the receptor and receptor-mediated activation of transcription. Ligand binding appears to permit proteolysis of the receptor; as a result, the receptor's intracellular domain can enter the nucleus and function as a transcriptional co-activator.

Genes required for GLP-1 asymmetry in the early Caenorhabditis elegans embryo.

The translation of maternal glp-1 mRNA is regulated both temporally and spatially in the early Caenorhabditis elegans embryo (T. C. Evans, S. L. Crittenden, V. Kodoyianni, and J. Kimble, Cell 77, 183-194, 1994). To investigate the control of embryonic glp-1 expression, we have examined the distribution of GLP-1 protein in selected maternal effect mutants that affect pattern or fate in the early embryo. We find that mutants that disrupt anterior-posterior asymmetry in the early embryo (par-1-par-6, emb-8, Par(q537)) disrupt the spatial but not temporal control of GLP-1 expression: GLP-1 is observed at the normal stage of embryogenesis in par-like mutants; however, it is uniformly distributed. In contrast, mutants that alter blastomere identity (skn-1, pie-1, mex-1, apx-1) do not affect the normal GLP-1 pattern. We conclude that genes controlling the asymmetry of cellular components, including P granules, also control GLP-1 asymmetry in the early embryo. The finding that mutants that disrupt anterior-posterior asymmetry translate GLP-1 in all blastomeres suggests that loss of embryonic asymmetry causes translational activation of GLP-1 in the posterior.

GLP-1 is localized to the mitotic region of the C. elegans germ line.

In C. elegans, germline mitosis depends on induction by the somatic distal tip cell (DTC) and on activity of the glp-1 gene. Using antibodies to GLP-1 protein, we have examined GLP-1 on western blots and by immunocytochemistry. GLP-1 is tightly associated with membranes of mitotic germline cells, supporting its identification as an integral membrane protein. Furthermore, GLP-1 is localized within the germ line to the mitotic region, consistent with the model that GLP-1 acts as a membrane receptor for the distal tip cell signal. Unexpectedly, GLP-1 and the zone of mitosis extend further than the DTC processes. We present three models by which the DTC may influence GLP-1 activity and thereby determine the zone of mitosis. The spatial restriction of GLP-1 appears to be controlled at the translational level in hermaphrodites. We suggest that down-regulation of GLP-1 may be required to effect the transition from mitosis into meiosis.

Evaluation and management of sialorrhea of pregnancy with concomitant hyperemesis.

This article describes two gravid patients who presented with first-trimester sialorrhea and hyperemesis. Although excessive salivation, especially when accompanied by protracted nausea and vomiting, is an unusual occurrence, it can have serious consequences for both the mother and fetus when left untreated. Initially, phenothiazine was prescribed and later belladonna alkaloid was added separately to the two treatment regimens. In order to successfully treat the excessive salivation, it was necessary to control the nausea and vomiting. Eradication of sialorrhea and hyperemesis were effected 10 days posttreatment. For both patients, pregnancy, delivery, and postpartum intervals proceeded uneventfully. Mothers and infants remain in good health 2 years posttreatment.

Translational control of maternal glp-1 mRNA establishes an asymmetry in the C. elegans embryo.

In C. elegans, the glp-1 gene encodes a membrane receptor that is required for anterior cell fates in the early embryo. We report that GLP-1 protein is localized to anterior blastomeres in 2- to 28-cell embryos. By contrast, glp-1 mRNA is present in all blastomeres until the 8-cell stage. Furthermore, the glp-1 3' untranslated region can restrict translation of a reporter mRNA to anterior blastomeres. Therefore, the translation of maternal glp-1 mRNA is temporally and spatially regulated in the C. elegans embryo. The regulation of maternal glp-1 mRNA has striking parallels to the regulation of maternal hunchback mRNA in the Drosophila embryo. Thus, the establishment of embryonic asymmetry in diverse organisms may involve conserved mechanisms of maternal mRNA regulation.

Cryptosporidiosis in veterinary students.

Cryptosporidiosis was diagnosed in 10 veterinary students. Exposure to the pathogen was associated with direct contact with infected calves and contact with contaminated materials. Affected students had fever (50%), headache (50%), nausea (70%), diarrhea (80%), and vomiting (40%). Clinical signs persisted for 30 hours to 16 days after the onset of clinical signs of disease. Although one student required hospitalization, the remaining students recovered without treatment.

Identification of two structural types of calcium-dependent adhesion molecules in the chicken embryo.

By using an immunological and peptide mapping approach two calcium-dependent cell-cell adhesion molecules (calCAMs) in the embryonic chicken are compared. A third closely related molecule is identified and compared to the two calCAMs. One of the calCAMs appears to be identical to the previously identified adhesion molecule N-cadherin, originally identified in chicken retina and localized to neural tissues. The second is the same as L-CAM, originally identified in chicken liver but localized to a variety of epithelial tissues. The third, also found in liver, is similar to L-CAM but is much closer in structure to N-cadherin. It is, however, immunologically distinct from N-cadherin. We therefore refer to this newly identified molecule as CRM-L for cadherin-related molecule in liver. CRM-L, N-cadherin, and L-CAM are all cell-surface proteins with a similar stability to tryptic digestion in the presence of calcium. CRM-L has the same molecular mass and isoelectric point as N-cadherin but is distinct from L-CAM in these properties. Two-dimensional peptide maps of complete tryptic digests reveal that CRM-L shares 69% of its peptides with N-cadherin and 20% with L-CAM. On the basis of these data, we suggest that there are at least two distinguishable types of calCAMs in the chicken embryo: one represented by the closely related molecules N-cadherin and CRM-L, and another represented by L-CAM.

Immunologically unique and common domains within a family of proteins related to the retina Ca2+-dependent cell adhesion molecule, NcalCAM.

Rabbit polyclonal antibodies raised to gp90, a fragment of the embryonic chick neural retina Ca2+-dependent adhesive molecule, gp130, recognize gp130 and inhibit Ca2+-dependent cell-cell adhesion. When tested against a panel of 10-day embryonic tissues, one of these antisera recognizes a component with a molecular weight identical to that of gp130 in embryonic chick cerebrum, optic lobe, hind brain, spinal cord and neural retina only; the second antiserum recognizes a similar component in all of the embryonic chick tissues tested. These data imply the existence of an extended family of closely related cell surface components with immunologically distinct subgroups each of which may mediate Ca2+-dependent cell-cell adhesion. As the term CAM, or cell adhesion molecule, has become common usage we propose to refer to these molecules as calCAMs, reflecting their calcium dependence. Analysis of fragments and endoglycosidase digests of NcalCAM have allowed a comparison of its structure with similar molecules from different tissues and species that have been implicated in Ca2+-dependent cell-cell adhesion.

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.