In sex determination, globally acting genes control a spectrum of tissue-specific regulators to coordinate the overall development of an animal into one sex or the other. In mammals, primary sex determination initially occurs in the gonad, with the sex of other tissues specified as a secondary event. In insects and nematodes, globally acting regulatory pathways have been elucidated, but the more tissue- and organ-specific downstream effectors of these pathways remain largely unknown. We focus on the control of sexual dimorphism in the C. elegans gonad. We find that the forkhead transcription factor FKH-6 promotes male gonadal cell fates in XO animals. Loss-of-function fkh-6 mutant males have feminized gonads and often develop a vulva. In these mutant males, sex-specific cell divisions and migrations in the early gonad occur in the hermaphrodite mode, and hermaphrodite-specific gonadal markers are expressed. However, sexual transformation is not complete and the male gonad is malformed. By contrast, fkh-6 mutant hermaphrodites exhibit no sign of sex reversal. Most fkh-6 hermaphrodites form a two-armed symmetrical gonad resembling that of the wild type, but differentiation of the spermatheca and uterus is variably abnormal. The function of fkh-6 appears to be restricted to the gonad: fkh-6 mutants have no detectable defects in extra-gonadal tissues, and expression of a rescuing fkh-6 reporter is gonad-specific. Genetic and molecular analyses place fkh-6 downstream of tra-1, the terminal regulator of the global sex determination pathway, with respect to the first gonadal cell division. We conclude that fkh-6 regulates gonadogenesis in both sexes, but is male specific in establishing sexual dimorphism in the early gonad.

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.Mammalian reoviruses (reoviruses) are prototypic members of the Reoviridae family, which includes the pathogenic rotaviruses, coltiviruses, and orbiviruses. Whereas reovirus causes infections that are generally asymptomatic in humans, it can induce respiratory, enteric, and nervous system diseases in animal models (reviewed in reference 67). Reovirus virions comprise a multilayered protein capsid that surrounds a segmented, double-stranded RNA genome (reviewed in reference 51). The outermost capsid layer consists of protein 3. The presence of 3 imparts environmental stability to the virion (54) but also appears to negatively regulate critical virion functions such as membrane penetration. The ability of reovirus to establish a productive infection requires proteolysis of the outer capsid (13,62,65). More recent data point toward 3 as the critical target for degradation (19,20).The first step in reovirus infection is attachment to cellular receptors through interactions with the viral receptor protein 1 (46, 75). Following attachment, virions are internalized by receptor-mediated endocytosis and delivered to endosomal compartments (12,13,65). In cell c...

Sex determination in C. elegans is controlled by the TRA-1 zinc finger protein, a Ci/GLI homolog that promotes female cell fates throughout the body. The regulatory hierarchy that controls TRA-1 is well established, but the downstream effectors that establish sexual dimorphism during larval development remain largely unknown. Here, we describe the use of cDNA microarrays to identify sex-enriched transcripts expressed during three stages of C. elegans larval development. By excluding previously identified germline-enriched transcripts, we focused on somatic sexual development. This approach identified a large number of sex-enriched transcripts that are good candidates to encode regulators of somatic sexual development. We found little overlap between genes with sex-enriched expression in early versus late larval development, indicating that distinct sexual regulatory programs operate at these times. Genes with sex-enriched expression are found throughout the genome, with no strong bias between autosomes and X chromosomes. Reporter gene analysis revealed that these genes are expressed in highly specific patterns in a variety of sexually dimorphic cells. We searched for TRA-1 consensus DNA binding sites near genes with sex-enriched expression, and found that most strongly sex-enriched mRNAs are likely to be regulated indirectly by TRA-1. These results suggest that TRA-1 controls sexual dimorphism through a small number of intermediary regulators rather than by acting directly on the full constellation of genes involved in sex-specific differentiation.

BackgroundThe C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure.ResultsWe made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants.ConclusionsThe results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.

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