Fog-2, a Germ-Line-Specific Sex Determination Gene Required for Hermaphrodite Spermatogenesis in Caenorhabditis Elegans

This paper describes the isolation and characterization of 16 mutations in the germ-line sex determination gene fog-2 (fog for feminization of the germ line). In the nematode Caenorhabditis elegans there are normally two sexes, self-fertilizing hermaphrodites (XX) and males (XO). Wild-type XX animals are hermaphrodite in the germ line (spermatogenesis followed by oogenesis), and female in the soma. fog-2 loss-of-function mutations transform XX animals into females while XO animals are unaffected. Thus, wild-type fog-2 is necessary for spermatogenesis in hermaphrodites but not males. The fem genes and fog-1 are each essential for specification of spermatogenesis in both XX and XO animals. fog-2 acts as a positive regulator of the fem genes and fog-1. The tra-2 and tra-3 genes act as negative regulators of the fem genes and fog-1 to allow oogenesis. Two models are discussed for how fog-2 might positively regulate the fem genes and fog-1 to permit spermatogenesis; fog-2 may act as a negative regulator of tra-2 and tra-3, or fog-2 may act positively on the fem genes and fog-1 rendering them insensitive to the negative action of tra-2 and tra-3.     

Genetic flexibility in the convergent evolution of hermaphroditism in Caenorhabditis nematodes

The self-fertile hermaphrodites of C. elegans and C. briggsae evolved from female ancestors by acquiring limited spermatogenesis. Initiation of C. elegans hermaphrodite spermatogenesis requires germline translational repression of the female-promoting gene tra-2, which allows derepression of the three male-promoting fem genes. Cessation of hermaphrodite spermatogenesis requires fem-3 translational repression. We show that C. briggsae requires neither fem-2 nor fem-3 for hermaphrodite development, and that XO Cb-fem-2/3 animals are transformed into hermaphrodites, not females as in C. elegans. Exhaustive screens for Cb-tra-2 suppressors identified another 75 fem-like mutants, but all are self-fertile hermaphrodites rather than females. Control of hermaphrodite spermatogenesis therefore acts downstream of the fem genes in C. briggsae. The outwardly similar hermaphrodites of C. elegans and C. briggsae thus achieve self-fertility via intervention at different points in the core sex determination pathway. These findings are consistent with convergent evolution of hermaphroditism, which is marked by considerable developmental genetic flexibility.     

Gain-of-Function Mutations of fem-3, a Sex-Determination Gene in Caenorhabditis elegans

We have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild-type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3(gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3(gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All nine fem-3(gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3(gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.     

Sex change by gene conversion in a Caenorhabditis elegans fog-2 mutant

Caenorhabditis elegans primarily reproduces as a hermaphrodite. Independent gene conversion events in mutant obligately outcrossing populations of C. elegans [fog-2(lf)] spontaneously repaired the loss-of-function mutation in the fog-2 locus, thereby reestablishing hermaphroditism as the primary means of reproduction for the populations.     

Fog-1, a Regulatory Gene Required for Specification of Spermatogenesis in the Germ Line of Caenorhabditis Elegans

In wild-type Caenorhabditis elegans, the XO male germ line makes only sperm and the XX hermaphrodite germ line makes sperm and then oocytes. In contrast, the germ line of either a male or a hermaphrodite carrying a mutation of the fog-1 (feminization of the germ line) locus is sexually transformed: cells that would normally make sperm differentiate as oocytes. However, the somatic tissues of fog-1 mutants remain unaffected. All fog-1 alleles identified confer the same phenotype. The fog-1 mutations appear to reduce fog-1 function, indicating that the wild-type fog-1 product is required for specification of a germ cell as a spermatocyte. Two lines of evidence indicate that a germ cell is determined for sex at about the same time that it enters meiosis. These include the fog-1 temperature sensitive period, which coincides in each sex with first entry into meiosis, and the phenotype of a fog-1; glp-1 double mutant. Experiments with double mutants show that fog-1 is epistatic to mutations in all other sex-determining genes tested. These results lead to the conclusion that fog-1 acts at the same level as the fem genes at the end of the sex determination pathway to specify germ cells as sperm.     

Specification of male development in Caenorhabditis elegans: the fem genes

Mutation of the gene fem-2 causes feminization of both sexes: hermaphrodites make no sperm, and males produce oocytes in an intersexual somatic gonad. A double mutant harboring ts alleles of both fem-1 (formerly named isx-1; G. A. Nelson, K. K. Lew, and S. Ward, 1978, Dev. Biol. 66, 386-409) and fem-2 causes transformation of XO animals (normally male) into spermless hermaphrodites at restrictive temperature. The phenotypes, temperature-sensitive periods, and maternal effects observed in mutants of each fem gene are found to be similar. It is suggested that the fem genes are centrally involved in specification of male development in Caenorhabditis elegans--both in the germ line of hermaphrodites and in somatic and germ line tissues of males.     

Use of a psoralen-induced phenocopy to study genes controlling spermatogenesis in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, spermatogenesis represents one of two alternative developmental pathways open to premeiotic germ cells. At least two genes, fem-1 and fem-2, control the initiation of spermatogenesis in XX (hermaphrodite) worms, and the entire spectrum of male differentiation in XO animals. Low-dose irradiation of worms treated with the light-activated DNA crosslinking drug trimethylpsoralen, at levels that do not affect cell division or growth rates, blocks spermatogenesis in C. elegans hermaphrodites and produces an identical phenotype to that of temperature-sensitive mutations in the fem genes. Psoralen treatment does not, however, produce corresponding phenotypes of these mutants in XO animals. The developmental age for phenocopy production is the same as the hermaphrodite temperature-sensitive period of the two mutants. The effects of pulses of restrictive temperature and psoralen treatment on fem-2 mutant hermaphrodites are additive, suggesting that psoralen crosslinking may reduce the level of the fem-2 gene product. Microbeam experiments localize the target for the psoralen effect to the primary germ cells in the first stage larvae, indicating that a critical step occurs in a small number of precursor cells prior to their commitment to spermatogenesis.     

Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2

To investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2's PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain. Received: 11 April 2001 / Accepted: 6 August 2001     

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.     

Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

Activity of the Sex-Determining Gene tra-2 Is Modulated to Allow Spermatogenesis in the C. ELEGANS Hermaphrodite

In the nematode C. elegans, there are two sexes, the self-fertilizing hermaphrodite (XX) and the male (XO). The hermaphrodite is essentially a female that makes sperm for a brief period before oogenesis. Sex determination in C. elegans is controlled by a pathway of autosomal regulatory genes, the state of which is determined by the X:A ratio. One of these genes, tra-2, is required for hermaphrodite development, but not for male development, because null mutations in tra-2 masculinize XX animals but have no effect on XO males. Dominant, gain-of-function tra-2 mutations have now been isolated that completely feminize the germline of XX animals so that they make only oocytes and no sperm and, thus, are female. Most of the tra-2(dom) mutations do not correspondingly feminize XO animals, so they do not appear to interfere with control by her-1, a gene thought to negatively regulate tra-2 in XO animals. Thus, these mutations appear to cause gain of tra-2 function in the XX animal only. Dosage studies indicate that 5 of 7 tra-2(dom) alleles are hypomorphic, so they do not simply elevate XX tra-2 activity overall. These properties suggest that in the wild type, tra-2 activity is under two types of control: (1) in males, it is inactivated by her-1 to allow male development to occur, and (2) in hermaphrodites, tra-2 is active but transiently inactivated by another, unknown, regulator to allow hermaphrodite spermatogenesis; this mode of regulation is hindered by the tra-2(dom) mutations, thereby resulting in XX females.     

Assessing the Viability of Mutant and Manipulated Sperm by Artificial Insemination of Caenorhabditis Elegans

We describe a protocol for artificial insemination of Caenorhabditis elegans which we used to evaluate the viability of sperm from different strains and of sperm activated in vitro. Worms can be artificially inseminated with almost 100% success. Both male and hermaphrodite sperm can be used for insemination. Sperm from a sterile hermaphrodite [fem-3(q23ts)] were found to be viable. As with normal mating, male sperm inseminated into hermaphrodites artificially outcompete the hermaphrodite's own sperm, even though they have not been ejaculated with seminal fluid. Spermatozoa that were activated in vitro from spermatids by the weak base triethanolamine were viable. In contrast, spermatozoa activated in vitro by protease treatment were not.     

MRG-1, a mortality factor-related chromodomain protein,is required maternally for primordial germ cells to initiate mitotic proliferation in C. elegans

We identified MRG-1, a Caenorhabditis elegans chromodomain-containing protein that is similar to the human mortality factor-related gene 15 product (MRG15). RNA-mediated interference (RNAi) of mrg-1 resulted in complete absence of the germline in both hermaphrodite and male adults. Examination of the expression of PGL-1, a component of P granules, revealed that two primordial germ cells (PGCs) are produced during embryogenesis in mrg-1(RNAi) animals, but these PGCs cannot undergo mitotic proliferation, and they ultimately degenerate during post-embryonic development. Zygotic RNAi experiments using RNAi-deficient hermaphrodites and wild-type males demonstrated that MRG-1 functions maternally. Moreover, immunoblot analysis using mutant animals with germline deficiencies indicated that MRG-1 is synthesized predominantly in oocytes. These results suggest that MRG-1 is required maternally to form normal PGCs with the potential to start mitotic proliferation during post-embryonic development.