Veena Prahlad et al. (2003) International Worm Meeting "Developmental plasticity in the sex determination mechanism of C.elegans requires mating"

Elizabeth B Goodwin, Dave Pilgrim, Veena Prahlad

[International Worm Meeting, 2003]

The X:A ratio is thought to be important in setting the sexual fate of C.elegans at fertilization. Our analysis indicates that the effect of the X:A ratio on sexual development can be overridden postembryonically by environmental factors, namely bacterial metabolites. Specifically, metablites from log phase bacteria can induce a proportion of XX L1 larvae to develop as males. This sexual transformation is accompanied by the loss of one of the X chromosomes, from some or all tissues of the adult worm. Importantly, this developmental plasticity requires mating. These conclusions are supported by several findings. L1s produced from a mating of wild-type XX and XO animals grown in the presence of metabolites from log phase bacteria had sex ratios skewed in favor of males: approximately 60-63% were males, suggesting that 20% of XX larvae developed as males instead of hermaphrodites. This distortion of sex ratio was not due to differences in viability, since all larvae were accounted for in these experiments. That XX larvae were sexually transformed by log phase bacterial metabolites was further confirmed by monitoring the development of individual XX cross-progeny L1s produced from the mating of a XO male that carried a GFP transgene integrated on the X and wild-type XX hermaphrodites. In these experiments 18% of the XX larvae identified by their GFP expression grown in the presence of log phase bacterial metabolites developed as males. An additional 10% developed as intersexual animals, possibly indicating incomplete transformation. Surprisingly, many of the sexually transformed animals lost GFP expression. PCR analysis and mating experiments using the transformed XX worms suggested that this was not simply due to the excision of the GFP transgene, but more likely due to the loss of the entire GFP containing chromosome. Finally, these effects were specific to cross-progeny, since L1 self-progeny from XX hermaphrodites, which did or did not carry a GFP containing X, did not undergo sexual transformation or X chromosome instability under the same conditions. Our findings suggest that the sex determination mechanism in C.elegans is more plastic than previoulsy thought. In addition, it appears that sexual reproduction promotes developmental plasticity in the sex determination mechanism of C.elegans. This plasticity may give cross-progeny an advantage over self-progeny since they may be more able to better adapt to changing environments. These findings might in part explain why C.elegans maintains males in a hermaphroditic species.

Phillip Grote et al. (2003) International Worm Meeting "tra-4 is necessary to ensure complete hermaphrodite development and encodes a zinc finger DNA binding protein"

BARBARA CONRADT, Phillip Grote, Claudia Huber

[International Worm Meeting, 2003]

During C. elegans development, the decision to develop a female (XX) or male (XO) soma is determined by the ratio of the number of X chromosomes to the number of sets of autosomes (X:A ratio). The resulting signal is transmitted through a cascade of interacting genes and determines the activity of the most downstream factor of this cascade, the transcription factor TRA-1A (TRA, transformer). Strong loss-of-function (lf) mutations in the tra-1 gene lead to a complete transformation of the XX soma into a male soma while weak tra-1(lf) mutations result in a partial masculinization of the XX soma. A partially masculinized XX soma overall resembles a wild-type XX soma, however, frequently the hermaphrodite-specific HSN neurons are absent and the male-specific CEM neurons are present. The HSNs and CEMs are the only neurons in C. elegans that are sex-specific as a result of programmed cell death. The specification of the cell death fate of these cells therefore seems to be particularly sensitive to changes in tra-1 dosage. In a screen for mutants, in which the CEMs inappropriately survive in XX animals, we identified mutations in a previously not described gene, referred to as tra-4, which is required for complete feminization of the XX soma. bc45, a lf mutation in the tra-4 gene, causes a partial masculinization of the XX soma: 73% of the HSNs undergo programmed cell death and 80% of the CEMs escape programmed cell death. In addition, in tra-4(bc45) L1 hermaphrodites the B cells and the ventral left coelomocytes have a male appearance or position, respectively, indicating weak masculinization. Double mutant analyses suggest that tra-4 acts upstream of fem-1, fem-2, and fem-3 (fem, feminization of XX and XO animals), and downstream of her-1 (her, hermaphrodization of XO animals) and possibly tra-2. tra-4 might therefore be a negative regulator of the fem genes. The tra-4 locus encodes a protein with predicted nuclear localization signals and C2H2 type Zinc finger domains, which indicates a role in transcriptional regulation. bc45 is a missense mutation leading to the exchange of a conserved amino acid in one of the C2H2 DNA-binding motives, suggesting that bc45 might not be a null allele. The analysis of the expression pattern of a functional Ptra-4GFP::TRA-4 construct indicates that tra-4 is expressed in most if not all somatic nuclei at all developmental stages in animals of both sexes. Using various approaches, we are currently searching for targets of the TRA-4 protein.

Stephanie Yonker et al. (2002) West Coast Worm Meeting "Molecular Characterization of the Dosage Compensation Gene, dpy-21"

Stephanie Yonker, Barbara J Meyer

[West Coast Worm Meeting, 2002]

Dosage compensation in Caenorhabditis elegans equalizes X-chromosome gene expression between XO males and XX hermaphrodites. DPY-21 is one of several proteins required for proper dosage compensation. dpy-21 mutations disrupt X gene expression less severely than other dosage compensation mutations and cause only 20% larval lethality compared to extensive maternal-effect lethality. Further, unlike other dosage compensation dpy mutations which affect gene expression only in XX animals, dpy-211 mutations appear to affect X-linked gene expression in XO animals. Assays of different X-linked genes show that dpy-21 mutations can cause both elevated and reduced gene expression in males.

Dawes HE et al. (1997) International C. elegans Meeting "sdc-2 TRIGGERS HERMAPHRODITE-SPECIFIC DEVELOPMENT IN XX ANIMALS"

Dawes HE, Matsubara Lapidus D, Meyer BJ, Davis TL

[International C. elegans Meeting, 1997]

sdc-2 is required during embryogenesis to activate the hermaphrodite modes of X chromosome dosage compensation and sex determination. sdc-2 directs the formation of a dosage compensation-specific protein complex on the X chromosomes of XX animals. Members of the dosage compensation machinery are not properly localized to X in the absence of sdc-2, and most XX animals die as a consequence. sdc-2 promotes hermaphrodite sexual differentiation by repressing the male-specific gene her-1. The rare sdc-2 mutant escapers are strongly masculinized. These data, along with the fact that sdc-2 is the only sdc gene that lacks a functional maternal component, suggest that sdc-2 might be sex-specifically activated to set the hermaphrodite fate. We have shown that SDC-2 is indeed expressed exclusively in XX embryos, where, like SDC-3, DPY-26, and DPY-27, it is localized to the X chromosomes. Furthermore, our observations suggest that sdc-2 acts as a switch to trigger XX-specific development when it is expressed in developing embryos. For example, the misexpression of sdc-2 in XO embryos inappropriately triggers the hermaphrodite mode of dosage compensation and leads to extensive (~82%) XO-specific lethality. Antibody staining revealed that misexpressed sdc-2 directs the localization of the dosage compensation machinery to the single X chromosome in XO animals. In addition, some XO animals that are rescued from lethality by a dpy-27 mutation develop as fertile hermaphrodites, indicating that the expression of sdc-2 in XO animals can also promote hermaphrodite sexual differentiation. We also found that ectopically expressed sdc-2 and sdc-3 act synergistically to promote the XX-specific fate, killing nearly 100% of XO embryos. In fact, SDC-2 may act directly with SDC-3 to trigger the X-speceific assembly of the dosage compensation complex in early XX embryos: SDC-2 is dependent upon the presence of SDC-3 (and other dosage compensation proteins) for its localization to the X chromosome, and immunoprecipitation experiments suggest that SDC-2 and SDC-3 stabily interact in vivo.

Kopczynski JB et al. (1995) International C. elegans Meeting "THE X:A RATIO, A MULTIGENIC SIGNALLING SYSTEM"

Akerib CC, Meyer BJ, Kopczynski JB

[International C. elegans Meeting, 1995]

The primary signal for sex determination is the X:A ratio. We found that three regions near the left end of X include dose-sensitive signal elements. Most XO animals with two doses of all three regions are dead; XX animals with a single dose of these regions are Dpy, indicating a dosage-compensation defect, and masculinized. Changing the dose of any one of these regions alone has little or no effect. Furthermore, other regions of X must also include signal elements. As one approach to identifying mutations in signal elements, we isolated suppressors of the XO-specific lethality caused by X duplications. Two strong suppressors, y303 and y304, map between dpy-3 and unc-2, suggesting that y303 and/or y304 might be mutations in fox-1. y303 and y304 XX animals are wild type, but XX animals heterozygous for both y303 or y304 and meDf6, which deletes at least two signal elements, are Dpy, as expected if y303 and y304 are loss-of-function mutations in a signal element. A second approach to identifying signal elements relies on the finding that xol-1, a switch gene and likely target of the X:A ratio, is sex-specifically regulated, with high levels in XO embryos and low levels in XX embryos. Using a xol-1:lacZ reporter gene as an assay, we screened for mutations that reduce the perceived X:A ratio in XX animals and thereby cause elevated expression of xol-1 in these animals. We identified two X-linked mutations: y263, which maps between unc-18 and dpy-6, and y264, which maps to the right of unc-3. y264 XX animals are slightly Dpy and Egl. y264 acts either as part of the X chromosome signal or as a regulator of xol-1, since y264 can suppress the XO-specific lethality caused by signal element duplications, but cannot suppress xol-1 mutations. Both of these approaches have proven powerful for identifying components of a multigenic signalling system.

Nusbaum C et al. (1991) International C. elegans Meeting "Molecular and Genetic Characterization of sdc-2, A Gene that Coordinately Controls Sex Determination and Dosage Compensation in XX Animals."

Brenner DS, Nusbaum C, Meyer BJ

[International C. elegans Meeting, 1991]

sdc-2 is required for proper X-chromosome expression and sexual development of XX animals. Analysis of 40 independent sdc-2 mutations reveals that they cause two distinct phenotypes in XX animals: masculinization, reflecting a defect in sex determination, and lethality or dumpiness, reflecting a disruption in dosage compensation. sdc-2 mutations have no apparent effect in XO animals. We interpret these phenotypes to indicate that sdc-2 mutations shift the sex deterrnination and dosage compensation pathways in XX animals to their XO modes of expression. We have cloned the sdc-2 gene. 18 kb of DNA spanning a region containing sdc-2 allele-specific DNA alterations is sufficient to confer rescue of the sdc-2 mutant phenotypes following germline transformation. Probes spanning most of this region identify a single transcript of approximately 12 kb in size. This transcript is eliminated in at least one sdc-2 mutant strain and reduced in size in another. Levels of the sdc-2 transcript are similar in XX animals of all stages and in XO adult males. This observation suggests that sdc-2 regulation takes place at the post-transcriptional level. DNA sequencing of the gene is in progress. Recently, we have isolated a strongly temperature-sensitive allele of sdc-2 that will be used to determine the sdc-2 TSP and to select extragenic suppressors.

Veena Prahlad et al. (2005) International Worm Meeting "Identification of a bacterial component that induces postembryonic sexual transformation and genomic instability in C. elegans"

Richard Morimoto, Veena Prahlad

[International Worm Meeting, 2005]

Previous work demonstrated a surprising plasticity in the sex determination mechanism of C. elegans: specific bacterial metabolites could cause XX larvae to post-embryonically change sexual fate and develop as males instead of hermaphrodites. This sexual transformation was accompanied by chromosomal instability and an apparent loss of an X chromosome. In these initial experiments conducted using crude bacterial metabolites, sexual transformation occurred only in XX cross-progeny larvae generated by mating males with hermaphrodites; XX self-progeny did not show a significant degree of sexual transformation. We have subsequently identified a component of bacterial metabolite preparations that appears responsible for the sexual transformation and apparent X chromosome loss. This component is bacterial DNA. Purified Op50 DNA not only induces the sexual transformation of XX cross-progeny, but can also cause 3.6% XX self-progeny larvae to post-embryonically change sexual fate and develop as males. Plasmid DNA, and synthetic single stranded oligonucleotides can also induce both XX cross- and self-progeny to undergo sexual transformation. This ability of DNA to induce sexual transformation appears to be, to some extent, sequence specific. Using synthetic oligonucleotides, we show that the presence of specific CpG motifs within the DNA, repetitive sequences and the oligonucleotide backbone all affect the efficiency of sexual transformation: certain sequences cause as many as 6-10% XX self-progeny larvae to undergo sexual transformation, while others are not effective. These findings are reminiscent of the ability of exogenous DNA to induce an immune response in mammals, and numerous other organisms. The mammalian immune response to specific DNA sequences is presumably a defense mechanism against bacteria and other pathogens. We are currently exploring the extent to which the sexual transformation of C. elegans larvae in response to specific exogenous DNA sequences also reflects the innate immune response of C.elegans to bacterial pathogens. Thus, we are in midst of screening mutants in candidate genes to identify the pathways involved in this process, and using RT-PCR to identify whether specific antibacterial genes known to upregulated during theC. elegans immune response are also specifically upregulated upon exposure to exogenous DNA. The observed DNA-induced generation of males is intriguing. Our studies may provide direct evidence for the Red-Queen Hypothesis that suggests that sexual reproduction in nature is advantageous due to its ability to combat the invasion of pathogens.

Chin Yee Loh et al. (2004) West Coast Worm Meeting "The X Chromosome Counting Mechanism of C. elegans Utilizes Multiple X-signal Elements to Determine Sex"

Jennifer Powell, Barbara J Meyer, John Gladden, Chin Yee Loh

[West Coast Worm Meeting, 2004]

Most organisms reproduce sexually, and to do so, they have evolved specialized sexes with distinct morphological characteristics and behaviors. We are interested in the molecular mechanisms by which an embryo specifies its sex and initiates the appropriate developmental pathways to ensure the proper development of each sex. C. elegans determines sex by an X chromosome counting mechanism that distinguishes one X chromosome from two by assessing the dose several X-linked genes called X-signal elements ( XSEs ), which act cumulatively to repress the master sex switch gene xol-1 . In XX animals, the XSEs repress xol-1 expression, promoting hermaphrodite development and X chromosome dosage compensation. In XO animals, with half the number of XSE genes, xol-1 is highly expressed promoting male development. How can a decrease in the copy number of each XSE by only half drastically affect xol-1 expression? The answer is that C. elegans uses multiple XSEs that act at different levels of xol-1 regulation. Although an individual XSE represses xol-1 only partially , XSEs act cumulatively to fully repress xol-1 in XX animals. When the copy number of every XSE is reduced by half, as in XO animals, the XSEs cannot repress xol-1 efficiently, and xol-1 becomes expressed at high levels. Consistent with individual XSEs being partial repressors of xol-1 , reducing the copy number of an XSE from two to one has no effect on XX animals. However, when the copy number of three or more XSEs is reduced by half, xol-1 repression is disrupted, causing sex determination and dosage compensation defects in XX animals. Thus far, two XSEs , sex-1 and fox-1, have been characterized extensively. sex-1 is a nuclear receptor that represses xol-1 transcriptionally , while fox-1 encodes an RNA binding protein that disrupts xol-1 via RNA splicing to cause retention of the 6 th intron , producing an inactive transcript. We have identified two new potential XSEs , ceh-39 and sex-2, and have shown that they regulate xol-1. ceh-39 encodes a ONECUT homeodomain protein and sex-2 ( pqn-65) encodes a protein containing poly-glutamine and asparagine stretches. Like SEX-1 and FOX-1, CEH-39 localizes to embryonic nuclei during the time when regulation of xol-1 is critical. sex-2 transcripts were detected in embryos, indicating that sex-2 is also expressed at the right time to act as a regulator of xol-1 . Not only do XSEs act cumulatively to repress xol-1, they also function synergistically . For example, while fox-1 XX animals have no phenotype and only a third of sex-1 XX animals are dead, virtually all fox-1 sex-1 XX animals die. Although a ceh-39(y414) deletion appears wild-type and sex-2(y326) is slightly Dpy , both ceh-39 and sex-2 synergize with fox-1 and sex-1 mutations in XX animals to enhance sex determination and dosage compensation phenotypes. Since XSEs act cumulatively to repress xol-1 , increasing the dose of one XSE can rescue the XX-specific phenotypes and lethality associated with another XSE mutation. For example, overexpression of ceh-39 or one extra copy of sex- 2 significantly restores viability to sex-1 mutants. In addition, an increase in the copy number of XSEs is detrimental to XO survival due to repression of xol-1 and inappropriate activation of the dosage compensation machinery. Overexpression of ceh-39 enhances the XO lethality caused by extra copies of XSEs . Since XSEs should act upstream of xol-1, a mutation in an XSE is expected to increase xol-1 expression, while overexpression of an XSE is expected to decrease xol-1 expression. To test this, we assessed whether changes in XSE dose could affect transcriptional and translational xol-1: :lacZ reporters. These reporters are repressed in wild-type XX animals but are derepressed by sex-1 mutations. ceh-39( y414) and a sex-2 mutation derepress the translational and transcriptional reporters, respectively. In the reciprocal experiment, a reduction of the translational reporter activity was observed in sex-1 XX animals overexpressing ceh-39 . These results demonstrate that ceh-39 functions upstream of xol-1. The C. elegans X chromosome counting mechanism creates a genetic switch that allows XX and XO animals to adopt distinct sexual fates. At least four XSEs amplify a two-fold difference in X chromosome dose between XX and XO animals to set the on or off state of xol-1 , ensuring proper control of sex determination and dosage compensation in the worm.

Spence AM et al. (1995) International C. elegans Meeting "Physical Interaction between the Sex Determining Proteins FEM-3 and TRA-2A."

Heck L, Spence AM, Mehra A, Kuwabara PE

[International C. elegans Meeting, 1995]

The fem genes promote male development in all tissues of C. elegans. In XX animals, they are prevented from doing so primarily by the activity of tra-2. The sequence of tra-2 predicts that it encodes a large integral membrane protein, TRA-2A, with a C-terminal cytoplasmic domain [TRA-2A(Cterm)]. Overexpression of TRA-2A(Cterm) partially feminizes X0 tra-2(+) and XX tra- 2(lf) males. The predicted products of the fem genes are intracellular proteins. We report that TRA-2A(Cterm) interacts specifically with FEM-3. Full-length FEM-3 and TRA-2A(Cterm) interact in the yeast two hybrid system. Coimmunoprecipitation of TRA- 2A(Cterm) with epitope-tagged FEM-3 following their synthesis in vitro confirms their ability to associate. Deletion analysis reveals that a 141 amino acid fragment of TRA-2A(Cterm) is sufficient to interact with FEM-3 in the two hybrid system. The first 345 amino acids of FEM- 3 are required for the interaction. Overexpression of FEM-3 in XX nematodes causes extensive somatic masculinization, suggesting that fem-3 activity is normally limiting in XX somatic tissues. Loss-of-function mutations in fem-1 are completely epistatic to FEM-3 overexpression. We suggest that regulation of fem activity in vivo involves binding of FEM-3 to the intracellular domain of TRA-2A and that male development requires the liberation of FEM-3 from this interaction.

Katlin B Massirer et al. (2005) International Worm Meeting "The role of Argonaute-like genes in the sex determination and dosage compensation"

John Gladden, Barbara Meyer, Katlin B Massirer, Amy Pasquinelli

[International Worm Meeting, 2005]

In C. elegans sex is determined by the ratio of X chromosomes to sets of autosomes. The X portion of this ratio is relayed by X signal elements (XSE) that act in a cumulative, dose-dependent, and synergistic manner to repress the master sex switch gene xol-1. In XX animals, the dose of XSEs is sufficient to repress xol-1, promoting the hermaphrodite fate and initiating dosage compensation, a process that equalizes X-linked gene expression between XX and XO animals by reducing transcription from both X chromosomes of XX hermaphrodites by half. In XO animals, a single dose of XSEs is unable to repress xol-1. Active xol-1 promotes the male fate and prevents dosage compensation. sex-1 is a well characterized XSE that represses xol-1 transcriptionally. In XX animals, sex-1 mutants cause 30% lethality due to disruptions in dosage compensation. We found that the argonaute-like gene, alg-2, genetically interacts with sex-1. alg-2 mutants appear wild type but exhibit synergistic lethality when combined with sex-1 mutations. Epistasis analysis of alg-2 indicates that it functions at the level of dosage compensation. Indeed, in many nuclei of the dead alg-2; sex-1 XX embryos, the dosage compensation complex in not properly localized to the X chromosome. However, it is unclear exactly how alg-2 affects dosage compensation. Worms with mutations in alg-2 are also compromised in processing of embryonic miRNA precursors, opening the possibility that alg-2 may affect dosage compensation through a miRNA. To help understand the role of alg-2 in sex-determination and dosage compensation, we are analyzing the spatial-temporal expression of the alg-2 gene, its miRNA substrates and their potential regulatory targets.