Genes That Implement the Hermaphrodite Mode of Dosage Compensation in Caenorhabditis Elegans

We report a genetic characterization of several essential components of the dosage compensation process in Caenorhabditis elegans. Mutations in the genes dpy-26, dpy-27, dpy-28, and the newly identified gene dpy-29 disrupt dosage compensation, resulting in elevated X-linked gene expression in XX animals and an incompletely penetrant maternal-effect XX-specific lethality. These dpy mutations appear to cause XX animals to express each set of X-linked genes at a level appropriate for XO animals. XO dpy animals are essentially wild type. Both the viability and the level of X-linked gene expression in XX animals carrying mutations in two or more dpy genes are the same as in animals carrying only a single mutation, consistent with the view that these genes act together in a single process (dosage compensation). To define a potential time of action for the gene dpd-28 we performed reciprocal temperature-shift experiments with a heat sensitive allele. The temperature-sensitive period for lethality begins 5 hr after fertilization at the 300-cell stage and extends to about 9 hr, a point well beyond the end of cell proliferation. This temperature-sensitive period suggests that dosage compensation is functioning in XX animals by mid-embryogenesis, when many zygotically transcribed genes are active. While mutations in the dpy genes have no effect on the sexual phenotype of otherwise wild-type XX or XO animals, they do have a slight feminizing effect on animals whose sex-determination process is already genetically perturbed. The opposite directions of the feminizing effects on sex determination and the masculinizing effects on dosage compensation caused by the dpy mutations are inconsistent with the wild-type dpy genes acting to coordinately control both processes. Instead, the feminizing effects are most likely an indirect consequence of disruptions in dosage compensation caused by the dpy mutations. Based on the cumulative evidence, the likely mechanism of dosage compensation in C. elegans involves reducing X-linked gene expression in XX animals to equal that in XO animals via the action of the dpy genes.     

sdc-1: a link between sex determination and dosage compensation in C. elegans

Mutations in the X-linked gene sdc-1 affect both sex determination and X-chromosome dosage compensation in C. elegans, providing evidence that these two pathways share a common step. In XX animals (normally hermaphrodites), sdc-1 mutations cause partial masculinization and elevated levels of X-linked gene expression, an apparent shift of both pathways toward their XO modes of expression. The masculinization occurs through effects on the major sex determination pathway, upstream of all previously identified sex-determining genes. XO animals are apparently unaffected by the sdc-1 mutations. We propose a model in which the wild-type sdc-1 activity is either a component of the primary sex-determining signal (the X/Autosome ratio) or involved in transmitting information about this signal to both the sex determination and dosage compensation pathways.     

The Dpy-30 Gene Encodes an Essential Component of the Caenorhabditis Elegans Dosage Compensation Machinery

The need to regulate X chromosome expression in Caenorhabditis elegans arises as a consequence of the primary sex-determining signal, the X/A ratio (the ratio of X chromosomes to sets of autosomes), which directs 1X/2A animals to develop as males and 2X/2A animals to develop as hermaphrodites. C. elegans possesses a dosage compensation mechanism that equalizes X chromosome expression between the two sexes despite their disparity in X chromosome dosage. Previous genetic analysis led to the identification of four autosomal genes, dpy-21, dpy-26, dpy-27 and dpy-28, whose products are essential in XX animals for proper dosage compensation, but not for sex determination. We report the identification and characterization of dpy-30, an essential component of the dosage compensation machinery. Putative null mutations in dpy-30 disrupt dosage compensation and cause a severe maternal-effect, XX-specific lethality. Rare survivors of the dpy-30 lethality are dumpy and express their X-linked genes at higher than wild-type levels. These dpy-30 mutant phenotypes superficially resemble those caused by mutations in dpy-26, dpy-27 and dpy-28; however, detailed phenotypic analysis reveals important differences that distinguish dpy-30 from these genes. In contrast to the XX-specific lethality caused by mutations in the other dpy genes, the XX-specific lethality caused by dpy-30 mutations is completely penetrant and temperature sensitive. In addition, unlike the other genes, dpy-30 is required for the normal development of XO animals. Although dpy-30 mutations do not significantly affect the viability of XO animals, they do cause them to be developmentally delayed and to possess numerous morphological and behavioral abnormalities. Finally, dpy-30 mutations can dramatically influence the choice of sexual fate in animals with an ambiguous sexual identity, despite having no apparent effect on the sexual phenotype of otherwise wild-type animals. Paradoxically, depending on the genetic background, dpy-30 mutations cause either masculinization or feminization, thus revealing the complex regulatory relationship between the sex determination and dosage compensation processes. The novel phenotypes caused by dpy-30 mutations suggest that in addition to acting in the dosage compensation process, dpy-30 may play a more general role in the development of both XX and XO animals.     

Feedback Control of Sex Determination by Dosage Compensation Revealed through Caenorhabditis Elegans Sdc-3 Mutations

In Caenorhabditis elegans, sex determination and dosage compensation are coordinately controlled through a group of genes that respond to the primary sex determination signal. Here we describe a new gene, sdc-3, that also controls these processes. In contrast to previously described genes, the sex determination and dosage compensation activities of sdc-3 are separately mutable, indicating that they function independently. Paradoxically, the sdc-3 null phenotype fails to reveal the role of sdc-3 in sex determination: sdc-3 null mutations that lack both activities disrupt dosage compensation but cause no overt sexual transformation. We demonstrate that the dosage compensation defect of sdc-3 null alleles suppresses their sex determination defect. This self-suppression phenomenon provides a striking example of how a disruption in dosage compensation can affect sexual fate. We propose that the suppression occurs via a feedback mechanism that acts at an early regulatory step in the sex determination pathway to promote proper sexual identity.     

Revisiting the X:A signal that specifies Caenorhabditis elegans sexual fate

In Caenorhabditis elegans, sex is determined by the opposing actions of X-signal elements (XSEs) and autosomal signal elements (ASEs), which communicate the ratio of X chromosomes to sets of autosomes (X:A signal). This study delves more deeply into the mechanism by which XSEs transmit X chromosome dose. We determined the relative contributions of individual XSEs to the X:A signal and showed the order of XSE strength to be sex-1 > sex-2 > fox-1 > ceh-39 >/= region 1 XSE. sex-1 exerts a more potent influence on sex determination and dosage compensation than any other XSE by functioning in two separate capacities in the pathway: sex-1 acts upstream as an XSE to repress xol-1 and downstream as an activator of hermaphrodite development and dosage compensation. Furthermore, the process of dosage compensation affects expression of the very XSEs that control it; XSEs become fully dosage compensated once sex is determined. The X:A signal is then equivalent between XO and XX animals, causing sexual differentiation to be controlled by genes downstream of xol-1 in the sex-determination pathway. Prior to the onset of dosage compensation, the difference in XSE expression between XX and XO embryos appears to be greater than twofold, making X chromosome counting a robust process.     

tra-2 encodes a membrane protein and may mediate cell communication in the Caenorhabditis elegans sex determination pathway.

The Caenorhabditis elegans sex-determining gene, tra-2, promotes female development in XX animals. In this paper we report the cDNA sequence corresponding to a 4.7 kb tra-2 mRNA and show that it is composed of 23 exons, is trans-spliced to SL2, and contains a perfect direct repeat in the 3' untranslated region. This mRNA is predicted to encode a 1475 amino acid protein, named pTra2A, that has a secretory signal and several potential membrane-spanning domains. The molecular analysis of tra-2 loss-of-function mutations supports our open reading frame identification and suggests that the carboxy-terminal domain is important for tra-2 activity. We propose that in XX animals the carboxy-terminal domain of pTra2A negatively regulates the downstream male promoting fem genes. In XO animals, tra-2 is negatively regulated by her-1, which acts cell nonautonomously. Because hydropathy predictions suggest that pTra2A is an integral membrane protein, pTra2A might act as a receptor for the her-1 protein. We propose that in XO animals, the her-1 protein promotes male development by binding and inactivating pTra2A. The role of cell communication in C. elegans sex determination might be to ensure unified sexual development throughout the animal. If so, then regulation of sexual fate by her-1 and tra-2 might provide a general model for the coordination of groups of cells to follow a single cell fate.     

Genetic Analysis of X-Chromosome Dosage Compensation in Caenorhabditis elegans

We have shown that the phenotypes resulting from hypomorphic mutations (causing reduction but not complete loss of function) in two X-linked genes can be used as a genetic assay for X-chromosome dosage compensation in Caenorhabditis elegans between males (XO) and hermaphrodites (XX). In addition we show that recessive mutations in two autosomal genes, dpy-21 V and dpy-26 IV, suppress the phenotypes resulting from the X-linked hypomorphic mutations, but not the phenotypes resulting from comparable autosomal hypomorphic mutations. This result strongly suggests that the dpy-21 and dpy-26 mutations cause increased X expression, implying that the normal function of these genes may be to lower the expression of X-linked genes. Recessive mutations in two other dpy genes, dpy-22 X and dpy-23 X, increase the severity of phenotypes resulting from some X-linked hypomorphic mutations, although dpy-23 may affect the phenotypes resulting from the autosomal hypomorphs as well. The mutations in all four of the dpy genes show their effects in both XO and XX animals, although to different degrees. Mutations in 18 other dpy genes do not show these effects.     

Extragenic suppressors of a dominant masculinizing her-1 mutation in C. elegans identify two new genes that affect sex determination in different ways

Summary: The her-1 regulatory switch gene in C. elegans sex determination is normally active in XO animals, resulting in male development, and inactive in XX animals, allowing hermaphrodite development. The her-1(n695gf) mutation results in the incomplete transformation of XX animals into phenotypic males. We describe four extragenic mutations that suppress the masculinized phenotype of her-1(n695gf) XX. They define two previously undescribed genes, sup-26 and sup-27. All four mutations exhibit semidominance of suppression and by themselves have no visible effects on sex determination in otherwise genotypically wild-type XX or XO animals. Analysis of interactions with mutations in the major sex-determining genes show that sup-26 and sup-27 influence sex determination in fundamentally different ways. sup-26 appears to act independently of her-1 to negatively modulate synthesis or function of tra-2 in both XX and XO animals. sup-27 may play a role in X-chromosome dosage compensation and influence sex determination indirectly.     

xol-1: a gene that controls the male modes of both sex determination and X chromosome dosage compensation in C. elegans

Loss-of-function mutations in the X-linked gene xol-1 cause the feminization and death of XO animals (normally males) by shifting the sex determination and dosage compensation pathways toward their hermaphrodite modes. XO-specific lethality most likely results from the reduction in X chromosome expression caused by xol-1 mutations. Mutations in genes required for the hermaphrodite mode of dosage compensation suppress lethality but not feminization, and restore X chromosome expression to nearly wild-type levels. Mutations in genes that control the hermaphrodite modes of both sex determination and dosage compensation fully suppress both defects. These interactions suggest that xol-1 is the earliest-acting gene in the known hierarchy controlling the male/hermaphrodite decision and is perhaps the gene nearest the primary sex-determining signal. We propose that the wild-type xol-1 gene product promotes male development by ensuring that genes (or gene products) directing hermaphrodite sex determination and dosage compensation are inactive in XO animals. Interestingly, in addition to feminizing XO animals, xol-1 mutations further masculinize XX animals already partially masculinized.     

Identification of X Chromosome Regions in Caenorhabditis Elegans That Contain Sex-Determination Signal Elements

The primary sex-determination signal of Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio). This signal coordinately controls both sex determination and X chromosome dosage compensation. To delineate regions of X that contain counted signal elements, we examined the effect on the X/A ratio of changing the dose of specific regions of X, using duplications in XO animals and deficiencies in XX animals. Based on the mutant phenotypes of genes that are controlled by the signal, we expected that increases (in males) or decreases (in hermaphrodites) in the dose of X chromosome elements could cause sex-specific lethality. We isolated duplications and deficiencies of specific X chromosome regions, using strategies that would permit their recovery regardless of whether they affect the signal. We identified a dose-sensitive region at the left end of X that contains X chromosome signal elements. XX hermaphrodites with only one dose of this region have sex determination and dosage compensation defects, and XO males with two doses are more severely affected and die. The hermaphrodite defects are suppressed by a downstream mutation that forces all animals into the XX mode of sex determination and dosage compensation. The male lethality is suppressed by mutations that force all animals into the XO mode of both processes. We were able to subdivide this region into three smaller regions, each of which contains at least one signal element. We propose that the X chromosome component of the sex-determination signal is the dose of a relatively small number of genes.     

X chromosome dosage compensation and its relationship to sex determination in C. elegans

A genetic regulatory hierarchy controls all aspects of Caenorhabditis elegans sex determination and X chromosome dosage compensation in response to the primary sex-determining signal, the X/A ratio. Initially, these two processes are coordinately regulated by a group of genes that transmit this primary signal to downstream genes that preferentially control either sex determination or dosage compensation. The relationship between these two processes is complex: not only are they coordinately controlled, a feedback mechanism operates to allow a disruption in dosage compensation to affect sexual fate. We describe our genetic and molecular understanding of the regulatory hierarchy, the feedback control and the dosage compensation process itself.     

X-linked gene expression and sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio between the number of X chromosomes and the number of sets of autosomes (the X/A ratio). Animals with an X/A ratio of 0.67 (a triploid with two X chromosomes) or less are males. Animals with an X/A ratio of 0.75 or more are hermaphrodites. Thus, diploid males have one X chromosome and diploid hermaphrodites have two X chromosomes. However, the difference in X-chromosome number between the sexes is not reflected in general levels of X-linked gene expression because of the phenomenon of dosage compensation. In dosage compensation, X-linked gene expression appears to be 'turned down' in 2X animals to the 1X level of expression. An intriguing and unexplained finding is that mutations and X-chromosome duplications that elevate X-linked gene expression also feminize triploid males. One way that this relationship between sex determination and X-linked gene expression may be operating is discussed.     

Dosage compensation: making 1X equal 2X

Animals that have XX females and XY or XO males have differing doses of X-linked genes in each sex. Overcoming this is the most immediate and vital aspect of sexual differentiation. A number of systems that accurately compensate for sex-chromosome dosage have evolved independently: silencing a single X chromosome in female mammals, downregulating both X chromosomes in hermaphrodite Caenorhabditis elegans and upregulating the X chromosome in male Drosophila all equalize X-linked gene expression. Each organism uses a largely non-overlapping set of molecules to achieve the same outcome: 1X = 2X.