The scute (T4) gene acts as a numerator element of the X:a signal that determines the state of activity of sex-lethal in Drosophila.

The ratio of X chromosomes to sets of autosomes (X:A) is the primary genetic signal that determines sex and dosage compensation in Drosophila. The gene Sex-lethal (Sxl) receives this signal and is responsible for the execution of the alternative developmental programmes of males and females. We have found that the scute (T4) gene, which is involved in neurogenesis, also plays a role in the activation of Sxl. The following results suggest that scute (T4) may be a numerator element of the X:A signal: scute (T4) mutations show female-specific lethality. There are female-specific lethal synergistic interactions between sis-a, a previously described numerator element, and mutants for T4. The female lethality is suppressed by SxlM1, a constitutive allele which expresses an active Sxl product independently of the X:A ratio. The Hw685 mutation, which overexpresses T4, is lethal to males with a duplication of sis-a. This lethality is suppressed by either Sxlf1, or the T4 point mutation sc10-1. There are female-specific lethal interactions between sc10-1 and daughter-less (da), a gene needed maternally for Sxl to become active. The sc10-1 mutation masculinizes triploid intersexes.     

Production of X0 clones in XX females of Drosophila

The experiments reported here are aimed at determining whether mutations deleting the function of the Sex-lethal (Sxl) gene are able to suppress the lethality of X0 clones, induced in females after the time when the state of activity of Sxl is irreversibly fixed by the ratio of the number of X chromosomes to sets of autosomes (X:A). This analysis was carried out by comparing the frequency of induced male clones (X0 constitution) in SxlfLS/+ and Sxl+/Sxl+ females, following irradiation at blastoderm and larval stages. The genotype used in these experiments, however, could also give rise to 2X; 2A cells homozygous for SxlfLS, and such cells would also differentiate male structures. To minimize this possibility, we have constructed a genotype made up of a ring and a rod X chromosome. In such ring-rod females the production of 2X; 2A clones homozygous for SxlfLS is a rather rare event, if possible at all. X0 male clones were produced in both types of females following irradiation at blastoderm stage, while X0 male clones were only observed in SxlfLS/+ females when irradiation took place at larval stage. In this latter case, the only X0 male clones were those that contained the SxlfLS mutation. These results support the idea of Sánchez & N?thiger (1983) that the X:A signal irreversibly sets the state of activity of Sxl at blastoderm stage, and in addition show that X0 clones generated after that time are viable if they contain a Sxl- mutation. These results are compatible with the idea of Sxl being the only gene that responds to the X:A signal.     

Isolation and characterization of a Drosophila gene essential for early embryonic development and formation of cortical cleavage furrows

We have isolated a new female sterile mutant from Drosophila melanogaster, which arrests the embryonic development during the transition from syncytial to cellular blastoderm. Cytological analysis of the mutant embryos indicates that pseudocleavage furrows in the syncytial blastoderm are abnormal but not completely disrupted. However, cleavage furrows during cellularization are totally disorganized, and no embryos can develop beyond this stage. Consistent with this observation, the expression of this gene peaks around the cellular blastoderm and not in any later developmental stages. Based on immunofluorescence experiments, the protein product of this gene is localized in both pseudocleavage furrows at the syncytial blastoderm and in the cleavage furrows during the cellularization stage. Sequence homology analysis demonstrates a modest, but statistically significant, similarity of this protein with the carboxyl-terminal domains of dystrophin and a family of proteins collectively known as apodystrophins. It is possible that this protein may play an essential role in organizing and maintaining a specialized cytoskeletal structure, a function also suggested for dystrophin and apodystrophins.     

Evidence That Sisterless-a and Sisterless-B Are Two of Several Discrete ``numerator Elements'''' of the X/a Sex Determination Signal in Drosophila That Switch Sxl between Two Alternative Stable Expression States

The primary signal for Drosophila sex determination is the number of X chromosomes relative to the number of sets of autosomes. The present report shows that the numerator of this X/A signal appears to be determined by the cumulative dose of a relatively limited number of discrete X-linked genetic elements, two of which are sisterless-a and sisterless-b. This discovery regarding the nature of the sex determination signal grew out of previous studies of both the likely X/A signal target (the feminizing switch gene, Sex-lethal) and two positive regulators of that target gene (sis-a and daughterless). Combinations of genetic perturbations in these three genes had been shown to have synergistic effects. A model proposed in part to account for these interactions generated a large variety of strong predictions for sex-specific synergistic interactions that would be diagnostic for X/A numerator elements and could distinguish them from other components of the sex determination system. All these predictions, as well as other predictions for X/A numerator elements, are shown here to be fulfilled. The most compelling observations involve sexually reciprocal viability effects of duplications of wild-type genes: combinations of sis-a+, sis-b+ and/or Sxl+ duplications are lethal to males but rescue females from the otherwise lethal effects of changes in other components of the sex determination machinery. The many interactions described here illustrate an important principle that may seem counter-intuitive: perturbations of the sex determination signal for Drosophila generally will not appear to affect adult sexual phenotype. This principle follows from the fact that Sxl is involved in dosage compensation as well as sex determination, and from important aspects of the nature and timing of Sxl's regulation both by the X/A signal and by Sxl's own products (positive autoregulation). These factors mask potential effects on adult sexual differentiation by causing the premature death of cells and/or individuals. The fact that the vast array of results presented here conform to this principle is strong evidence in favor of a "binary state" model for Sxl regulation by the X/A signal. This model is favored over an alternative "multiple state" hypothesis that was proposed by others in a different study of the X/A signal. In that same study it was concluded that region 3E8-4F11 of the X chromosome contained especially potent X/A numerator elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors

We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.     

The interaction between daughterless and sex-lethal in triploids: a lethal sex-transforming maternal effect linking sex determination and dosage compensation in Drosophila melanogaster

Regulation of Drosophila sex determination and X-chromosome dosage compensation in response to the X-chromosome/autosome (X/A) balance of the zygote is shown to require proper functioning of both the da+ gene in the mother and the Sxl+ gene in the zygote. Previous studies led to the hypothesis that zygotic Sxl+ alleles are differentially active in females (XXAA) vs males (XYAA) in response to the X/A balance, and that maternal da+ gene product acts as a positive regulator in this connection. Sxl+ activity was proposed to impose the female developmental sequence on cells which would follow the male sequence in its absence. Important predictions of this proposal are verified. This study focuses primarily on the phenotype of triploid intersexes (XXAAA, X/A = 0.67). They are shown here to survive effects of da and Sxl mutations that would be lethal to diploids. The ambiguous X/A signal of intersexes normally causes them to develop as phenotypic mosaics of male and female tissue. Loss of maternal da+ or zygotic Sxl+ gene function shifts their somatic sexual phenotype to the male alternative. A gain-of-function mutation at Sxl has the opposite effect, imposing female development regardless of the maternal genotype with respect to da. It also reduces their rate of X-linked gene expression. The effects of a duplication of Sxl+ resemble those of the constitutive Sxl allele, but are less extreme. The role of these genes in the process of X-chromosome dosage compensation is inferred indirectly from the strict dependence of the mutations' lethal effects on the X/A balance in haploids, diploids, and triploids, and more directly from the effects of the mutations on the phenotypes of the X-linked neomorphic mutations, Bar and Hairy-wing. The relationship of da+ and Sxl+ gene functions to those of other sex-specific lethal loci in D. melanogaster, and to sex determination mechanisms in other species, is discussed.     

Stage-specific chromosomal association of <Emphasis Type="Italic">Drosophila</Emphasis> dMBD2/3 during genome activation

The Drosophila gene dMBD2/3 encodes a protein with significant homologies to the mammalian methyl-DNA binding proteins MBD2 and MBD3. These proteins are essential components of chromatin complexes involved in epigenetic gene regulation. Because the available in vitro data on dMBD2/3 are conflicting we have started an in vivo characterization of dMBD2/3. We detected expression of two isoforms specifically during embryonic development. Staining of whole embryos combined with high-resolution confocal microscopy revealed a highly regulated spatial distribution. During the syncytial blastoderm stage, dMBD2/3 formed speckles that localized to the cytoplasm. Shortly after, during the cellular blastoderm stage, the protein entered the nucleus and formed bright foci that associated with DNA. This rapid transition coincided with the activation of the embryonic genome. A similar observation was made during activation of the spermatocyte genome as dMBD2/3 formed distinct foci associated with the activated Y chromosome. Our results indicate that dMBD2/3 forms specialized nuclear compartments to keep certain genes epigenetically silenced during genome activation.