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.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

Evidence of a Dual Function in Fl(2)d, a Gene Needed for Sex-Lethal Expression in Drosophila Melanogaster

In Drosophila melanogaster, the female sexual development of the soma and the germline requires the activity of the gene Sxl. The somatic cells need the function of the gene fl(2)d to follow the female developmental pathway, due to its involvement in the female-specific splicing of Sxl RNA. Here we report the analysis of both fl(2)d1 and fl(2)d2 mutations: (1) fl(2)d1 is a temperature-sensitive mutation lethal in females and semilethal in males; (2) fl(2)d2 is lethal in both sexes; (3) the fl(2)d1/fl(2)d2 constitution is temperature-sensitive and lethal in females, while semilethal in males. The temperature-sensitive period of fl(2)d1 in females expands the whole development. SxlM1 partially suppresses the lethality of fl(2)d1 homozygous females and that of fl(2)d1/fl(2)d2 constitution, whereas it does not suppress the lethality of fl(2)d2 homozygous females. The addition of extra Sxl+ copies does not increase the suppression effect of SxlM1. The fl(2)d1 mutation in homozygosis and the fl(2)d1/fl(2)d2 constitution, but not the fl(2)d2 in homozygosis, partially suppress the lethality of SxlM1 males. This suppression is not prevented by the addition of extra Sxl+ copies. The semilethality of both fl(2)d1 and fl(2)d1/fl(2)d2 males, and the lethality of fl(2)d2 males, is independent of Sxl function. There is no female synergistic lethality between mutations at fl(2)d and neither at sc or da. However, the female synergistic lethality between mutations at Sxl and either sc or da is increased by fl(2)d mutations. We have analyzed the effect of the fl(2)d mutations on the germline development of both females and males. For that purpose, we carried out the clonal analysis of fl(2)d1 in the germline. In addition, pole cells homozygous for fl(2)d2 were transplanted into wild-type host embryos, and we checked whether the mutant pole cells were capable of forming functional gametes. The results indicated that fl(2)d mutant germ cells cannot give rise to functional oocytes, while they can form functional sperm. Moreover, SxlM1 suppresses the sterility of the fl(2)d1 homozygous females developing at the permissive temperature. Thus, with respect to the development of the germline the fl(2)d mutations mimic the behavior of loss-of-function mutations at the gene Sxl. Females double heterozygous for fl(2)d and snf1621 are fully viable and fertile. fl(2)d2 in heterozygosis partially suppresses the phenotype of female germ cells homozygous for snf1621; however, this is not the case with the fl(2)d1 mutation. The fl(2)d mutations partially suppress the phenotype of the female germ cells homozygous for ovoDIrSI.(ABSTRACT TRUNCATED AT 400 WORDS)     

Requirement offlex (femalelethal onX) in the development of the female germ line ofDrosophila melanogaster

Drosophila melanogaster females homozygous forflex, an X-linked recessive mutation, do not survive. Hemizygous males are unaffected. Homozygous embryos appear to lack SXL, the product of theSex-lethal (Sxl) gene, apparently as a result of disruption ofSxl splicing. It is known that bothSxl and its somatic splicing regulators [snf andfl(2)d] also function in the development of the female germ line. For this reason, we investigated the role offlex in the germ line by generatingflex/flex clones inflex/+ females. Females carrying such clones in their germ lines do not lay eggs whereas females carryingflex+ eggs lay viable eggs. Additionally, DAPI staining of ovarioles showed that diploid germ cells that are homozygous mutant forflex do not complete oogenesis. These results indicate that theflex+ gene product may be required for the development of the female germ line.     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

     

Analysis of the Drosophila maternal effect mutant MAT(3)1 by pole cell transplantation experiments

Summary Pole cell transplantations were used to construct germ line mosaics of the Drosophila melanogaster maternal effect mutant mat(3)1. The mutant is of particular interest since the development of embryos derived from homozygous mat(3)1 females is arrested at the pole cell stage. Such embryos form exclusively pole cells and no blastoderm cells. By means of germ line mosaics we could demonstrate the primary target tissue of mutant gene expression. For normal development the mat(3)1 ⁺gene has to be expressed in the germ line. Pole cells formed in defective embryos derived from homozygous mutant mothers were transplanted into normal recipient embryos to test their developmental potential. Heterozygous mat(3)1 pole cells were found to form fertile gametes in both sexes whereas homozygous mat(3)1 pole cells form fertile gametes only in males. The lack of progeny derived from homozygous mat(3)1 donor pole cells in recipient females further demonstrates the germ line autonomy of the mat(3)1 mutation. Pole cells from defective embryos that are transplanted into normal hosts colonize the gonads with the same frequency as donor pole cells derived from normal embryos. This indicates that mat(3)1 derived pole cells are normal with respect to their function as germ cells and that the mat(3)1 mutant might therefore offer a convenient source for the mass isolation of functional pole cells.     

Sex determination in Drosophila: the X-chromosomal gene liz is required for Sxl activity.

In Drosophila, females require products of the gene Sxl for sex determination, dosage compensation and fertility. I show here that the X-chromosomal gene liz, located in 4F1 to 4F11 and previously called fs(1)1621, provides maternal and zygotic functions necessary for Sxl activity in germ line and soma. In XX animals, the mutation SxlM1 which was reported to express the female-specific functions of Sxl constitutively can rescue all phenotypes resulting from lack of liz product. XY animals carrying SxlM1 and lacking maternal or zygotic liz activity survive as males with some female traits. A stock was constructed in which the females are liz SxlM1/liz SxlM1 and males liz SxlM1/Y. This shows that SxlM1 is not truly expressed constitutively in animals with an X:A ratio of 0.5, but requires activity of liz for initiation or maintenance.     

Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.     

Post-implantation development and cytogenetic analysis of diandric heterozygous diploid mouse embryos

Diandric heterozygous diploid mouse embryos were produced by standard micromanipulatory techniques using eggs from female mice with a normal chromosome constitution and fertilised by homozygous Rb(1.3)1Bnr males containing a pair of large metacentric marker chromosomes in their karyotype. The constructed diandric eggs were transferred to the oviducts of pseudopregnant recipients and subsequently autopsied midday on the eighth day of gestation. From a total of 85 eggs transferred to females that subsequently became pregnant, 30 implanted. Eighteen implantation sites were found to contain resorptions, and 12 egg cylinder stage embryos were recovered. These were cytogenetically examined. In two cases, no mitoses were observed, and in a third embryo of normal size, only a single paternally-derived marker chromosome was present in its mitoses, indicating that this embryo had a normal chromosome constitution. This presumably resulted from a technical error during the micromanipulatory procedure. The remaining nine morphologically small but normal embryos were diploid, and each had two paternally-derived marker chromosomes, thus establishing their ploidy and confirming their diandric origin. G-banding analysis revealed that all of these embryos had an XY sex chromosome constitution. Since the expected XX:XY:YY ratio of 1:2:1 was not observed, it is clear that the XX class embryos were lost at some stage during the pre- or early post-implantation period, though whether they are represented by the resorption sites is not yet established. The YY class would not be expected to be recovered in any case, as these embryos are believed to be lost during early cleavage. The cytogenetic findings reported here are therefore similar to the results of the chromosomal analyses of the human complete hydatidiform moles of dispermic origin, all of which apparently have an XY karyotype. It is unclear why, both in the human and in the mouse, the XX diandric heterozygous diploid group should develop poorly compared to similar embryos with an XY karyotype.     

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.     

The fused toes locus is essential for somatic-germ cell interactions that foster germ cell maturation in developing gonads in mice

Ovarian development absolutely depends on communication between somatic and germ cell components. In contrast, it is not until after birth that interactions between somatic and germ cells play an important role in testicular maturation and spermatogenesis. Previously, we discovered that Irx3 expression was localized specifically to female gonads during embryonic development; therefore, we sought to determine the function of this genetic locus in developing gonads of both sexes. The fused toes (Ft) mutant mouse is missing 1.6 Mb of chromosome 8, which includes the entire IrxB cluster (Irx3, Irx5, Irx6), Ftm, Fts, and Fto genes. Homozygote Ft mutant embryos die around embryonic day 13.5 (E13.5); therefore, to assess later development, we harvested gonads at E11.5 and transplanted them into nude mouse hosts. Our results show defects in somatic and germ cell maturation in developing gonads of both sexes. Testis development was normal initially; however, by 3-wk posttransplantation, expression of Sertoli and peritubular myoid cell markers were decreased. In many cases, gonocytes failed to migrate to structurally impaired basement membranes of seminiferous cords. Developmental abnormalities of the ovary appeared earlier and were more severe. Over time, the Ft mutant ovary formed very few primordial or primary follicles, which contained oocytes that failed to grow and were surrounded by scarce granulosa cells that expressed low levels of FOXL2. By 3 wk after transplantation, it was difficult to identify ovarian tissue in Ft mutant ovary transplants. In summary, we conclude that the Ft locus contains genes essential for somatic-germ cell interactions, without which the germ cell niche fails to mature in both sexes.     

Akodon sex reversed females: the never ending story

The existence of fertile A. azarae females with a chromosome sex pair indistinguishable from that of males was reported more than 35 years ago. These heterogametic females were initially thought to occur due to an extreme process of dosage compensation in which X inactivation was restricted to Xp and complemented by a deletion of Xq (Xx females). Later on, a C-banding analysis of A. mollis variant females showed that these specimens were in fact XY* sex reversed and not Xx females. The finding of positive testing for Zfy and Sry multiple-copy genes in Akodon males and heterogametic females confirmed the XY* assumption. At the present time, XY* sex reversed females have been found to exist in nine Akodon species. Akodon heterogametic females produce X and Y* oocytes, which upon sperm fertilization give rise to viable XX (female), XY* (female), and XY (male) embryos, and to non-viable Y*Y zygotes. Heterozygous females exhibit a better reproductive performance than XX females in order to compensate the Y*Y zygote wastage. XY* sex reversed females are assumed to occur due to a deficient Sry expression resulting in the development of ovaries instead of testes. Moreover, the appearance of Y* elements is a highly recurrent event. It is proposed that homozygosity for an autosomal or pseudoautosomal recessive mutation (s-) inhibits Sry expression giving rise to XY* embryos with ovary development. Location of the Y* chromosome in the female germ cell lineage produces an ovary-specific imprinting of the Sry* gene maintaining its defective expression through generations independently from the presence or absence of s- homozygosity. By escaping the ovary-specific methylation some Y* chromosomes turn back to normal Ys producing Y oocytes capable of generating normal male embryos when fertilized by an X sperm. Fluctuations in the rate of variant females in field populations and in laboratory colonies of Akodon depend on the balance between the appearance of new variant females (s-/s-, XY* specimens) and the extinction of sex reversed specimens due to imprinting escape.     

Developmental arrest of fertilized eggs from the B6.YDOM sex-reversed female mouse

When the Y chromosome of a Mus musculus domesticus mouse strain is placed onto the C57BL/6J (B6) inbred background, the XY progeny develop ovaries or ovotestes but never normal testes during fetal life. While some of the hermaphroditic males become fertile, none of the XY females produces litters. Here, we examined the fertility and development of oocytes derived from the XY female mouse. With or without preceding injection of gonadotropins, female mice were mated with normal B6 males, and their embryos were recovered at various developmental stages. In vitro fertilization was performed with the eggs recovered from the oviduct after treatment with go-nadotropins. Development of embryos was examined by both light and electron microscopy. The results indicate that the oocytes released from the B6.Y^DOM ovary were efficiently fertilized and often initiated the first cell cleavage, but all embryos died during early preimplantation periods. Even when oocytes were fertilized in vitro, minimizing their exposure to the XY oviduct/uterus environment, most embryos died at the 1- or 2-cell stage. A few exceptional embryos reached the 4- or 8-cell stage, but abnormalities were evident in both nuclear and cytoplasmic structures of all embryos. After cleavage, neighbouring blastomeres were only loosely associated, and microvilli were abundant at the intercellular interfaces. We postulate that oocytes of the B.6.Y^DOM female mouse become defective during XY ovarian differentiation, and, hence, fail to proceed through normal embryonic development. © 1994 Wiley-Liss, Inc.     

泥鳅雄核发育纯合二倍体的产生

以机械方法挑去泥鳅(Misgurnus anguillicaudatus)×大鳞副泥鳅(Paramisgurnus dabryanus)(♀)属间杂交受精卵的雌核,得到泥鳅雄核发育单倍体胚胎。将这种单倍体胚胎的囊胚细胞核移植到大鳞副泥鳅去核卵中,获得了243个原肠胚胎,其染色体鉴定表明,29.6%的核移植体的染色体发生了加倍。在另一实验组中,从769个核移植卵得到了5尾2cm以上的个体。尾鳍染色体鉴定、肌肉LDH同工酶电泳和形态鉴别表明,这5尾核移植体为泥鳅雄核发育纯合二倍体。     

Ectopic formation of primordial germ cells by transplantation of the germ plasm: Direct evidence for germ cell determinant in Xenopus

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus.EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.     

An ultrastructural study of the ovary cord organization and oogenesis in the amphibian leech Batracobdella algira (Annelida,Clitellata, Hirudinida)

This study reveals the ovary micromorphology and the course of oogenesis in the leech Batracobdella algira (Glossiphoniidae). Using light, fluorescence, and electron microscopies, the paired ovaries were analyzed. At the beginning of the breeding season, the ovaries were small, but as oogenesis progressed, they increased in size significantly, broadened, and elongated. A single convoluted ovary cord was located inside each ovary. The ovary cord was composed of numerous germ cells gathered into syncytial groups, which are called germ-line cysts. During oogenesis, the clustering germ cells differentiated into two functional categories, i.e., nurse cells and oocytes, and therefore, this oogenesis was recognized as being meroistic. As a rule, each clustering germ cell had one connection in the form of a broad cytoplasmic channel (intercellular bridge) that connected it to the cytophore. There was a synchrony in the development of the clustering germ cells in the whole ovary cord. In the immature leeches, the ovary cords contained undifferentiated germ cells exclusively, from which, previtellogenic oocytes and nurse cells differentiated as the breeding season progressed. Only the oocytes grew considerably, gathered nutritive material, and protruded at the ovary cord surface. The vitellogenic oocytes subsequently detached from the cord and filled tightly the ovary sac, while the nurse cells and the cytophore degenerated. Ripe eggs were finally deposited into the cocoons. A comparison of the ovary structure and oogenesis revealed that almost all of the features that are described in the studied species were similar to those that are known from other representatives of Glossiphoniidae, which indicates their evolutionary conservatism within this family.