Sex determination in sciarid flies: a model for the control of differential X-chromosome elimination

In sciarids, all zygotes start development with the 3X;2A chromosome constitution, two of the three X chromosomes being of paternal origin. The elimination of either one or two paternal X chromosomes produces the X:A signal which determines development along the female (2X;2A) or male (X0;2A) pathway, respectively. A model is proposed in which a chromosomal factor (CF) positively interacts with the X chromosome(s) causing its/their elimination. The number of X chromosomes to be eliminated is controlled by a maternal factor (MF) which regulates the amount of free CF factor interacting with the X chromosomes. Imprinting refers to the inability of maternal X chromosomes to bind CF factor. Copyright 1999 Academic Press.     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of <Emphasis Type="Italic">Sciara coprophila</Emphasis>

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.     

Meiotic pairing constraints and the activity of sex chromosomes

The state of activity and condensation of the sex chromosomes in gametocytes is frequently different from that found in somatic cells. For example, whereas the X chromosomes of XY males are euchromatic and active in somatic cells, they are usually condensed and inactive at the onset of meiosis; in the somatic cells of female mammals, one X chromosome is heterochromatic and inactive, but both X chromosomes are euchromatic and active early in meiosis. In species in which the female is the heterogametic sex (ZZ males and ZW females), the W chromosome, which is often seen as a condensed chromatin body in somatic cells, becomes euchromatic in early oocytes. We describe an hypothesis which can explain these changes in the activity and condensation of sex chromosomes in gametocytes. It is based on the fact that normal chromosome pairing seems to be essential for the survival of sex cells; chromosomal anomalies resulting in incomplete pairing during meiosis usually result in gametogenic loss. We argue that the changes seen in the sex chromosomes reflect the need to avoid pairing failure during meiosis. Pairing normally requires structural and conformational homology of the two chromosomes, but when the regions is avoided when these regions become heterochromatinized. This hypothesis provides an explanation for the changes found in gametocytes both in species with male heterogamety and those with female heterogamety. It also suggests possible reasons for the frequent origin of large supernumerary chromosomes from sex chromosomes, and for the reported lack of dosage compensation in species with female heterogamety.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara. It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara. Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

Abstract. In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara . It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara . Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Presence of an Allocyclic Early Replicating X Chromosome in Female Embryos of the Rat, Chinese Hamster and Rabbit

Previous studies on early female mouse embryos revealed the presence of two kinds of inactive X chromosomes, one replicating late and the other early in the DNA synthetic period. The X chromosome that replicates early is of special interest because of its paternal origin, preferential occurrence in trophectoderm and primitive endoderm derivatives, and programmed shift to the late replicator. This study by BrdU labeling and acridine orange fluorescence staining was undertaken to examine whether the inactive X chromosome behaves in a similar manner in other laboratory mammals. In rat embryos the paternal X chromosome was found to show the same behavior in extraembryonic tissues. Early replicating chromosomes were also found in the extraembryonic regions of Chinese hamster and rabbit embryos, although their parental origin could not be determined due to the absent of X chromosome polymorphism in these species. Probably the early replicating X chromosome occurs commonly in mammals. Its functional significance is unknown.     

Paternal chromosome incorporation into the zygote nucleus is controlled by maternal haploid in Drosophila

maternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternal chromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia.     

The elimination and differentiation of chromosomes in the germ line of sciara

The germ line chromosomes of S. coprophila have been followed from the time of origin of the germ cells up to the time of meiosis in the male and up to first larval molt in the female. The mechanism which prevents the accumulation of L (limited) chromosomes in the germ line is a unique process of chromosome elimination: it occurs in male and female embryos after the germ cells have migrated from the pole plasm to the definitive gonad site, and it involves the movement of whole L chromosomes through the nuclear membrane into the cytoplasm. The extra paternal X chromosome is eliminated from the germ cells at the same time and in the same manner. Following this elimination there is a cytological differentiation of the chromosomes remaining inside the nucleus. First, the 4 paternal homologues of the regular complement undergo a loosening of coils and become light-staining whereas the maternal homologues remain condensed like the L's. Next, the L chromosomes undergo a process of extreme attenuation and dispersion following which they return to the condensed state. H³-thymidine autoradiography on gonial and premeiotic cells in the testis reveals that the L chromosomes undergo DNA replication at the end of the S period, also that there are asynchronies in DNA synthesis among the regular chromosomes. The phenomena of differential chromosome staining and asynchronous DNA replication are considered in the light of current theory regarding heterochromatization and gene inactivation, also in relation to the phenomenon of chromosome imprinting encountered in this genus.The studies reported here were supported by the National Science Foundation grants GB-42 and GB-2857, and in part by Contract No. AT-(40-1)-2690 under the Division of Biology and Medicine, U.S. Atomic Energy Commission.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Botany, Columbia University. This work was carried out in the laboratory of Professor J. Herbert Taylor and has been supported in part by U.S. Public Health Training Grant No. 2 T 1-GM-216-05. Grateful acknowledgement is made to Professor Spencer W. Brown, Department of Genetics, University of California, Berkeley, in whose laboratory the final studies were completed.     

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.     

Pattern of Formation of Nuclear Chromosomal Apparatus after Remote Hybridization of Wheat and Buckwheat: Effect of Low Temperature

We studied the pattern of embryo formation in wheat and buckwheat interspecific hybrids. Formation of nonreduced gametes in the embryo sac was induced by low temperature (4–7°C). Changed thermal conditions during meiosis proved to favor formation of hybrid embryos. Vital embryos have been obtained. Karyotypic analysis on the metaphase plates from the root meristem demonstrated different genotypes. Heterogeneous embryos caring both maternal and paternal chromosomes as well as the embryos caring solely maternal or paternal chromosomes have been revealed. Spermatozoa penetrate the embryo sac as a cell, which increases aminopeptidase activity. Sometimes low temperature induces formation of somatic embryos from nucellus cells in the plants.     

Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation

Complete uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat (Triticum aestivum) x pearl millet (Pennisetum glaucum) hybrid embryos. All pearl millet chromosomes were eliminated in a random sequence between 6 and 23 d after pollination. Parental genomes were spatially separated within the hybrid nucleus, and pearl millet chromatin destined for elimination occupied peripheral interphase positions. Structural reorganization of the paternal chromosomes occurred, and mitotic behavior differed between the parental chromosomes. We provide evidence for a novel chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei is different, and heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin is the final step during haploidization.     

Genome size and determination of DNA content of the X chromosomes, autosomes, and germ line-limited chromosomes of Sciara coprophila

The unique chromosome biology of the fungus fly Sciara coprophila has fascinated investigators for over 80 years. Male meiosis exhibits a monopolar spindle, nonrandom segregation of imprinted chromosomes and nondisjunction of the X chromosome. The unusual mechanism of sex determination requires selective elimination of X chromosomes in embryogenesis. Supernumerary (L) chromosomes are also eliminated from the soma during early cleavage divisions. Distinctive DNA puffs on the larval salivary gland chromosomes are sites of DNA amplification. As a foundation for future genome studies to explore these many unusual phenomena, we have used DNA-Feulgen cytophotometry to determine genome size from hemocyte nuclei of male (X0) and female (XX) larvae and adults. The DNA content of the X chromosome is approximately 0.05 pg DNA and the autosomal complement is approximately 0.45 pg DNA. Measurements of DNA levels for individual sperm from adults showed that the DNA contribution of the germ line-limited (L) chromosomes constitutes as much as 35% of the DNA of the male gamete. A parallel study using Sciara ocellaris, a related species lacking L chromosomes, confirmed the presence of two X chromosomes in the sperm of this species.     

Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare x H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.     

Chromosomal instability in lemon grass,Cymbopogon flexuosus (Steudel) Wats

Somatic mitoses in C. flexuosus exhibit a significant degree of chromosomal instability leading to nearly 33% cells with chromosome elimination. A range of chromosome numbers between 20-8 (most common being 2n=20, the somatic number for this species) was encountered from root tip cells. The course of variation suggests a gradual elimination of somatic chromosomes. The larger chromosomes are less stable and are eliminated earlier. The variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result from weaker spindle operation and minute chromosomes.CIMAP Publication No. 571 (1984)     

X chromosome reactivation and regulation in cloned embryos

Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.     

Histone macroH2A1.2 is concentrated in the XY-body by the early pachytene stage of spermatogenesis

The pairing of sex chromosomes during meiosis in male mammals is associated with ongoing heterochromatinization and X inactivation. This process occurs in a specific area of the nucleus that can be discerned morphologically: the sex vesicle or XY-body. In contrast to X inactivation in the somatic cells of female mammals the reasons for X inactivation in the male germline remain obscure. We have recently demonstrated that the inactive X chromosome in somatic cells of female mammals is marked by a high concentration of histone macroH2A. Here we investigate X inactivation in the meiotic cells of the male germline. We demonstrate here that macroH2A1.2 is present in the nuclei of germ cells starting first with localization that is largely, if not exclusively, to the developing XY-body in early pachytene spermatocytes. Our results suggest that inactivation of sex chromosomes in the male germ cell includes a major alteration of the nucleosomal structure.     

Disruption of imprinted X inactivation by parent-of-origin effects at Tsix

In marsupials and in extraembryonic tissues of placental mammals, X inactivation is imprinted to occur on the paternal chromosome. Here, we find that imprinting is controlled by the antisense Xist gene, Tsix. Tsix is maternally expressed and mice carrying a Tsix deletion show normal paternal but impaired maternal transmission. Maternal inheritance occurs infrequently, with surviving progeny showing intrauterine growth retardation and reduced fertility. Transmission ratio distortion results from disrupted imprinting and postimplantation loss of mutant embryos. In contrast to effects in embryonic stem cells, deleting Tsix causes ectopic X inactivation in early male embryos and inactivation of both X chromosomes in female embryos, indicating that X chromosome counting cannot override Tsix imprinting. These results highlight differences between imprinted and random X inactivation but show that Tsix regulates both. We propose that an imprinting center lies within Tsix.