XO Embryos Research Articles

Dose-dependent action of the RNA binding protein FOX-1 to relay X-chromosome number and determine C. elegans sex.

We demonstrate how RNA binding protein FOX-1 functions as a dose-dependent X-signal element to communicate X-chromosome number and thereby determine nematode sex. FOX-1, an RNA recognition motif protein, triggers hermaphrodite development in XX embryos by causing non-productive alternative pre-mRNA splicing of xol-1, the master sex-determination switch gene that triggers male development in XO embryos. RNA binding experiments together with genome editing demonstrate that FOX-1 binds to multiple GCAUG and GCACG motifs in a xol-1 intron, causing intron retention or partial exon deletion, thereby eliminating male-determining XOL-1 protein. Transforming all motifs to GCAUG or GCACG permits accurate alternative splicing, demonstrating efficacy of both motifs. Mutating subsets of both motifs partially alleviates non-productive splicing. Mutating all motifs blocks it, as does transforming them to low-affinity GCUUG motifs. Combining multiple high-affinity binding sites with the twofold change in FOX-1 concentration between XX and XO embryos achieves dose-sensitivity in splicing regulation to determine sex.

Studying Early Lethality of 45,XO (Turner's Syndrome) Embryos Using Human Embryonic Stem Cells

Turner's syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos.

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.

Roles for mating and environment in C. elegans sex determination.

In Caenorhabditis elegans the two sexes, hermaphrodites and males, are thought to be irreversibly determined at fertilization by the ratio of X chromosomes to sets of autosomes: XX embryos develop as hermaphrodites and XO embryos as males. We show instead that both sex and genotype of C. elegans can be altered postembryonically and that this flexibility requires sexual reproduction. When grown in specific bacterial metabolites, some XX larvae generated by mating males and hermaphrodites develop as males and lose one X chromosome. However, XX larvae produced by hermaphrodite self-fertilization show no such changes. We propose that sexual reproduction increases developmental flexibility of progeny, allowing for better adaptation to changing environments.

Developmental retardation of XO mouse embryos at mid-gestation.

It is not known why XO mouse embryos, which develop more slowly than XX embryos until early mid-gestation, reach the same stage in their growth and development as their XX littermates at the mid-gestation stage. It is hypothesized that there is an effect of 'litter size' that causes an acceleration of the development of XO embryos at mid-gestation. The present study was performed to determine whether the development of XO embryos is retarded compared with that of their XX litermates at early mid-gestation (day 8 of gestation), before reduction of litter size. The percentage of pre-somite stage XO embryos was greater than the percentage of pre-somite stage XX embryos, and the mean number of somites was greater in XX embryos than it was in XO embryos. These findings indicate that the development of XO embryos was retarded when compared with that of their XX litermates at early mid-gestation. This result is discussed with respect to the compensatory development of XO embryos at mid-gestation and the reduction of litter size shortly after early mid-gestation.

The nuclear hormone receptor SEX-1 is an X-chromosome signal that determines nematode sex

Organisms in many phyla determine sexual fate by distinguishing one X chromosome from two. Here we use the model organism Caenorhabditis elegans to dissect such an X-chromosome-counting mechanism in molecular detail. In this nematode, several genes on the X chromosome called X signal elements communicate X-chromosome dose by controlling the activity of the sex-determination gene xol-1. xol-1 specifies male (XO) fate when active and hermaphrodite (XX) fate when inactive. The only X signal element described so far represses xol-1 post-transcriptionally, but xol-1 is repressed in XX animals by transcriptional and post-transcriptional mechanisms. Here we identify a nuclear-hormone-receptor homologue, SEX-1, that regulates the transcription of xol-1. We show that sex-1 is vital to X-chromosome counting: changing sex-1 gene dose in XX or XO embryos causes sexual transformation and death from inadequate dosage compensation (the hermaphrodite-specific process that equalizes X-gene expression between the sexes). The SEX-1 protein acts directly on xol-1, associating with its promoter in vivo and repressing xol-1 transcription in XX embryos. Thus, xol-1 is the direct molecular target of the primary sex-determination signal, and the dose of a nuclear hormone receptor helps to communicate X-chromosome number to determine nematode sex.

Viability, development and incidence of chromosome anomalies of preimplantation embryos from XO mice.

We examined the viability and developmental status of XO embryos at preimplantation stage (day 3 of gestation) by assessing blastocyst formation and counting cell number using our XO mouse colony. We also examined the incidence of chromosome anomalies. Embryos from XO mice (XY, XX and XO) developed more slowly (in cell numbers and blastocoele formation) than those from XX mice (XY and XX). XO embryos also tended to develop more slowly than XX embryos in the XO group. Although litter size at the preimplantation stage of gestation was almost twice as large (12.7) as those at mid-gestation (7.6) and near-term (7.2) in this colony, the adjusted XY:XX:XO ratio (2.8:2.0:1.0) did not differ greatly. This indicates that almost half of the embryos must have been eliminated during the first half of gestation in the XO group, probably regardless of sex chromosome complements. Thus, we consider that maternal XO sex chromosome constitution is disadvantageous for the intrauterine development of the embryo during the early period of gestation. This may be related to precocious aging of XO mice. Further, we confirmed that a high incidence of abnormal karyotypes occurs in embryos from our XO mouse colony.

DPY-27: A chromosome condensation protein homolog that regulates C. elegans dosage compensation through association with the X chromosome

dpy-27 is an essential dosage compensation gene that acts to reduce expression of both hermaphrodite X chromosomes. The DPY-27 protein becomes specifically localized to the X chromosomes of wild-type XX embryos, but remains diffusely distributed throughout the nuclei of male (XO) embryos. In xol-1 mutant XO embryos that activate the XX mode of dosage compensation and die from inappropriately low X chromosome transcript levels, DPY-27 becomes localized to X. Therefore, sex specificity of the dosage compensation process is regulated at the step of DPY-27 X chromosome localization. DPY-27 exhibits striking similarity to proteins required for assembly and structural maintenance of Xenopus chromosomes in vitro and for segregation of yeast chromosomes in vivo. These findings suggest a link between global regulation of gene expression and higher order chromosome structure. We propose that DPY-27 implements dosage compensation by condensing the chromatin structure of X in a manner that causes general reduction of X chromosome expression.

Function of the her-1 gene is required for maintenance of male differentiation in adult tissues of C. elegans.

Function of the sex-determining gene her-1 is required in XO embryos of C. elegans to specify male development. Using a temperature-sensitive mutant of her-1, we show that when XO males reared at a permissive temperature are shifted as adults to a nonpermissive temperature, they initiate vitellogenin synthesis in the intestine and oocyte production in the germline. A similar shift has no effect on her-1(+) males. We conclude that sexual differentiation of the intestine and germline is plastic, requiring her-1 expression throughout adulthood for maintenance of the male state.

Growth and development of 39, X mouse embryos at mid-gestation

We examined the growth and development of XO embryos at mid-gestation (day 10) by assessing crown-rump length, head length, somite number, and morphological score (Van Maele-Fabry et al., 1990), using an XO mouse colony established from the Jcl/ICR strain. Contrary to expectation, there were no differences between the XO and XX embryos in any parameter measured. Also, no particular region was specifically retarded in XO embryos during development. We therefore concluded that the growth and development of XO embryos were not retarded when compared with their XX littermates at day 10 of gestation.

The Caenorhabditis elegans sex determining gene fem-3 is regulated post-transcriptionally.

The fem-3 gene of Caenorhabditis elegans is required for male development. Both maternal and zygotic fem-3 activities are required for spermatogenesis in the XX hermaphrodite germline and for male development in somatic and germline tissues XO (male) animals. Here we show that fem-3 RNA is contributed to embryos as a maternal product and that this RNA is degraded early in embryonic development. The poly(A) tail of embryonic fem-3 RNA is substantially longer than that of adult hermaphrodites which indicates that poly(A) tail lengthening probably occurs at or soon after fertilization. During subsequent development, fem-3 poly(A) tails shorten. The amount of fem-3 RNA in XX and XO embryos is equivalent, suggesting sex-specific regulation of maternal fem-3 activity occurs post-transcriptionally. The sequence of fem-3 predicts an open reading frame that could encode a soluble protein; putative fem-3 null mutants truncate this open reading frame. We discuss the implications of these results for the regulation and function of fem-3.

Teratogen susceptibility of XO mothers and XO embryos in mice.

We examined whether the chromosomal imbalance inherent in an XO constitution in mice is more susceptible to teratogenic influence of biotin deficiency using a newly established mouse colony with pure X monosomy. We hypothesized that XO mothers or XO embryos might be more susceptible to certain teratogens. Contrary to our expectation, the incidence of external malformations induced by biotin deficiency did not differ either between XX dams and XO dams or between XX fetuses and XO fetuses.

Molecular cloning and transcript analysis of fem-3, a sex-determination gene in Caenorhabditis elegans.

The fem-3 gene is required for specification of the male fate in the nematode Caenorhabditis elegans: XO males need fem-3 for male differentiation in both soma and germ line; XX hermaphrodites need it for the production of sperm. We have cloned fem-3 by transposon tagging. Among eight spontaneous fem-3 mutations generated in a strain in which the transposable element Tc1 is mobile, six contain Tc1 insertions in a 2-kb region of the genome. From this region, we have identified three fem-3 transcripts. Two, 1.7 kb and 1.62 kb, are presented in embryos, and two, 1.62 kb and 1.55 kb, are present in L4s and adults. The fem-3 transcripts are not XO specific; however, in XX adult hermaphrodites, they appear to be limited to the germ line--a tissue involved in male development (both for spermatogenesis and for the maternal contribution of fem-3 to the embryo). The amount of fem-3 RNA in XO embryos is approximately sixfold greater than in XX embryos. The significance of this difference in specifying male development in XO but not in XX embryos is discussed.

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice

The meiotic segregation pattern of the X chromosome in XO mice was determined by counting second meiotic metaphase chromosomes. A total of 584 oocytes was collected from superovulated but unmated XO and XX mice. Second meiotic metaphase (MII) counts indicated that the X chromosome in XO mice did not segregate randomly but remained preferentially in the oocyte. Increased maternal age had no effect upon this segregation pattern. Selection against XO and YO preimplantation embryos was evaluated by observing their relative frequencies during first and second cleavage divisions. A combined total of 1650 preimplantation eggs was collected from superovulated and mated XO and XX female mice. Eggs were obtained during metaphase of first (CI) or second cleavage (CII) divisions by injecting mated females with colchicine at 28 or 49 hr, respectively, after human chorionic gonadotrophin (HCG) administration. Prior to analysis, chromosomes were banded by trypsin-Giemsa, which facilitated specific chromosome identification, including the X and Y. The proportion of XX, XY, XO, and YO embryos observed at CI from XO females was not significantly different from expected values based on MII counts. At CII, a significant difference was observed from the expected proportion of embryo classes resulting from a reduction in XO and YO embryos. The results provide cytogenetic evidence emphasizing that, in order for development to continue, at least one functional X chromosome is necessary as early as the two-cell stage. Triploid one- and two-cell embryos were observed more frequently in embryos from XO than XX females. The sex ratio was not significantly different from 1:1 during either cleavage stage regardless of the type of female from which the embryos were derived.

The mortality of human XO embryos.

Summary. Because of the high mortality of human XO embryos, it was questioned whether the single X in a proportion of cells was inactivated. If genetic inactivation of the single X in XO conceptuses was of significant importance for reduced viability and subsequent abortion, a proportion of cells grown from XO fetuses should carry an inactivated late-labelling X chromosome. This hypothesis was tested by autoradiography and analysis of the late DNA-labelling pattern of XO cells derived from a 15-week-old spontaneous human abortus. None of the analysed cells had a late-labelling X chromosome. Consequently, the XO cells from this aborted fetus were not different from XO cells from liveborn individuals.

Gonadal dysgenesis in a newborn with XO karyotype.

Few reports of karyotype-verified Turner's syndrome in neonates are available. This report is the third such case in which normal primordial follicles were not present in the ovary at the time of birth. In contrast, germ cells have been identified in all except one neonate with XO Turner's syndrome. These findings and the published data in XO embryos support the concept that developmental arrest at the stage of primordial follicle formation and subsequent regression of germinal structures is the most likely morphological basis for the ovarian dysgenesis which accompanies the XO state.