Extraembryonic Cell Lineages Research Articles

Homeobox gene HB24, a regulator of haematopoiesis, is a candidate for regulating differentiation of the extra-embryonic trophoblast cell lineage.

Expression of the human homeobox gene HB24 in the cytotrophoblast stem cell population in first-trimester human placenta was investigated. HB24 expression was downregulated after cytotrophoblast had differentiated into syncytiotrophoblast. Expression of HB24 was also detected in first-trimester invasive extravillous cytotrophoblast cells. In term placenta, HB24 expression was detected in residual cytotrophoblast cells and in syncytiotrophoblast. Northern analysis revealed two HB24 transcripts in first-trimester placenta of approximately 2.5 kb and 5.3 kb. Ribonuclease protection assays were used to confirm expression of HB24 in first-trimester and term placenta and also to provide evidence that HB24 is downregulated in choriocarcinoma cell lines. In addition to being required for differentiation of the haematopoietic cell lineage, HB24 may be required for differentiation of the extra-embryonic trophoblast cell lineage.

Confined placental mosaicism for trisomies 2, 3, 7, 8, 9, 16, and 22: their incidence, likely origins, and mechanisms for cell lineage compartmentalization.

Analysis of confined placental mosaicism (CPM) for trisomies 2, 3, 7, 8, 9, 16, and 22, in diagnostic chorionic villus sampling procedures, demonstrates apparent incidences of CPM for individual trisomies of between 9 and 91 cases per 100,000 pregnancies, with trisomy 7 being the most common. More detailed analysis of the percentage of aneuploid cells present, and the distribution of abnormality between the cytotrophoblast and extra-embryonic mesoderm cell lineages, shows a highly specific pattern for each chromosome. Theoretical considerations, in conjunction with direct observations, indicate that the overriding influence on the patterns of cell distribution seen in CPM is the distribution of aneuploid cells laid down during blastogenesis. This in turn reflects closely the origin of mosaicism from either correction of a trisomic conception or post-fertilization somatic error. The pattern of aneuploid cells for each trisomy, as seen at the end of the first trimester and later in pregnancy, can therefore be used to predict the relative contribution of meiotic and mitotic errors to CPM, and hence the likely incidences of uniparental disomy from this source, upd(16)mat being the most common (1 in 10,000 continuing pregnancies). In addition, CPM for trisomies 2, 3, and 8 shows strong evidence of a non-random distribution of aneuploid cells between the different extra-embryonic cell lineages. Analysis of comparable data from spontaneous abortion material repeats this non-random pattern for trisomies 2 and 3, and suggests that a similar phenomenon may also be occurring for trisomy 22. A non-random distribution could be attributable to selection for or against, or intolerance of, particular trisomic cells in certain lineages, but is more probably a result of either cell lineage-specific non-disjunction or consistent uneven compartmentalization of aneuploid cells during blastocyst development.

X Chromosome Inactivation and Imprinting

In contrast to the random inactivation of either maternal or paternal X-chromosome in the somatic cells of eutherian mammals, in marsupials the paternal X-chromosome is preferentially inactivated in all cells. Similar exclusively paternal X-inactivation occurs in two extraembryonic cell lineages of mice and rats. Thus, genetic imprinting is an important feature of X-inactivation. In embryonic development the initiation of X-inactivation is thought to occur through the X-inactivation centre, located on the X-Chromosome, and thus imprinting probably acts through this centre. A candidate gene for a role in the inactivation centre is Xist (X inactive specific transcript) which is expressed only from the inactive X-Chromosome. The expression of Xist in the mouse embryo is appropriate for it to be a cause rather than a consequence of inactivation. It appears before inactivation, and only the paternal allele is expressed in the extraembryonic lineages. In the germ cells also changes in X-chromosome activity are accompanied by changes in Xist expression. Studies of methylation of the Xist gene have shown that in male tissues where Xist is not active it is fully methylated, whereas in the female the allele on the active X-chromosome only is methylated. In male germ cells, where Xist is expressed, it is demethylated and the demethylation persists in mature spermatozoa. Thus a methylation difference in germ cells could possibly be the imprint. In androgenotes, with paternally derived chromosomes, Xist is expressed at the 4-cell stage, whereas in gynogenotes and parthenogenotes expression does not appear until the blastocyst stage. Thus, Xist expression shows imprinting. When expression appears in parthenogenotes it is random, suggesting that the imprint has been lost. The Xist gene has no open reading frame and is thought to act through mRNA but its function is unknown.

Oct-4 Transcription Factor Is Differentially Expressed in the Mouse Embryo during Establishment of the First Two Extraembryonic Cell Lineages Involved in Implantation

Oct-4 is the earliest expressed gene known to encode a transcription factor which is developmentally regulated during mammalian embryogenesis. In order to understand the role of Oct-4 in early murine embryogenesis, we carried out an analysis of the temporal and spatial pattern of protein expression. We report the presence of Oct-4 protein in cultured cells and murine embryos as determined by immunohistochemistry using confocal microscopy. Oct-4 protein is present in both embryonic stem cells and embryonal carcinoma cells, but is down-regulated following differentiation of these cells by culture in the absence of leukemia inhibitory factor or in the presence of retinoic acid, respectively. In embryos, the protein is found at low levels in unfertilized eggs and is localized predominantly to the pronuclei upon their formation following fertilization. The protein is present in the nuclei in all cleavage stages, but following differentiation of the trophectoderm at the blastocyst stage, Oct-4 protein is only expressed in the inner cell mass. The pattern of protein expression to this stage correlates well with previously reported in situ hybridization results; however, a striking difference was seen in primitive endoderm cells which had begun to differentiate and migrate along the inner surface of the trophectoderm. In direct contrast to RNA localization studies which demonstrate that there are only low levels of Oct-4 transcripts in primitive endoderm cells, protein expression in these early migrating cells is higher than in the inner cell mass.

Abnormal development of embryonic and extraembryonic cell lineages in parthenogenetic mouse embryos.

Parthenogetically activated, diploid mouse oocytes can develop to midgestation stages in utero. However, even these advanced parthenogenones appear to die because of much reduced trophoblast and yolk sac development. Previous studies have compared the general features of parthenogenetic and androgenetic development and determined the fate of uniparental cells in chimeras with normal embryos. These studies led to the concept of genomic imprinting as the cause for developmental failure when either the maternal or the paternal genome is duplicated, with the corresponding deficiency of the other. Genomic imprinting appears to arise during gametogenesis and to act through dosage effects in a set of imprinted genes, whose expression depends on their parental origin. In this study we undertook a more detailed morphological analysis of parthenogenetic development in the mouse and established a classification system to quantify the developmental extent of parthenogenones. We found that the failure of parthenogenones occurred at different times during early postimplantation development, generating a spectrum of concepti which had developed to different extents, with only a small fraction of the embryos reaching advanced somite stages. In all parthenogenones differentiation and proliferation of the trophectoderm and primitive endoderm lineages (both extraembryonic) was abnormal, and in all, even the best-developed parthenogenones, we observed similar deficiencies in the embryonic lineages, especially the mesoderm. Common to all abnormally developed lineages was that the proportion of undifferentiated precursor cells was much reduced, while their differentiated descendants were relatively abundant. We propose, therefore, that the failure of parthenogenones to develop to term is due to abnormal regulation of differentiation and proliferation in both embryonic and extraembryonic lineages. In this hypothesis, the apparent tissue specific defects observed in parthenogenones arise as a consequence of the functional importance of certain tissues (like the trophoblast) early in development. The spectrum of parthenogenones thus appears to reflect critical events in early development, whose regulation are affected by genomic imprinting.

Development of early cell lineages in marsupial embryos: an overview.

All major embryonic and extra-embryonic cell lineages are established before implantation in marsupials. In reptiles, birds, monotremes and most marsupials, the zygote is polarized, sometimes markedly so, and the cleavage pattern in relation to the polarized state provides the mechanism for the generation of positional signals. These ensure that the embryonic cell lineages develop in the centre of the developing blastoderm or blastocyst epithelium and the extra-embryonic lineages at the periphery. The evolution of the vertebrate yolky egg was accompanied by a decreasing dependence on maternal determinants and increasing dependence on positional signals to determine cell fate. It is proposed that when a less yolky egg evolved, the mechanisms for determination of cell fate in a developing epithelium were retained. It is proposed that in marsupials, positional signals are involved in the determination of cell fate of embryonic and trophectoderm cells but do so in a two-dimensional epithelium not a three-dimensional morula. The next lineage formed is the primary endoderm which separates off from the primitive ectoderm in the embryoblast and eventually lines the blastocyst cavity. Positional signals are responsible for the determination of primary endoderm in eutherian mammals, birds and probably also marsupials. Order of cell division during cleavage provides a mechanism whereby some cells in the embryoblast of marsupials have earlier and greater contact with their neighbouring cells. The mechanism for determination of primary endoderm cells in the blastocyst epithelium is examined in the Virginia opossum and the stripe-faced dunnart.

The X inactivation centre and X chromosome imprinting.

Genetic imprinting is an important component of X chromosome inactivation, since in marsupials and extraembryonic cell lineages of mice and rats, the paternally derived X chromosome is preferentially inactivated. This imprinting is thought to be mediated via the X inactivation centre. The gene symbolized Xist is a strong candidate for a role in the function of the X inactivation centre and the paper reviews the evidence that Xist shows imprinted behaviour and that differential methylation is the possible basis of the imprint. This paper is the text of the speech given by Dr. Mary Lyon after the awarding of the Mauro Baschirotto prize at the meeting of the European Society of Human Genetics in Paris, June 1994 (see page 305).

Ontogeny of hyaluronan secretion during early mouse development.

The ontogeny of hyaluronan (HA) secretion during early mouse embryogenesis has been investigated using a biotin-labelled HA-binding complex from cartilage proteoglycan. HA is first secreted by visceral endoderm cells of the early egg cylinder on day 5.5 post coitum (p.c.), predominantly into the expanding yolk cavity. On day 6.5 p.c., HA is present in both the yolk and proamniotic cavities, but pericellular staining is restricted to the visceral endoderm and a population of embryonic ectoderm cells at the antimesometrial end of the proamniotic cavity. By the primitive streak stage, HA is secreted into the ectoplacental, exocoelomic, amniotic and yolk cavities, whilst the only cells exhibiting pericellular staining are those of the embryonic and extraembryonic mesoderm, including the allantois. Comparisons of HA-staining patterns of cultured whole blastocysts, microdissected trophectoderm fragments and immunosurgically isolated inner cell masses, revealed no trophoblast-associated HA secretion during outgrowth in vitro but significant synthetic activity by the endodermal derivatives of differentiating inner cell masses. To identify the cell lineages responsible for secretion of HA into the embryonic cavities and to investigate the origin of the HA observed around migrating mesoderm cells, day 7.5 p.c. primitive streak stage conceptuses were dissected into their various embryonic and extraembryonic cell lineages. HA secretion was observed after short-term suspension culture of mesoderm, embryonic ectoderm and embryonic endoderm, but was undetectable in fragments of ectoplacental cone, parietal yolk sac (primary giant trophoblast and parietal endoderm), extraembryonic ectoderm or extraembryonic endoderm. The level of synthesis by the HA-positive tissues was markedly enhanced by culture in medium containing serum, compared with that obtained following culture in medium supplemented with a defined serum substitute containing insulin, transferrin, selenous acid and linoleic acid. This suggests that additional growth factors, present in serum but absent from the serum substitute, are required for optimal HA synthesis by the HA-secreting tissues in vitro, and probably also in vivo. The implications of these events for implantation and the development of peri- and early post-implantation mouse embryos are discussed, and a new role for HA in the initial formation and expansion of the embryonic cavities is proposed.

Karyotypes and X chromosome inactivation in segregants of a murine X-autosome translocation, T(X;4)37H.

Karyotypes and X chromosome inactivation were studied in embryos obtained from female mice carrying T(X;4)37H translocation on day 6 to 8 of gestation by a BrdU-acridine orange method. A total of 18 different karyotypes were found in 477 embryos examined: 90.0% embryos were products expected from 2:2 alternate or adjacent 1 disjunction. 3:1 and adjacent 2 disjunctions accounted for approximately 8.0% and 0.7% conceptuses, respectively. In the embryo proper of balanced T37H/ + conceptuses, inactivation was random with respect to the normal X and the larger translocation X (4x) chromosome. In all the cells with the 4x inactive, the late replication apparently did not spread to the attached autosomal portion, although black/brown coat variegation implies spreading of inactivation into the autosomal region. The X chromosome segment deprived of the inactivation center remained active in all the cells examined and it exerted deleterious effects on embryonic or fetal development. Observation in embryos having two maternally derived X chromosomes showed that they were indeed resistant to inactivation in early extraembryonic cell lineages, and two copies of active X chromosomes in the trophectoderm fatally affected embryonic development due to inability to form the extraembryonic ectoderm and ectoplacental cone from the polar trophectoderm. In unbalanced X aneuploids the X chromosomes with the deletion were preferentially inactivated due to strong selection against nullisomy X.

Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development.

This paper shows stage- and tissue-specific global demethylation and remethylation occurring during embryonic development. The egg genome is strikingly undermethylated and the sperm genome relatively methylated. Following a loss of genomic methylation during preimplantation development, embryonic and extraembryonic lineages are progressively and independently methylated to different final extents. Methylation continues postgastrulation and hence could be a mechanism initiating, or confirming, differential programming in the definitive germ layers. It is proposed that much of the methylation observed in somatic tissues acts to stabilize and reinforce prior events that regulate the activity of specific genes, chromosome domains or the X chromosome (in females). Fetal germ cell DNA is markedly undermethylated and we favour the idea that the germ lineage is set aside before the occurrence of extensive methylation of DNA in fetal precursor cells.

Cell lineage-specific undermethylation of mouse repetitive DNA.

Several distinct cell lineages are established during mouse embryogenesis. The trophectoderm and primitive endoderm give rise to extraembryonic structures alone, while the primitive ectoderm becomes the fetus proper. Recent studies suggest that the levels of DNA modification are lower in inactive X chromosomes from extraembryonic tissues than in embryonic and adult somatic tissues. Using HpaII/MspI isoschizomers, Southern blots and cloned probes, we show here that repetitive DNA sequences from all derivatives of the two extraembryonic lineages, trophectoderm and primitive endoderm, are substantially undermethylated compared with primitive ectoderm derivatives. This contrasts with the highly methylated state of these repetitive elements observed in adult somatic tissues. Specific demethylation or inhibition of de novo methylation, or a combination of both mechanisms, may be involved. These findings suggest that elements of gene regulation dependent on DNA modification may be different in extraembryonic cell lineages.