A chimaeric animal model for confined placental mosaicism

The reason for chromosome mosaicism being sometimes confined to only part of the conceptus is unknown. To address this problem, we produced tetraploid diploid chimaeric mouse conceptuses. At 12 1/2 days, no tetraploid cells were detected in the fetus. They rarely contributed to other derivatives of the primitive ectoderm lineage but were commonly found in the primitive endoderm and trophectoderm lineages. This provides a useful animal model of human confined placental mosaicism and suggests that the primitive endoderm (hypoblast) lineage should be included in future studies of human mosaic conceptuses.     

Maternal X chromosome expression in mouse chorionic ectoderm

An electrophoretic variant of the X-linked enzyme phosphoglycerate kinase (PGK-1) has been used to study regulation of X chromosome expression in the diploid derivatives of the trophectoderm at 8–8.5 days post coitum in the mouse. These derivatives included the chorionic ectoderm and the polar trophoblast. The biochemical analysis suggests that only the maternally derived X chromosome (X^m) is expressed in the diploid trophectoderm derivatives. Cell selection and maternal tissue contamination were ruled out as possible causes of the observed X^m expression. From these and other results, we conclude that all derivatives of the trophectoderm, along with the primitive endoderm, express only X^m, whereas derivatives of the primitive ectoderm show random X chromosome expression.     

Use of triple tissue blastocyst reconstitution to study the development of diploid parthenogenetic primitive ectoderm in combination with fertilization-derived trophectoderm and primitive endoderm

Diploid mouse conceptuses lacking a paternal genome can form morphologically normal but small fetuses of up to 25 somites, but they invariably fail to develop beyond mid-gestation. Such conceptuses differ from normal most notably in the poor development of extra-embryonic tissues which are largely of trophectodermal and primitive endodermal origin. However, it is not clear whether the demise of diploid parthenogenetic (P) or gynogenetic (G) conceptuses is attributable entirely to the defective development of these two tissues or whether differentiation of the primitive ectoderm, the precursor of the foetus, extra-embryonic mesoderm and amnion, is also impaired by the absence of a paternal genome. Therefore, a new blastocyst reconstitution technique was used which enabled primitive ectoderm from P blastocysts to be combined with primitive endoderm and trophectoderm from fertilization-derived (F) blastocysts. One third of the 'triple tissue' reconstituted blastocysts that implanted yielded foetuses. However, all foetuses recovered on the 11th or 12th day of gestation were small and, with one exception, either obviously retarded or arrested in development. The exception was a living 44 somite specimen which is the most advanced P foetus yet recorded. Foetuses were invariably degenerating in conceptuses recovered on the 13th day. In contrast, at least 16% of control reconstituted blastocysts with primitive ectoderm as well as primitive endoderm and trophectoderm of F origin developed normally on the 13th day of gestation or to term. Hence, the presence of a paternal genome seems to be essential for normal differentiation of all 3 primary tissues of the mouse blastocyst. The P foetuses that developed from reconstituted blastocysts were so closely invested by their membranes that they often showed abnormal flexure of the posterior region of the body. Several also showed a deficiency of allantoic tissue. Therefore, the possibility that the defect in development of P primitive ectoderms resided in their extra-embryonic tissues was investigated by analysing a series of chimaeras produced by injecting them into intact F blastocysts. The foregoing anomalies were not discernible even when P cells made a large contribution to the extra-embryonic mesoderm or amnion plus umbilical cord. Furthermore, selection against P cells was no greater in extra-embryonic derivatives of the primitive ectoderm than in the foetus itself.     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.     

Expression of glucose-phosphate isomerase in relation to growth of the mouse oocyte in vivo and in vitro

The activity of the enzyme glucose-phosphate isomerase (GPI-1) in mouse oocytes is subject to regulation by the cis-acting gene Gpi-lt^a. Electrophoretic analysis of oocytes from 9- and 10-day-old mice showed that oocyte-specific regulation of GPI-1 is not observed in germ cells that have not started to grow (20 μm diameter) but appears as soon as oocyte growth begins (30 μm or larger). Three in vitro culture systems were used to examine the relation of GPI-1 expression to oocyte growth: culture of intact neonatal ovaries, and co-culture of dissociated oocytes and somatic cells from neonatal and from 13-day foetal ovaries. In all three systems modification of GPI-1 expression always occurred when oocyte growth began, showing that the presence of a normal follicle is not necessary for the expression of the gene Gpi-lt^a.     

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.     

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus × Mus caroli)

Hybrid Mus musculus × Mus caroli embryos were produced by inseminating M. musculus (C57BL/Ola Ws) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3½ days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preim-plantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 31/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 31/2-day samples (12 samples of compacted morulae) but were consistently detected at 4½ days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 elec-trophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele. © 1992 Wiley-Liss, Inc.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Clonal analysis of early mammalian development

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.     

High activity of an unstable form of glucose phosphate isomerase in the mouse

Quantitative electrophoretic studies of the three allozymes of glucose phosphate isomerase (GPI-1) produced byGpi-1s ^a/Gpi-1s^c heterozygous mice revealed two opposing influences on GPI-1 activity. First, the GPI-1 AC heterodimer is less stable than GPI-1 AA but more stable than the GPI-1 CC homodimer. Second, a genetic determinant that maps close to or within theGpi-1s structural gene causes elevated activity of GPI-1 AC and probably also GPI-1 CC dimers. The relative lability of these allozymes masks this elevated activity in some tissues but the effect is probably ubiquitous. The significance of these observations is discussed.This study was begun while JDW was at the MRC Radiobiology Unit and continued at the Department of Obstetrics and Gynaecology of the University of Edinburgh, where it was supported, in part, by a grant from the Moray Endowment Fund.     

Differential localization of TGF-beta 2 in mouse preimplantation and early postimplantation development

The localization of transforming growth factor type beta 2 (TGF-beta 2) has been followed during preimplantation and early postimplantation murine development using an anti-peptide antibody that specifically recognizes TGF-beta 2. The staining pattern showed that TGF-beta 2 is expressed from the four-cell stage onward and is differentially regulated as cells diverge to various lineages. High levels of staining were found in the trophectoderm of the blastocyst but no staining was observed in the inner cell mass. During postimplantation development the primitive and embryonic ectoderm also lacked detectable staining while visceral endoderm stained well. Parietal endoderm cells also showed positive staining reaction although to a lesser extent than visceral endoderm cells. These findings were confirmed in model systems of the embryo, namely, embryonal carcinoma and embryonic stem cells differentiated to to cells with either visceral or parietal endoderm characteristics. The possible regulatory role of this factor in early embryogenesis is discussed.     

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.     

Scanning Electron Microscopy (SEM) of the Chick Embryo Primitive Streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2–5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the primitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arranged cells with long, thin processes.
Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

Gastrulation and the establishment of the three germ layers in the early horse conceptus

Experimental studies and field surveys suggest that embryonic loss during the first 6 weeks of gestation is a common occurrence in the mare. During the first 2 weeks of development, a number of important cell differentiation events must occur to yield a viable embryo proper containing all three major germ layers (ectoderm, mesoderm, and endoderm). Because formation of the mesoderm and primitive streak are critical to the development of the embryo proper, but have not been described extensively in the horse, we examined tissue development and differentiation in early horse conceptuses using a combination of stereomicroscopy, light microscopy, and immunohistochemistry. Ingression of epiblast cells to form the mesoderm was first observed on day 12 after ovulation; by Day 18 the conceptus had completed a series of differentiation events and morphologic changes that yielded an embryo proper with a functional circulation. While mesoderm precursor cells were present from Day 12 after ovulation, vimentin expression was not detectable until Day 14, suggesting that initial differentiation of mesoderm from the epiblast in the horse is independent of this intermediate filament protein, a situation that contrasts with other domestic species. Development of the other major embryonic germ layers was similar to other species. For example, ectodermal cells expressed cytokeratins, and there was a clear demarcation in staining intensity between embryonic ectoderm and trophectoderm. Hypoblast showed clear α1-fetoprotein expression from as early as Day 10 after ovulation, and seemed to be the only source of α1-fetoprotein in the early conceptus.     

Boundary cells of endodermal origin define the mouth of Hydra vulgaris (Cnidaria)

We investigated morphology, dynamics and origin of cells surrounding the mouth of Hydra vulgaris using the monoclonal antibody L96. This antibody recognises a one cell-thick ring of endodermal epithelial cells exactly at the boundary between endoderm (gastrodermis) and ectoderm (epidermis). L96⁺ cells can stretch considerably without any cell rupture during mouth opening. Thus, our data prove the existence of a distinct cell population defining hydra's mouth. A model for mouth opening is proposed and the significance of L96⁺ cells for boundary formation between ectoderm and endoderm is discussed.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Description of an embryonic lethal gene, l(5)-1, linked to Wsh

A recessive lethal mutant linked to Wsh causes the death of homozygous embryos between 4.5 and 5.5 days postcoitum (pc). Histological examination of implantation sites from intercross and backcross matings indicates that homozygotes are not all evident at 4.5 days pc, when embryos have begun to form trophectoderm giant cells and primitive endoderm, but are degenerating by 5.5 days pc, with only a few primary giant cells remaining after this time. The mutants thus form blastocysts that initiate the implantation process but the inner cell mass and polar trophectoderm fail to develop further. In vitro examination and culture of blastocysts indicated that the mutant homozygotes hatch from the zona pellucida and outgrow, although they do so somewhat more slowly than normal embryos. After 3 days of culture, the inner cell masses of mutant outgrowths may be smaller than normal. Since the gene has no known heterozygous effect and the primary gene function remains unknown, the mutant has been given the provisional symbol l(5)-1 for the first lethal on chromosome 5.     

Developmental potency of mouse primitive ectoderm cells, embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.)mouse embryos and primordial germ cells from 12.50-day p.c. male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c. blastocysts. Sixteen (61.5%) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells; four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts. However, no overt chimaera was obtained with either 5.25-day or 6.25day embryonic ectoderm cells or 12.50-day male primordial germ cells. GPI analysis of mid-gestation conceptuses developed from injected blastocysts showed that 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus. Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells, and their developmental potency becomes limited within a short period of time in normal development.