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Background: Rather than being prepatterned, orientation of the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been claimed to depend on the conceptus being constrained by its zona pellucida (ZP) during blastulation. This hypothesis merited closer scrutiny, because it seemed at variance with observations on living conceptuses., Methods: Two-cell conceptuses with an oil drop injected into the lesser diameter (LD) of the ZP at the first cleavage plane were cultured until shortly before blastulation when the blastomere underlying the drop was labelled with carbocyanine dye. After removing the ZP, conceptuses were re-cultured to the blastocyst stage for recording the position along the axis of the centres of the patches of labelled cells., Results: These centres showed significant bias towards the equatorial (Eq) region of the axis compared with those resulting from labelling a blastomere at random, even following softening of the ZP at the 2-cell stage. This was also true if conceptuses were denuded at the 2-cell stage and the blastomere underlying an intact second polar body (PB) labelled in morulae., Conclusions: These findings further support the view that the Em-Ab axis of the mouse blastocyst is normally prepatterned and provide no evidence of a role for the ZP in its specification.

Background: Preliminary observations revealed that advanced zygotes of the PO strain mouse are often bilaterally symmetrical, and suggested that both the plane of first cleavage and features of the blastocyst bear a consistent relationship to the zygote's bilateral plane., Methods: Spaced oil drops were injected into the zona pellucida to delineate the bilateral plane in pronuclear zygotes, and a distinct cluster of drops then placed over the second polar body. Such non-invasive marking was combined with gelation of the perivitelline space to prevent rotation of the zygotes within the zona pellucida., Results: Nearly two-thirds of advanced pronuclear stage zygotes were bilaterally symmetrical and, regardless of whether first cleavage was meridional, it was almost invariably orthogonal to the bilateral plane. Moreover, both the axis of polarity and bilateral plane of the blastocyst bore a consistent relationship to the zygote's bilateral plane. Haploid parthenotes also exhibited bilateral symmetry, although in the absence of fertilization, first cleavage was less consistently orthogonal to the bilateral plane., Conclusions: Bilateral symmetry may be an intrinsic property of the oocyte that is induced by its activation and, from the reproducible way it maps on both the 2-cell conceptus and blastocyst, seems to play a role in early patterning.

Once experimental embryological studies revealed the striking ability of mammals to regulate their early development, the notion that pattern-formation might depend on information already present in the egg before cleavage was generally regarded as untenable. Mammals were therefore assumed to differ from almost all other animals in the way in which their embryonic patterning was set up. This view was justified by the profound way in which their early development is modified to meet the requirements of viviparity. However, it ignored various findings showing that exposure of gametes and very early conceptuses to altered conditions could perturb organisation of the fetus. Recent studies that place particular emphasis on non-invasive approaches have revealed hitherto overlooked regularities in early mouse development. They clearly show that specification of embryonic axes normally begins before cleavage in this species. Moreover, the relevant patterning processes seem to depend on intrinsic organisation of the egg rather than, as claimed recently, the site of entry of the fertilizing sperm. These new findings are of interest for two reasons. First, from an evolutionary perspective, it means that mammals retain common features with other animals in how their early development is controlled. Second, it raises the practical question whether the increasing use of in vitro manipulation of gametes and zygotes for assisting human reproduction carries a risk of perturbing development.

Although specification of the antero-posterior axis is a critical intial step in development of the fetus, it is not known either how, or at what stage in development, this process begins. Such information is vital for understanding not only normal development in mammals but also monozygotic twinning, which, at least in man, is associated with a significantly increased incidence of birth defects. According to recent studies in the mouse, specification of the fetal anteroposterior axis begins well before gastrulation, and probably even before the conceptus implants. Moreover, evidence is accruing that the origin of relevant asymmetries depends on information that is already present in the zygote before it embarks on cleavage. Hence, early development in mammals does not differ as markedly from that in other animals as has generally been assumed. Consequently, at present, the possibility of adverse effects of techniques used to assist human reproduction cannot be disregarded.

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Although overall polarity is discernable morphologically in both the growing and mature oocyte in mammals, it is typically relatively inconspicuous in the zygote. Furthermore, the conceptus exhibits an essentially radial organization during cleavage which was long held to persist until the primitive streak forms at the onset of gastrulation. This view has been challenged by various recent studies which clearly show that asymmetries are evident both morphologically and at the molecular level from very early in development. Collectively, these new findings argue that specification of the anterior-posterior axis of the fetus depends on information that is localized extra-embryonically in cells which begin to differentiate before the conceptus implants in the uterus.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Several findings challenge the notion that specification of cell types and embryonic axes in mammals are rooted entirely in the temporal and spatial relations between cleaving blastomeres. They raise the question as to whether, as in most non-mammalian species, these processes depend on information already present in the egg. However, experiments designed to investigate this possibility directly by perturbing the organization of the zygote or, very recently, by deleting one or other of its polar regions [M. Zernicka-Goetz. Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles. Development 1998;125:4803-4808 (Ref. 1)], have been interpreted to mean that such a role for the egg can be discounted. This conclusion seems premature in view of continuing uncertainty regarding the developmental potential of individual blastomeres in mammals.

Two pentasaccharides suitable for conjugation, namely 3-aminopropyl glactosylgloboside and its beta-D-GalNAc-(1-->4)-alpha-D-Gal-linked positional isomer, were synthesized from 3III,4III-di-O-unprotected globotrioside and the trichloroacetimidate of beta-D-Gal-(1-->3)-beta-D-GalNPhth derivative. Glycosylation at both positions led to the formation of beta-D-GalNPhth-(1-->4)-alpha-D-Gal and beta-D-GalNPhth-(1-->3)-alpha-D-Gal-linked products in a ratio of 1:1 without selectivity. Complete NMR spectral assignments are also described.

The purpose of this study was to measure the effect of insulin-like growth factor-2 deficiency on the growth of the mouse conceptus. Initial observations on normal development in the 129J/Sv strain established that wet and dry weights were reduced by 35% when the insulin-like growth factor-2 gene was inactive. The DNA contents were reduced by only 15%. We exchanged the inner cell mass and trophectoderm between mouse blastocysts that had or lacked an active insulin-like growth factor-2 gene. At embryonic Day 16.5, lack of this gene's activity in the derivatives of either tissue decreased the fluid volumes of the exocelomic and amniotic cavities. The wet weights of the "fetal placentas," the yolk sacs, and the fetuses were also decreased. However, the tissue wet weight decrease could not be accounted for by the change in DNA content, indicating that cell-associated biomass had changed. The conclusions are 1) that insulin-like growth factor-2 levels regulate the composition of the fetus and extra-embryonic tissues and 2) that trophectoderm and inner cell mass derivatives cooperate to control extra-cellular fluid volume in the conceptus.

This is a personal account of why the author chose to focus on devising techniques for micromanipulating the blastocyst stage conceptus as a way of investigating early development in mammals. Its aim is to provide insight into what such technical innovations entailed and how they have contributed to present understanding of both embryology and the analysis of gene function in mammals. The ability to dissect and reconstitute mouse blastocysts, and to inject cells or tissue into them, enabled genes to be harnessed as markers for elucidating the lineage of cells and interactions between tissues from the stage when differentiation is first evident. Most importantly, it made it possible to apply clonal analysis to the study of cell fate in mammals. The scope of blastocyst micromanipulation was further enhanced when embryonal carcinoma (EC) cells and, particularly, embryonic stem (ES) cells were found to be able to participate in normal development and contribute to the germ line following injection into the blastocyst.

Rather little is known about the changes in orthodontic treatment results exceeding a decade after treatment. The purpose of this study was to quantify changes in tooth relationships in a series of cases (n = 36) at 6 years and again at 15 years after treatment. The rate of change decreased with time, supporting the contention that most "relapse" occurs soon after treatment; continued change generally cannot be distinguished from normal aging processes that occur, regardless of whether a person had been treated orthodontically. There were minor, but statistically significant, associations between increased incisor irregularity ("relapse") and parasagittal growth of the jaws. Greater irregularity occurred when mandibular growth exceeded that of the maxilla, decreasing overjet and crowding the lower incisors within the containing arch of the maxilla. Overall, relapse tended to be less in these cases treated by a single experienced specialist that in university-based samples treated by multiple, orthodontic residents.

Remarkably little is known about mammalian embryonic stem (ES) cells despite their very widespread use in studies on gene disruption and transgenesis. As yet, it is only in the mouse that lines of ES cells which retain the ability to form gametes following reintroduction into the early conceptus have been obtained. Even in this species, most strains have so far proved refractory to the derivation of such cell lines. Apart from persisting ignorance as to how the various procedures that have been claimed to improve success actually do so, even the tissue of origin of ES remains uncertain. Furthermore, it is doubtful whether retention of pluripotency or expression of so-called "stem cell" marker molecules provide an adequate basis for classifying cells as genuine ES cells. This is because epiblast cells, their presumed precursors, lose the capacity to colonize the preimplantation conceptus well before they become restricted in the types of cell they can form or cease to express such marker molecules. In addition, it has yet to be established whether heterogeneity of cells within individual ES cell lines arises entirely during culture or is at least partly attributable to lack of homogeneity among their precursors. Finally, it has yet to be explained why ES chimeras evidently differ from those obtained by combining cells from different conceptuses in showing greater variation between tissues in the level of chimerism.

Mutation of the APC (adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carrying Min (multiple intestinal neoplasia), a mutant allele of Apc, develop intestinal and mammary tumors as adults. To study the role of the Apc gene in development, we have investigated the phenotype of embryos homozygous for ApcMin (Min). Development of the primitive ectoderm fails prior to gastrulation in homozygous Min embryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives.

An impressive feature of cellular differentiation in metazoa is its stability. This has led to widespread acceptance of the view that the determined state is a heritable property of individual cells. There are, in fact, rather few types of cell for which this has been demonstrated convincingly. Not only is it clear that gene expression is subject to continuous regulation but there is also evidence for an increasing variety of cells that changes in differentiation can be induced by manipulating their environment. Such findings suggest that extrinsic factors may play a more significant role in maintaining the differentiated state of cells than is generally assumed.

One of the problems in studying early acting recessive lethal genes is recognizing the homozygotes prior to their demise. Molecular probes can assist in this task, but their use generally requires removal of cells and consequent damage to the embryo, which may compromise its subsequent development. The present study was undertaken in an attempt to distinguish intact, living, lethal yellow (Ay/Ay) embryos from Ay/ae and ae/ae littermates before implantation by placing them in implantation delay or diapause. After 2 days in the reproductive tract of prepubertal females, the great majority of presumptive lethal Ay/Ay embryos has failed to hatch from the zona pellucida and they exhibited a marked deficiency of cells relative to controls, particularly in the inner cell mass. This argues against a stage-specific role for the gene in implantation.

We have previously developed a technique of trophectoderm biopsy to obtain cells from human blastocysts for preimplantation genetic diagnosis. To determine whether this technique affects the subsequent development of the blastocyst, 45 manipulated blastocysts were observed from days 3 to 14 in culture, the amount of human chorionic gonadotrophin (HCG) secreted by each embryo was measured and these results were compared with those of 26 non-manipulated controls documented in a previous study. A slit was made in the zona pellucida opposite the inner cell mass in 18 of the 45 blastocysts. This increased the rate of hatching but the other morphological changes up to day 14 were similar to those seen in the non-manipulated controls. There was no difference in the mean cumulative HCG secretion by these zona-slit controls (149.8 +/- 45.7 mIU/ml) compared to the non-manipulated controls (146.2 +/- 23.7 mIU/ml). A slit was also made in the zona pellicida of the other 27 blastocysts. Approximately 12-18 h later, in 18 blastocysts a biopsy of the herniating trophectoderm cells (5-30) was performed. The rate of hatching and adherence to the culture dish was similar to the non-manipulated controls. The mean cumulative HCG secretion decreased significantly (57.5 +/- 16.2 mIU/ml, P less than 0.01) after the biopsy procedure. However, if a small biopsy was performed (less than 10 cells removed) the decrease in HCG secretion (87.6 +/- 24.8 mIU/ml) was not significant, whereas when a large biopsy was performed (greater than 10 cells), HCG levels fell to 19.9 +/- 9.1 mIU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Conditions were found for staining whole mid-gestation capsular parietal endoderms and visceral yolk sacs for malic enzyme activity that gave excellent discrimination between wild-type (Mod-1+/Mod-1+) cells and mutant (Mod-1n/Mod-1n) cells that lack the cytoplasmic form of the enzyme. Reciprocal blastocyst injection experiments were undertaken in which single primitive endoderm cells of one genotype were transplanted into embryos of the other genotype. In addition, Mod-1+/Mod-1+ early inner cell mass (ICM) cells were injected into Mod-1n/Mod-1n blastocysts, either in groups of two or three singletons or as daughter cell pairs. A substantial proportion of the resulting conceptuses showed mosaic histochemical staining in the parietal endoderm, visceral yolk sac, or in both these membranes. Stained cells were invariably intimately intermixed with unstained cells in the mosaic parietal endoderms. In contrast, one or both of two distinct patterns of staining could be discerned in mosaic visceral yolk sacs. The first, a conspicuously 'coherent' pattern, was found to be due to endodermal chimaerism; the second, a more diffuse pattern, was attributable to chimaerism in the mesodermal layer of this membrane. The overall distribution of cells with donor staining characteristics resulting from primitive endoderm versus early ICM cell injections was consistent with findings in earlier experiments in which allozymes of glucosephosphate isomerase were used as markers. The conspicuous lack of phenotypically intermediate cells in predominantly stained areas of mosaic membranes suggested that the histochemical difference between Mod-1+/Mod-1+ and Mod-1n/Mod-1n genotypes was cell-autonomous. This conclusion was strengthened by the results of staining mixed in vitro cultures of parietal endoderm in which presence or absence of phagocytosed melanin granules was used as an independent means of distinguishing wild type from null cells. By substituting tetranitro blue tetrazolium for nitro blue tetrazolium in the incubation medium, satisfactory differential staining was obtained for both the extraembryonic endoderm and other tissues of earlier postimplantation wild type versus null embryos. Finally, absence of cytoplasmic malic enzyme activity does not appear to have a significant effect on the viability or behaviour of mutant cells.

Cloning of cells from peri-implantation embryos by blastocyst injection was used to investigate the time of X-chromosome inactivation in that part of the ectoderm lineage giving rise to foetal tissues of the mouse. Matings were arranged so that the two X-chromosomes of female donor cells controlled two distinct coat colours and host blastocysts were of a third colour genotype. No coat chimaeras were obtained in experiments using donor cells from the primitive ectoderm of 6th or 7th day embryos or from lactationally delayed implanting or reactivated blastocysts. In contrast, a minimum of 80 unequivocal coat chimaeras were obtained in experiments in which primitive ectoderm cells from 5th day implanting blastocysts were used for injection. The majority of these chimaeras that had received a female cell exhibited both donor colours in addition to host colour in their coats, suggesting that the donor cell had not undergone X-inactivation until one or more cycles after transplantation. The remainder of such chimaeras exhibited only one or other donor coat colour. Determination of the parental origin of the allocyclic X-chromosome in donor metaphase preparations in internal tissues of several chimaeras revealed that the coat pattern did not always reflect the X-activity status of the donor cell clone as a whole. Nevertheless, the findings suggest that X-inactivation takes place shortly after implantation in the primitive ectoderm cell population from which the foetus is derived. Of the 68 chimaeras in which the sex of both the donor and host component was established 62 proved to be fertile. Furthermore, 21 of the 37 fertile chimaeras whose sex corresponded with that of the donor cell yielded functional gametes of donor origin. Injection of cells from a single donor blastocyst into a series of host blastocysts established that at least 2 cells in 5th day primitive ectoderm can give rise to both somatic cells and functional germ cells among their mitotic descendants.

Chimaeric mice were obtained by injecting embryonic (CBA/H-T6 X PDE)F1 cells into PDE blastocysts. Three of 15 young were overt chimaeras. One female and one male chimaera survived to adulthood and after completion of test breeding, which demonstrated chimaerism in the germ cells of both, they were killed for study when aged 32 and 33 weeks respectively. Chromosome spreads were scored for the presence or absence of the T6 marker chromosome in direct preparations from bone marrow, spleen, thymus, lymph nodes, Peyer's patches, corneas, gut epithelium and testes. Preparations from monolayer cultures of skin, kidney, ovary and gut from mitogen-stimulated blood cultures were scored in the same way. Both components of the chimaeras were identified in every one of 53 specimens studied, some of which, such as single lymph nodes, corneas, and segments of gut, may not have contained more than 10(5) proliferating cells. This result complements published evidence for fine-grained mixture obtained in morula-aggregation chimaeras by other methods and implies extensive cell movement during embryogenesis. Results obtained from the lymphomyeloid tissues show a clear partition into two groups in respect of the proportions of host-type to donor-type cells indentified. The one group consists of bone marrow, thymus and Peyer's patches, the other of spleen and lymph nodes. This result would be most simply explained in terms of two distinct stem cell pools and appears to conflict with the currently favoured hypothesis of a single stem cell pool for the whole lymphomyeloid complex located in bone marrow. Four groups of factors may, however, modify the relative representation of the two components in different lymphomyeloid sites: (1) The magnitude of embryonic founder populations. (2) Limited recruitment from the stem cell pool in post-natal life. (3) Variable size of clones produced by individual stem cells. (4) Differential cellular behaviour determined by genotypic differences.

Mouse blastocysts were microsurgically injected with embryonal carcinoma cells from in vitro teratocarcinoma cell lines C17, C86, SIKR-OSB, and PCC3/A/I. The embryos were allowed to develop to term and the resulting offspring were analysed for chimaerism using coat colour markers and isozyme differences of the enzyme glucose phosphate isomerase. When injected into blastocysts, cell line C86 produced tumours in six of 74 animals born. The tumours were detected at birth and were poorly differentiated neuroectodermal teratocarcinomas. Cell line C17 gave 13 chimaeras in 77 mice born, five of which showed chimaerism only in normal tissues, mainly melanocytes of the coat and eye. The other eight chimaeras developed tumours. Seven of these developed in adult animals and were mainly fibrosarcomas. Cell line SIKR-OSB resulted in one normal chimaera in 44 mice born. Of 86 animals born following injection of cell line PCC3/A/1, there was one chimaera with a small tumour and three normal chimaeras. The levels of chimaerism were generally very low. The mice were test bred but with no evidence of germ line chimaerism. The karyotypes of all the cell lines were abnormal. How this and other factors such as cell cycle times might affect the incorporation of embryonal cells into the developing embryo is discussed.

Mammalian chimaeras can be produced experimentally by aggregating early embryos or by injecting cells into them. They have been used to study several aspects of early development. However, lack of a genetic marker enabling unequivocal identification of all cells of either genotype in situ has frustrated full exploitation of the experimental possibilities offered by these organisms. Hence interspecific chimaeras have been produced between rat and mouse embryos in which cells of the two species can be identified in sectioned embryos by immune fluorescence. These chimaeric embryos have been used to study differentiation of the trophoblast and inner cell mass, and the deployment of cells during morphogenesis. Preliminary results suggest that the two tissues are determined by the blastocyst stage, and that the trophoblast forms part of the extra-embryonic membranes originally presumed to be derived from the inner cell mass. Also, rat inner cell mass cells can induce mitosis in mouse trophoblast. Futhermore, the distribution of rat cells in implanted embryos suggests that the embryo may grow in a coherent clonal manner from a very early stage. Very recently, chimaerism has been induced by transplanting single rat cells, which may allow a more critical analysis of morphogenesis and determination than was possible hitherto. An obvious question raised by crossing the species barrier is the extent to which results may be applicable to normal development. Adverse effects of immunological interaction between the mouse uterine foster-mother and fetal rat cells, and sorting out of cells according to species, are two of the problems that might complicate interpretation of these experiments.

Delayed implanting blastocysts recovered from both ovariectomized and lactating pregnant mice were examined for the presence of primitive endoderm. One or more of three different criteria were used to identify this tissue which was found to be present in nearly all such blastocysts, regardless of the length of time for which their implantation had been delayed. Furthermore, the tissue appeared to differentiate in blastocysts from ovariectomized females at approximately the same postcoital stage as in those from sham-operated controls. Formation of the parietal endoderm layer was not observed in a single instance during delay, but began approximately 10 h after it had been terminated by injecting ovariectomized females with oestradiol benzoate. Cells in mitosis were found both at longer intervals after the onset of implantation delay and at shorter intervals after its termination than reported previously. It is concluded that, contrary to what might have been anticipated from certain earlier studies, there seem to be no obvious advantages in using delayed implanting rather than nondelayed blastocysts for investigating initial steps in differentiation of the primitive endoderm. Delayed blastocysts may, nevertheless, be of value in elucidating factors controlling the differentiation or onset of migration of parietal endoderm cells.

In the early mammalian embryo, initially asymmetric cell contacts appear to induce blastomere polarization, elements of which can persists through cytokinesis. This leads to the generation of inner and outer populations of blastomeres which may subsequently diverge as a result of residing in distinct microenvironments. Similar processes may account for the generation of primitive endoderm versus primitive ectoderm, and that of trophectoderm versus inner cell mass. However, if this is the case, the response of cells to positional cues must change as a function of either their previous positional history or the number of cycles they have completed. Once these primary tissues have been established, specific interactions between them lead to further cellular diversification.

Chimaeric conceptuses have been produced by injection of 5 1/2-and 6 1/2-day extra-embryonic ectoderm and 5 1/2-day embryonic and extra-embryonic endoderm into 3 1/2-day mouse blastocysts. Extra-embryonic ectoderm cells contributed only to the ectoplacental cone and/or trophoblast giant cell fractions, reflecting the probable trophectoderm origin of these cells. Proximal (visceral) endoderm cells overlying both the embryonic and extra-embryonic ectoderm contributed cells only to the endoderm of the visceral yolk sac, indicating that the definitive embryonic endoderm has not formed by 5 1/2 days p.c.

The administration of GnRH agonists to shrink fibroids, albeit temporarily, may be of benefit to those patients in whom tubal blockage by the fibroids has given rise to infertility. Tubal patency has been restored in two of our patients, resulting in pregnancy in one.

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.

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

Two distinct patterns of chimaerism were found in conceptuses produced by injecting dissociated 4.5-day inner cell mass cells into genetically dissimilar blastocysts. Pattern 1: donor cells were found in the endoderm layer of the visceral yolk sac, but not in the adjacent mesoderm layer of this organ or in the foetus itself. Pattern 2: donor cells were found in the mesoderm layer of the visceral yolk sac and/or foetus, but never in the yolk-sac endoderm as well. Primitive endoderm cells of donor inner cell masses are responsible for the first pattern and primitive ectoderm cells for the second. These results, together with those of previous studies, suggest that the entire foetus, including its endodermal components, is formed from the primitive ectoderm, and that primitive endoderm forms only extra-embryonic endoderm of the conceptus.

Several cell and tumour lines were isolated from a single-embryo-derived teratocarcinoma and their karyotypes and differentiation in adult hosts recorded. The majority of cells contained normal karyotypes by banding. The cells were injected into blastocysts and although they sometimes colonized the yolk sac, they never colonized the embryo. Thus the possession of a normal karyotype is not a sufficient condition for embryo colonization. The loss of growth capacity was investigated by studying differentiation and tumourigenicity in a variety of circumstances. The change in appearance from an EC cell morphology to a big flat cell in culture leads to retardation of growth in adult hosts. When EC cells are injected into a blastocyst, the ability to grow progressively both in culture and in adult hosts is lost.

Inner cell masses (ICMs) were isolated from early blastocysts by immunosurgery and incubated in a dense suspension of melanin granules for 3 h after 21 h in culture. The majority of such labelled ICMs subsequently formed outgrowths in vitro in which either giant cells or small solitary cells contained melanin granules. However, a substantial minority produced outgrowths in which both types of cell were unequivocally labelled. Labelled cells appeared from the results of control experiments to have originated within the external layer of the ICM. The giant cells were indistinguishable morphologically from those formed by authentic trophectodermal tissue. The small cells were identified as belonging to the extraembryonic endodermal lineage on the basis of their distribution in host conceptuses following injection into blastocysts. These findings support the conclusion reached in previous studies that early ICM cells can engage in trophectodermal differentiation under certain conditions. In addition, by providing evidence that both trophectoderm and endoderm cells can differentiate from the outer layer of the same ICM, they argue that loss of cellular lability is not coordinated throughout this tissue. Heterogeneity in the differentiation of external cells may depend on differences in both the stage of the mitotic cycle and the number of such cycles that they have completed since fertilization. Finally, cell number in isolated early ICMs was found to increase approximately two-fold during the first 24 h of culture in the present experiments. This contrasts with the results of previous experiments in which cell number either increased more modestly or failed to do so altogether.

The capacity of immunosurgically (IS) treated inner cell masses (ICMs) versus microsurgically (MS) isolated primitive ectoderms from blastocysts recovered on the 5th day of gestation to regenerate an external layer of endoderm cells in vitro was investigated. While the majority of IS-treated ICMs regenerated such a layer, MS-isolated ectoderms seldom did so. Examination of the two types of tissue fragments revealed that IS-treated ICMs almost invariably retained viable endoderm cells whereas MS-isolated ectoderms did so only exceptionally. The endoderm was found to be more than one cell layer thick in ICMs from 5th day blastocysts, suggesting that some endoderm cells survive IS because they are protected from exposure to antiserum. Typing of the endoderm layer that regenerated following IS treatment of recombinant ICMs composed of genetically dissimilar endoderm and ectoderm provided direct evidence that it originated from residual endoderm cells rather than the underlying ectoderm. Finally, blastocyst injection experiments confirmed that IS-treated ICMs behave like a mixture of ectoderm and endoderm tissue in vivo, and provided no support for the view that cells of the original and regenerated endoderm differ in developmental potential. These findings challenge earlier conclusions concerning cell lineage and determination in the primitive ectoderm that were based on development in vitro of IS-treated ICMs from giant blastocysts.

Chimaeric combinations of normal and mutant embryonic tissues were used to investigate the lethal effect of the yellow gene. The homozygous mutant embryos could not be identified before implantation. Therefore, embryos from both intercross matings and control backcross matings were used to provide inner cell masses (ICMs) for injection into genetically marked blastocysts of the CFLP random bred stock. All conceptuses obtained from reimplanted blastocysts were analysed at mid-gestation for the presence of donor isozyme of glucose phosphate isomerase. A similar proportion of chimaeras were found in the experimental and control series, indicating rescue of the lethal Ay/Ay ICM tissue. The reciprocal experiment also produced a similar proportion of chimaeras but there was a 25% postimplantational loss of injected embryos evidenced by empty decidual swellings. The results suggest that the yellow mutation primarily affects the trophectoderm which cannot be rescued by a normal ICM, whereas Ay/Ay ICM is capable of survival in a chimaera at least until mid-gestation.