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Biomedical subjects

J Rossant

Publications and source records attributed to J Rossant.

At least 163 records · Page 9Linked to original sources
PubMed1984

Effect of delaying DNA replication on blastocyst formation in the mouse.

Differentiation in the early mouse embryo involves cellular responses to both spatial and temporal signals. The temporal signals that trigger blastocyst formation, the first differentiative event, are not yet understood, but it has been suggested that the numbers of DNA replications undergone since fertilization might act as the timing mechanism. Preimplantation mouse embryos were treated with aphidicolin, an inhibitor of eukaryotic DNA polymerase alpha, for eight hours during the S phase of the fourth cleavage division. This treatment produced a delay in cell division but the morphologic event of cavitation, which signals the onset of blastocyst formation, was not delayed. Treated embryos actually cavitated a few hours ahead of control embryos at approximately half the cell number. This result indicates that blastocyst formation is not timed by the number of DNA replicative cycles completed since fertilization, but by some other intrinsic cellular clock.

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PubMed1984

Local active suppression by suppressor cells in the decidua: a review.

The immunological survival of the antigen-bearing mammalian feto-placental unit is determined by the functional properties of the tissues at the feto-maternal interface. Antigen-specific systemic suppressor mechanisms such as suppressor T cells and nonantigen-specific suppressive serum factors appear not to play a major role in protection of the fetus. A novel type of non-MHC specific suppressor cell accumulates locally in the decidua of successfully allopregnant mice. This decidua-associated suppressor is a small lymphocytic cell possessing cytoplasmic granules, lacks T cell markers, and is deficient in number and activity at the implantation sites of viable xenogeneic Mus caroli embryos gestating in the uterus of Mus musculus animals at the time that maternal lymphoid cells begin to infiltrate the xenoembryos. These Mus caroli embryos subsequently resorb. Further experimental studies suggest that the trophoblast cells associated with successful pregnancy recruit bone-marrow derived maternal non-T suppressor cells to the decidua and thus, by an indirect mechanism, may act to protect the fetus from effector cells of the mother's immune system.

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PubMed1983

Interspecific hybrids and chimeras in mice.

Interspecific hybrids and chimeras in mammals provide unique tools for investigating problems in genetics and embryology, because of the degree of disparity between the two component genotypes. We have attempted to produce hybrids and chimeras between Mus musculus, the laboratory mouse, and Mus caroli, a wild species of mouse from Southeast Asia. M. musculus and M. caroli do not normally interbreed, although sterile hybrids can be produced at a low rate by artificial insemination. Extrinsic problems of genotypic incompatibility between the fetus and the maternal environment seem to be involved in poor hybrid survival, since M. caroli blastocysts also die when transferred to the M. musculus uterus. Death is associated with the generation of maternal T-cells which are cytotoxic to M. caroli target cells in vitro. It is not yet clear whether this immune response is the primary cause of death or is secondary to breakdown of some other components of the fetal-maternal interaction. It is clear, however, that it is the trophoblast layer that mediates survival or death of the foreign embryonic cells in the M. musculus juterus, since M. caroli inner cell mass cells can survive to term after injection into M. musculus blastocysts: Viable interspecific chimeras result. Even more convincing evidence is provided by the production of viable M. caroli offspring by trophoblast vesicle reconstitution using trophoblast of M. musculus genotype and inner-cell mass of M. caroli type. Studies of properties of isolated trophoblast tissues have indicated that M. caroli trophoblast may differ from M. musculus in both its antigenic and immunosuppressive properties. Elucidation of trophoblast-uterine interactions in these various interspecific pregnancies is being aided by the development of an in situ marker system, which can distinguish cells of the two species in sectioned material by in situ hybridization with a M. musculus satellite DNA probe. This same marker is also proving a very powerful tool for analyzing cell lineage development in chimeras.

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PubMed1983

Somatic and germline mosaicism in interspecific chimaeras between Mus musculus and Mus caroli.

Detailed analysis of mosaicism in interspecific chimaeras between Mus musculus and Mus caroli revealed that cells of the two species could coexist and interact normally in all tissues studied. No selection occurred against M. caroli cells during gestation of chimaeras in the M. musculus uterus, but some tissue-specific differential growth of M. musculus and M. caroli cells occurred during postnatal development. Similar effects have, however, been reported in interstrain M. musculus chimaeras. The similarity between inter- and intraspecific chimaeric growth patterns supports the use of this interspecific system as a model for analysing cell lineage relationships during development.

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PubMed1983

Use of repetitive DNA sequences to distinguish Mus musculus and Mus caroli cells by in situ hybridization.

Mammalian chimaeras have proved useful for investigating early steps in embryonic development. However, a complete clonal analysis of cell lineages has been limited by the lack of a marker which is ubiquitous and can distinguish parental cell types in situ. We have developed a cell marker system which fulfils these criteria. Chimaeric mice were successfully produced from two mouse species which possess sufficient genetic differences to allow unequivocal identification of parental cell types. DNA-DNA in situ hybridization with cloned, species-specific sequences was performed to distinguish the parental cell types. We have identified a cloned, Mus musculus satellite DNA sequence which shows hybridization differences between Mus musculus and Mus caroli DNA. This clone was used a a probe in in situ hybridizations to bone marrow chromosomes from Mus musculus, Mus caroli, and an interspecific F1 hybrid. The clone could qualitatively distinguish Mus musculus from Mus caroli chromosomes after in situ hybridization, even when they were derived from the same F1 hybrid cell. Quantitation of this hybridization to interphase nuclei from bone marrow spreads indicates that the probe can successfully distinguish Mus musculus from Mus caroli cells and can determine the percentage contribution of Mus musculus in mixtures of bone marrow cells of these species and in chimaeric bone marrow cell preparations.

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PubMed1983

Identification of embryonic cell lineages in histological sections of M. musculus in-equilibrium M. caroli chimaeras.

An in situ cell marker system has been developed which allows identification of Mus caroli and Mus musculus cells in interspecific chimaeras. A radioactively labelled, cloned DNA probe to M. musculus satellite DNA was hybridized in situ to sections of M. musculus and M. caroli adult tissues. Autoradiography revealed high levels of hybridization to the nuclei of M. musculus cells, but little or no label bound to M. caroli cells. The DNA probe could also distinguish M. musculus and M. caroli cells in the same tissue section. Patches of labelled and unlabelled cells were clearly identified in sections of adult chimaeric tissues and also in the embryonic ectoderm of 6.5-day embryonic chimaeras. The ability to recognize M. musculus and M. caroli cells in sections of chimaeras should provide a powerful new tool in analyses of cell lineages in both embryonic and adult mouse chimaeras. The marker system has several advantages over other marker systems so far developed, the most important of which is its ubiquity. Since it is a nuclear marker, only cells without nuclei should be unsuited to its use. The potential of the marker system has been shown by its use in demonstrating directly for the first time the postimplantation derivatives of inner cell mass and trophectoderm in blastocysts 'reconstituted' with M. musculus trophectoderm and M. caroli inner cell mass.

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PubMed1982

Histological and immunological studies of post implantation death of Mus caroli embryos in the Mus musculus uterus.

Mus caroli blastocysts surgically transferred to the Mus musculus uterus implant and develop successfully during the first 9.5 days of gestation. At this time, the trophoblast giant cell area becomes infiltrated with lymphocytes and, subsequently, massive hemorrhage occurs, associated with embryo death. This process is specific for the xenogeneic embryos and does not affect adjacent allogeneic embryos present in the same uterus. Cells taken from the resorbing tissues contain a population of large Thy 1+, Lyt 1+, Lyt 2+ cytotoxic cells capable of lysing Mus caroli concanavalin A blasts in vitro. It is suggested that failure of the Mus caroli embryos in the Mus musculus uterus is mediated by the maternal immune system and that the xenogeneic blastocyst transfer system provides a suitable animal model system for studies of mechanisms that prevent rejection of the fetal allograft.

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PubMed1982

Interspecific chimeras in mammals: a new experimental system.

The first viable interspecific chimeras in mammals have been made by mixing embryonic cells from two species of mouse, Mus musculus and Mus caroli. These chimeras resemble intraspecific chimeras in M. musculus in their patterns of mosaicism. They provide an interesting experimental system for studying interactions between cells of the two species. Three separate but related areas of investigation utilizing these chimeras are described. 1) In situ DNA-DNA hybridization using a cloned repetitive DNa sequence from M. musculus has enabled M. caroli and M. musculus cells to be distinguished in bone marrow spreads from chimeras. Further refinement of this technique should allow use of this in situ marker system for detailed analysis of cell distribution in chimeras. Such a ubiquitous marker system is not yet available in M. musculus. 2) Breeding interspecific chimeras has increased production of interspecific hybrids, which are very useful for studies of genetic interactions between the two species. 3) Mus caroli embryonic cells can survive in the M. musculus uterus in a chimera, although M. caroli embryos alone die around d 11 to 16 of pregnancy in M. musculus. Investigation of this phenomenon has implicated immune effector mechanisms in death of M. caroli embryos and has revealed the importance of trophoblast genotype for survival of foreign cells in the uterine environment.

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PubMed1982

Importance of trophoblast genotype for survival of interspecific murine chimaeras.

Interspecific chimaeras between Mus musculus and Mus caroli were made by injection of M. musculus inner cell masses (ICMs) into M. caroli blastocysts, and by aggregation of 8-cell embryos. Both types of chimaera were transferred to M. musculus recipients: the former did not survive to term, but viable chimaeras were produced following embryo aggregation. Previous experiments have shown that chimaeras produced by injection of M. caroli ICMs into M. musculus blastocysts are viable, whereas M. caroli blastocysts cannot survive in the M. musculus uterus. There results indicate that the presence of trophoblast cells of maternal uterine genotype allows M. caroli in equilibrium M. musculus chimaeras to survive in the M. musculus uterus. It is suggested that M. musculus trophoblast components may protect the M. caroli embryonic cells from maternal immune rejection.

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PubMed1982

The developmental potential of a euploid male teratocarcinoma cell line after blastocyst injection.

A karyotypically normal male embryonal carcinoma (EC) cell line, P19, produced large numbers of chimaeras in midgestation after groups of cells were injected into mouse blastocysts. A wide variety of apparently normal tissues were colonized by the EC cells but most chimaeras were also morphologically abnormal. Few live chimaeras were produced and all contained tumours of EC cell origin as well as EC contributions to normal tissues. This apparently incomplete regulation of the EC cells by the embryonic environment was not due to EC cell variation, since a clonal subline. P19S18, produced similar patterns of colonization. It was also not caused by the inability of the blastocyst to regulate large numbers of injected EC cells, since a single P19S18 cell could contribute to both normal and tumour tissue in the same mouse. Neither a high rate of colonization of the embryo nor a normal karyotype is, therefore, sufficient to ensure reversion of EC cells to normal embryonic behaviour.

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PubMed1982

Mechanism of size regulation in mouse embryo aggregates.

A detailed comparison of the postimplantation development of normal and double-sized mouse embryos, produced by aggregating two 8-cell stage eggs, revealed that size regulation occurred in the double embryos between 5 days, 16 h post coitum (p.c.) and 6 days, 16 h p.c. Size regulation occurred simultaneously in all tissues, suggesting that a single regulatory mechanism may control size in the early embryo. Size regulation appeared to be brought about by alteration in cell cycle length. There was no obvious increase in cell death in the double embryos nor an increase in the non-dividing cell population. However, colcemid treatment revealed a significant difference in mitotic index between double and control embryos over the period of size regulation. Control embryos showed a proliferative burst around 6 days, 8 h p.c. which did not occur in the double embryos. It is not yet clear whether this control of proliferative activity in double embryos is exerted by the embryo itself or by the uterine environment. Histological analysis also suggested that proamniotic cavity formation, which occurs before size regulation, was dependent on total cell number and not on the number of cell cycles undergone since fertilization. Proamniotic cavity formation was observed to occur at different times but at similar cell numbers in double, control and half embryos.

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PubMed1981

Molecular studies on cells of the trophectodermal lineage of the postimplantation mouse embryo.

Embryonic ectoderm (EmE), extraembryonic ectoderm (EE), ectoplacental cone diploid cells (EPC) and secondary giant cells (GC) were isolated from 7 1/2-day mouse embryos and their polypeptide synthetic profile assessed by fluorography of 2D polyacrylamide gels. Fifty polypeptides showed different distributions amongst the tissues, permitting characterization of each tissue by an array of polypeptide markers typical for the tissue at that developmental stage. The three tissues on the presumptive trophectoderm lineage did not show identical synthetic patterns. However, culture of EE cells in vitro resulted in conversion of their polypeptide synthetic profile to that of EPC after 2 days and of GC after 6 days, whilst culture of EPC cells converted their polypeptide synthetic profile to that of GC after only 4 days. These changes in polypeptide synthesis correlated well with the ploidy levels of the tissues at different times in culture.

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PubMed1981

Effect of culture conditions on diploid to giant-cell transformation in postimplantation mouse trophoblast.

Diploid extraembryonic ectoderm and ectoplacental cone from the 7.5-day mouse embryo were grown in vitro under a variety of culture conditions in an attempt to discover conditions which maintain trophoblast in a diploid state and prevent giant-cell formation. It was found that maintenance of tissue integrity was not enough to keep the tissues dividing and diploid, but that the presence of inner-cell-mass derivatives did have some effect. This effect was only apparent when trophoblast cells were entirely enclosed by embryonic tissues. Monolayers of embryonic or embryonal carcinoma cells did not prevent giant-cell formation. Diploid extraembryonic ectoderm and ectoplacental cone responded differently: ectoplacental cells eventually formed trophoblast giant cells even when enclosed by embryonic cells whereas extraembryonic ectoderm cells apparently could be maintained in a diploid condition. This and other differences in properties between extraembryonic ectoderm and ectoplacental cone are discussed with reference to a new model for the postimplantation trophoblast lineage in the mouse.

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PubMed1981

In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation.

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6.5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8.5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5.5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives. It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

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