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R L Gardner

Publications and source records attributed to R L Gardner.

At least 73 records · Page 4Linked to original sources
PubMed1985

Clonal analysis of X-chromosome inactivation and the origin of the germ line in the mouse embryo.

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.

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PubMed1984

An in situ cell marker for clonal analysis of development of the extraembryonic endoderm in the mouse.

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.

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PubMed1984

Heterogeneous differentiation of external cells in individual isolated early mouse inner cell masses in culture.

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.

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PubMed1982

Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo.

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.

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PubMed1979

Investigation of the fate of 4-5 day post-coitum mouse inner cell mass cells by blastocyst injection.

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.

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PubMed1979

Investigation of the lethal yellow Ay/Ay embryo using mouse chimaeras.

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.

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PubMed1979

Growth and differentiation of an embryonal carcinoma cell line (C145b).

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.

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PubMed1978

The cellular origin of chemically induced tumours.

Tumours were induced by chemical carcinogens in chimaeric mice made by the amalgamation of two embryos each producing a different electrophoretic variant of the enzyme glucose phosphate isomerase. Although, in these chimaeras, the smallest samples of normal tissue that could be analysed almost invariably contained both isoenzymes, almost all the tumours contained only one. An analysis of the size of the clones that formed the chimaeric epidermis permitted the conclusion that the epidermal tumours produced could not have risen from more than 8 cells; and the most probable interpretation of the data is that these tumours were clonal growths.

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PubMed1978

Investigation of the potency of cells from the postimplantation mouse embryo by blastocyst injection: a preliminary report.

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.

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PubMed1978

Participation of cultured teratocarcinoma cells in mouse embryogenesis.

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.

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PubMed1975

Investigation of cellular interaction and deployment in the early mammalian embryo using interspecific chimaeras between the rat and mouse.

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.

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PubMed1975

Marker chromosome analysis of two mouse chimaeras.

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.

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