Showing papers by "Richard L. Gardner published in 1984"

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

Abstract: Conditions were found for staining whole mid-gestation capsular parietal endoderms and visceral yolk sacs for malic enzyme activity that gave excellent discrimination between wildtype ( Mod-1 + /Mod-1 + ) cells and mutant ( Mod-l n /Mod-1 n ) 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-1 n /Mod-1 n 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-1 n /Mod-l n 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.

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

Abstract: Inner cell masses (ICMs) were isolated from early blastocysts by immunosurgery and incubated in a dense suspension of melanin granules for 3h 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.