Gardner R L
J Embryol Exp Morphol. 1984 Apr;80:251-88.
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.
已找到对整个妊娠中期的包膜壁内胚层和脏卵黄囊进行苹果酸酶活性染色的条件,该条件能很好地区分野生型(Mod-1+/Mod-1+)细胞和缺乏该酶细胞质形式的突变型(Mod-1n/Mod-1n)细胞。进行了反向囊胚注射实验,即将一种基因型的单个原始内胚层细胞移植到另一种基因型的胚胎中。此外,将Mod-1+/Mod-1+早期内细胞团(ICM)细胞以两个或三个单个体一组或作为子细胞对的形式注射到Mod-1n/Mod-1n囊胚中。相当比例的所得胚胎在壁内胚层、脏卵黄囊或这两层膜中均显示出镶嵌组织化学染色。在镶嵌的壁内胚层中,染色细胞总是与未染色细胞紧密混合。相比之下,在镶嵌的脏卵黄囊中可辨别出两种不同染色模式中的一种或两种。第一种是明显的“连贯”模式,发现是由于内胚层嵌合体所致;第二种是更弥散的模式,归因于该膜中胚层的嵌合体。由原始内胚层与早期ICM细胞注射产生的具有供体染色特征的细胞的总体分布与早期实验结果一致,早期实验中使用磷酸葡萄糖异构酶的同工酶作为标记。在镶嵌膜的主要染色区域明显缺乏表型中间细胞,这表明Mod-1+/Mod-1+和Mod-1n/Mod-1n基因型之间的组织化学差异是细胞自主的。壁内胚层体外混合培养物的染色结果强化了这一结论,在该培养物中,吞噬的黑色素颗粒的存在与否被用作区分野生型和无效细胞的独立方法。通过在孵育培养基中用四硝基蓝四唑代替硝基蓝四唑,对植入后早期野生型与无效胚胎的胚外内胚层和其他组织均获得了满意的差异染色。最后,细胞质苹果酸酶活性的缺失似乎对突变细胞的活力或行为没有显著影响。