In Drosophila melanogaster the primor- dial germ are normally formed at the posterior tip of the egg during the preblastoderm stage. In order to determine whether the posterior polar plasm is capable of inducing the formation of primordial germ in another region of the embryo, portions of this cytoplasm were transferred from wild-type embryos of the early cleavage stage to the anterior tip of mwh e embryos of the same age. At various times after the injection (15-200 min), embryos were fixed for histological analysis. Alternating thick and thin sections were examined for the presence of experimentally induced cells. In more than half of the embryos analyzed in this way, one to six were found containing the polar granules as well as round nuclear structures, both of which are characteristic of normal and are not present in blastoderm cells. In order to determine whether these pole cells function normally, i.e., develop further into germ cells, the induced at the anterior tip of mwh e blastoderm embryos were introduced into the posterior region of y w sn3 hosts of the same age. The flies resulting from these embryos were mated to y w sn3 partners. In addition to the expected y w sn3 progeny, wild-type flies heterozygous for mwh e and, therefore, descended from the experimentally in- duced were found in 4% of the crosses. Such flies did not appear in the control experiments after trans- fer of normal anterior from noninjected blastoderm embryos. These results demonstrate that the posterior polar plasm can be transferred to the anterior tip of the embryo and that in this presumptive somatic region it still re- tains its capacity to determine the formation of the pri- mordial germ cells.
Mouse teratocarcinoma cells from the OTT6050 ascites tumor were established in tissue culture and selected for 5-bromodeoxyuridine (BrdUrd) resistance. The embryonal carcinoma cells grew without a feeder layer, remained deficient for thymidine kinase (EC 2.7.1.75), and differentiated like the original tumor into various tissues after subcutaneous injection into 129 mice. We fused the BrdUrd-resistant mouse teratocarcinoma cells with HT1080-6TG human diploid fibrosarcoma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and selected for hybrid cells in hypoxanthine/aminopterin/thymidine medium. The resulting hybrid cells segregated human chromosomes quickly and retained one to three human chromosomes including chromosome 17 that carries the human genes for thymidine kinase and galactokinase (EC 2.7.1.6). Single hybrid cells from five independent clones containing human chromosome 17 were injected into mouse blastocysts bearing several genetic markers that affect the coat color phenotype and strain-specific enzyme variants in order to detect tissue differentiation derived from the injected cells. After the injection of single hybrid cells into a total of 103 experimental blastocysts that had been surgically transferred to pseudopregnant foster mothers, 49 mice were born and 2 of them clearly revealed coat mosaicism. In 2 of 17 mice thus far analyzed, the injected hybrid cells proved to be capable of participating substantially in development of seven different organs. However, human gene products have not yet been detected unequivocally in those tissues and weak human-specific galactokinase activity could be recovered only from two mosaic tissues. Our results demonstrate that, after in vitro culture and selection, at least some of the human-mouse hybrid cells still retain their in vivo potential to differentiate and become functionally integrated in the living organism. It now seems feasible to cycle mouse teratocarcinoma cells carrying human genetic material through mice via blastocyst injection to study human gene expression during differentiation.
Recent progress in experimental mouseembryology has provided new approaches to the genetic manipulation of the mammalian embryo. The production of uniparental embryos enables one to compare maternal and paternal gene activity during development, to study the biological consequences of homozygosity of mutant genes, and to further elucidate the unsolved problem of X chromosome inactivation. Transplantation of nuclei from somatic cells into mouse eggs is considered the most vigorous functional test for the developmental capacity of the nuclear genome and provides a bioassay for the study of possible genomic changes during cellular differentiation. Transplantation of cloned eukaryotic genes into mouse eggs will permit the molecular and genetic analysis of their integration and regulation during development and, eventually, their germ line transmission as new heritable elements.
Posterior polar plasm of the Drosophila egg has been shown to function autonomously in germ cell determination after transplantation to either the anterior or mid-ventral region of the early embryo. By means of similar transplantations, we have tested the ability of polar plasm of Drosophila immigrans to induce the formation of pole cells in a Drosophila melanogaster embryo. After the transplantation of polar plasm, "hybrid" pole cells were found in which both pole cell-specific organelles, the polar granules and nuclear body, were structurally similar to those characteristic of the transplanted cytoplasm. In order to determine whether these hybrid cells can function as germ cell precursors, these cells were transplanted to the posterior tip of genetically marked embryos. Approximately 5% of the flies obtained from embryos receiving potential pole cells produce offspring derived from the induced pole cells. This result demonstrates that polar plasm can function in interspecific species combinations and indicates that the molecular mechanisms of germ cell determination are conservative in evolution. Finally, in order to test whether there is any evidence for cytoplasmic inheritance of polar granules, embryos derived from hybrid pole cells were examined for their polar granule morphology. The fine structure of the granules conformed to that of the nucleus. Thus, no evidence was found for the cytoplasmic inheritance of these particular organelles.
