Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro.

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5-18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro.

Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus.

The p53 gene is rearranged in a high proportion of erythroleukemic cell lines derived from the spleens of mice infected with Friend leukemia virus. These rearrangements result in either the synthesis of a truncated protein or the inactivation of the p53 gene. Here we have molecularly characterized the rearrangements in two murine erythroleukemic cell lines induced by Friend leukemia virus, DP20-1 and CB3, that contain a rearranged p53 gene and fail to express p53 protein. The rearrangement in the DP20-1 cell line is due to the insertion of Friend spleen focus-forming provirus (SFFV) in the 3' end of the p53 gene in intron sequences between exons 9 and 10. Transfection of molecular clones of this SFFV provirus into NIH3T3 cells results in the generation of infectious virus as determined by its ability, in the presence of helper virus, to induce rapid splenomegaly and polycythemia when injected into adult DBA/2J mice. Insertion of SFFV in DP20-1 cells resulted in the expression of an aberrant 2.9 kb RNA species. Analysis of a molecular clone of the rearranged p53 gene in a second cell line, CB3, revealed that the p53 gene in this clone has sustained a large deletion within the p53 gene resulting in the loss of coding sequences between exons 4 and 8. The 5' end of the deletion originates within exon 4 and extends 3' to within the eighth intron. The significance of these findings with regard to the multi-stage nature of Friend virus induced erythroleukemia is discussed.

Polytene chromosomes in mouse trophoblast giant cells.

Mouse trophoblast giant cells undergo successive rounds of DNA replication resulting in amplification of the genome. It has been difficult to determine whether giant cell chromosomes are polyploid as in liver cells or polytene as in Dipteran salivary glands because the chromosomes do not condense. We have examined the pattern of hybridization of mouse giant cells with a variety of in situ chromosome markers to address this question. Hemizygous markers displayed one hybridization signal per nucleus in both diploid and giant cells, while homozygous markers displayed two signals per nucleus in both cell types. These patterns are consistent with cytological evidence indicating that giant cell chromosomes are polytene rather than polyploid. However, in contrast to the situation in Dipteran salivary glands, the two homologues do not appear to be closely associated. We conclude that the mechanism of giant cell DNA amplification involves multiple rounds of DNA replication in the absence of both karyokinesis and cytokinesis, and that sister chromatids, but not homologous chromosomes, remain closely associated during this process.