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Expression of cytokeratin endo A has been analyzed during mouse blastocyst formation and embryonal carcinoma cell differentiation. To study the regulation of endo A expression, nuclease S1 mapping experiments have been performed on RNA extracted from two-cell to 7.5-day embryos. Low levels of endo A mRNA begin to be detectable in eight-cell embryos. The amount of this mRNA increases at the blastocyst stage, suggesting that endo A expression is regulated at the mRNA level during blastocyst formation. At this stage, in situ hybridization studies show that endo A mRNA is present in the trophectoderm but not in the inner cell mass. In 7.5-day embryos, endo A mRNAs are also detectable in the endoderm layer and in the amnion.

The recombinant cDNA RecXVI, which hybridizes to the endo A mRNA, detects two specific bands on a Southern blot of EcoRI-digested genomic mouse DNA. We have screened a mouse genomic library with this cDNA and isolated these two sequences. Endo A is encoded by a 7.5-kilobase gene and by a 1.6-kilobase pseudogene devoid of introns. A repetitive sequence belonging to the B2 family is located in the third intron of the gene. We have observed that transcription of B2 sequences and of the endo A gene are mutually exclusive.The first detectable morphological differentiation of mouse embryonic cells takes place during blastocyst formation, in the course of which two different types of cells, the trophectoderm and the inner cell mass cells, become differentiated (1). This differentiation is accompanied by modifications in the pattern of protein synthesis, revealing a difference in the regulation of gene expression (2). Among these differences, some intermediate filament proteins are synthesized in the trophectoderm but not in inner cell mass cells (3, 4). Analysis of the onset of synthesis of these proteins might provide an insight into molecular events directly related to the cell commitment program.We describe here the isolation and characterization of the genes encoding endo A (5), also referred to as cytokeratin A (6). Endo A appears during blastocyst formation and has been identified in trophoblast but not in inner cell mass cells (7). It is therefore a valuable marker of the modifications in gene expression during the first binary choice made by embryonic cells. Later in development, endo A is still expressed in a tissue-specific manner; it is expressed in visceral and parietal endoderm and in epithelial cells derived from mesoderm and endoderm (i.e., in liver and kidney) but not in fibroblasts, myoblast, neural tissues, or keratinocytes (8). In vitro, endo A is found in trophoblastoma cell line TDM-1, but not in embryonal carcinoma cells.A cDNA clone, RecXVI, was isolated (9) from a cDNA library prepared from TDM-1. This cDNA hybridizes to a specific 18S mRNA encoding endo A. We have used this cDNA to isolate the endo A genes to analyze the regulation signals involved in the expression of this protein during the first embryonic differentiation. We describe here the structure of the genes and discuss the possible regulatory role of a repetitive sequence that is included in one of them.MATERIALS AND METHODS Cells. Teratocarcinoma cell lines F9 and PCC3 (10) were cultured under standard conditions. TDM-1 is a trophoblastoma cell line (10).Isolation of Mouse Genomic Clones. A genomic library of BALB/c mouse DNA restricted with EcoRI and cloned into phage X Charon 4A was plated to a density of 15,000 phages per 10-cm plate and screened as described (11) using the cDNA RecXVI.Electron Microscopy of Heteroduplexes. Heteroduplexes were prepared by renaturing the DNAs in 50% formamide/0.58 M NaCl/50 mM Pipes, pH 6.8/1 mM EDTA at 640C for 1 hr. After fixation with glyoxal for 2 hr at 12'C, a solution was prepared f...

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The major transplantation (or H-2) antigens in the mouse are cell-surface glycoproteins composed of a heavy chain and a light chain, the beta-2 microglobulin (beta 2m). The expression of these proteins is regulated during development. Embryonic cells at early stages of development do not express these proteins. On the other hand, these molecules are present on the surface of all adult somatic cells. We investigated whether the expression of both chains was coordinately regulated. Using specific single-stranded DNA probes in an S1 nuclease analysis, we compared the relative amounts of H-2D and beta 2m transcripts in normal tissues, in transformed cells, and during embryonic development. Our results show that (1) the steady state level of beta 2m transcripts varies from one adult organ to another, while that of H-2D transcripts stays approximately the same; (2) upon transformation, the amount of H-2D-specific mRNA increases drastically, while the beta 2m mRNA level remains constant; (3) whereas the quantity of beta 2m mRNA increases during early development, the amount of H-2D mRNA remains at a very low level. These data suggest that the regulation of H-2D and beta 2m genes are not identical and that their activation during development is not synchronous.