The vimentin promoter as a tool to analyze the early events of retinoic acid-induced differentiation of cultured embryonal carcinoma cells.

The vimentin gene encodes an intermediate filament protein expressed in the parietal endoderm, mesodermal, and early neural cells in vivo but by most in vitro-cultured cells regardless of their embryonic origin. Here we show that the vimentin gene promoter is very active in F9 embryonal carcinoma cells and increases in activity during differentiation. Using a series of 5'-deletion mutants, we provide evidence that the regions of the promoter involved in F9 cell activity are different from those previously demonstrated to be active in differentiated cell lines. Furthermore, we show that in differentiating F9 cells the activities of two different regions of the promoter are significantly enhanced. A distal region (-1710/-957) appears to contain functional binding sites for the murine Hox-A5 homeoprotein as demonstrated by band shift and footprinting experiments. A proximal region (-140/-78) contains a 30-bp repetitive sequence found in other genes activated during differentiation of F9 cells. Using band shift assays and methylation interference, we present evidence that a sequence-specific single-stranded DNA-binding protein(s) specifically interacts with the minus strand of the 30-bp sequence.

What can be learned from intermediate filament gene regulation in the mouse embryo.

In recent years, intermediate filaments (IFs) have attracted much interest, largely because their constitutive polypeptide units are specifically expressed in various cell types and thus represent excellent differentiation markers. Data obtained through biochemical studies and molecular cloning have allowed the classification of IFs into five types according to their protein structure. The expression of most IF types is characteristic of a given cell type: cytokeratins (IF types I and II) are produced in epithelia, neurofilaments and alpha-internexin (type IV) in neurons and nestin (type IV) in neuroblast and myoblast. On the other hand the four type III IFs are highly related proteins which are expressed in different cell types. Thus the study of type III IF gene regulation provides an excellent approach towards the analysis of cell-specific transcription. This review focuses on type III IF gene regulation during mouse embryogenesis and describes the latest data obtained through the combination of both in vitro (in cell lines) and in vivo (in transgenic mice) approaches. It appears that, while intragenic sequences play a major role in the regulation of the expression of the genes encoding other types of IFs, a major contribution to the transcriptional regulation of type III IF genes is brought by 5' upstream sequences. However, recent evidence obtained through the use of transgenic mice indicate that upstream sequences must cooperate with intragenic elements to establish the complex and dynamic expression pattern characteristic of type III IF genes. The very high similarity between the coding sequences of type III IF genes raises the question of the significance of the occurrence of four members of this class. We propose a model for the amplification of this small gene family based on the increasing complexity of expression patterns in higher organisms. This could have led first to the requirement for a highly sophisticated control region in an ancestral type III IF gene, followed by two successive gene duplications, thus leading to the appearance of four different regulatory regions directing the cell-specific transcription of nearly identical genes in different cell types.

Control of skeletal muscle-specific transcription: involvement of paired homeodomain and MADS domain transcription factors.

In the last few years, many aspects of skeletal muscle-specific gene regulation have been explained by the activity of the helix-loop helix (HLH) myogenic regulatory factors of the MyoD family, which are sequentially expressed during skeletal muscle formation. However, evidence is accumulating that muscle specific transcription requires functional interactions of these muscle-specific HLH factors with other regulatory proteins whose expression is not only restricted to skeletal muscle. These regulators include the SRF and MEF2 MADS domain and the MHox paired homeodomain transcription factors. Together with the aforementioned HLH factors, they build an increasingly complex network of regulatory factors. Two members of the Pax multigenic family of developmental control transcription factors, Pax-3 and 7, have been shown to be expressed not only in nervous tissue but also in skeletal muscle precursor cells. Their possible involvement in the control of muscle-specific transcription is discussed in light of known molecular properties of Pax gene products described in other biological systems.

Expression of a zebrafish caudal homeobox gene correlates with the establishment of posterior cell lineages at gastrulation.

This paper deals with the first identification of a caudal cDNA containing a homeobox of the Drosophila caudal family in the zebrafish. A cDNA library from late gastrula stage embryos was constructed and screened with a mouse Cdx 1 homeobox probe. A 1.6 kb cDNA clone containing a homeobox related to other caudal homeoboxes was isolated and called cdx[Zf-cad1]. Analysis of the predicted 301 amino acid translation product reveals additional regions of homology outside the homeodomain with other members of the caudal family. Particularly, the cdx[Zf-cad1] putative protein shares a conserved N-terminal region with its chicken homolog CHox-cad. Transcripts are first detected just before the onset of gastrulation. At the beginning of gastrulation, a single 1.8 kb cdx[Zf-cad1] transcript is located near the blastoderm margin with a high level of expression restricted to the epiblast. At this stage, the hypoblast is clearly negative. At the end of gastrulation, cdx[Zf-cad1] is widely expressed in vegetal (i.e. prospective posterior) epiblast and hypoblast, with a somewhat weaker expression in the dorsal hypoblast. During somitogenesis, cdx[Zf-cad1] exhibits a posterior regionalization in the neurectoderm. In contrast, no expression is detected in the mesoderm of 22 h embryos (late somitogenesis). Posterior endoderm is also positive at this stage. cdx[Zf-cad1] transcripts cease to be detected about 48 h after fertilization. They are undetectable in the adult, particularly in female gonads. The pattern of cdx[Zf-cad1] expression during and after gastrulation is consistent with its possible involvement in the regionalization of the embryo at these stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory elements of the human vimentin gene: activation during proliferation.

We investigated the constitution of the vimentin regulatory region through the use of cloned deletion mutants and the nucleotide sequence analysis in order to determine the elements which are implicated in the various physiological stimulations. We report that the vimentin promoter is constituted of a juxtaposition of at least 20 different putative regulatory elements illustrating the molecular tinkering theory. Fifty-eight motifs were found, representing 20 different sequences. Each of these mini-elements displays a consensus sequence homologous to or closely related to that found in regulatory regions of different genes correlated with processes of cell activation and proliferation.

Chromosomal localization of the mouse gene coding for the 68 kDa neurofilament subunit.

The chromosomal localization of the mouse gene coding for the 68 kDa intermediate filament subunit of neurones (NF-L) was determined by in situ hybridization using specific 3H-labelled DNA probes. There is only one copy of the NF-L gene. The gene encoding NF-L is located on chromosome 14 region (D1-E1).

A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine.

A mouse gene, Cdx-1, was isolated from an embryonic cDNA library using a Drosophila caudal gene probe. The deduced amino acid sequence of Cdx-1 contains conserved sequence domains along the entire gene, as well as a highly conserved caudal-type homeo box. A structural comparison suggests a common ancestral origin of mouse Cdx-1 and Drosophila caudal. The expression of Cdx-1 during embryogenesis was analyzed by Northern blotting and in situ hybridization. Cdx-1-specific transcripts are localized in the epithelial lining of the intestines beginning at 14 days' gestation. The expression of Cdx-1 in the intestine continues into adulthood, but cannot be detected in any other tissues. The Cdx-1 gene is the first homeo-box-containing gene expressed in cells derived from the embryonic endoderm.

A labile inhibitor blocks endo A gene transcription in murine undifferentiated embryonal carcinoma cells.

The endo A gene encoding for an intermediate filament protein, a cytokeratin is usually expressed in epithelial cells. The regulation of this gene, probed by using cycloheximide, an inhibitor of protein synthesis was studied in various cell lines. The lines explored were undifferentiated embryonal carcinoma PCC4 cells which normally do not express endo A gene, PCC4 cells cultivated permanently at 31 degrees C (PCC4-31), which are epithelial-like cells derived by differentiation from PCC4 cells, but which do express endo A gene, TDM1 cells, an epithelial teratocarcinoma cell line, and 3T6 mouse fibroblasts. Treatment of undifferentiated PCC4 cells by cycloheximide led to transcriptional induction of the endo A gene, and the same effect was observed after this treatment in PCC4-31 cells. By contrast, cycloheximide did not induce endo A gene expression in 3T6 cells, and reduced the transcriptional activity of this gene in TDM1 cells. We conclude that a labile inhibitor (or several) blocks endo A gene expression in undifferentiated PCC4 cells. We suggest that in these cells, the expression of the endo A gene is regulated both positively and negatively, possibly by a cellular E1A-like activity, as we previously demonstrated it for Py virus (C. Crémisi and C. Babinet, 1986 J. Virol. 59; 761-763). We further suggest that negative regulatory factors involved in this regulation are absent in TDM cells and reduced in PCC4-31 cells.

Spontaneous differentiation of murine teratocarcinoma stem cells at low temperature.

Embryonal carcinoma cells differentiate in vitro after treatment with different drugs or under certain culture conditions. We show here that at 31 degrees C PCC-aza-1 cells differentiated spontaneously into cells with characteristics resembling those of epithelial cells. With this temperature, we observed morphological changes; reorganization of the cytoskeleton with polymerization of actin cables, tight junctions, and cytokeratin endo A messenger RNA and protein expression. The cells also became totally permissive to Py virus but remained resistant to SV40 infection. This new pathway of differentiation has the advantage of being reversible at 37 degrees C.

Mesenchymal-epithelial conversions induced by 5-azacytidine: appearance of cytokeratin Endo-A messenger RNA.

When mouse-teratocarcinoma-derived fibroblasts (1246 cell line) are subjected to treatment with the inhibitor of DNA methylation, 5-Azacytidine (5 AzaC), they transiently express at 55-kilodalton intermediate-filament protein recognized by the epithelial-specific monoclonal antibody, TROMA-1, although they retain a fibroblastic morphology. However, rare clones (e.g., the 1339 cell line) that permanently express the antigen recognized by TROMA-1 can be derived from the 5 AzaC-treated 1246 population, and these clones have an epithelial phenotype. In the present study, we used cloned DNA probes to demonstrate that, in 1246 fibroblasts, 5 AzaC induces the appearance of Endo-A mRNA. High levels of Endo-A mRNA were also detected in the epithelial derivative, cell line 1339. In both cases, the capping site of the Endo-A mRNA was found to be the same as that in epithelial cells which normally express this RNA.

Expression of the cytokeratin endo A gene during early mouse embryogenesis.

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.

RNAs containing B2 repeated sequences are transcribed in the early stages of mouse embryogenesis.

An in situ hybridization technique was used to detect RNAs containing B2 sequences in the early mouse embryo. Accumulation of B2 sequences occurs early from the one cell stage. The level of B2 RNA decreases in the late two cell embryo, and then increases at the moment of second cleavage. In the blastocyst, inner cell mass cells contain more B2 transcripts than trophectoderm cells. In 7.5-day embryos the expression of B2 sequences is restricted to ectoderm and mesoderm. At all stages, transcription of the B2+ strand is greater than B2- strand. We detected B2+ RNAs in the nucleus and cytoplasm, whereas B2- RNAs were present only in the nucleus.

One gene and one pseudogene for the cytokeratin endo A.

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.

Asynchronous regulation of mouse H-2D and beta-2 microglobulin RNA transcripts.

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.

Collagen synthesis in mouse embryonal carcinoma cells: effect of retinoic acid.

In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected. In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.

New method for obtaining uncontaminated urine from women.

Midstream sampling and colony counts have greatly improved precision in diagnosing urinary tract infections. In women, however, contamination by vaginal flora and cells still poses a diagnostic problem. We have devised an instrument for helping collect uncontaminated urine from women and have tested it in three sets of observations. Approximately 96% of 200 women were able to use the device successfully after minimal instruction. Bacterial colony counts and quantitative estimation of vaginal epithelial cells were performed on urine collected by 18 female volunteers using conventional clean-catch technics and on urine collected by the same 18 women using the Clean Streamer. The average bacterial colony count in conventionally collected urine was 8,100/ml while in Clean Streamer collected urine it was 1,722/ml. The average number of vaginal epithelial cells in conventionally collected urine was 22.4/ml while in Clean Streamer collected urine it was 14.1/ml. The difference in both comparisons is statistically significant (P = .01). We believe that use of the Clean Streamer greatly facilitates the ability of a woman to collect a urine sample uncontaminated by vaginal secretions and flora.