Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues

We have obtained cDNA clones coding for the A, B1, and B2 chains of laminin by screening a cDNA library prepared from mouse EHS tumor poly(A)RNA in the lambda gt11 expression vector with polyclonal antibody against denatured laminin. These cDNA clones were used in combination with a cDNA clone coding for the alpha 1 type IV collagen chain to study the regulation of genes for these basement membrane proteins in retinoic acid-induced differentiating mouse F9 teratocarcinoma cells and in various adult murine tissues. The levels of mRNA for the laminin A, B1, and B2 chains and for the alpha 1 type IV collagen chain were increased simultaneously and reached a maximum at almost the same time during the differentiation of F9 cells, suggesting coordinate expression in these cells. The tissue levels of mRNA encoding for the basement membrane components, however, varied considerably. The highest level of the B1 chain mRNA was observed in kidney, whereas, the levels of mRNA for A and B2 chains were highest in heart. Almost the same levels of expression of the alpha 1(IV) collagen mRNA were found in kidney, lung, and heart. The results indicate that the expression of genes for the basement membrane proteins is not coordinately regulated in these tissues. It is thus possible that different subunit structures of the laminin molecule may exist in tissues.     

Murine cyclophilin-S1: a variant peptidyl-prolyl isomerase with a putative signal sequence expressed in differentiating F9 cells.

Fractionation of differentiating murine teratocarcinoma F9 cells and extraction of the nuclear/microsomal pellets with ethidium bromide led to the purification and microsequencing of the protein mCyP-S1, a novel cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase (PPIase). mCyP-S1 is a new member of the cyclophilin class of proteins. Cloning and sequencing of the mCyP-S1 cDNA revealed extended coding capacity for a putative N-terminal signal sequence, suggesting processing of mCyP-S1 during intracellular translocation across the membrane of the endoplasmic reticulum. mCyP-S1 is abundantly expressed in a variety of mouse organ tissues and its mRNA levels increase during F9 cell differentiation. Specific subcellular localization of PPIases is postulated to contribute to functional specificities of this class of enzymes.     

Two-stage hormonal control of type IV collagen mRNA levels during differentiation of F9 teratocarcinoma cells

A cDNA clone related to mouse Type IV collagen has been prepared from F9 teratocarcinoma cells induced to differentiate with retinoic acid and dibutyryl-cAMP. This cDNA clone has been used to investigate the regulation of Type IV collagen mRNA during differentiation. The level of this mRNA is very low in untreated F9 cells, increases substantially after treatment of the cells with retinoic acid, and is further increased by addition of dibutyryl-cAMP. In contrast, dibutyryl-cAMP has no effect on the mRNA level in cells that have not been previously exposed to retinoic acid. These results demonstrate that these two compounds regulate in a sequential manner the steady-state level of Type IV collagen mRNA. This cDNA clone should allow a detailed examination of the mechanism of the two-stage regulation of collagen expression by retinoids and cyclic AMP.     

Expression of mouse histone H1(0) promoter sequences following microinjection into Xenopus oocytes and developing embryos

Evidence from expression studies using transfected F9 teratocarcinoma stem cells indicates that the synthesis of the H1(0) histone is turned on very soon after the cells have been treated with retinoic acid, which causes them to differentiate into murine parietal endoderm. This increase in H1(0) at the time of commitment would allow a reorganization of the chromatin with a reprogramming of the gene activity of undifferentiated F9 cells to the differentiated state. The particular interest of the further development of this differentiation model is to answer the question whether this specific stimulation of expression can be induced only in homologous differentiation systems, or whether the identified H1(0) promoter sequences can also be specifically stimulated by heterologous factors. We therefore injected the mammalian H1(0) promoter sequences into Xenopus oocytes and fertilized eggs. The results of oocyte injection experiments indicate that H1(0) promoter sequence elements similar to those used in transfected F9 cells are specifically expressed in the oocyte. For analysis of H1(0) expression in Xenopus embryos we used promoter constructs ligated to beta-galactosidase sequences for microinjection. This procedure allows a particularly rapid and complete detection of expressed promoter clones within the differentiated tissues of the early Xenopus embryo.     

Isolation of cDNA clones for basal lamina components: type IV procollagen

We have isolated cDNA clones for mouse type IV procollagen from a library constructed from total poly A+RNA of 13.5 day mouse embryo parietal endoderm (PE) cells. In Northern analysis these clones hybridise to a 6.8 kb RNA which is abundant in embryonic PE cells and in differentiated F9 teratocarcinoma cells. Hybrid selection and in vitro translation of the cDNA specific mRNA produced a single polypeptide of Mr = 165 000. This polypeptide was specifically immunoprecipitated with mouse type IV procollagen antisera and comigrated on SDS-gel electrophoresis with one of the two in vitro synthesised chains of type IV procollagen. Undifferentiated F9 teratocarcinoma cells can be induced by retinoic acid and dibutyryl cAMP to differentiate in vitro into endoderm-like cells which resemble mouse PE cells in synthesising large amounts of basement membrane proteins, including type IV procollagen. Here we show, using one of the cDNA clones as a probe for type IV procollagen, that an increase in cellular concentration of type IV procollagen mRNA occurs within 24 to 48 hours of induction, reaching a constant high level by 72 hours.     

Cloning and characterization of keratin D,a murine endodermal cytoskeletal protein induced during in vitro differentiation of F9 teratocarcinoma cells

Summary We have identified a cDNA coding for the murine keratin D from a collection of clones representing F9 teratocarcinoma stem cell mRNA sequences. These sequences are synthesized specifically after the addition of retinoic acid and cAMP to the culture medium. The clone is 1,382 nucleotides long and contains the entire information for the active polypeptide, the complete 3 end and most, if not all, of the 5 non-coding region. The mRNA is found in hepatocytes, in PYS-2 cells (an endodermal cell line) and in differentiated (retinoic-acid-treated) F9 cells, but not in untreated F9 cells. The length of the mRNA is 1.4 kb, as estimated by Northern blot hybridization. Southern hybridization performed under very stringent conditions detects a single fragment hybridizing strongly with the cloned cDNA, suggesting that the mouse genome contains only one or very few copies of this gene. We present the first complete sequence of a keratin expressed in simple epithelia, i.e. keratin D, and discuss its structural features.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.     

Molecular cloning of a novel retinoic acid-responsive gene, HA1R-62, which is also up-regulated in Hoxa-1-overexpressing cells.

Using a PCR-based cDNA subtractive hybridization method (L. Diatchenko et al., Proc. Natl. Acad. Sci. USA, 93: 6025-6030, 1996), we cloned a cDNA fragment of a novel gene that is highly expressed in F9-10; F9-10 is an F9 teratocarcinoma stem cell line that expresses high levels of exogenous Hoxa-1 mRNA and protein in comparison to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of retinoic acid (RA). Rapid amplification of cDNA ends was used to clone the full-length cDNA of this gene, designated HA1R-62 (Hoxa1 regulated-62). We have shown that HA1R-62 is also a RA-responsive gene and that it is expressed (mRNA size, approximately 4.3 kb) in adult mouse thymus, lung, kidney, and ovary as well as in 12.5-day mouse embryos. DNA sequence analysis and in vitro translation experiments have shown that HA1R-62 encodes a protein with a molecular mass of approximately 26 kDa. Elucidation of the function of the HA1R-62 gene product will provide new insights into the functions of RA and homeobox genes.     

Isolation and characterization of the CRIPTO autosomal gene and its X-linked related sequence.

We have previously reported on the identification of a cDNA clone encoding a novel human growth factor, named "CRIPTO," that is abundantly expressed in undifferentiated human NTERA-2 clone D1 (NT2/D1) and mouse (F9) teratocarcinoma cells. We now report the organization and nucleotide sequence of two related genomic sequences. One (CR-1) corresponds to the structural gene encoding the human CRIPTO protein expressed in the undifferentiated human teratocarcinoma cells, and the other (CR-3) corresponds to a complete copy of the mRNA containing seven base substitutions in the coding region representing both silent and replacement substitutions. The 440 bp 5' to the CAP site of CR-1 are preserved in CR-3. CR-1 maps to chromosome 3, and CR-3 maps to Xq21-q22. Southern blot analysis reveals that multiple CRIPTO-related DNA sequences are present in the human as well as in the mouse genome.     

Characterization of cloned sequences complementary to F9 cell double-stranded RNA and their expression during differentiation

In a cDNA library prepared from the RNA of cultured murine F9 teratocarcinoma cells, we identified sequences exhibiting strong hybridization to double-stranded RNA (dsRNA) of F9 cells but weak hybridization to mouse liver dsRNA. Northern-blot hybridization of RNA extracted from F9 cells with or without treatment with retinoic acid revealed differences in the expression of some of these sequences in undifferentiated and differentiated cells. As shown by Southern-blot hybridization experiments, these differences of expression were not related to a gross rearrangement of the corresponding genomic DNA sequences of the differentiated cells. When RNA from F9 cells was used, one of the cloned dsRNA-related sequences selected mRNA which was translated in vitro to a polypeptide with an Mr of 24,000; the level of this mRNA was reduced in F9 cells that had been treated with retinoic acid. Our results show that the differentiation of F9 cells induced by retinoic acid results in the differential expression of some middle-repetitive sequences.     

Reversible effects of sodium butyrate on the differentiation of F9 embryonal carcinoma cells

We have studied effects of sodium butyrate on embryonal carcinoma F9 cell differentiation. In the presence of sodium butyrate, F9 cells underwent rapid and drastic morphological changes and expressed marked increases in mRNA levels of various differentiation markers. When sodium butyrate was removed from the cultures, all the examined phenotypes of F9 cell differentiation rapidly reverted to the characteristics of undifferentiated stem cells. However, under the same conditions, when cycloheximide or actinomycin D was added to the cultures, such phenotypic reversion was not observed, but high mRNA levels of the differentiation markers as well as altered cell morphology were retained. These results indicated that the effects of sodium butyrate on induction of teratocarcinoma cell differentiation were reversible and that de novo syntheses of some mRNA(s) and protein(s) were necessary for the reversion of differentiated cells to stem cells.     

Synthesis and secretion of beta-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Isolation of differentially expressed human cDNA clones: similarities between mouse and human embryonal carcinoma cell differentiation

The study of early human development is of great importance but has been limited by the lack of suitable reagents. Recently, however, the human embryonal carcinoma (EC) cell line NT2D1 has been isolated. This cell line will differentiate upon exposure to retinoic acid (RA). A cDNA library was constructed from poly(A)+ RNA derived from NT2D1 cells treated with 10(-5) M-RA for 7 days (delta NT2D1 cells). By differential cDNA screening, it was found that 1.12% of delta NT2D1 cDNA recombinants screened detected an increase in signal with 32P-cDNAs derived from delta NT2D1 as compared with NT2D1. To compare RA-induced differentiation of mouse and human EC cells, the delta NT2D1 cDNA library was rescreened with 32P-cDNAs derived from the mouse EC cell line F9 and the result compared with 32P-cDNA derived from F9 differentiated to parietalendoderm (F9PE)-like cells and visceral-endoderm (F9VE)-like cells. Approximately 1.2% of the delta NT2D1 cDNA recombinants detected a differential increase in signal following differentiation of mouse EC cells to F9VE and/or F9PE. Of these homologous regulated sequences, 0.3% were common to both mouse and human EC cell RA-induced differentiation. Five different cDNA clones were isolated that detect a marked increase (5- to 75-fold) in mRNA abundance following RA-induced differentiation of NT2D1. Of these five clones, three detect homologous mRNAs which also increase in abundance following differentiation of the mouse EC cell line F9 to PE- and/or VE-like cells; the other two clones do not detect sequences in the mouse mRNAs tested. One clone shows homology to SPARC, a gene known to be regulated during mouse embryonic development. While another clone, SO5A, has a limited range of expression, being detected in F9VE and in a human parietal-endoderm-like cell, but not in F9PE and a human visceral-endoderm-like cell. This work shows that there are both similarities and differences in mouse and human EC cell differentiation, and these cDNA clones provide some of the first reagents for studying the molecular biology of human development.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

Synthesis and secretion of β-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, β-nerve growth factor (βNGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature βNGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.