Gene expression in visceral endoderm: a comparison of mutant and wild-type F9 embryonal carcinoma cell differentiation.

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.

Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue.

Stem cells generate many differentiated, short-lived cell types, such as blood, skin, and sperm, throughout adult life. Stem cells maintain a long-term capacity to divide, producing daughter cells that either self-renew or initiate differentiation. Although the surrounding microenvironment or "niche" influences stem cell fate decisions, few signals that emanate from the niche to specify stem cell self-renewal have been identified. Here we demonstrate that the apical hub cells in the Drosophila testis act as a cellular niche that supports stem cell self-renewal. Hub cells express the ligand Unpaired (Upd), which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in adjacent germ cells to specify self-renewal and continual maintenance of the germ line stem cell population.

An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells.

The message for the zinc finger gene Rex-1 (Zfp-42) is expressed in undifferentiated murine F9 teratocarcinoma cells and embryonic stem cells. Expression of Rex-1 is reduced at the transcriptional level when F9 cells are induced by the addition of retinoic acid (RA) to differentiate. We have isolated genomic DNA for the Rex-1 gene (Zfp-42), characterized the gene's structure, and mapped the gene to mouse chromosome 8. Promoter elements contributing to the regulation of the Rex-1 promoter in F9 cells have been identified. A region required for Rex-1 promoter activity in F9 stem cells contains an octamer motif (ATTTGCAT) which is a binding site for octamer transcription factor members of the POU domain family of DNA-binding proteins. Rex-1 reporter plasmids including this octamer site also exhibited reduced expression in F9 cells treated with RA. Thus, the octamer motif is a regulatory element required for the activity of the Rex-1 promoter in F9 stem cells, and this motif contributes to the negative regulation by RA of the transcription of the Rex-1 gene. As an initial confirmation of the in vivo relevance of the isolated fragment, a larger Rex-1 promoter fragment, also containing the octamer site, was able to promote expression of the bacterial lacZ gene in mouse embryos at the morula stage.

Bone morphogenetic proteins-2 and -4 are involved in the retinoic acid-induced differentiation of embryonal carcinoma cells.

Bone morphogenetic proteins-2 and -4 (BMPs-2 and -4) are transforming growth factor beta-related proteins that can induce bone formation in vivo. We observed that the level of endogenous BMP-2 mRNA increased an average of 11-fold on differentiation of F9 embryonal carcinoma cells into parietal endoderm after treatment with retinoic acid (RA) and cAMP, whereas the message for the closely related BMP-4 decreased 12-fold after this treatment. Therefore, the effects of exogenous recombinant BMP-2 protein on the RA-induced differentiation of F9 embryonal carcinoma cells were investigated. BMP-2 addition altered the growth and morphology of RA-treated but not untreated cells. Moreover, the abundance of several messages was affected by exogenous BMP-2 treatment. Notably, the BMP-2 and -4 messages themselves were reduced by the addition of exogenous BMP-2. The observations suggest that RA, which is known to affect bone morphogenesis, may regulate the osteoinductive proteins, BMP-2 and -4. Furthermore, BMP-2 and -4 may be involved in preimplantation embryogenesis.

The oncofetal gene Pem encodes a homeodomain and is regulated in primordial and pre-muscle stem cells.

The oncofetal gene, Pem, is expressed in a stage specific manner during murine ontogeny. The carboxy terminal portion of the predicted Pem protein has significant similarity to homeodomains of the Drosophila prd family. The Pem gene is expressed in undifferentiated embryonal stem (ES) and embryonal carcinoma (EC) cell lines. Pem mRNA is induced 35-fold in ES cells differentiated in the absence of retinoic acid. Pem mRNA is increased in EC cells differentiated towards parietal or visceral endoderm, consistent with the abundant Pem expression in embryonic yolk sac. In 10T mesenchymal stem cells committed to muscle cell differentiation, Pem mRNA expression is dramatically increased. The elevation in Pem expression preceded the induction of the muscle master regulatory gene, myoD. We conclude that the Pem gene encodes a candidate transcription factor which is developmentally regulated.

Induction of altered gene expression in early embryos.

This review focuses on known genes whose expression may be perturbed by teratogens during early embryogenesis (preorganogenesis). Teratogens may disrupt embryogenesis by modifying positional information. Genes controlling positional information include those specifying the primary body axes: anterior-posterior, dorsal-ventral, or left-right. These genes often encode transcription factors, whose regulation or activation can stimulate aberrant tissue differentiation and morphogenesis. Alternatively, teratogens may directly affect cell differentiation, proliferation, or apoptosis. Hydrophilic signalling molecules such as growth factors and hydrophobic molecules such as retinoids regulate these processes. The signalling pathways activated often induce the coordinate regulation of tissue specific gene expression. In addition to modifying individual signalling pathways, teratogens can synergize with or antagonize the effects of other teratogens through inappropriate interactions between signal transduction pathways. Since teratogens may often directly or indirectly perturb the expression of known or as yet undescribed developmentally critical genes, this review also provides a short description of techniques to identify genes whose expression is altered by teratogens.

MYTHSEEKER Project Status Summary.

This is a summary of the present status of The MYTHSEEKER Project. The first section provides a brief synopsis of the project's purpose and approach. The second section summarizes present activity in a number of directions-including decisions that are shaping development. The third section outlines the next steps for development programming, the initial software product, and a communications network of MYTHSEEKER Centers. Some extended viewpoints are mentioned.

Virtual reality in psychotherapy: the MYTHSEEKER software.

This chapter outlines the use in psychotherapy and medical diagnosis of an intelligent software system that helps clients to explore Personal Myth within virtual reality environments. Patented MYTHSEEKER software will allow clients to work with mythic analogues of lifeshapes and aspirations. This can help to focus therapy directions, find ways to participate with the person's world, and allow a kind of personal expression not previously possible. The software phases of assessment, facilitation, and enaction are described by which the client is assisted to explore systems of mythology or spirituality (called Depth Systems) that are traditional, ancient or newly-arising. The client builds a Personal Depth System representing Personal Myth, based on experiencing other Depth Systems, which can itself be experienced in the virtual environment. This paper outlines our methodology and technology to realize these operations. Space limitations prevent further description in the present chapter of MYTHSEEKER software technology or psychotherapy scenarios of involvement.

Receptor-selective retinoids implicate retinoic acid receptor alpha and gamma in the regulation of bmp-2 and bmp-4 in F9 embryonal carcinoma cells.

The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.

Retinoids. A window into vertebrate development.

Vitamin A is required for maintaining healthy epithelial tissues, e.g., skin and gut, normal reproductive capacity, and vision. Vitamin A deficiency also causes premalignant changes in epithelial tissues. These observations led to the use of retinoids (vitamin A-related compounds) in treating various skin diseases and more recently to inhibit cancer growth. Retinoids are extremely teratogenic to developing vertebrate embryos, a fact which limits their clinical usefulness. In particular, specific malformations of skeletal structures are often observed; for instance, retinoic acid induces craniofacial and limb deformities in avian and mammalian embryos. Recent technological advances have increased our understanding of how retinoids affect vertebrate development. Some newly discovered mechanisms underlying these actions are reviewed.

Specific induction of apoptosis in P19 embryonal carcinoma cells by retinoic acid and BMP2 or BMP4.

Retinoic acid (RA) affects the response of many cells to growth factors, including the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-beta, family of growth factors, originally identified by their bone-inducing activities. Their widespread expression suggests many roles other than that in osteogenesis. Because RA modulates the cell's response to growth factors, this may be a means by which the retinoids exert some of their known teratogenic effects. One such cellular response may be apoptosis. While apoptosis is required for normal development, the location and timing of its induction must be carefully controlled. Recently, several TGF-beta family members have been implicated in the induction of apoptosis in certain cell types. We show here, using P19 embryonal carcinoma cells, that the combination of RA and BMP2 or BMP4 synergistically induces apoptosis in 40% of the population within 24 hr. In contrast, RA alone induces apoptosis in only 10-15% of the population and each of the BMPs alone minimally induces apoptosis. Apoptosis depends on the dose of both the RA and the BMP as well as on new protein synthesis. Further, the induction of apoptosis prevents the formation of fully differentiated neurons and glial cells and instead leads to primarily smooth muscle cell differentiation. These results suggest that some of the malformations caused by retinoids may be due to the induction of inappropriate apoptosis in cells exposed to BMPs.

BMP4- and RA-induced apoptosis is mediated through the activation of retinoic acid receptor alpha and gamma in P19 embryonal carcinoma cells.

Some growth factors, for example, members of the transforming growth factor-beta family, can induce apoptosis in a variety of cells. Retinoic acid (RA) also causes apoptosis in several malignant cell types. We have previously demonstrated that, although BMP2 or BMP4 cannot induce apoptosis alone, BMP2 or BMP4 and RA synergize to induce apoptosis in 95% of P19 embryonal carcinoma cells within 4 days of treatment. Such treatment also prevents neuronal differentiation of these cells. Retinoids exert their many effects through any of six distinct nuclear receptors. These retinoid-activated transcription factors directly regulate genes involved in cellular response such as apoptosis. Complete understanding of how BMP and RA specifically induce cell death requires identification of the retinoid receptors controlling apoptosis. By using receptor-selective retinoid agonists and antagonists, we have obtained evidence suggesting that activation of RAR alpha or gamma is sufficient to induce apoptosis in BMP4-treated cells.

Retinoic acid- and bone morphogenetic protein 4-induced apoptosis in P19 embryonal carcinoma cells requires p27.

During development, many cells are specifically eliminated. Therefore, programmed cell death must be understood to fully elucidate embryogenesis. Retinoic acid (RA) and bone morphogenetic protein (BMP) 4 induce rapidly dividing P19 embryonal carcinoma cells to undergo apoptosis. RA alone minimally induces apoptosis, while BMP4 alone induces none. RA and BMP4 exposure also elevates the number of cells in the G1 phase of the cell cycle. Because many cell cycle proteins control both proliferation and apoptosis, we determined the role of these proteins in inducing apoptosis. Although the mRNA levels of cyclins D1 and D2 are reduced in cells undergoing apoptosis, the protein levels are not. In contrast, RA and BMP4 induce the Cdk inhibitor p27. This protein binds Cdk4 in RA- and BMP4-treated cells and inhibits Cdk4-dependent kinase activity. We used p27 antisense oligonucleotides to rescue the P19 cells from RA and BMP4 apoptosis thus proving that p27 is necessary. The Cdk4 substrate, retinoblastoma (Rb) protein, is also induced in apoptotic cells. Consistent with the decreased kinase activity of the apoptotic cells, this Rb protein is hypophosphorylated and presumably active. These data support the hypothesis that RA and BMP4 together induce the p27 protein leading to Rb activation and ultimately apoptosis.

Cloning of Tetrahymena genomic sequences whose message abundance is increased during conjugation.

A molecular and biochemical inquiry into protein regulation during Tetrahymena thermophila conjugation was carried out in two ways: a two-dimensional gel analysis of newly translated proteins and the molecular cloning of genes whose message abundance is increased. The two-dimensional gel analysis indicated that the synthesis of 32 predominantly basic proteins was stimulated in conjugating cells. The induction of these proteins could not be correlated with length of starvation or with mating type. The transcription pattern and molecular organization of three clones of T. thermophila genomic DNA, selected on the basis of differential hybridization to conjugating or control cell RNA, were investigated. Two of the clones, which were homologous to transcripts detected in conjugating cells, showed no rearrangements between micro- and macronuclear DNA. A third clone was divided into three segments. One segment was homologous to sequences limited to the micronucleus. A second segment hybridized to a large number of restriction fragments of micronuclear DNA digested with HindIII but to only two fragments of macronuclear DNA. A third segment, which was complementary to one transcript in conjugating cells and to two different transcripts in control cells, hybridized to two fragments in micronuclear DNA and one fragment in macronuclear DNA.

Adolescence in Tetrahymena thermophila.

The development of sexual maturity in Tetrahymena thermophila has been shown to include an intermediate stage, adolescence, during which cells are capable of mating with mature cells but not other adolescent cells. When the progeny of successfully mated cells are grown logarithmically and tested frequently for the ability to mate, they are unable to form mating pairs for about 65 generations. This period is known as immaturity. During the next stage, the progeny pair with mature cells but not with other adolescent cells despite the presence of complementary mating types. Adolescence persists for 20-25 generations before the cells attain maturity, which is defined as the ability to mate with any cell of different mating type. Once paired with mature cells, adolescents successfully complete conjugation. Cytological preparations show that both members of the pair undergo meiosis and form macronuclear anlagen. The proteins synthesized during a mating between adolescents and mature cells are similar to those synthesized during a mating between mature cells as determined by two-dimensional gel analysis. Both the adolescent cell and the mature partner contribute genetic markers to the progeny.

Cloning and characterization of the endogenous cephalosporinase gene, cepA, from Bacteroides fragilis reveals a new subgroup of Ambler class A beta-lactamases.

Bacteroides fragilis CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. beta-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this beta-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The beta-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A beta-lactamase-deficient mutant strain of B. fragilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal beta-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other beta-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of beta-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A beta-lactamases.

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.

A putative G-protein-coupled receptor, H218, is down-regulated during the retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor beta or gamma signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes.

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.