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J C Cross

Publications and source records attributed to J C Cross.

At least 37 records · Page 2Linked to original sources
PubMed2000

Posttranscriptional regulation of human leukocyte antigen G during human extravillous cytotrophoblast differentiation.

Human maternal tolerance to a semiallogenic fetus may be maintained, in part, by the unusual expression pattern of antigen-presenting molecules in placental trophoblast cells. Extravillous cytotrophoblast (EVC) cells, which invade the maternal decidua, express high levels of human leukocyte antigen G (HLA-G), a nonclassical, major histocompatibility complex (MHC) class I molecule. HLA-G transcripts have been detected in tumors and other tissues, yet protein accumulation is rare. We show that, within EVC cells themselves, the mRNA is more broadly expressed than the protein. Specifically, accumulation of HLA-G protein was markedly delayed during EVC cell differentiation. To elucidate this mechanism, we performed a comprehensive analysis comparing the expression of HLA-G and proteins essential for MHC class I expression at the cell surface. The transporter for antigen processing proteins TAP1 and TAP2, as well as tapasin and beta(2)-microglobulin, appeared to be coordinately expressed throughout EVC cell columns. Strikingly, they all accumulated well in advance of the HLA-G protein but concurrently with its mRNA. A similar delay in the accumulation of the HLA-G protein was observed in vitro, using cultures of chorionic villi. We conclude that posttranscriptional regulation of HLA-G is fundamental to EVC cell development and is achieved independently of the peptide loading system. This represents a novel mechanism of MHC class I regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

The HAND1 basic helix-loop-helix transcription factor regulates trophoblast differentiation via multiple mechanisms.

The basic helix-loop-helix (bHLH) transcription factor genes Hand1 and Mash2 are essential for placental development in mice. Hand1 promotes differentiation of trophoblast giant cells, whereas Mash2 is required for the maintenance of giant cell precursors, and its overexpression prevents giant cell differentiation. We found that Hand1 expression and Mash2 expression overlap in the ectoplacental cone and spongiotrophoblast, layers of the placenta that contain the giant cell precursors, indicating that the antagonistic activities of Hand1 and Mash2 must be coordinated. MASH2 and HAND1 both heterodimerize with E factors, bHLH proteins that are the DNA-binding partners for most class B bHLH factors and which are also expressed in the ectoplacental cone and spongiotrophoblast. In vitro, HAND1 could antagonize MASH2 function by competing for E-factor binding. However, the Hand1 mutant phenotype cannot be solely explained by ectopic activity of MASH2, as the Hand1 mutant phenotype was not altered by further mutation of Mash2. Interestingly, expression of E-factor genes (ITF2 and ALF1) was down-regulated in the trophoblast lineage prior to giant cell differentiation. Therefore, suppression of MASH2 function, required to allow giant cell differentiation, may occur in vivo by loss of its E-factor partner due to loss of its expression and/or competition from HAND1. In giant cells, where E-factor expression was not detected, HAND1 presumably associates with a different bHLH partner. This may account for the distinct functions of HAND1 in giant cells and their precursors. We conclude that development of the trophoblast lineage is regulated by the interacting functions of HAND1, MASH2, and their cofactors.

Animals↗
PubMed2000

Id-2 regulates critical aspects of human cytotrophoblast differentiation, invasion and migration.

During early human placental development, the conceptus attaches itself to the uterus through cytotrophoblast invasion. Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop-helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. We therefore examined the importance of this family of factors in the human placenta. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain (Inhibitor of DNA binding proteins (Id-1 to Id-4)). During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, we first characterized Id expression in cytotrophoblast cells. The cells expressed Id-3 constitutively; Id-2 was downregulated, at the mRNA and protein levels, as the cells differentiated in culture and in situ, respectively. In cases when cytotrophoblast differentiation was compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression was maintained. To assess the functional relevance of these correlations, we used an adenovirus vector to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retained characteristics of undifferentiated cells: (alpha)1 integrin expression was low and cyclin B expression was retained. Furthermore, invasion through Matrigel was partially inhibited and migration was strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development.

Animals↗
PubMed1999

Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E.

The stability of many proteins is controlled by the ubiquitin proteolytic system, which recognizes specific substrates through the action of E3 ubiquitin ligases [1]. The SCFs are a recently described class of ubiquitin ligase that target a number of cell cycle regulators and other proteins for degradation in both yeast and mammalian cells [2] [3] [4] [5] [6]. Each SCF complex is composed of the core protein subunits Skp1, Rbx1 and Cul1 (known as Cdc53 in yeast), and substrate-specific adaptor subunits called F-box proteins [2] [3] [4]. To understand the physiological role of SCF complexes in mammalian cells, we generated mice carrying a deletion in the Cul1 gene. Cul1(-/-) embryos arrested around embryonic day 6.5 (E6.5) before the onset of gastrulation. In all cells of the mutant embryos, cyclin E protein, but not mRNA, was highly elevated. Outgrowths of Cul1(-/-) blastocysts had limited proliferative capacity in vitro and accumulated cyclin E in all cells. Within Cul1(-/-) blastocyst cultures, trophoblast giant cells continued to endocycle despite the elevated cyclin E levels. These results suggest that cyclin E abundance is controlled by SCF activity, possibly through SCF-dependent degradation of cyclin E.

Animals↗
PubMed1999

Murine Gcm1 gene is expressed in a subset of placental trophoblast cells.

The gcm gene of Drosophila melanogaster encodes a transcription factor that is an important component in cell fate specification within the nervous system. In the absence of a functional gcm gene, progenitor cells differentiate into neurons, whereas when the gene is ectopically expressed the cells produce excess glial cells at the expense of neuronal differentiation. Recent searches of databases have uncovered high sequence similarity between the Drosophila gem gene and an anonymous human placental cDNA clone (Altschuller et al., 1996; this communication). Here we report the molecular organization of the murine Gcm1, its spatio-temporal pattern of expression in developing placenta, and its map position at E1-E3 on murine chromosome 9. The murine gene is composed of at least 6 exons. The promoter region contains an "initiation sequence" and is GC rich, characteristics of the promoters of several transcription factors. The mRNA has a modest 5'UTR (ca. 200 bases) but an extensive 3' UTR (ca. 2 kb). Northern blot and mRNA in situ hybridization studies showed that Gcm1 expression was readily detectable only in the placenta. It began at embryonic day 7.5 within trophoblast cells of the chorion and continued to about embryonic day 17.5 within a subset of labyrinthine trophoblast cells. Comparison with other transcription factors revealed that Gcm1 expression defines a unique subset of trophoblast cells.

Animals↗
PubMed1999

A repertoire of differentially expressed transcription factors that offers insight into mechanisms of human cytotrophoblast differentiation.

During human placental development, specialized cells allocated to the extraembryonic lineage (cytotrophoblasts) invade the uterus, anchoring the conceptus to the decidua and tapping a supply of maternal blood. This unusual behavior requires cytotrophoblasts to assume highly specialized characteristics; some are commonly associated with tumor cells, while others are typical of endothelia. Here we investigated the transcriptional mechanisms that control cytotrophoblast differentiation/invasion. Specifically, we examined the cells' expression of a number of transcription factors, at the RNA level, as they differentiated along the invasive pathway in vitro. Since basic helix-loop-helix (bHLH) proteins play important roles in murine trophoblast differentiation, we first examined their expression by cytotrophoblasts. As in murine placental development, expression of the human homologue of Mash-2 was confined to progenitor cells. But expression of Hand-1, which promotes differentiation of murine trophoblast giant cells, was not detected. We also found that cytotrophoblasts upregulated the expression of bHLH/PAS factors that function in adaptive responses to hypoxia, including hEPAS-1, which is expressed primarily in endothelial cells. Quite unexpectedly, we discovered that cytotrophoblasts express high levels of mRNA encoding the human homologue of the Drosophila neuronal fate gene, glial cells missing-1 (gcm-1). We also found evidence of crosstalk between the bHLH and GCM-1 regulatory networks. Together, these results offer insights into the transcriptional mechanisms that govern cytotrophoblast differentiation/invasion. Interestingly, these mechanisms suggest analogies with those that govern differentiation of murine stem cells allocated to both the intra- and extraembryonic lineages.

Animals↗
PubMed1999

Defective induction of the transcription factor interferon-stimulated gene factor-3 and interferon alpha insensitivity in human trophoblast cells.

During pregnancy, trophoblast cells of the placenta contact maternal immune cells and yet are protected from attack. One mechanism that may account for this is that trophoblasts show altered expression of major histocompatibility complex (MHC) antigens. The gene for human leukocyte antigen G (HLA-G), a nonclassical gene, is expressed at high levels in trophoblast. Unlike other MHC class I genes, the HLA-G gene lacks an interferon (IFN) response element. Moreover, we demonstrate here that IFN, which regulates classical MHC class I genes in other cell types, does not affect these genes in trophoblast, owing to inactivation of an IFNalpha signaling pathway. Trophoblast cells (JEG-3 and JAR) were found to be selectively refractory to IFN. Specifically, although IFNalpha induced the transcription factors STAT1, STAT2, and IFN regulatory factor-1, and a protective response against encephalomyocarditis virus, it failed to protect the cells from vesicular stomatitis virus, activate a transfected MHC class I gene promoter, and induce the transcription factor IFN-stimulated gene factor (ISGF)-3. The lack of ISGF3 DNA-binding activity apparently was due to diminished p48/ISGF3gamma subunit activity since ISGF3 DNA-binding activity and IFNalpha induction of MHC class I promoter activity were reconstituted by p48/ISGF3gamma supplementation. These data indicate that a specific IFN signaling pathway is inactive in JEG-3 trophoblast cells because of altered activity of p48/ISGF3gamma, and they suggest IFN insensitivity as a mechanism that may help promote feto-placental survival.

Choriocarcinoma↗
PubMed1999

Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development.

We have identified a novel gene in a gene trap screen that encodes a protein related to the DnaJ co-chaperone in E. coli. The gene, named Mrj (mammalian relative of DnaJ) was expressed throughout development in both the embryo and placenta. Within the placenta, expression was particularly high in trophoblast giant cells but moderate levels were also observed in trophoblast cells of the chorion at embryonic day 8.5, and later in the labyrinth which arises from the attachment of the chorion to the allantois (a process called chorioallantoic fusion). Insertion of the ROSAbetageo gene trap vector into the Mrj gene created a null allele. Homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, which precluded formation of the mature placenta. At embryonic day 8.5, the chorion in mutants was morphologically normal and expressed the cell adhesion molecule beta4 integrin that is known to be required for chorioallantoic fusion. However, expression of the chorionic trophoblast-specific transcription factor genes Err2 and Gcm1 was significantly reduced. The mutants showed no abnormal phenotypes in other trophoblast cell types or in the embryo proper. This study indicates a previously unsuspected role for chaperone proteins in placental development and represents the first genetic analysis of DnaJ-related protein function in higher eukaryotes. Based on a survey of EST databases representing different mouse tissues and embryonic stages, there are 40 or more DnaJ-related genes in mammals. In addition to Mrj, at least two of these genes are also expressed in the developing mouse placenta. The specificity of the developmental defect in Mrj mutants suggests that each of these genes may have unique tissue and cellular activities.

Allantois↗
PubMed1998

Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation.

Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, we have introduced a null mutation into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2-Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Formation of the placenta and extraembryonic membranes.

In eutherian mammals, the first cell types that are specified during embryogenesis are committed to form extraembryonic (placenta and fetal membranes) rather than embryonic structures. The trophoblast cell lineage, for example, forms at the morula-to-blastocyst transition: cells at the periphery of the morula become trophoblast, whereas cells on the inside remain undifferentiated embryonic ectoderm, which later gives rise to the fetus as well as the endodermal and mesodermal components of the placenta and extraembryonic membranes. Genetic studies in mice are beginning to identify growth factors and cell adhesion molecules that mediate interactions between cell types that are essential for morphogenesis of the placenta and fetal membranes, as well as transcription factors that control the differentiation of extraembryonic cell types.

Animals↗
PubMed1998

The transition to endoreduplication in trophoblast giant cells is regulated by the mSNA zinc finger transcription factor.

Terminal cell differentiation is usually associated with cell cycle exit. In some lineages, however, cells undergo continued rounds of DNA synthesis without intervening mitoses (endoreduplication) resulting in polyploid nuclei. This is striking in rodent trophoblast giant cells which contain up to 1000N of DNA. In Drosophila, the Escargot gene has been implicated in regulating the transition from mitotic cell cycles to endocycles during development. We found that a murine homologue, mSna, was expressed in mouse trophoblast and was downregulated during giant cell differentiation. The mSNA zinc finger protein bound to E-box DNA elements and, in transfected C3H10T1/2 fibroblasts, acted as a transcriptional repressor. The maximal repressive effect was dependent on both the zinc finger DNA-binding domain and the N-terminal, seven-amino-acid SNAG domain. Misexpression experiments in Rcho-1 trophoblast cells revealed that mSna regulates the transition from replicating precursor cells to committed giant cells: overexpression blocked, whereas antisense RNA-mediated underexpression promoted trophoblast giant cell differentiation. Overexpression of mSna in precursor cells had no effect on cell cycle kinetics, but did increase cyclin A and B levels, implying actions during G2. These effects were dependent on both the zinc finger and SNAG domains. Together, these data suggest that mSNA has an ESCARGOT-like function to repress the transcription of genes that promote the transition from mitotic to endoreduplicative cell cycles in rodent trophoblast.

Amino Acid Sequence↗
PubMed1998

Elucidation of the genetic basis of the antigen presentation defects in the mutant cell line .220 reveals polymorphism and alternative splicing of the tapasin gene.

Antigenic peptides are presented to cytotoxic T lymphocytes by heterodimers of MHC class I molecules and beta2-microglobulin. Peptides are generated in the cytosol and translocated into the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP). Optimal binding of peptides to class I molecules is facilitated by the physical association between class I heterodimers and TAP. This association is mediated largely by the glycoprotein tapasin. Analysis of tapasin function has relied on a mutant cell line, .220, which is defective in tapasin expression and antigen presentation. We have investigated the genetic basis of these defects. In .220 cells, Tapasin transcripts lack exon two. This is caused by a single nucleotide substitution, disrupting the 5' splice site of the second intron. A tapasin protein is produced in .220 cells, but has a truncated signal peptide and lacks the N-terminal 49 amino acids encoded by full-length transcripts. Nonetheless, this truncated form is translocated into the ER and interacts with TAP. As a result of alternative splicing, transcripts lacking exon two are also present in wild-type cells, although no truncated protein was detected. Additionally we describe a polymorphism in the Tapasin gene, with two alleles encoding arginine or threonine at peptide position 240.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis.

The placenta and cardiovascular system are the first organ systems to form during mammalian embryogenesis. We show here that a single gene is critical for development of both. The Hand1 gene, previously called Hxt, eHAND and Thing1, encodes a basic helix-loop-helix (bHLH) transcription factor that starts to be expressed during pre-implantation development. After implantation, Hand1 expression is restricted to placental trophoblast cells and later to embryonic cardiac and neural crest cells. We generated Hand1-null mutant mice by gene targetting. Homozygous mutant embryos arrested by embryonic day (E) 7.5 of gestation with defects in trophoblast giant cell differentiation. This early mortality could be rescued by aggregation of mutant embryos with wild-type tetraploid embryos, which contribute wild-type cells to the trophoblast, but not the embryo. By E10.5, however, the Hand1-null fetuses derived from tetraploid chimaeras died due to cardiac failure. Their heart tubes showed abnormal looping and ventricular myocardial differentiation. Therefore, Hand1 is essential for differentiation of both trophoblast and cardiomyocytes, which are embryologically distinct cell lineages.

Animals↗
PubMed1998

Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells.

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Animals↗
PubMed1997

Developmental restriction of Mash-2 expression in trophoblast correlates with potential activation of the notch-2 pathway.

Mash-2 expression begins during preimplantation development, but is restricted to trophoblasts after the blastocyst stage. Within the trophoblast lineage, Mash-2 transcripts are first expressed in the ectoplacental cone and chorion, but not in terminally differentiated trophoblast giant cells. After day 8.5 of gestation, Mash-2 expression becomes further restricted to focal sites within the spongiotrophoblast and labyrinth. Downregulation is probably important for normal development since overexpression of Mash-2 reduces giant cell formation. We have investigated the role that the Notch signaling pathway may play in trophoblast development. Mash-2 is a homologue of Drosophila achaete/scute complex genes. In Drosophila, activation of the Notch receptor induces transcriptional repressors encoded by the hairy/Enhancer of split (HES) genes, which interact with the Groucho protein to shut off achaete-scute transcription. In the developing mouse placenta, we found that all elements of the Notch pathway were expressed. In particular, the Notch-2, HES-2, and HES-3 genes were coexpressed in trophoblast giant cells and in foci within the spongiotrophoblast at day 10.5 when Mash-2 transcription becomes restricted. Two members of the mammalian Groucho family were expressed in trophoblasts; TLE3 was expressed broadly in the giant cell, spongiotrophoblast, and labyrinthine regions, whereas TLE2 was limited to giant cells and focal regions of the spongiotrophoblast. These data suggest that Notch signaling through activation of HES transcriptional repressors may play a role in murine placental development.

Animals↗
PubMed1997

Molecular genetics of implantation in the mouse.

Embryo implantation is a complex developmental process requiring precise coordination between mother and offspring to ensure success. Implantation failure is clinically relevant to in vitro fertilization programs and to an understanding of diseases of pregnancy like preeclampsia. Basic and clinical research have identified a number of proteins involved in peri-implantation development, but an understanding of the implantation process and its cellular and molecular components is just beginning. This review will focus on the implantation and development of the murine embryo and placenta. The significance of ectopic expression and targeted mutagenesis models to these processes will be discussed.

Animals↗
PubMed1997

Activin is a local regulator of human cytotrophoblast cell differentiation.

Cytotrophoblast cells of the first trimester placenta are highly invasive when removed from villi and cultured in vitro. In vivo, however, only selected cytotrophoblast cells break through the overlying syncytiotrophoblast to form cytotrophoblast columns and ultimately invade the endometrium. To explore the role of paracrine growth factors in regulating cytotrophoblast development, we cultured explants of first trimester chorionic villi in vitro. Both activin and inhibin, as well as the activin binding protein follistatin, are produced by various trophoblast cells throughout pregnancy. We found that addition of activin-A, but not inhibin-A, stimulated the outgrowth of cytotrophoblast cells into the surrounding matrix. This outgrowth was characteristic of that observed in extravillous cytotrophoblast cells in vivo; it was accompanied by cell division within the proximal region of the cytotrophoblast outgrowth, synthesis of fibronectin, as well as the expression of markers characteristic of invasive cytotrophoblast cells, human leukocyte antigen-G and matrix metalloproteinase (MMP)-9. Activin also specifically induced the early expression of MMP-2 within villous cytotrophoblast cells. Addition of the activin binding protein, follistatin, blocked all of the effects of exogenous activin. The morphological and biochemical effects of activin were similar to those observed when signaling of endogenous transforming growth factor-beta was blocked. Interestingly, the latter effects were also reversed by the addition of follistatin. These data suggest that activin plays a local role in promoting cytotrophoblast column formation, likely by regulating the differentiation of villous cytotrophoblast into extravillous cytotrophoblast cells.

Activins↗