Gestational folate deficiency alters embryonic gene expression and cell function.

Folic acid is a nutrient essential for embryonic development. Folate deficiency can cause embryonic lethality or neural tube defects and orofacial anomalies. Folate receptor 1 (Folr1) is a folate binding protein that facilitates the cellular uptake of dietary folate. To better understand the biological processes affected by folate deficiency, gene expression profiles of gestational day 9.5 (gd9.5) Folr1-/- embryos were compared to those of gd9.5 Folr1+/+ embryos. The expression of 837 genes/ESTs was found to be differentially altered in Folr1-/- embryos, relative to those observed in wild-type embryos. The 837 differentially expressed genes were subjected to Ingenuity Pathway Analysis. Among the major biological functions affected in Folr1-/- mice were those related to 'digestive system development/function', 'cardiovascular system development/function', 'tissue development', 'cellular development', and 'cell growth and differentiation', while the major canonical pathways affected were those associated with blood coagulation, embryonic stem cell transcription and cardiomyocyte differentiation (via BMP receptors). Cellular proliferation, apoptosis and migration were all significantly affected in the Folr1-/- embryos. Cranial neural crest cells (NCCs) and neural tube explants, grown under folate-deficient conditions, exhibited marked reduction in directed migration that can be attributed, in part, to an altered cytoskeleton caused by perturbations in F-actin formation and/or assembly. The present study revealed that several developmentally relevant biological processes were compromised in Folr1-/- embryos.

Tandem sequence repeats in transmembrane channel proteins.

The flow of ions and small molecules out of and between cells is mediated by various classes of transmembrane proteins. One group of putative channel proteins, including the abundant lens protein MIP, is widely distributed from prokaryotes to vertebrates. This article suggests that these proteins contain a structural twofold repeat and may have arisen by gene duplication. Such a model has implications for the tertiary structures of these important proteins.

Teratogen-induced eye defects mediated by p53-dependent apoptosis.

BACKGROUND: Many birth defects are believed to involve gene-environment interactions, although the mechanisms involved are poorly understood. Apoptosis is a common effect of many kinds of environmental stresses on the developing embryo; therefore, mechanisms of teratogenesis may be approached within the context of the cell death program. The p53 tumor suppressor gene encodes a transcription factor which functions as a critical regulator of apoptosis in response to environmental stress. RESULTS: To investigate the relationship between p53-dependent apoptosis and teratogenesis, we subjected day 8 mouse embryos with different p53 gene backgrounds to a genotoxic stress, 2-chloro-2'-deoxyadenosine. Treatment rapidly stimulated nuclear p53 accumulation and triggered apoptosis in some (head-fold) but not other (primitive heart) developing structures. Induced cell death was p53 gene-dose dependent, as shown by the intermediate sensitivity of 4-5 somite stage embryos bearing only a single effective p53 allele and the lack of sensitivity of p53-null mutants. Abnormal development was manifested as eye defects by day 11, particularly lens agenesis. Overall the incidences of these defects at term were 73.3% for p53 wild-type fetuses, 52.5% for heterozygous mutants, and 2.2% for p53-null mutants. Statistical analysis indicated that the interaction between teratogen and genotype was highly significant (P < or = 0.001) for cell death on day 8 and eye defects on day 17. CONCLUSIONS: We conclude that teratogen induction of p53-dependent apoptosis in the developing embryo is positively coupled to the determination of congenital eye defects.

Nuclear convergence of the TGFbeta and cAMP signal transduction pathways in murine embryonic palate mesenchymal cells.

Transforming growth factors beta (TGFbeta) and cyclic AMP (cAMP) both participate in growth and differentiation of the developing mammalian secondary palate and elicit similar biological responses. Cross-talk between these two signal transduction pathways in cells derived from the embryonic palate has been demonstrated previously. In the present study, we have examined nuclear convergence of these signalling pathways at the level of transcriptional complex formation. Biotinylated oligonucleotides encoding a consensus Smad binding element (SBE), or a cyclic AMP response element (CRE), were mixed with cell extracts from murine embryonic palate mesenchymal (MEPM) cells that were treated with either TGFbeta or forskolin. Protein-oligonucleotide complexes were precipitated with streptavidin-agarose, and analysed by Western blotting to identify proteins in the complex bound to each consensus oligonucleotide. TGFbeta treatment of MEPM cells increased the levels of phosphorylated Smad2, phosphorylated cAMP response element binding protein (CREB), and the coactivator, CREB binding protein (CBP), that were part of a complex bound to the SBE. Treatment of cells with forskolin, a stimulator of adenylate cyclase, increased the amount of phosphorylated CREB and CBP, but not the amount of phosphorylated Smad2 bound in a complex to the SBE. Additionally, the presence of the co-repressors, c-Ski and SnoN, was demonstrated as part of a complex bound to the SBE (but not the CRE). Amounts of c-Ski and SnoN found in the SBE-containing complex increased in response to either TGFbeta or forskolin. These results demonstrate that phosphorylated CREB forms a complex with the co-activator CBP, phosphorylated Smad2 and the co-repressors c-Ski and SnoN on a consensus SBE. This suggests cooperative regulation of genes with SBE-containing promoters by the cAMP and TGFbeta signalling pathways in the developing palate.

Expression of the E2F family of transcription factors during murine development.

The E2F family of transcription factors plays a crucial role in the control of cell cycle progression and regulation of cellular proliferation, both processes fundamental to mammalian development. In the present study, we have examined the levels of expression of the six currently identified E2F proteins in murine embryos/fetuses as a function of gestational age, compared the expression of these six proteins in selected developing and adult tissues, and examined E2F expression in the embryonic murine palate, a tissue in which perturbation of proliferation is associated with induction of cleft palate. Our results indicate that: 1) multiple forms of individual E2F family members are present in embryonic, fetal and adult cells/tissues; 2) each of the six E2Fs is expressed in a tissue specific manner in both adult and embryonic/fetal organs; 3) certain forms of individual E2F family members are preferentially detected in adult tissues, whereas others are preferentially expressed in embryonic/fetal tissues; 4) expression of the various E2Fs and their isoforms follows distinct temporal patterns during murine gestation; and 5) individual E2F family members also exhibit differential patterns of temporal expression during murine palatogenesis.

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse.

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.

Isolation and characterization of the 5'-flanking sequence of the human ocular lens MIP gene.

The MIP (major intrinsic protein) gene, a member of an ancient family of membrane channel genes, encodes the predominant fiber cell membrane protein of the ocular lens. Its specific expression in the lens fibers is temporally and spatially regulated during development. To study the regulation of expression of MIP and delineate the regulatory elements underlying its tissue specificity and ontogenic profile, we have cloned 2840 bp of the human MIP 5'-flanking sequence. The human MIP 5'-flanking sequence contains three complete Alu repetitive elements in tandem at position between nt -1699 and -2684 (nt -1699/-2684). These Alu elements appear to have had a complex evolutionary history with insertions at different times. We have fused DNA fragments containing MIP 5'-flanking sequences to the bacterial cat reporter gene encoding chloramphenicol acetyltransferase and assayed them in primary cultures of chicken lens cells. We have mapped two negative regulatory regions in the human MIP 5'-flanking sequences -1564/-1696 and -948/-1000. We demonstrated that the human MIP 5'-flanking sequence -253/+42 contains a functional promoter in lens cells but is inactive in kidney epithelial cells or mouse fibroblasts, suggesting that this sequence contains regulatory elements responsible for the lens-specific expression of MIP.

Expression of the E2F and retinoblastoma families of proteins during neural differentiation.

Development of the brain is determined by a strictly orchestrated program of proliferation, migration, apoptosis, differentiation, synaptogenesis, tract formation, and myelination. The E2F family of transcription factors, whose activity and functions are regulated in large part through interactions with the retinoblastoma (Rb) family of tumor suppressor proteins, has been implicated as a key regulator of proliferation, differentiation, and apoptosis in a variety of tissues. We have examined levels of the E2F and Rb families of proteins during both brain development and neural differentiation of P19 cells, and found the expression profiles during these two processes of neural development and maturation to be quite similar, i.e., strong up-regulation of p130, pronounced down-regulation of p107, moderate up-regulation of pRb, and significant down-regulation of most species of E2F and dimerization protein (DP). However, several specific isoforms, namely a 30 kDa form of DP-2, a 57 kDa species of E2F-3, a 59 kDa form of E2F-5 and the isoforms of E2F-1 recognized by the E2F-1 (KH-95) antibody were up-regulated suggesting that these particular isoforms of E2F and DP play a tissue-specific function in differentiation and maturation of nervous tissue. The potential role of the E2F/DP family of transcription factors in aspects of neural development and differentiation are considered.

Convergence of cAMP, TGF-beta and retinoic acid signaling pathways in cells of the embryonic palate.

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-beta) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-beta1, cyclic adenosine 3', 5'-monophosphate (cAMP) and RA signaling systems. TGF-beta1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-beta mRNA in MEPM cells, though only TGF-beta1 inhibited RA-induced RAR-beta protein expression. Forskolin, but not TGF-beta1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-beta gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-beta-responsive, element. Thus, a putative TGF-beta Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-beta promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.

Regulation of TGF beta 3 gene expression in embryonic palatal tissue.

The TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the nuclear coactivators CBP and p300 in developing craniofacial tissue.

cAMP regulatory element-binding protein (CREB)-binding protein (CBP) and its functional homolog, the adenovirus E1A -associated 300-kDa protein (p300) are nuclear coactivators and histone acetyltransferases that integrate signals from disparate pathways by bridging specific transcription factors to the basal transcription apparatus. Their role in patterning and development was suggested by studies in mice in which CBP and p300 expression was disrupted and by the human Rubinstein-Taybi syndrome, which is associated with mutations of CBP. The cAMP signal transduction pathway plays a critical role during development of the palate. The linkage between cAMP and expression of specific genes is mediated via activation of trans-acting deoxyribonucleic acid-binding proteins such as the nuclear CREB. For genes regulated by CBP- or p300-containing transcriptional complexes, rates of transcription will depend in part on cellular levels and distribution of CBP/p300. We have thus determined the temporal and spatial expression of CBP and p300 in murine embryonic palatal tissue. Both CBP and p300 proteins and messenger ribonucleic acids are expressed in palatal tissue on each d of palate development (days 12-14 of gestation), as measured by Western blotting and reverse-transcription polymerase chain reaction. Expression of both CBP and p300 was greatest on day 12 of gestation, suggesting that these transcriptional coactivators are developmentally regulated. Immunohistochemical analysis of CBP and p300 expression in the murine embryonic craniofacial region revealed a ubiquitous distribution for both proteins. These studies lay the groundwork for further investigations into the role of CBP and p300 in cellular signaling during craniofacial development.

Release of targeted p53 from the mitochondrion as an early signal during mitochondrial dysfunction.

Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protein led by a sense (m53-EGFP) or antisense (c53-EGFP) mitochondrial import signal. Rotenone exposure (100nM, 1h) triggered the translocation of m53-EGFP from the mitochondrion to the nucleus, thus shifting the transfected cells from a mitochondrial p53 to a nuclear p53 state. Antibodies for p53 serine phosphorylation or lysine acetylation indicated a different post-translational status of recombinant p53 in the nucleus and mitochondrion, respectively. These data suggest that cycling of p53 through the mitochondria may establish a direct pathway for p53 signaling from the mitochondria to the nucleus during mitochondrial dysfunction. PK11195, a pharmacological ligand of mitochondrial TSPO (formerly known as the peripheral-type benzodiazepine receptor), partially suppressed the release of mitochondria-sequestered p53. These findings support the notion that p53 function mediates a direct signaling pathway from the mitochondria to nucleus during mitochondrial dysfunction.

Differential programming of p53-deficient embryonic cells during rotenone block.

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53-efficient embryonic fibroblasts compared to p53-deficient cells. These cell lines differed with respect to NADH/NAD(+) balance. This ratio constitutes a driving force for NAD- and NADH-dependent reactions and is inversed upon exposure to Rotenone (complex I inhibitor). Rotenone perturbed the structure of the elongated fibrillar tubulin network and decreased mRNA expression of tubulin genes both suggesting reprogramming and reorganization of the cytoskeleton in both cell lines. These changes were reflected in the abundance of specific mRNA and microRNA (miRNA) species as determined from genome-based analysis. Changes in mRNA and miRNA expression profiles reflected differences in energy utilizing pathways, consistent with the notion that the p53 pathway influences the cellular response to mitochondrial dysfunction and that at least some control may be embedded within specific mRNA/miRNA networks in embryonic cells.

Functional analysis of CBP/p300 in embryonic orofacial mesenchymal cells.

CREB binding protein (CBP) and the close structural homolog, p300, are nuclear coactivators of multiple signaling pathways that play important roles in embryonic development and cellular homeostasis. TGFbeta regulates the proliferation rate of many cell types and has been demonstrated to inhibit the growth rate of mouse embryonic maxillary mesenchymal (MEMM) cells. The role of CBP and p300 in TGFbeta-mediated control of proliferation of MEMM cells was thus investigated using an in vitro gene knockdown approach. TGFbeta reporter assays demonstrated that p300 mRNA knockdown via targeted siRNAs led to a reduction in the response to TGFbeta, whereas knockdown of CBP by the same approach had an insignificant effect. In MEMM cell proliferation assays, siRNA-mediated knockdown of CBP and/or p300 had little impact upon TGFbeta-mediated growth inhibition; however, the basal rate of proliferation was increased. Inhibition of p300 activity via overexpression of a dominant-negative mutant (p300deltaC/H3) led to significant inhibition of TGFbeta-mediated activation of p3TP-lux. As with the siRNA knockdown approach, p300deltaC/H3 also increased the basal rate of cell proliferation of MEMM cells. CBP/p300 siRNA knockdown had a significant but incomplete inhibition of TGFbeta-induction of matrix metalloproteinase-9 (gelatinase B) expression. These data demonstrate that p300 is involved in Smad-mediated transcription of p3TP-lux, however, its role (and that of CBP) in biological processes such as the control of cell proliferation and extracellular matrix metabolism is more complex and may be mediated via mechanisms beyond coactivator recruitment.

Molecular fingerprinting of BMP2- and BMP4-treated embryonic maxillary mesenchymal cells.

OBJECTIVE: To determine the differences in gene expression between control-, bone morphogenetic protein (BMP)2- and BMP4-treated murine embryonic maxillary mesenchymal (MEMM) cells. DESIGN: Transcript profiles of BMP2-, BMP4- and vehicle-treated MEMM cells were compared utilizing the murine high-density GeneChip arrays from Affymetrix. The raw chip data (probe intensities) were pre-processed using robust multichip averaging with GC-content background correction and further normalized with GeneSpring v7.2 software. Cluster analysis of the microarray data was performed with the GeneSpring software. Changes in the gene expression were verified by TaqMan quantitative real-time PCR. RESULTS: Expression of approximately 50% of the 45 101 genes and expressed sequence tags examined in this study were detected in BMP2-, BMP4- and vehicle-treated MEMM cells and that of several hundred genes was significantly altered (up or downregulated) in these cells in response to BMP2 and BMP4. Expression profiles of each of the 26 mRNAs tested by TaqMan quantitative real-time PCR were found to be consistent with the microarray data. Genes whose expression was modulated following BMP2 or BMP4 treatment, could be broadly classified based on the functions of the encoded proteins such as the growth factors and signaling molecules, transcription factors, and proteins involved in epithelial-mesenchymal interactions, extracellular matrix synthesis, cell adhesion, proliferation, differentiation, and apoptosis. CONCLUSION: Utilization of the Affymetrix GeneChip microarray technology has enabled us to delineate a detailed transcriptional map of BMP2 and BMP4 responsiveness in embryonic maxillary mesenchymal cells and offers revealing insights into crucial molecular regulatory mechanisms employed by these two growth factors in orchestrating embryonic orofacial cellular responses.

Interaction between Smad 3 and Dishevelled in murine embryonic craniofacial mesenchymal cells.

OBJECTIVES: To determine the in vivo interaction between Smad 3 and Dishevelled-1. DESIGN: Cell culture transfection followed by immunoprecipitation with specific antibodies. SETTING AND SAMPLE POPULATION: The Department of Molecular, Cellular, and Craniofacial Biology, Birth Defects Center, University of Louisville. EXPERIMENTAL VARIABLE: Overexpression of myc-Smad 3. OUTCOME MEASURE: Western blotting of anti-Dishevelled immunoprecipitates for Smad 3. RESULTS: Smad 3 and Dishevelled isoforms-1, -2, and -3 all bind Smad 3 in glutathione-S-transferase (GST) pull-down assays and Smad 3 binds to Dishevelled-1 in vivo. Stimulation of the transforming growth factor beta (TGFbeta) pathway leads to increased binding of Smad 3 and Dishevelled-1 in vivo. CONCLUSION: Smad 3 binds all three known isoforms of Dishevelled and binds Dishevelled 1 in vivo. TGFbeta signaling modulates the interaction between Smad 3 and Dishevelled-1.

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis.

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.

Genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (MIP) of the lens.

Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5'-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.