* Birth Defects Research Laboratory, Division of Genetics and Developmental Medicine, Departments of Pediatrics and Environmental Health;
Fred Hutchinson Cancer Research Center; and
Department of Biostatistics; University of Washington, Seattle, Washington 98195
Received September 17, 2003; accepted January 20, 2004
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ABSTRACT |
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Key Words: day-9 mouse embryos; hyperthermia; 4-hydroperoxycyclophosphamide; cDNA microarrays; gene expression profiling.
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INTRODUCTION |
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To identify new human teratogens and to develop measures to prevent teratogen-induced birth defects will require, in part, an understanding of the molecular mechanisms by which teratogens cause birth defects. Although mechanisms of teratogenesis are known to involve a variety of molecular and cellular processes (NRC, 2001), research published over the past 40 years has shown that teratogen-induced alterations in gene expression play an important role in the genesis of malformations in animals. For example, retinoic acid is a known teratogen that has been shown to activate specific receptors (e.g., retinoic acid receptor, RAR), culminating in the modulation of the expression of specific genes, e.g., the homeobox transcription factors (Chambon, 1996
; Collins and Mao, 1999
). Embryos exposed to exogenous retinoic acid exhibit dysregulation of homeobox genes, thereby leading to the abnormal expression of other genes regulated by homeobox transcription factors (Marshall et al., 1996
). Presumably this dysregulation of gene expression is subsequently translated into the abnormal differentiation, migration, proliferation, and apoptosis known to be associated with retinoic acid-induced developmental toxicity (Collins and Mao, 1999
). Another example is TCDD, an environmental pollutant that causes structural malformations including cleft palate. TCDD activates the aryl hydrocarbon receptor (AHR) that in turn modulates the expression of genes suspected to play a role in palate development; for example, in epidermal growth factor, epidermal growth-factor receptor, and transforming growth factors a, b1, and b2 (Abbott et al., 1994
). These examples show that, at least for these two developmental toxicants, the known mechanisms of developmental toxicity include toxicant-induced alterations in gene expression.
In addition to the studies just cited, a variety of studies have shown that other developmental toxicants also induce alterations in gene expression (NRC, 2001). Thus, toxicant-induced alterations in gene expression represent a mechanism of developmental toxicity common to developmental toxicants in general. In all of the cited studies, only a small number of developmental toxicant-responsive genes have been identified. Assuming that toxicant-induced alterations in gene expression represent a common step in the overall mechanistic pathway leading to abnormal development, what is needed now is a comprehensive assessment of developmental toxicant-induced alterations in global gene expression patterns. Such assessments could identify genes that potentially play a role in toxicant-induced abnormal development or patterns of gene expression that could serve as a biomarker of exposure to a specific developmental toxicant or class of developmental toxicants.
The recent development of DNA microarrays now offers the opportunity to monitor global changes in gene expression and therefore the potential to obtain significant new information concerning both normal and abnormal development. For example, Arbeitman et al. (2002) have used DNA microarrays to study the expression of nearly one-third of all Drosophila genes during the complete time course of normal development, and Tanaka et al. (2000)
have profiled gene expression patterns in E12.5 mouse embryos. With one exception (Knudsen et al., 2003
), there have been no similar global gene expression studies during teratogen-induced abnormal development. Thus, one of the objectives of the studies described in this paper was to begin to document global changes in gene expression in day-9 mouse embryos exposed to two classical teratogens, hyperthermia and cyclophosphamide, which were chosen because they have been extensively studied both in vitro and in vivo. As one example, both of these teratogens induce an episode of cell death in embryonic tissues that subsequently undergo abnormal development. Moreover, they both activate the mitochondrial apoptotic pathway with the same kinetics, i.e., between 0 and 5 h after initiation of exposure, and in similar populations of cells within the embryo, i.e., neuroepithelial and neural crest cells (Mirkes and Little, 2000
; Umpierre et al., 2001
). Thus, a second objective was to identify changes in gene expression that are common to HS and 4CP and thereby might have common pathways of teratogenesis. A final objective was to determine whether specific changes in gene expression could serve as biomarkers of HS or 4CP exposure.
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MATERIALS AND METHODS |
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Exposure conditions.
Hyperthermia (HS): On the morning of day 9, one group of embryos was cultured at 43°C for 15 min, then returned to culture at 37°C and incubated for 1 or 5 h. A parallel group was incubated at 37°C for 1 or 5 hours (untreated control).
4-Hydroperoxycyclophosphamide (4CP): Another group of embryos was cultured in medium containing 40 mM 4CP (freshly prepared in HBSS), a pre-activated analogue of cyclophosphamide (a gift from Michael Colvin, Johns Hopkins University). 4CP-Treated embryos were cultured with drug continuously for 1 or 5 h. For each treatment, a parallel group was incubated at 37°C without 4CP for 1 or 5 h (untreated control). Thus, each group of treated embryos (HS-1 h, designated: HS1; HS-5 h, designated HS5; 4CP-1 h, designated CP1; and 4CP-5 h, designated CP5) had a parallel, companion group of untreated embryos (CT1 and CT5). At 1 and 5 h after the initiation of exposure to HS or 4CP, groups of treated and control embryos were removed from culture, dissected free of associated membranes (yolk sac and amnion), and rinsed in HBSS. For each treatment and time point, 2575 embryos were pooled, snap-frozen, and stored at 80°C. In addition, for each treatment and time point, three independent experiments were performed, except for the 4CP, 5-h exposure, for which five independent experiments were performed. This resulted in three independent pools of embryos for each treatment and time point, except for the 4CP, 5-h group for which there were 5 independent pools of embryos. Finally, the exposures used in our studies produced elevated levels of cell death and abnormal development in 100% of treated embryos (data not shown).
DNA microarrays.
Mouse 15 K cDNA microarrays (on two slides constituting a slide-set) were obtained from the Center for Expression Arrays (CEA), Department of Microbiology, University of Washington. The Mouse_1-20_MU-HD-1 slide contained 7680 cDNAs and the Mouse_1-20_MU-HD-2 slide contained 7567 cDNAs spotted in duplicate. Duplicate spots were located such that one spot was on the left side of the slide and the other was on the right side. A complete list of genes can be found at http://ra.microslu.washington.edu/genelist/genelist.html. These cDNA microarrays were constructed using PCR products generated from the mouse 15,000-clone set from the National Institute on Aging (NIA; http://lgsun.grc.nia.nih.gov/cDNA/15k.html). The cDNA clones were PCR amplified, purified, and run on agarose gels for quality control (images can be viewed at http://ra.protocol.documents/15k%20mouse%PCR. The cDNAs were spotted in duplicate in a 50% DMSO solution onto Amersham type-7, mirror-coated slides. The spotting was done using an Amersham Molecular Dynamics GenIII arrayer and the hybridized slides were scanned with an Amersham/Molecular Dynamics GenIII scanner.
RNA isolation, probe labeling, and microarray hybridization.
Total RNA was separately isolated from each pool of embryos with RNeasy Midi kit (Qiagen). Poly(A)+ RNA was extracted from total RNA samples using Oligotex mRNA Midi kit (Qiagen). First-strand cDNA probes were prepared by direct incorporation of CyDye-labeled dCTP through reverse transcription of mRNA. Protocols for probe preparation and hybridization conditions are available at: http://expression.washington.edu/protocol/protocol.html. Labeling reactions were performed separately with Cy3 and Cy5-nucleotides; i.e., RNA isolated from one group of treated embryos was used to prepare cDNA labeled with Cy3 and RNA from a parallel group of control embryos was used to prepare cDNA labeled with Cy5. Fluorescently labeled cDNAs were then combined and hybridized simultaneously to our cDNA microarrays. For each experiment, duplicate slide sets were hybridized with probes generated from the same mRNAs (treated and control) but with the fluorescent labels reversed (Fig. 1). Thus, for each exposure/time point, at least 6 microarray slide sets were hybridized (10 sets were hybridized for the 4CP-5-h exposure).
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Microarray data were normalized using the regional Loess algorithm from the R/maanova package (Wu et al., 2002; http://www.jax.org/staff/churchill/labsite/software/), which runs in the statistical software program R (Becker et al., 1988
; http://www.r-project.org). This normalization accounts for systematic intensity-dependence and spatial variation in microarray log ratios.
Data were analyzed by extending the ANOVA approach of Kerr et al. (2000) to include random effects. We analyzed each treatment condition (HS 1 h, HS 5 h, 4CP 1 h, and 4CP 5 h) separately. For each gene in a given experiment, we have:
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Northern-blot analysis.
Total RNA extracted from embryos was dissolved in formamide. RNA samples (25 µg) were fractionated on 1.1% agarose -formaldehyde gel and capillary transferred to a GeneScreen-Plus nylon membrane (DuPont NEN, MA) with 10x SSC. Membranes were washed with 2x SSC and fixed by UV cross-linking. Mouse cDNAs were obtained from bacterial gene constructs pSPORT1-Cyclin G1, -Hsp 105/110, and -Jun, purchased from ATCC (American Type Culture Collection, VA). To generate probes, plasmids were digested with SalI and NotI restriction enzymes. Fragments that encode cDNAs were purified from a 1.2% agarose gel with Amicon Ultrafree-MC Centrifugal filter (Millipore, MA) and concentrated by ethanol precipitation. cDNA probes (30 ng) were labeled with 40 µCi of [a32P]-dCTP (DuPont NEN, MA) by random priming. Unincorporated nucleotides were removed using a G-25 Sephadex column. Membranes were prehybridized for 1 h at 650C in 1 M NaCl with 0.1% SDS and 1 µg/ml of sheared calf thymus DNA and denatured probes were added to the solution for an additional 18 h. Blots were washed in 0.1 M NaCl with 0.1% SDS at 650C and exposed to X-OMAT Blue XB-1 film (Eastman-Kodak, Rochester, NY). Signals were quantified by densitometry with KODAK 1D Image Analysis Software (Kodak). For an RNA loading control, membranes were hybridized with a 115 bp cDNA fragment of 28S ribosomal RNA gene obtained from pTRI-RNA-28S plasmid (Ambion, TX) by digestion with RsaI.
Western-blot analysis.
Frozen embryos were sonicated in 25 µl/embryo of either p53 and Hsp105/110 lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM benzamidine, 1.4 µg/ml mM pepstatin A) or Jun lysis buffer (20 mM Tris, pH 6.8, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.5 mM DTT, 10% glycerol, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM benzamidine, 25 mM ß-glycerophosphate, pH 7.0, 2 mM NaPPi, and 1 mM Na3VO4). Protein concentrations were determined using the BCA Protein Assay Reagent Kit (Pierce, IL). Equal amounts of proteins (25 µg of Hsp105/110 and 12 µg of Jun) were separated by SDS-PAGE gel (8% for hsp105/110 and 10% for Jun) and electro-transferred onto Immobilon Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), using a semidry transfer apparatus (Ellard Instrumentation, LTD, Seattle, WA) for Jun and a transblot apparatus (Bio-Rad, Hercules, CA) with 10 mM CAPS, pH 11.0, 10% methanol buffer for Hsp105/110. Membranes were blocked with 2% non-fat Dry milk (NFDM) in TBST at room temperature (RT) for 1 h and then incubated overnight at 40C with primary antibodies: Jun (rabbit anti-human polyclonal antibody, 1:1000 dilution), Cell Signaling Technology, Beverly, MA); Hsp105/110 (goat antimouse pAb, 1:80 dilution), Santa Cruz Biotechnology, Inc., Santa Cruz, CA); pan p53 (clone 240, 1:200 dilution), Oncogene, LaJolla, CA); and ser-15 p53 (rabbit polyclonal antibody, 1:1000 dilution), Cell Signaling Technology, Beverly, MA. They were then washed with TBST and incubated for 2 h at RT with horseradish peroxidase (HRP)-conjugated secondary antibodies: Jun (donkey antirabbit HRP at 1:5000 dilution, Amersham, Piscataway, NJ); Hsp105/110 (donkey antigoat IgG-HRP at 1:3000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA); pan-p53 (sheep antimouse HRP at 1:3000 dilution, Amersham); ser-15 p53 (donkey antirabbit HRP at 1:3000 dilution, Amersham). After washing with TBST, signals were detected on X-OMAT Blue XB-1 film (Kodak, Rochester, NY) by using the enhanced chemiluminescence (ECL) PLUS Western-blot Detection System (Amersham) and quantified by densitometry with Kodak 1D Image Analysis Software. For a protein loading control, the membranes were stripped with Re-Blot Plus Western Blot Recycling Kit (Chemicon International, Inc., Temecula, CA) and incubated with either mouse anti-ß-actin monoclonal antibody (1:8000, Sigma-Aldrich Co., St. Louis, MO) for the Jun blot or goat anti-human ß-Actin (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for the Hsp105/110 blot. After washing with TBST, membranes were incubated with secondary antibodies: Jun (sheep antimouse HRP conjugated pAb (1:3000), Amersham), Hsp105/110 (donkey anti-goat IgG-HRP (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA).
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RESULTS |
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DISCUSSION |
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A second issue involves the use of whole day-9 embryos as the source of RNA to probe our 15 K cDNA microarrays. Day-9 mouse embryos already contain a number of tissues undergoing rapid development, e.g., the nervous system and heart. Thus, homogenizing whole embryos to isolate RNA precludes us from directly associating the alteration in the expression of a particular gene with the development of a particular population of cells. Despite this limitation, our analysis does identify specific genes whose expression is altered by HS and/or 4CP somewhere in the embryo. Once genes of interest are identified, subsequent studies using, for example, laser-capture microdissection can be employed to obtain RNA from specific populations of cells.
Assuming that the changes in gene expression revealed by our microarray analyses approximate the true changes in gene expression induced by HS and 4CP, our expression profiles tell us the following about HS- and 4CP-induced teratogenesis. First, our data show that the changes in gene expression induced in day-9 mouse embryos by HS and 4CP are complex, i.e., multiple genes associated with a variety of intracellular functions show altered expression within the first 5 h after exposure to HS or 4CP. For example, data showing that several p53 transcriptional target genes are coordinately upregulated by HS and 4CP relate directly to on-going studies showing that HS and 4CP induce a transient increase in cell death in treated embryos, particularly in areas of normal programmed cell death (Menkes et al., 1970; Milaire and Rooze, 1983
; Sulik et al., 1988
). Recent studies show that this teratogen-induced cell death involves activation of the mitochondrial apoptotic pathway, i.e., release of cytochrome c, activation of caspases, and proteolysis of various cellular proteins (Little and Mirkes, 2002
; Mirkes and Little, 1998
, 2000
). Although it is known that several teratogens induce the activation of this pathway, little is known concerning how different cells in the embryo make the decision to activate the mitochondrial apoptotic pathway or not to. However, research using cultured cells has shown that the tumor suppressor gene, p53, plays an important role in a cell's decision to live (cell cycle arrest) or die (apoptosis). In part, the decision to either live or die is mediated by stress-induced activation of p53 (increased levels) and subsequent p53-mediated transcription of target genes such as p21 and Bax, respectively (Oren et al., 2002
). Although the activation of p53 is complex, site-specific phosphorylation, e.g., ser-15, is known to play a role in the stabilization and accumulation of p53 (Dumaz and Meek, 1999
). Given the importance of p53 in regulating life/death decisions, our microarray data showing significant upregulation of several p53 transcriptional target genes (upregulation of cyclin G1 mRNA was independently verified by Northern-blot analysis) immediately led us to hypothesize that HS and 4CP activate the p53 pathway. Preliminary follow up studies confirming that HS and 4CP do indeed activate p53 (increased ser-15 phosphorylation and p53 accumulation), highlight the power of microarray-based gene expression profiling studies to identify proteins and associated pathways that may play a role in teratogenesis. We are currently pursuing studies to determine the significance of these findings in terms of HS- and 4CP-induced cell death and abnormal development.
As another example, we previously showed, using Northern and/or Western blot analyses, that Hsp70 and Hsp25/27 are induced when embryos are exposed to hyperthermia (Mirkes et al., 1996; Thayer and Mirkes, 1997
); however, these analyses did not provide information about other heat shock proteins. Our microarray data confirm our Hsp25/27 results (the inducible Hsp70s are not contained on our microarray) and extend our knowledge base concerning the heat shock responses to several additional heat shock proteins including Hsps10, 40, 60, 841, 861, and 105/110. Although our microarray data indicate that 4CP does not significantly upregulate the expression of Hsps 10, 40, 60, 841, and 861, Hsps 25/27 and 105/110 are significantly upregulated. Both Hsp25/27 and 105/110 have been shown to play a role in protecting cells from the deleterious effects of different stressors (Fortin et al., 2000
; Oh et al., 1997
). Additional experiments will be required to determine whether the HS- and 4CP-induced upregulation of Hsps 25/27 and 105/110 are biologically relevant in the context of HS- and 4CP-induced teratogenesis. Nonetheless, our microarray data confirm and significantly extend our understanding of teratogen-induced activation of the stress response pathway in early post implantation mouse embryos. Similarly, our microarray data confirmed that Jun is upregulated by HS (Mirkes et al., 2000
; unpublished data) and extended our understanding of HS-induced activation of genes involved in signal transduction pathways (Fos, Gtpbp4, Iqgap1, and Lats1). The challenge now is to determine what role, if any, the alteration of specific genes play in teratogen-induced abnormal development. For example, we know that Hsp70 plays a role in protecting embryos from some of the deleterious effects of heat shock (Mirkes et al., 1999
); however, we do not know what, if any, role other heat shock proteins identified by our microarray analysis perform in preventing or facilitating teratogen-induced abnormal development.
Unlike the stress response pathway, which was already known to be activated in embryos exposed to HS, our microarray data also identified genes and an associated pathway that would not have been expected given existing information concerning HS-induced teratogenesis. Seven genes (Hmgsc1, Hmgcr, Mvd, IdI1, Fdft1, Sqle, and Dhcr7) that encode proteins in the cholesterol biosynthesis pathway are coordinately downregulated by HS. Hmgrc encodes the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. HMGCoA reductase catalyzes the synthesis of mevalonic acid from HMGCoA and is the rate-controlling enzyme in the cholesterol biosynthetic pathway. Furthermore, Nguyen et al. (1990) have shown that HMGCoA reductase activity is related to Hmgrc mRNA levels. On the basis of this information, we would predict that the reduction in Hmgrc mRNA observed is translated into reduced HMGCoA reductase activity and thereby reduced cholesterol levels in embryos exposed to HS. In addition, our data show that Dhcr7 gene expression is also downregulated in HS-treated embryos. Dhcr7 encodes the enzyme 7-dehydrocholesterol reductase that converts 7-dehydrocholesterol to cholesterol. This gene is known to be mutated in the autosomally inherited Smith-Lemli-Opitz (SLO) syndrome, which is characterized by an accumulation of 7-dehydrocholesterol and reduced serum and tissue cholesterol (Nowaczyk and Waye, 2001
). Thus, the observed reduction in Dhcr7 mRNA should further diminish cholesterol levels in HS-treated embryos. More important, the reduced cholesterol observed in SLO and in animals exposed to teratogens that are 7-dehydrocholesterol reductase inhibitors (AY9944 and BMI15.766) is linked to holoprosencephaly in humans and a variety of malformations, including holoprosencephaly, in animals (Kolf-Clauw et al, 1996
, 1997
). Recent work has shown that the link between reduced cholesterol and abnormal development is mediated, at least in part, through the sonic hedgehog signaling pathway. Cholesterol plays a dual role in this pathway by limiting the spatial extent of sonic hedgehog signaling (Beachy et al., 1997
; Porter et al., 1996
) and by facilitating transduction of the hedgehog signal within target cells (Beachy et al., 1997
; Cooper et al., 1998
). Thus, on the basis of our microarray data, we hypothesize that the decreased expression of key genes in the cholesterol pathway induced by HS results in a general, or perhaps tissue-specific, reduction in cholesterol levels in the early post implantation mouse embryo. This, in turn, contributes to HS-induced teratogenesis, at least in part, by disrupting sonic hedgehog signaling. We are currently investigating this hypothesis.
In addition to understanding mechanisms of teratogenesis, another goal of microarray-based expression profiling is to identify gene expression patterns that can serve as biomarkers of exposure. Realizing this goal will require large experiments (multiple exposures, time points, and developmental stages) with many more biological replicates than were included in this study. However, analysis of our HS and 4CP data sets have suggested genes that might be specific to each of the two teratogens studied. For example, we have identified 2 genes that encode DNA repair enzymes that are upregulated in embryos exposed to 4CP but not in embryos exposed to HS, i.e., RAD 51, which encodes protein involved in homologous recombinational repair (HRR) of DNA double-strand breaks (Bernstein et al., 2002; Grenon et al., 2001
; Baumann and West, 1998), and Msh6, which encodes a protein involved in DNA mismatch repair (MMR) (Kolodner, 1996
). These results extend the work of Vinson and Hales (2001)
in which they assessed the effects of 4CP on 17 genes encoding different DNA repair genes. In their study, 4CP induced a downregulation of RAD51, whereas in our studies, 4CP up-regulates the expression of this gene. Although the reason for this discordance is not known, it likely results from the fact that our data were collected after a 5-h exposure to 40 mM 4CP, whereas in the Vinson and Hales study, data were collected after a 44-h exposure to 10 mM 4CP. The difference in findings highlights the fact that development is a dynamic process, such that the effects of teratogens on gene expression are not only dose-dependent but also time-dependent.
Similarly, HS induces the expression of the DNA repair-associated gene Brca2 that is not induced by 4CP. Brca2, like Rad51, is involved in HRR of double-strand breaks (Bernstein, 2002). Thus, HS and 4CP induce different genes involved in HRR, Brca2 and Rad51, respectively. It is unclear whether the differential upregulation of these two genes by HS and 4CP reflect different effects on DNA repair pathways induced or different levels of DNA damage induced by these two teratogens. In contrast, only 4CP induces the expression of Msh6, which is involved in MMR. Finally, HS and 4CP both induce the expression of Ercc5, a gene encoding an enzyme involved in nucleotide excision repair (NER) (Habraken et al., 1994
). Although additional research is required, our initial microarray data suggest that 4CP and HS may induce different types of DNA damage and consequently different groups of DNA repair genes. Thus, specific DNA repair genes or combinations of DNA repair genes may be useful biomarkers of exposure to teratogens that damage DNA or that induce specific types of DNA damage. Similarly, our microarray results show that Hsps 10, 40, 60, 841, and 861 are upregulated in embryos exposed to HS but not in embryos exposed to 4CP. Heat shock proteins constitute one class of stress response proteins that also include receptor-mediated response proteins (e.g., retinoid receptors), sensor-mediated response proteins (e.g., metallothionein), and damage-specific response proteins (e.g., PARP). Perhaps gene expression profiling can be used to identify stress response gene signatures specific to individual teratogens or groups of teratogens. Developing teratogen-specific gene signatures need not be limited to stress response pathways discussed with respect to HS and 4CP. As indicated in Tables 1 and 2, differential effects of HS and 4CP are also observed for genes related to the cell cycle and signal transduction. In addition, further data mining, particularly related to as yet unidentified genes (ESTs), could very well identify other genes and their related pathways that will help identify teratogen-specific gene expression signatures.
In summary, we have used DNA microarrays and gene expression profiling to show that 4CP and HS significantly alter gene expression in day-9 mouse embryos, i.e., hundreds to thousands of genes exhibit altered expression compared to untreated embryos. Using bioinformatic tools, we have grouped a subset of these genes into functional categories, and show that although some genes show altered expression after exposure to HS and 4CP (common genes), others appear to be specific to HS or 4CP. Results obtained in our study pose two major challenges for future research: first, to investigate further the "biologically significant" genes identified in our microarray experiments and then to link these to an abnormal phenotype; second, to generate a teratogen expression-profiling database that can be mined to identify biomarkers of exposure. These are daunting challenges; however, preliminary analyses of our microarray data provide the basis for cautious optimism.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Department of Pediatrics-Box 356320, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195
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