Human Molecular Genetics, 2002, Vol. 11, No. 15 1763-1773
© 2002 Oxford University Press
EDA targets revealed by skin gene expression profiles of wild-type, Tabby and Tabby EDA-A1 transgenic mice
1Laboratory of Genetics, NIH/National Institute on Aging, Baltimore, Maryland 21224, USA, 2J. C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, South Carolina 29646, USA, 3Department of Dermatology, Kinki University School of Medicine, Osaka 589, Japan and 4Department of Dermatology and Syphilology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA
Received April 10, 2002; Accepted May 24, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the EDA gene cause anhidrotic ectodermal dysplasia (EDA), with lesions in skin appendage formation. To begin to analyze EDA pathways, we have used expression profiling on 15 000-gene mouse cDNA microarrays, comparing adult mouse skin from wild-type, EDA-defective (Tabby) mice, and Tabby mice supplemented with the EDA-A1 isoform, which is sufficient to rescue multiple Tabby phenotypes. Given the sensitivity of the current microarray system, 8500 genes (60%) were estimated to be expressed, including transcription factors and growth-regulatory genes that had not previously been identified in skin; but only 24 (0.16%), one-third of them novel, showed significant differences between wild type and Tabby. An additional eight genes not included in the 15 000 gene set were shown to have expression differences by real-time RT–PCR. Sixteen of 32 affected genes were restored significantly toward wild-type levels in EDA-A1 transgenic Tabby mice. Significant up-regulation in Tabby skin was observed for several dermal matrix genes, including Col1a1, Col1a2, Col3a1 and Sparc. In contrast, down-regulation occurred for the NEMO/NF-kB pathway, already implicated in skin appendage formation, and even more markedly for a second pathway, JNK/c-jun/c-fos and their target genes, that has not previously been clearly associated with skin development. These data are consistent with the regulation of the NF-kB pathway by EDA, and support its involvement in the regulation of the JNK pathway as well.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in either the EDA or EDAR genes lead to anhidrotic ectodermal dysplasia (EDA) (1–5). One of the most frequent of 170 clinically distinguishable ectodermal dysplasias, EDA is characterized by the absence of hair follicles and sweat glands, and abnormal tooth formation in affected males (6). Consistent with a role in initiation and possibly in maintenance of skin appendages, EDA expression is detected in early embryonic ectoderm and continues in adult skin epidermis, hair follicles and sweat glands (1,2,7). The X-linked EDA gene encodes a type II transmembrane protein, ectodysplasin, which has both a tumor necrosis factor (TNF) ligand motif and 19 Gly–X–Y collagen repeats in its extracellular region (1,2,8,9). Mutation in either domain causes the disorder (10,11). Several groups have shown that the collagen domain forms an extracellular triple helix (11,12), which can be cleaved from cell membranes by a furin-like serine protease (11–14). The resultant soluble trimer or cell-bound EDA can then bind through its TNF–ligand moiety to TNF-family receptors, EDAR or XEDAR, to activate the NEMO/NF-kB pathway (4,15–18) through the adapter protein EDARADD (19,20). Consistent with this formulation, mutations in mouse EDAR, EDARADD or IKK-gamma (NEMO) can give rise to EDA-like disorders.
The downstream effectors of the TNF motif-based EDA pathway in the skin and skin appendages are largely unknown. To find candidate target genes, we have made use of a model system. The Tabby mouse, which we have proven to be the mouse counterpart of human EDA (2), has a frameshift mutation resulting in truncated ectodysplasin lacking both collagen and TNF–ligand motifs, and, in a complementary study, we have shown that a transgenic Tabby mouse bearing the single EDA-A1 isoform restores most skin appendages (21). Because the Tabby transgenic mouse is a direct control to see whether effects in Tabby are specifically reversed, these mutant mice provide unique materials to explore genes directly or indirectly affected by EDA. We have performed cDNA microarray hybridization experiments using RNAs from wild-type, Tabby and EDA-A1 transgenic Tabby adult mouse skin, and report candidate genes for EDA-specific gene expression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Microarray assessment of expressed and differentially expressed genes in wild-type, Tabby and Tabby transgenic skin
As judged by normalized signal intensities greater than 5000 on microarrays (see Methods), adult wild-type C57BL/6 mouse skin expressed around 8500 (60%) of the 15 000 genes assayed. Figure 1 represents the classification of genes expressed in adult wild-type mouse skin. Fifty per cent of these are novel genes, and 25%, or about half of those previously described, have defined functions. Twenty known genes with intensities >50 000 (10-fold greater than the dependable sensitivity of the microarray analyses) are newly inferred to be highly expressed in skin. They include transcription factors and some signal transduction molecules (Table 1).
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To assess the differential expression of genes, we first plotted intensities for all genes on the microarrays, based on probes from wild-type (abscissa) and Tabby back skin (ordinate) (Fig. 2A). The intensities cluster tightly along the diagonal line of equal expression, indicating that the overall expression profile of wild-type and Tabby mice is very similar. Twenty-four genes (0.16%) showed significant differences between wild-type and Tabby, both in statistical analysis and in image views of reaction spots. Seven genes were down-regulated in Tabby, whereas 17 genes were up-regulated (Fig. 2B). The down-regulated genes include several regulatory proteins: c-fos and c-jun; Adm (adrenomedullin); and Znf216 (22). Up-regulated genes include a number of dermal matrix proteins, Col1a1, Col1a2, Col3a1 and Sparc (Table 2). A further indication that these genes are responding to the level of EDA comes from their expression level in skin of Tabby mice bearing the EDA-A1 isoform as a transgene. The expression levels of 11 of the 24 were restored significantly toward wild-type levels in the transgenic Tabby mice (Table 2). These inferences are further supported by direct observation of microarray images, as well as by northern blot assays and real-time RT–PCR (see below).
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The qualitative results are evident, for example, in enlarged sample portions of microarrays hybridized with different RNA probes (Fig. 3A). Visual inspection as well as densitometric analysis clearly shows the lower expression of c-fos and Znf216 in Tabby mice, whereas Col3a1 and Sparc are more highly expressed. The restoration of expression toward wild-type level in the EDA-A1 Tabby transgenic mice is illustrated for c-fos, Col3a1 and Sparc, whereas Znf216 expression remains at the level seen in Tabby. The sensitivity of microarrays is similar to that of northern blot analyses, and Figure 3B shows the consistent qualitative results obtained using c-fos cDNA as a hybridization probe against RNAs extracted from wild-type, Tabby and EDA-A1 transgenic Tabby skin. As in the microarray assays, c-fos mRNA was sharply reduced in Tabby and restored in EDA-A1 transgenic Tabby mice. Figure 3C shows a sample confirmatory assay by RT–PCR with a primer set specific for Adm on serial dilutions of cDNA mixtures. As in microarray analyses, Adm was down-regulated in Tabby skin and restored toward wild-type levels in EDA-A1 transgenic Tabby mice.
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Real-time RT–PCR confirms and extends gene expression changes in Tabby and EDA-A1 transgenic Tabby skin
One would expect that EDA receptors (1–4,16) and downstream effector genes might be differentially expressed in epidermis. A number of these genes are among the 24 significantly altered in expression in microarrays; and of these, all six tested by real-time RT–PCR gave concordant results (Fig. 4 and Table 2). In addition, we tested 13 more genes that might be involved in EDA signaling but were not in the 15 000 gene set (Tables 2 and 3).
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We first tested a series of genes in the early steps of the EDA-NF-kB regulatory scheme (Figs 5 and 4A) by real-time RT–PCR. The Edar gene, encoding the receptor for EDA-A1, was slightly reduced (to 66% of wild-type) in Tabby, and was sharply up-regulated to higher than wild-type levels in the EDA-A1 transgenic Tabby mice. In contrast, the expression of the gene Edaradd, encoding the adapter protein EDARADD, was unchanged in the mutant and transgenic mice. Of the immediately downstream genes in the NF-kB branch of the regulatory pathway, expression of Ikk-gamma was essentially unchanged, but the p50 heterodimer component of NF-kB was slightly reduced (to 74% of wild-type) in Tabby mice, and was essentially restored (to 96% of wild-type) in the EDA-A1 transgenic mice. Adrenomedullin, another regulatory gene in skin, which has an NF-kB binding site in its promoter region (23,24), was also confirmed by real-time RT–PCR: it was reduced by 60% in Tabby skin, and significantly rescued in the EDA-A1 transgenic (data not shown).
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More consistent and notable reductions were seen in genes that are involved in JNK-based regulation (Figs 5 and 4B). Expression of c-jun and c-fos was significantly reduced in Tabby mice, with c-jun reduced to 40% of wild-type and c-fos showing an especially marked 10-fold reduction—the largest change we observed in the entire set of 15 000 genes. Furthermore, both c-jun and c-fos were appreciably restored toward wild-type levels (to 69% and 75%, respectively) in the transgenic mice. Jnk, which was not included in the microarray, showed a less marked reduction in Tabby skin by real-time RT–PCR (to 65% of wild-type), and remained at about that level in transgenic mice. Collectively, these results are consistent with previous inferences that EDA-A1 regulates downstream effectors in skin through EDAR and NEMO/NF-kB, and suggest the additional participation of another pathway, JNK/c-jun/c-fos (see Discussion).
We also assessed the expression levels of selected genes from four other pathways critically involved in skin appendage formation but not represented in the 15 000 gene set. By real-time PCR assays, the expression of Bmp-4 and Shh in Tabby and EDA-A1 transgenic Tabby mice was indistinguishable from wild-type (data not shown). In contrast, and in good agreement with a previous study of protein levels (25), Egfr mRNA was significantly reduced in Tabby and was restored to higher than wild-type levels in the transgenic mice, while Egf ligand mRNA was not significantly changed (Fig. 4C). The Wnt pathway transcription factor Lef1 mRNA was also significantly reduced in Tabby mice and higher than wild-type in transgenic animals, but Catnb (beta-catenin) and other downstream targets were unchanged (Fig. 4C and Discussion).
Real-time RT–PCR assays were also consistent for the group of dermal matrix proteins that show opposite up-regulation in Tabby animals (Fig. 4D). In particular, Col1a1, Col3a1 and Sparc jumped to 2.5-fold the wild-type levels, and were significantly lowered toward wild-type levels in the transgenic animals.
Table 2 summarizes the genes significantly changed in expression level in Tabby skin, as determined by microarray analysis, real-time PCR, or both.
Genomic analysis of novel genes differentially expressed in Tabby mice
Mouse chromosome assignments for 29 of the 32 genes with unique locations were determined (Table 2), as deduced from public (GenBank) and Celera genomic sequences. The eight ‘novel’ genes detected as differentially expressed in Tabby versus wild-type mouse skin (Table 2) are derived from embryos and fetal tissues, six from 2–16 cell embryos, and two from 12.5 days post conception (dpc) female mesonephros. All these novel genes are largely represented by 3'-sequences, with no open reading frame. Details of these genes were further assessed. Based on searches in the NIA and GenBank cDNA/EST databases, and in the Celera sequence for mouse genomic DNA, cDNA/EST sequences were identified that are complementary to each of the eight novel genes, and contain poly(A) tails as well as genomic sequence tracts.
Two putative novel transcripts with unique genomic locations (H3104B08 and H3109D12; see Table 2) were both down-regulated and were significantly restored toward wild-type values in the Tabby EDA-A1 transgenic animals. H3104B08 corresponds exactly to sequence in a full-length RIKEN cDNA (GenBank AK014920 and mCG22663 in the Celera database). This gene encodes a 288 residue novel protein and contains no recognizable motif or functional cue. The second down-regulated novel gene, H3109D12 (GenBank BG072343), maps within a putative intron of a predicted gene, mCG1060 (Celera database). Comparison of cDNA and the corresponding genomic sequence revealed the presence of two putative exons, most likely representing two additional or alternatively spliced exons of the mCG1060 gene that have not previously been inferred.
The other six novel transcripts (listed ‘Unknown’ in Table 2) are all up-regulated in Tabby skin. Of these six transcripts, expression of only one transcript (H3091E04, Table 2) is restored toward wild-type levels in the EDA-A1 transgenic mice. That cDNA clone, H3091E04 (GenBank BG070896), maps within another predicted gene, mCG20629 (Celera database). Identification of this corresponding cDNA further confirms the identity of a gene at this locus. Comparison of cDNA sequence to the corresponding genomic sequence predicts at least two exons of 290 and 144 bp.
Of the other five up-regulated novel transcripts, H3117B08 (GenBank BG072975) and H3123B01 (GenBank BG073481) were only partly analyzed, because they showed no clear-cut locations in draft genomic sequences. H3111G08, which corresponds exactly to a RIKEN cDNA (GenBank AK002443), is gene mCG19093 in the Celera database. H3101G01 (GenBank BG071661) matches a gene that is included in an orphan segment of mouse genomic DNA, ‘GAx5JB7TUFHY’. The cDNA sequence is split into three inferred exons. H3121A01 (GenBank BG073296) falls into gene mCG22436 and contains three putative exons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The pathophysiology of EDA must be explicated in parallel with an understanding of normal development. One starting point comes from patient studies indicating that specific lack of the long protein isoform EDA-A1 is sufficient to produce the disorder (10–13,26). In accord with that observation, when we supplied EDA-A1 to Tabby mice—which lack all EDA isoforms—it largely restored wild-type histology and natural history (21). The results here complement the phenotypic analyses. They provide a first approximation of the repertoire of gene expression in skin. In addition, significant changes in regulatory and structural gene transcription were observed when the EDA gene was ablated in Tabby mice, and a trend to restoration of gene expression levels in EDA-A1 transgenic mice (Table 2) is obviously correlated with their recovery of active sweat glands and productive tail and back skin hair follicles (21). We note that these data are based on adult skin samples, and the gene-expression pattern observed may represent some functions in the maintenance of skin appendages in mature mice rather than the full action of EDA during fetal life. Here we focus on the findings that only a small cohort of genes involved in the regulation of skin and its appendages are significantly affected, and, in contrast to the all-or-none phenotype of the Tabby mice, the observed changes in transcription level are only partial.
Common and differentially expressed genes in Tabby and wild-type mouse skin
There is no reported or apparent defect in skin structure or barrier function in Tabby mice, and Tabby mice retain one of three types of mouse hair follicle (27). Thus, it is not surprising that the genes expressed in skin are largely the same as in the wild-type, including the wide range found in essentially all tissues (Fig. 1). In this initial study, however, we have assayed 15 000 genes assembled from cDNAs expressed predominantly or exclusively in embryonic and early fetal stages (28). At these times, initiating steps of organogenesis, including the first invaginations that lead to hair follicle, sweat gland and tooth bud formation, would be occurring, and we see changes in the levels of corresponding control genes and skin matrix. The relatively small number of genes significantly affected—32 of 15 000—is likely to reflect a limited change in the expression profile, for several reasons:
- First, the results are internally consistent, with high values of the Pearson correlation coefficient (0.9) in replicate trials, and significant t-test values with fold differences ranging from 1.5–10; this indicates that the significant results are not inferred from intrinsically highly variable trials.
- Second, the genes that show significant differences in intensity on microarray are confirmed by northern analyses (Fig. 3B) and quantitative real-time RT–PCR (Fig. 4); this indicates that the level of false positives is low.
- Third, the expression of 16 of 32 genes significantly affected in Tabby mice is reverted significantly toward wild-type expression levels by the introduction of the transgenic A1 isoform.
- Fourth, and most critical, genes that show significant differences include regulatory genes that cluster in pathways and are supported as involved in skin/skin appendage formation by other approaches.
Targets reduced in expression in absence of EDA
Not all regulatory genes involved in skin appendage formation are affected in Tabby mice. For example, in the Results, and according to additional data not shown, bone morphogenetic protein (BMP) and sonic hedgehog (SHH) pathway genes (Bmp-2, Bmp-4, Shh and Gli2) and transforming growth factor (TGF) receptors showed no detectable changes in Tabby mice. BMP-4 and SHH were found to be down-regulated in Tabby and Downless mice in early embryonic stages [embryonic day (E) 15]; however, their expression was largely recovered at a later stage (E17) (4,29,30), and is fully recovered in adult mice, as seen here. The dynamic pattern is thus complex, and both the down-regulation and subsequent restoration must be explicated by further studies.
Interestingly, for another important pathway, Wnt, which is active in the regulation of EDA (31,32) and has other effects on hair follicle numbers (33,34), the major effectors Gsk3-beta, Apc and Catnb are unchanged. One downstream transcription factor, Lef1, is appreciably down-regulated (Fig. 4C), but may be modulated by downstream factors (35) rather than being a primary target of EDA (32).
In contrast, Figure 5 schematizes two regulatory pathways in which multiple factors show significantly reduced expression in Tabby mice. EDA has been shown to act through the EDAR receptor and EDARADD to activate the NEMO/NF-kB pathway (see Introduction). At first, significant reduction is most marked for Edar, and is fully restored in EDA-A1 transgenic Tabby mice. Edaradd is not detectably affected, but the more distal Nf-kb (p50) and Ikk-gamma again showed a reduction in Tabby and recovery in EDA-A1 animals, based on microarray and real-time RT–PCR assays. A more distal response gene, Adm, which is specifically expressed in keratinocytes of skin epidermis, hair follicles, and sweat glands (23), and largely overlaps EDA in its pattern of expression in situ, was significantly down-regulated in Tabby skin (Fig. 3C, Table 2).
The most striking changes, however, were seen in a second downstream pathway, involving activator protein 1 (AP-1) genes c-fos and c-jun, themselves targets of JNK. Jnk was moderately down-regulated, and both c-jun and c-fos were sharply down-regulated in Tabby skin (Figs 3 and 4). AP-1 genes have not previously received much emphasis in discussions of EDA; and any role for JNK downstream of EDA has been problematic, suggested by some authors (36) but not supported by others (37). However, earlier studies have shown that c-fos and c-jun are expressed in the skin epidermis, hair follicles and sweat glands, where they are involved in the generation of skin tumors (38,39). They also function in tooth formation (40–42), a process in which the EDA pathway also plays an important role (15,21,31). Targets of AP-1 genes in skin include several matrix metalloproteases (MMPs) and keratins (43). It may be relevant that c-fos facilitates expression of MMPs in response to epidermal growth factor (EGF) signaling (44,45). Like c-fos, Mmp-9 is down-regulated in Tabby skin (see below), and, interestingly, the EGF receptor is also down-regulated in Tabby skin (25, Fig. 4C). Ectopic application of EGF has been reported to rescue part of the Tabby phenotype (46), suggesting possible cross-talk between the JNK and EGF pathways under EDA control (Fig. 5). As for c-jun, it has been suggested to control mesenchyme–epidermal interactions by regulating keratinocyte growth factor (KGF) expression in skin fibroblasts (47), and may thereby also contribute to skin appendage formation.
The currently detected distal EDA targets will surely be considerably augmented in further work. This is because the genes assayed are predominantly recovered from early embryonic tissues. Thus, they do not contain cDNAs for the bulk of structural genes that would be expressed in the maturing appendages (e.g. amelogenin of teeth, keratins of hair), which would currently have to be assessed by other techniques, one-by-one in RNA from developing or adult skin. As examples of additional candidate targets for c-fos and NF-kB that are expressed in epidermis, we tested the expression level of Plau [urokinase-type plasminogen activator (U-PA)] and Mmp-9 (matrix metalloproteinase 9/gelatinase B). Both genes are regulated by c-fos and NF-kB (48,49), and have been shown to be expressed in skin keratinocytes (50,51). They were not included in the 15 000 gene set, but could be assayed by real-time RT–PCR. Their expression levels were down-regulated to 35% and 52% of wild-type, respectively, in Tabby skin (Table 2). It is thus conceivable that the EDA pathway, via an action on specific protease levels, may act in part by affecting the migration of keratinocytes.
Overall, our results strongly support the notion that, in addition to NEMO/NF-kB, JNK/c-fos/c-jun is a second major EDA-dependent pathway (Fig. 5). However, its role is unlikely to be decisive on its own, because c-fos-null mice show no gross skin appendage phenotype (39–41), and additional regulatory signals, particularly from EGF receptor (EGFR), also are known to participate.
Downstream targets with increased expression in the absence of EDA
In contrast to the decreased expression of some effectors, the expression of a second, intriguing group of distal targets, particularly dermal markers, is up-regulated in the absence of EDA. Histological study of the mouse strains confirms that the collagen layer of Tabby mouse skin is appreciably more dense and thicker than in the wild-type, with the EDA-A1 transgenic mouse skin being intermediate (see Fig. 5 of 21 and Fig. 4C). That layer is also likely to have increased vascularization, which could account for the higher level of globin mRNA detected in the microarray analyses (Table 2). Formally, NF-kB and c-jun/c-fos may be repressors of some dermal genes. Alternatively, such changes could result from compensatory reactions or secondary effects when EDA is lost. It may be relevant that ectodysplasin with an in-frame deletion of the collagen domain is still able to form triple helices and activate NF-kB-based transcription, but nevertheless causes EDA (12,36), so that the collagen domain apparently has a distinct important function. Speculatively, when interactions of the extracellular EDA collagen domain with matrix elements like integrins are lost, other collagens might be augmented in the dermis to substitute for the missing EDA, in response to indirect activation signals from the c-fos and c-jun pathways coming through the basement membrane of the epidermis.
Penetrance of the Tabby mutation
Presumably the EDA phenotype is correlated with only partial disruption of the NF-kB and JNK pathways. Consistent with this view, total ablation of the NEMO gene in the NF-kB pathway produces multiple additional phenotypes (17,52); and reversion of the Tabby phenotype by the EDA-A1 isoform is accompanied by corresponding partial recoveries of the levels of many NF-kB and JNK RNAs; full reversion of all genes may require other isoforms, particularly EDA-A2 (21).
There are at least three possible ways in which moderate changes in expression levels of regulatory genes could lead to the penetrant Tabby phenotype. In one, downstream effectors of skin appendages could require relatively high levels of NF-kB or c-fos/c-jun activity, so that partial reductions in critical components result in a loss of function. In an alternative, the signaling pathway could be ‘fractionated’, so that a portion specifically allocated to skin appendages—e.g. by interaction of EDA with the equivalent of grb-like proteins (53)—would be selectively lost in Tabby animals. The residual levels of significantly altered genes may then be correlated with the retention in Tabby of large numbers of one of the three types of hair follicle, ‘awl’ (21). Finally, at least part of EDA action could be post-transcriptional—based, for example, on the extracellular collagen-like domain of the protein. Interacting follicular proteins might then still form, but, lacking an EDA partner, would be turned over. Tests of these conjectures should be possible in further physiological studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Materials
The majority of hair follicles and sweat glands are located in the epidermis, dermis and subcutis. We used whole back skin of 2 to 3-month-old mice as starting material. Total RNA samples from wild-type, Tabby and EDA-A1 transgenic Tabby mice (three samples for each strain) were used in microarray hybridization, Northern blotting, RT–PCR and quantitative real-time RT–PCR. Mouse skin, after a 70% alcohol washing, was sampled by biopsy without cutting into underlying tissues and was immediately rinsed with phosphate-buffered saline (PBS) and frozen in dry ice. Trizol (Gibco, Grand Island, NY, USA) was used to isolate total RNA. The RNA samples were treated with a ‘DNA-free kit’ (Ambion, Austin, TX, USA) to eliminate genomic DNA contamination. Total RNAs from wild-type C57BL mouse, 16.5 dpc placenta and wild-type adult skin were used in real-time RT–PCR to generate a standard curve. The mouse 15 000 gene set (NIA 15K) printed on seven individual nylon membranes was used in hybridizations (28).
Microarray hybridization
Probe preparation.
RNA probes were prepared as previously described (28). Three probes each for wild-type, Tabby and EDA-A1 transgenic Tabby mice were prepared. Briefly, 66 µg of heat-denatured total RNA was used as a template to incorporate [
-33P]dCTP (Amersham Pharmacia, Piscataway, NJ, USA) during cDNA synthesis using SuperScript II (SSTII) reverse transcriptase (Gibco) and oligo(dT) primer. Each labeling reaction contained 1xfirst-strand reaction buffer, 1 mM DTT, 0.5 mM each of dATP, dGTP and dTTP (Amersham Pharmacia), 40 units of RNAse Inhibitor (Boehringer Mannheim, Mannheim, Germany), 0.05 mCi/mmol [-33P]dCTP, and 800 units of SSTII. After incubation at 42°C for 2 h, the reaction mixture was treated with 0.2 M NaOH/0.5 M EDTA at 65°C for 30 min, and then neutralized with 1 M Tris-HCl, pH 7.5. Before hybridization, RNA probes were purified with a BioSpin P-6 chromatography column (BioRad, Hercules, CA, USA).
Hybridization and data analysis.
Prehybridization was done at 65°C for 3 h in MicroHyb solution (Research Genetics, Carlsbad, CA, USA) supplemented with heat-denatured yeast tRNA (1 mg/ml; Amersham Pharmacia) and poly(A) RNA (1 mg/ml; Amersham Pharmacia). For hybridization, purified RNA probe, 10% dextran sulfate (Sigma, St. Louis, MO, USA) and mouse Cot1 DNA (0.07 mg/ml; Gibco) were added to the prehybridization solution and incubated at 65°C for 20 h. Membranes were washed thoroughly in 2xSSC/0.1% sodium dodecyl sulfate (SDS) and 0.1xSSC/1% SDS, and exposed to a Phosphoscreen for 14 days at room temperature, after which they were scanned with a Storm 860 instrument (Amersham Pharmacia) at a maximum resolution of 0.05 mm/pixel. Results from two independent hybridiza-tions were obtained for each probe. Images were analyzed by Imagequant 5.0 (Amersham Pharmacia).
To correct for the variability in background, local levels were determined at 54 locations that bear no DNA, distributed at intervals on each filter. The local background was then subtracted from each spot in a corresponding region. The sum of signal intensities on each filter was then normalized to 109 arbitrary units. The intensity of signals for the spots on filters ranged from 1 to 3.7x106, with an average of 6.6x104 after normalization.
To analyze the data, a first pass used the Student t-test to score DNA spots as statistically significantly different between Tabby and wild-type, as in 28. To ensure that cDNAs scored as different on microarrays were truly positive, we further focused on those that showed >1.5-fold differences and had expression values >5000 over background. At this level, positive clones can be clearly distinguished by eye. For example, in the experiments of Figure 2, 40 cDNA spots satisfied both criteria. Four of the genes were duplicated one to several times on the filters; the replicates gave the same difference between Tabby and wild-type in all cases. Finally, several genes were set aside as false positives, based on overlap from neighboring strong spots, seen in visual rescoring of 16- and 32-fold enlarged views of the hybridization membrane results (using ImageQuant 5 at maximum contrast levels to compare replicate trials; see Fig. 3). Based on these observations, 24 genes were chosen for further study.
Northern blotting analyses
Total RNA from wild-type, Tabby and EDA-A1 transgenic Tabby mice, 11 µg/lane, was fractionated by 1% agarose gel electrophoresis, and transferred to Hybond-N+ membrane (Amersham Pharmacia) by capillary action. Prehybridization was performed in ULTRAhyb solution (Ambion) for 2 h at 42°C. Hybridization was performed in ULTRAhyb solution with 2.5 mg/ml PolyA (Amersham Pharmacia) and 0.5 mg/ml salmon sperm (Sigma) at 42°C overnight. Washing was done thoroughly in 2xSSC/0.1% SDS at room temperature, and followed by 0.1xSSC/0.1% SDS and 0.1xSSC/1% SDS at 65°C.
Probes were synthesized with the Radprime DNA Labeling System (Gibco) according to the standard manufacturer's protocol. About 100 ng of cDNA was used as template for probe synthesis in each reaction. The final activity of -32P in each hybridization solution was 1x106 cpm. The templates for probe synthesis were cDNA inserts from NIA mouse cDNA libraries. All cDNA inserts were excised from plasmids with NotI and SalI, and their sequences verified before use.
Real-time RT–PCR
One-step quantitative real-time RT–PCR with Taqman probes and primers (ABI Prism 7700 Sequence Detection System, Applied Biosystems, Foster, CA, USA) was performed to confirm microarray results and to study genes that are not included in the 15 000 microarray. PCR was carried out for 40 cycles according to the manufacturer's protocols. Reactions were normalized to glyceraldehyde-3-phosphate dehydrogenase and 18S ribosomal RNA. Each set of PCR reactions included RT-minus or non-template samples to control for non-specific amplification. Detailed probe–primer sets for each gene are listed in Table 3.
RT–PCR was performed with serially diluted cDNAs (200 ng, 100 ng, 20 ng and 4 ng) as template with a specific primer set for adrenomedullin: forward primer 5'-CGCAGTTCCGAAAGAAGTGGA-3'; reverse primer 5'-TGCCGTCCTTGTCTTTGTCTG-3'. Reaction mixtures were treated at 95°C for 3 min, followed by cycles (35 for adrenomedullin and 25 for beta-actin control) of 95°C for 30 s, 62°C for 45 s and 72°C for 1 min.
Genomic analysis for novel genes
The sequences of novel genes were subjected to BLSTN in the Celera Discovery System (www.celera.com), to assign them to mouse chromosome locations and infer features of genomic structure for each gene. The cDNA/ESTs and genomic information for eight novel genes are from GenBank (www.ncbi.nlm.nih.gov), NIA mouse cDNA libraries (lgsun.grc.nia.nih.gov), and Celera, as indicated.
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ACKNOWLEDGEMENTS |
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The authors thank Ramaiah Nagaraja for helping with Celera database searches and critical reading of the manuscript, Kazuhiro Aiba for advice on statistical analysis, Yong Qian for implementing background subtraction protocols, and Carlos Galaviz for providing mouse placenta RNA. Celera databases were searched under a license to David Schlessinger, under the terms of an agreement with NIA.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 4105588337; Fax: +1 4105588331; Email: SchlessingerD{at}grc.nia.nih.gov
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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