Identification of developmentally regulated mesodermal-specific transcript in mouse embryonic metanephros

Yashpal S. Kanwar1,2, Anil Kumar1, Kosuke Ota3, Sun Lin1, Jun Wada3, Sumant Chugh2, and Elisabeth I. Wallner2

Departments of 1 Pathology and 2 Medicine, Northwestern University Medical School, Chicago, Illinois 60611; and 3 Third Department of Medicine, Okayama University, Okayama, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Mesodermal-specific cDNA or transcript (MEST) was identified by suppression subtractive hybridization-PCR of cDNA isolated from embryonic day 13 vs. newborn mice kidneys. At day 13 of mouse gestation, a high expression of MEST, with a single ~2.7-kb transcript that was exclusively localized to the metanephric mesenchyme was observed. The MEST mRNA expression gradually decreased during the later stages and then abruptly decreased in the newborn kidneys and subsequent postnatal life, after which a very mild expression persisted in the glomerular mesangium. Regression in mRNA expression during embryonic renal development appears to be related to methylation of the MEST gene. Treatment of metanephroi, harvested at day 13 of gestation with MEST-specific antisense oligodeoxynucleotide resulted in a dose-dependent decrease in the size of the explants and the nephron population. This was associated with a selective decrease in MEST mRNA expression and accelerated apoptosis of the mesenchyme. These findings suggest that MEST, a gene with a putative mesenchymal cell-derived protein, conceivably plays a role in mammalian metanephric development.

renal development; mesenchyme gene expression


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ORGANOGENESIS OF THE MAMMALIAN kidney proceeds by intercalation of the ureteric epithelial bud into the loose metanephric mesenchyme. This results in conversion of mesenchyme to an epithelial phenotype, which, in turn, stimulates the branching morphogenesis of the ureteric bud branches (12, 44). These inductive interactions are followed by a series of developmental events leading to the formation of glomeruli and tubules. During the later part of gestation, the glomeruli are capillarized by the intricate processes of vasculogenesis and angiogenesis (1). Fundamental to the understanding of the inductive neotransformation of the mesenchyme are the molecules that are expressed at the epithelial-mesenchymal interface or ligands expressed in the mesenchyme and receptors in the ureteric bud epithelium or vice versa. In this scenario, the molecules formulate juxtacrine-paracrine interactions reminiscent of cell-matrix interactions that are critical for the morphogenesis of various organs during embryonic life, including the metanephros. Development of the metanephros is modulated by a number of molecules that include extracellular matrix (ECM) glycoproteins, ECM receptors (i.e., integrins), cell adhesion molecules (CAMs), intracellular cytoskeletal proteins, growth factors or hormones and their receptors, DNA-binding proteins, protooncogenes, and ECM-degrading enzymes and their inhibitors (5, 14, 18, 23, 38, 46, 47). As expected, the activities of such a diverse group of molecules in embryonic development are intertwined. The concept of epithelial-mesenchymal interactions in organogenesis originated from studies performed with various ECM proteins. A large number of ECM proteins, their receptors (i.e., integrins), and functionally interactive molecules (i.e., CAMs), which play a role in the development of the metanephros, have been described (5, 14, 23, 38), whereas the biology of other novel molecules in embryonic processes remains to be defined.

For the search for new molecules that may play a role in various developmental processes, a number of subtractive hybridization techniques have been employed in recent years (20, 33, 35, 50) with the premise that their time-specific expression would be relevant to that particular stage of embryonic development for a given organ system. In other words, the relevant molecule would be upregulated at a given stage of embryonic development and can be differentially subtracted from the stage at which its expression, in relative terms, is diminished or downregulated. Among the various techniques, the suppression subtractive hybridization (SSH)-PCR seems to be suitable when small amounts of mRNA (0.5-2 µg) are available at a particular stage of embryonic development. In addition, by SSH-PCR, genes with low-abundance mRNA transcripts can be readily identified and characterized. Another advantage of this PCR-based method is that one can identify differentially expressed genes before the development of any phenotype, and this becomes relevant for the search for new molecules, the expression of which may precede reciprocal inductive interactions in the metanephros. We describe the identification of a molecule by SSH-PCR that conceivably belongs to the family of mesenchymal cell-derived proteins and may be relevant to the development of the mammalian metanephros.


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Animals. Paired male-female mating of ICR mice (Harlan Sprague Dawley) was carried out, and appearance of the vaginal plug was designated day 0 of fetal gestation. Kidneys from fetuses at days 13 (E13), 15 (E15), 16 (E16), 17 (E17), and 19 (E19) of gestation and from newborn and 1- and 3-wk-old mice were obtained. The terms E19 and newborn are interchangeably used, since the gestational period in mice is ~19 days, and E19 and newborn kidneys exhibit minimal morphological differences.

PCR-select cDNA subtraction. Total RNA was extracted from ~2,000 E13 and ~20 newborn kidneys by guanidinium isothiocyanate-CsCl centrifugation (8). The RNA was digested with RNase-free DNase (1 U/ml) in the presence of RNase inhibitor (1 U/µl) for 60 min at 37°C. A chloroform-phenol extraction was performed, and the RNA was reprecipitated with ethanol in the presence of RNase-free glycogen as a carrier (1 µg/µl). Poly(A)+ RNA was purified by employing an Oligotex mRNA minikit (Qiagen) and used for cDNA synthesis. First-strand cDNA was synthesized by using 200 U of Superscript II RNase H- reverse transcriptase (GIBCO BRL) in a total volume of 20 µl of the reaction mixture containing 1 µg of poly(A)+ RNA, 10 µM 3' random hexamer primer (GIBCO BRL), 1 µl of dNTP mix (10 mM each), and 4 µl of 5× reverse transcriptase buffer [250 mM Tris · HCl, pH 8.5, 150 mM KCl, 40 mM MgCl2, and 100 mM dithiothreitol (DTT)] at 42°C for 1.5 h. The second-strand cDNA synthesis, RsaI endonuclease enzyme digestion, adapter ligation, hybridization, and PCR amplification were preformed as described in the SSH-PCR kit manual (Clontech) with the following modifications: 1) the amount of driver cDNA during the second hybridization was five times the amount recommended; 2) cDNA prepared from mRNA isolated from E13 kidneys was employed as the "tester," and cDNA of newborn kidneys mRNA served as the "driver"; and 3) primary amplification included 30 PCR cycles and 20 secondary cycles. The PCR products were analyzed by 1.5% agarose gel electrophoresis, with a 100-bp DNA ladder as a marker. The differential PCR products in the tester cDNA population subtracted from the driver were gel purified and cloned into plasmid vector pCR2.1 (Invitrogen). The products were sequenced and subjected to homology search by using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).

Northern blot analyses. A number of genes were found to be differentially regulated by SSH-PCR. One of the clones was a 363-bp fragment with nucleotide sequences identical to a "putative mesenchymal-derived protein" that has been recently described as mesodermal-specific cDNA or transcript (MEST) (22, 41, 50). In view of the role of mesenchymal proteins in embryonic developmental processes, an effort was made to investigate the biology of MEST in fetal nephrogenesis. Total RNA was isolated from kidneys of E13, E15, E17, E19 (newborn), and 1- and 3-wk-old mice by the acid guanidinium isothiocyanate-phenol-chloroform extraction method (9). Total RNA (~10 µg) was subjected to 1.5% agarose gel electrophoresis containing 2.2 M formaldehyde and capillary transferred to Hybond N+ nylon membranes (Amersham). After ultraviolet cross-linking of the RNA to the membranes (UV Stratalinker 2400, Stratagene), prehybridization was carried out at 65°C for 4 h in a solution containing 5× saline-sodium citrate (SSC), 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml herring sperm DNA. The membranes were then hybridized with [32P]dCTP-labeled (1 × 106 cpm/ml) partial-length cDNA 363-bp fragment, derived from SSH-PCR, at 65°C for 18 h. The same membranes were also hybridized with radiolabeled beta -actin cDNA. The blots were washed under high-stringency conditions, and autoradiograms were prepared. The integrity of RNAs and their equal loading in various lanes were monitored by visualization of the intact 18S and 28S bands on the nylon membrane stained with 0.05% methylene blue in 0.5 M sodium acetate.

Tissue expression of MEST by in situ hybridization. To evaluate the spatiotemporal gene expression of MEST in developing kidneys, in situ hybridization was performed as previously described (24-27, 49, 50). Briefly, 363-bp MEST cDNA was subcloned into pBluescript KS(+) at the EcoRI site and used as a template to generate [35S]UTP-labeled sense and antisense riboprobes by employing T7 and T3 RNA polymerase included in the Riboprobe In Vitro Transcription System kit (Promega). The riboprobes were subjected to limited alkaline hydrolysis to yield 100- to 150-bp polynucleotide fragments, which were then purified by ethanol precipitation. The purified riboprobes were used for hybridizing with tissue sections of kidneys harvested from E13-E19 embryos, and 1- and 3-wk-old mice. The kidneys were fixed by immersion in 4% phosphate-buffered paraformaldehyde for 3 h at 4°C. They were dehydrated in a graded series of ethanols and embedded in paraffin. Tissue sections (3 µm thick) were prepared and mounted on glass slides coated with Vectabond (Vector Laboratories). The tissue sections were deparaffinized, hydrated, treated with 0.2 N HCl, deproteinated by proteinase K treatment, and acetylated with 0.1 M triethanolamine and 0.25% acetic anhydride. After the sections were washed with 2× SSC, they were prehybridized with hybridization solution (50% formamide, 10% dextran sulfate, 1× Denhardt's solution, 10 mM Tris · HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA, and 10 mM DTT) at 55°C for 4 h and then hybridized with [35S]UTP-labeled MEST riboprobes at 55°C for 15 h. After hybridization, the tissue sections were washed successively with 2×, 1×, and 0.5× SSC in the presence of 1 mM DTT. The sections were dehydrated in graded series of ethanols, air-dried, and coated with NTB2 photographic emulsion (Eastman Kodak), and tissue autoradiograms were prepared after 1-2 wk of exposure. The autoradiograms were photographed with a light microscope equipped with dark-field illumination.

Methylation experiments. Because MEST expression dramatically decreases after birth (see below), we investigated the methylation status in the 5' region of the MEST gene during development. A GenomicWalker kit (Clontech) was used to generate a PCR product that included sequences flanking upstream of the 5' region. With the use of genomic library as the target DNA, the first PCR product was generated by employing activator protein (AP)-1 adapter primer (5'-GTAATACGACTCACTCACTATAGGGC-3') and MEST-specific antisense primer (5'-GAGGCCCCGCAGCGATACAGGATCGGAGG-3'). This was followed by another round of PCR amplification to generate a second PCR product in which AP-2 nested adapter primer (5'-ACTATAGGGCACGCGTGGT-3') and nested MEST-specific antisense primer (5'-GGCGTCTGCGCAGCCTACGGTCCGCG-3') were employed. The resulting 1,429-bp product was cloned into a pCR II vector (Invitrogen) and used as the hybridization probe in Southern blot analysis. Genomic DNAs were isolated from kidneys of E15 and E19 (newborn) mice as previously described (52). The DNAs were successively digested with SacI and EcoRI (37) and purified by chloroform-phenol extraction followed by ethanol precipitation. The purified DNAs were digested with HpaII (methylation-sensitive) or MspI (methylation-insensitive) restrictive enzymes and subjected to 1% agarose gel electrophoresis. The gels were treated successively with denaturing and neutralizing solutions. Separate nylon membrane blots were prepared for HpaII- and MspI-digested genomic DNAs and hybridized with a [32P]dCTP-labeled 1,429-bp DNA fragment as generated above. The blots were washed under high-stringency conditions, and autoradiograms were prepared.

Metanephric organ culture system. About 1,000 explants were harvested on E13 and maintained in an organ culture for 5 days in a humidified incubator with 95% air-5% CO2 at 37°C, as described in detail previously (24-26). The culture medium consisted of equal volumes of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 (Sigma-Aldrich Chemical) supplemented with transferrin (50 µg/ml), penicillin (100 µg/ml), and streptomycin (100 µg/ml), and pH was maintained at 7.4. The medium was devoid of insulin, serum, selenium, or any other growth factor. About 200 explants were collected every day for 5 days and processed for Northern blot analyses to assess the MEST mRNA expression as described above.

Antisense experiments. The antisense experiments were performed to determine the role of MEST in organogenesis of the mammalian embryonic kidney. A sense-, two nonsense-, and an antisense-phosphorothioated oligodeoxynucleotide (ODN) were synthesized by an automated solid-phase synthesizer (Biotech Facility, Northwestern University) and purified by HPLC. The following 36-mer antisense ODN sequence was selected from the 3' end of the MEST cDNA: 5'-CAGGTACGCAGCCAGCAAGGGCACAGCCAGGAGCCC-3'. Its specificity for target nucleotide sequences was established by S1 nuclease protection assays as described previously (27, 49). The two nonsense 31-mer phosphorothioated ODNs had the following nucleotide sequences: 5'-TAATGATAGTAATGATAGTAAT- GATAGTAAT-3' and 5'-GATCGATCGATCGATCGATCGA- TCGATCGAT-3'.

About 900 mouse embryonic kidneys, harvested on E13, were maintained in culture for 2-5 days, and the ODNs were added to the medium daily at 0.5-1.5 µM. At <= 2.5 µM, the ODNs usually retain the translational blockade specificity with no discernible cytotoxic effects in the mouse metanephros (7, 27, 49). The metanephric explants (300 kidneys per variable, i.e., sense, antisense, and nonsense) were processed for light microscopy, quantitative RT-PCR analyses, in situ hybridization, and apoptosis studies. For light microscopy, the sections from the midplane of the embryonic kidneys with a maximum number of ureteric bud iterations, including both poles and the hilus, were evaluated as described previously (27, 49).

To assess the status of cell proliferation in the untreated control, sense-treated (1.5 µM), and antisense-treated (1.5 µM) explants, 75 additional embryonic kidneys (25 explants/ variable) were labeled with [3H]thymidine (25 µCi/ml) for 12 h and processed for determination of total incorporated radioactivity. The explants were individually washed with cold medium and treated with 5% trichloroacetic acid for 30 min at 90°C. The hydrolysate was cooled and microfuged, and the supernatant was saved. The DNA content was measured in the supernatant by the diphenylamine method as previously described (25). An aliquot was used for determination of radioactivity in a scintillation spectrophotometer. The total incorporated radioactivity (disintegrations per minute) was then normalized against micrograms of DNA.

MEST mRNA expression in antisense ODN-treated metanephroi by competitive RT-PCR. To assess the effect of antisense ODNs on mRNA expression, competitive RT-PCR analyses were carried out on explants treated for 3 days as described previously (27, 49). The explants were harvested on day 3 of culture, because MEST expression tends to diminish normally during the later stages of the culture when the nascent nephrons have relatively matured in the metanephros. Total RNAs were isolated from 50 explants per variable by the acid guanidinium isothiocyanate-phenol-chloroform extraction method (9). Extracted RNAs were treated with RNase-free DNase (Boehringer Mannheim) and subjected to ethanol precipitation. About 5 µg of total RNAs from each variable were subjected to first-strand cDNA synthesis with Maloney's murine leukemia virus reverse transcriptase and oligo(dT) used as a primer. The cDNAs from different variables were suspended in 5 µl of deionized and autoclaved water and stored at -70°C until further use. For the analysis of MEST mRNA in the ODN-treated explants, the sense and antisense primers were 5'-CCATGGTGCGCCGAGATCGCTTGC-3' and 5'-CCAGCTCAGAAGGAGTTGATGAAGCCC-3', respectively. For beta -actin, the sense and antisense primers were 5'-GACGACCATGGAGAAGATCTGG-3' and 5'-GAGGATGCGGCAGTGCGGAT-3', respectively (48). With the use of these primers and "wild-type" renal cDNA, the expected PCR product sizes would be 1,015 bp for mouse MEST and 461 bp for beta -actin. A competitive "mutant" DNA construct containing MEST sequences was synthesized by inserting the sense and antisense primers in the "minigene" construct. The latter construct, containing sequences of various other proteins, is available in our laboratory (25). Using these primers, the expected size of the competitive MEST-cDNA PCR product would be 565 bp. This modified minigene construct was used for the mRNA analyses of the MEST. The unmodified original minigene construct, containing primer sequences for beta -actin, was used as the competitive "mutant" DNA template with an expected 224-bp size of the PCR product (25). The beta -actin expression was used as a control in these RT-PCR studies.

For quantitative RT-PCR analyses, a fixed amount (1 µl) from antisense and nonsense ODN-treated metanephroi and serial logarithmic dilutions of the competitive template DNA (0.5 µg/µl) of MEST cDNAs were coamplified (17). The reaction mixture included 5 µl of 10× PCR buffer, dNTPs (250 µM each), sense and antisense primers (10 µM), and 2.5 U of Taq polymerase (Perkin-Elmer) in a total volume of 50 µl. The amplification reaction was carried out for a total of 30 cycles in a DNA Thermal Cycler (Perkin-Elmer). Each cycle consisted of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. The PCR products of wild-type and mutant MEST were analyzed by 2% agarose gel electrophoresis and photographed using an instant positive/negative film (Polaroid). The negatives were analyzed by a scanning densitometer (Hoefer Scientific Instruments), and the relative area under the traces was computed. Similarly, the wild-type and mutant beta -actin were analyzed. The ratios of the densitometric readings of wild-type to mutant PCR-DNA products were plotted using a logarithmic scale on the ordinate (y-axis) against the logarithmic dilutions of the competitive template DNA on the abscissa (x-axis).

MEST mRNA expression in antisense ODN-treated metanephroi by in situ hybridization. The in situ hybridization studies were performed to assess the antisense ODN effect on the spatiotemporal expression of MEST as described above. The antisense- and sense-treated explants were harvested on day 3 of culture and immersed in 4% phosphate-buffered paraformaldehyde for 2 h at 4°C. They were dehydrated in graded series of ethanol and embedded in paraffin. Tissue sections (3 µm thick) were prepared and mounted on glass slides. The sections were deparaffinized, hydrated, treated with 0.2 N HCl, deproteinated, and acetylated with 0.1 M triethanolamine and 0.25% acetic anhydride. The sections were prehybridized and hybridized with radiolabeled riboprobes as described above, and tissue autoradiograms were prepared and photographed.

Apoptosis in metanephric mesenchyme in antisense ODN-treated metanephroi. It is believed that metanephric mesenchyme normally undergoes apoptosis during development. In view of the strong expression of MEST in the metanephric mesenchyme on E13 and its absence in adult life (see below), we investigated the effect of the premature disruption of the MEST gene by antisense treatment on apoptosis. An in situ cell death detection kit (Boehringer Mannheim Biochemicals) (25), in which nicked or blunt ends of DNA strands or the 3'-OH ends of genomic DNA are labeled with fluorescein dUTP and the reaction is catalyzed by TdT, was used. This procedure is known as the TUNEL (TdT-mediated dUTP nick end labeling) method. For these studies, the explants were treated with antisense ODN for 3 days. They were immersion fixed in 4% phosphate-buffered (pH 7.4) paraformaldehyde solution for 3 h at 24°C and embedded in paraffin blocks. Sections (4 µm thick) were prepared and mounted on clean glass slides. The tissue sections were deparaffinized and rehydrated with decreasing concentrations of ethanols. The sections were incubated with proteinase K (20 µg/ml in 10 mM Tris · HCl, pH 7.4) for 20 min at 37°C. They were then washed with PBS and processed for TUNEL reactions by following instructions given in the manual. The sections were examined with an ultraviolet microscope equipped with epi-illumination. Appropriate positive (DNase-digested) and negative (TdT-untreated) controls were performed during the TUNEL reaction.


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Developmental expression of MEST in mouse kidneys. Gene expression of MEST was investigated in the kidney at various stages of gestation. A single ~2.7-kb mRNA transcript was observed on E13 (Fig. 1A). With the application of 10 µg of total RNA, a strong signal for MEST mRNA was observed. The MEST mRNA transcript could be readily observed even with the application of 1 µg of total RNA isolated from E13 mouse metanephric explants (not shown). The mRNA expression slowly decreased during the various stages of gestation, extending into the postnatal period, and was barely detectable in kidneys of 3-wk-old mice, suggesting that MEST is developmentally regulated. Interestingly, a steep decrease in the MEST renal mRNA expression was observed between E17 and newborn (E19) mice. No alternative spliced isoform of MEST mRNA transcript was seen throughout the embryonic development and in the postnatal period (Fig. 1A). Also, no mRNA transcript could be visualized in adult mouse kidneys (not shown). The mRNA expression of beta -actin in mouse kidneys was constant throughout the embryonic and postnatal periods (Fig. 1B). The loading of equal amounts of total RNA from various developmental and neonatal stages was confirmed by methylene blue staining of the blot, where densities of 28S and 18S RNA were similar in all six lanes (Fig. 1C).


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Fig. 1.   Developmental mRNA expression of mesodermal-specific transcript (MEST). Northern blot analysis of total RNA of kidneys from embryonic, neonatal, and postnatal mice reveals a single ~2.7-kb transcript (A, arrow). A heavy mRNA expression of MEST is seen in embryonic day 13 (E13) kidneys and slowly decreases during the later stages of gestation. A steep decrease in MEST mRNA expression is observed between embryonic day 17 (E17) and newborn (day 19, E19) mice, and mild expression is detectable at 1 and 3 wk of postnatal life. The mRNA expression of beta -actin remains constant throughout the course of development of the kidney (B). Loading of equal amounts of total RNA in all 6 lanes is confirmed by staining the blot with methylene blue, where the intensity of 28S and 18S bands seems to be similar (C).

To confirm the developmental mRNA expression and to delineate the spatiotemporal distribution of MEST, in situ hybridization studies were performed. On E13, a heavy mRNA expression, which seemed to be localized mainly to the metanephric mesenchyme, was observed (Fig. 2, A and F). No signal was noted in the ureteric bud branches and in the epithelial elements of the maturing nascent nephrons. On E16, the MEST mRNA expression was reduced and was localized in the residual subcapsular metanephric mesenchyme (Fig. 2, B and G). No expression was seen in developing cortical or medullary tubules. In E19 or newborn kidneys, the expression was further reduced but was still visible in the subcapsular region of the renal cortex (Fig. 2, C and H). In kidneys of 1-wk-old mice, a very weak mRNA signal could be visualized under the renal cortex, whereas expression in the cortical or medullary tubules remained absent (Fig. 2, D and I). Very mild mRNA expression of MEST above the background level was also seen in the interstitium. In kidneys of 3-wk-old mice, no expression was seen in the subcapsular cortex and a very mild expression was seen in the interstitium (Fig. 2, E and J). Interestingly, mild MEST mRNA expression was seen in the renal glomeruli (Fig. 2J, arrowheads), and it seemed to be confined to the mesangium.


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Fig. 2.   Spatiotemporal MEST gene expression in embryonic, neonatal, and postnatal kidneys, as assessed by in situ autoradiography. At E13, MEST is heavily expressed in the metanephric mesenchyme (A and F). No mRNA expression is seen in the ureteric bud branches (U) and epithelial elements of the developing nephrons. At embryonic day 16 (E16), mRNA expression can be seen in the subcapsular metanephric mesenchyme, and it is absent in the medulla (B and G). In newborn (E19) kidneys, mRNA expression is seen in the residual mesenchyme of the renal cortex (C and H) but is not detectable in the medulla. At 1 wk, mRNA expression is barely detectable under the subcapsular cortex (D and I), and no expression is seen in the medulla. At 3 wk, MEST mRNA expression is absent in the subcapsular renal cortex. However, very mild expression is seen in the interstitium and glomeruli (J, arrowheads); in glomeruli, it seems to be confined to the mesangium. Insets in F and G: high magnification of areas adjacent to *; delineation of radioactive grains is enhanced, although the colors are different.

Methylation experiments were performed to understand the mechanism(s) that could explain the abrupt decrease of MEST expression in kidneys during the neonatal period. The blot that included MspI-digested products revealed ~1.0- and ~0.6-kb bands in the genomic DNA isolated from E15 and E19 (newborn) mouse kidneys (Fig. 3A, arrowheads). In contrast, the blot containing HpaII-digested products had two bands in the E15 genomic DNA only and a single ~4.1-kb band in E19 (newborn) mice (Fig. 3B, arrow), suggesting that methylation could potentially be one of the mechanisms responsible for the downregulation of MEST expression in embryonic life.


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Fig. 3.   DNA methylation status of MEST gene 5'-upstream SacI-EcoRI region assessed by Southern blot analysis of the genomic DNA isolated from mouse kidneys at embryonic day 15 (E15) and E19 (newborn). Genomic DNA was digested with methylation-insensitive MspI (A) or methylation-sensitive HpaII (B) restriction enzymes. Blot was probed with a 1,429-bp fragment of the MEST gene that hybridizes with the genomic DNA segments containing MspI/HpaII restriction sites. Blot with MspI-digested products revealed ~1.0- and ~0.6-kb bands (arrowheads) in the E15 and E19 (newborn) kidneys. Blot containing HpaII-digested products had 2 bands only in the E15 kidneys, and in E19 (newborn) kidneys, a single ~4.1-kb band was observed (arrow), suggesting that the MEST gene at this stage of development is methylated. Control (CON) included the SacI-EcoRI fragment, which was not digested with MspI/HpaII.

Role of MEST in mammalian metanephrogenesis (antisense experiments). Because, similar to some of the other mesenchymal proteins, e.g., interstitial collagens and fibronectin, the MEST is developmentally regulated and is selectively expressed in the metanephric mesenchyme, we proceeded to investigate its role in metanephric development utilizing antisense technology and the organ culture system. However, one needs to be certain that the time frame chosen for the antisense experiments does not influence the basal expression of MEST during various stages of the in vitro organ culture system. The MEST mRNA expression, similar to that of beta -actin, remained constant in the metanephroi, harvested on E13, for 5 days in culture (Fig. 4, A and B), and thus this time frame was considered appropriate to perform the antisense experiments. The explants treated with antisense ODN for 5 consecutive days exhibited dose-dependent notable alterations in the metanephroi. There was an overall reduction in the size of the explants (Fig. 5, B-D, vs. A). At 0.5 µM antisense ODN in the culture medium, the nephron population was reduced and the mesenchyme was expanded (Fig. 5, B and G) compared with the untreated control explants (Fig. 5, A and F). Tubular and glomerular elements were reduced. The mesenchyme in deeper portions of the explant was loose and expanded. At 1.0 µM, a further reduction in the number of nephrons was observed, and the mesenchyme was more expanded (Fig. 5, C and H). At 1.5 µM, the size of explants was significantly reduced (Fig. 5D). The number of tubules and glomeruli was remarkably reduced (Fig. 5, D and I). The ureteric bud branches were swollen and deformed, and their normal pattern of dichotomous iterations was disrupted. The acuteness of the tips of ureteric bud branches was lost. Interestingly, foci of cells undergoing apoptosis could be observed in the metanephric mesenchyme (Fig. 5I, inset, arrowheads). The extent of apoptosis was readily noticeable in explants treated with antisense ODN for 3 days (see below). The embryonic renal explants treated with 1.5 µM nonsense or sense ODN did not reveal any major morphological change compared with the untreated metanephroi (Fig. 5, E vs. A and J vs. F). A mild reduction in the size of the metanephric explants and a slight expansion of the mesenchyme were observed in explants treated with nonsense/sense ODN (Fig. 5, E and J). However, the ureteric bud branches exhibited normal iterations, and only ~5% loss of nascent nephrons was observed. The morphological changes described above in the control, untreated, and sense- and antisense-treated explants are summarized in Table 1. Because the antisense ODN induced a reduction in the population of nephrons, [3H]thymidine uptake, a measure of cell proliferative activity, was assessed. A mild reduction in [3H]thymidine incorporation in the antisense-treated metanephric explants was observed compared with the controls. The respective total incorporated radioactivities in the untreated control and explants treated with sense and antisense ODN were 50.57 ± 5.29, 48.36 ± 4.57, and 36.29 ± 4.03/µg DNA, respectively.


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Fig. 4.   MEST mRNA expression in metanephric explants at various stages of organ culture. Explants were harvested on E13 and maintained in culture for 5 days. Northern blot analysis of total RNA isolated from metanephroi at days 1 (d1), 2 (d2), 3 (d3), 4 (d4), and 5 (d5) of culture reveals a single ~2.7-kb transcript (A, arrow). A heavy mRNA expression of MEST is observed, and no significant change in its expression is seen at different stages of the culture. mRNA expression of beta -actin remains constant throughout the metanephric culture (B). Loading of an equal amount of total RNA in all 5 lanes is confirmed by staining the blot with methylene blue, where intensity of 28S and 18S bands seems to be similar (C).



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Fig. 5.   Low-magnification (A-E) and high-magnification (F-J) photomicrographs of untreated (A and F), sense/nonsense oligodeoxynucleotide (ODN)-treated (E and J, 1.5 µM), and MEST antisense ODN-treated [B and G (0.5 µM), C and H (1.0 µM), and D and I (1.5 µM)] E13 explants at 5 days in culture. Antisense-treated explants reveal dose-dependent alterations (B-D and G-I). Explants treated with 0.5 µM antisense ODN reveal a mild reduction in the number of tubules and glomeruli, and mesenchyme is mildly expanded (B and G). Treatment with 1.0 µM antisense ODN caused a notable decrease in the size of explants associated with loss of developing nephrons, expansion of the mesenchyme, and blunting of the tips of the ureteric bud branches (C and H). Treatment with 1.5 µM antisense ODN induced further reduction in the size of the metanephric explants and the population of the nascent nephrons (D and I). A few foci of apoptosis in antisense ODN-treated explants are also observed (H and I, arrowheads). Sense/nonsense ODN-treated explants reveal a mild reduction in the size of the metanephric explants and a ~5% decrease in the population of the nascent nephrons (E and J) compared with the untreated explants (A and F). Inset: high-magnification photograph of an area of I, in which visualization of apoptotic nuclei (arrowheads) in the metanephric mesenchyme is clearly enhanced.


                              
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Table 1.   Morphological changes in sense- and antisense-treated metanephric explants compared with untreated control

Gene expression studies in antisense ODN-treated metanephric explants. To correlate the morphological alterations in the explants with MEST mRNA expression, competitive RT-PCR and in situ hybridization studies were performed. The method of competitive RT-PCR was used, since Northern blot analyses require large amounts of RNA for which a huge number of E13 metanephroi are needed. In the sense/nonsense ODN-treated mice (MEST control), a linearity in the ratios of wild-type to mutant MEST DNA could be maintained when plotted against the 10-1-10-6 serial logarithmic dilutions of the competitive (mutant) template DNA. Within this range of dilutions, the bands of wild-type and mutant DNA were discernible for densitometric analyses to obtain a ratio. The densitometric graphic plots are not included here, because they have been published previously (24-27, 49, 50), and thus only the raw data, i.e., electrophoretograms, are included (Fig. 6). A ratio of 1 was obtained at dilutions of 10-3-10-4 of the competitive mutant DNA for the control group (Fig. 6A, lanes 3 and 4). In the MEST antisense ODN-treated group, a ratio of 1 was obtained at dilutions of 10-4-10-5 of the competitive DNA (Fig. 6B, lanes 4 and 5). This suggests a decrease on the order of ~1-2 logarithms of the mRNA expression in the antisense ODN-treated explants. However, for beta -actin, no significant differences in the linearity relationship in the range of logarithmic dilutions of the competitive DNA between the two groups (beta -actin control and antisense) were observed. A ratio of 1 was obtained at dilutions of 10-3-10-4 of the competitive mutant DNA for the control and antisense-treated groups (Fig. 6, C and D, lanes 3 and 4).


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Fig. 6.   Competitive RT-PCR analyses of MEST (A and B) and beta -actin (C and D) cDNAs prepared from sense/nonsense-treated (control) and MEST antisense-treated metanephroi. In MEST control (A), a ratio of 1 is observed between wild-type and mutant DNA at 10-3-10-4 log dilutions of the competitive (mutant) DNA (lanes 3 and 4); in the antisense-treated group (B), a ratio of 1 is observed at 10-4-10-5 log dilutions (lanes 4 and 5). This indicates a 10- to 100-fold reduction in MEST mRNA expression in the antisense ODN-treated metanephroi. No significant differences in wild-type-to-mutant ratios are observed in the beta -actin control (C) and antisense-treated group (D), and a ratio of 1 is observed at 10-3-10-4 log dilutions of the competitive DNA (lanes 3 and 4) in both groups.

Gene expression in the sense/nonsense and antisense ODN-treated explants was also evaluated by in situ hybridization. With antisense ODN (1.5 µM) treatment for 3 days in culture, a remarkable decrease in the expression of MEST in the metanephric mesenchyme was observed (Fig. 7, C and D). Some scattered MEST mRNA expression could be seen in the deeper regions of the antisense-treated metanephric explants (Fig. 7D). A mild reduction in the overall size and nephron population was observed, suggesting that the antisense ODN treatment resulted in disruption of the MEST gene before the morphological changes described in Fig. 5. The metanephroi treated with sense/nonsense ODNs revealed minimal reduction in the mRNA expression of MEST (Fig. 7, A and B).


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Fig. 7.   Low-magnification (A and C) and medium-magnification (B and D) photographs of in situ tissue autoradiograms of E13 explants treated with 1.5 µM sense/nonsense (A and B) or MEST antisense ODN (C and D) for 3 days in culture. At 3 days in culture, overall size of the metanephric explants treated with antisense ODN is mildly reduced. In control (CON) explants treated with sense/nonsense ODN, heavy mRNA expression of MEST gene is seen in the metanephric mesenchyme (A and B). No expression is seen in the ureteric bud branches (U). A notable loss of MEST mRNA expression is seen in the antisense-treated (AS) explants (C and D).

Apoptosis in antisense ODN-treated metanephric explants. The extent of apoptosis, which is usually best assessed by electron microscopy, was evaluated by the TUNEL procedure, which employs labeling of apoptotic cells with fluorsceinated dUTP. A time point of 3 days in culture was chosen, since at that stage basal apoptosis in metanephroi can be seen as well. The control metanephroi exposed to sense/nonsense ODNs did not exhibit any significant increase in apoptotic nuclei, as seen in the Epon-embedded and toluidine-stained sections (Fig. 8D, arrowhead). The metanephroi exposed to MEST antisense, however, revealed a notable increased concentration of darkly stained nuclei that were confined to the nephrogenic zone of the explants (Fig. 8H, arrowheads). These findings were reinforced in the TUNEL assay studies, where an accentuated apoptosis was observed in metanephroi exposed to MEST antisense (Fig. 8, E-G, arrowheads) compared with the controls (Fig. 8, A-C, arrowheads). Interestingly, the dUTP-labeled cells were exclusively confined to the metanephric mesenchyme, and a very few cells exhibiting apoptosis were localized to the ureteric bud branches or epithelial elements of the nascent nephrons.


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Fig. 8.   A-C and E-G: fluorescence micrographs showing dUTP-labeled apoptotic cells of metanephric explants treated with sense/nonsense (CONTROL, A-C) or antisense (ANTISENSE, E-G) ODN at 3 days in culture. Sections of the explants were incubated with fluorescein-labeled dUTP, and the reaction was catalyzed by TdT. Compared with control, antisense-treated explants reveal markedly accentuated apoptosis that is readily seen in high-magnification photographs (G vs. C, arrowheads). Accentuation of apoptosis can also be appreciated in Epon-embedded toluidine-stained sections (D and H). Many cells with apoptotic darkly stained nuclei are seen in the MEST antisense ODN-treated explants (H, arrowheads). A basal level of apoptosis is also seen in explants treated with sense/nonsense ODN (D, arrowhead) at this stage of the metanephric culture.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The MEST gene was originally discovered in a mouse embryonal carcinoma cell line, MC12, and is exclusively expressed in the embryonic and extraembryonic mesoderm in early postimplantation embryos (41). The mRNA expression of MEST is solely confined to the mesoderm-derived tissues during midgestation, while its expression declines rapidly during the subsequent stages and eventually disappears in adult life. This is also the pattern of many other genes and especially of the imprinted genes characterized by the expression of mRNA from a single parental allele (21). Imprinted genes are believed to be involved in embryonic growth and behavioral development and at times, because of their inappropriate expression, may exert effects similar to those of protooncogenes and tumor suppressor genes (21). In this regard, a target mutation of MEST, a paternally imprinted gene, seems to cause a general growth retardation of embryonic and extraembryonic structures, which may otherwise appear normal (34). Taking advantage of systemic screening and cDNA subtraction hybridization between normal and parthenogenetic embryos, Kaneko-Ishino et al. (22) isolated several paternally expressed genes (Pegs), one of which, i.e., Peg1, had coding sequences identical to that of MEST. With the application of similar technology, i.e., SSH-PCR, we were also able to isolate MEST from fetal renal tissue, which contains mesodermal or mesenchymal elements during embryonic life. Although MEST may play a role in placental angiogenesis during embryonic life (36) and has partial nucleotide sequence homology with the alpha /beta -hydrolase fold family (29), no definitive functions have been assigned to this molecule. The MEST cDNA has an open reading frame of 335 amino acids, and a ~35-kDa recombinant protein has been recently isolated in our laboratory (unpublished data). It does not appear to have any signal peptide, but hydropathic analysis suggests that it is a transmembrane protein with two transmembrane helices, suggesting that it may be involved in various cellular interactions. Nevertheless, identification of MEST in the fetal kidney at a time when epithelial-mesenchymal interactions are prevalent led us to investigate its developmental regulation and role in fetal nephrogenesis.

As in whole mouse embryos (41), MEST expression was highest during midgestation in the developing metanephros (Fig. 1). Interestingly, the spatiotemporal studies revealed that at that stage mRNA expression is exclusively confined to the metanephric mesenchyme (Fig. 2). During the same time frame, the mesenchyme, an intriguing component of the metanephros, undergoes inductive transformation with a change to an epithelial phenotype from which glomerular and tubular elements are formed (12, 23, 44, 47). In other words, the genes or their putative proteins expressed in the mesenchyme, in conjunction with those expressed in the ureteric epithelial bud, could modulate epithelial-mesenchymal interactions in a paracrine or juxtacrine manner, which is essential for the formation of nascent nephrons. Similar to MEST, several glycoproteins exhibit stage-specific expression in the metanephric mesenchyme. Among the classic mesenchymal cell-derived proteins that are expressed in the mesenchyme are interstitial collagens (types I and III), fibronectin, tenascin, fibrillin-1, and nidogen (14, 23, 38, 44, 47). Although they may follow the inductive events in the metanephric mesenchyme, their mRNA expression is somewhat asynchronous. For instance, the interstitial collagens disappear at day 11 after the initial induction, while the expression of fibronectin and its splice variants EIIIA, EIIIB, and V persists to day 15/16 of murine gestation (39). Similar to tenascin, a mesenchymal protein containing fibronectin-like domains, such as fibrillin-1, is transiently expressed in the induced mesenchyme during midgestation around the metanephric epithelial condensates, S-shaped bodies, and tubules (3, 26). In addition to these structural proteins, some of the other macromolecules relevant to the biology of the mesoderm and metanephric development and expressed in the mesenchyme include some of the secretory proteins, i.e., matrix metalloproteinase-2 (40), integrins, i.e., alpha beta and alpha 4beta 1 (30, 31), and cell adhesion molecules, i.e., N-CAM (28) and BMP-7 (10). Although spatiotemporal expression of mesenchymal proteins has been well described in the literature, the mechanisms of their transcriptional regulation with respect to embryonic development have not been clearly defined.

Transcription regulation during embryonic development has been investigated mainly for the genes that exhibit maternal or paternal imprinting (4). Embryonic development in mammals is distinct from that in other vertebrates, because it depends on a small number of imprinted genes that are expressed from the maternal or paternal genome. Gene inactivation experiments suggest that imprinted genes regulate embryonic and placental growth and that DNA methylation is part of the imprinting mechanism (4). This means that many of the genes that are expressed in earlier stages of mammalian development have their CpG sequences progressively methylated so that their expression disappears in later life. An exception may be the housekeeping genes. Such a mechanism may be responsible for the gene inactivation of MEST, the evidence of which was derived from various cell lines derived from embryonic and adult tissues (37). This contention is further strengthened by the present studies, where DNA methylation could be demonstrated in the EcoRI-ScaI-digested fragments of the genomic DNA isolated from E15 and E19 (newborn) kidneys (Fig. 3). Although one has to take into account other mechanisms involved in gene inactivation, it appears that DNA methylation certainly contributes, to a certain degree, to the loss of MEST mRNA expression during the development of the mammalian metanephros.

Because MEST is expressed in the mesenchyme with a potential role for involvement in cell-cell or epithelial-mesenchymal interactions, the next question would be the identity of its interacting partner. Among the various epithelial-mesenchymal or cell-matrix interactions, the best prototype is the dystroglycan-integrin (cell-matrix) nidogen-laminin complex, which is responsible for signal transduction and modulation of nephrogenesis. Interference in these interactions, for example, antibody-mediated disruption of binding of laminin-1gamma 1 chain with nidogen, laminin alpha 1-beta 1-gamma 1 chains with alpha 6beta 1-integrin receptor, or laminin alpha 1 chain with dystroglycan results in the inhibition of nephrogenesis and formation of polarized epithelium (11-16, 45). Another pertinent aspect of cell-matrix interactions is the spatiotemporal concentration of a given morphogenetic modulator that is relevant to organogenesis. In this regard, it has been shown that interference in the concentration of cell surface proteoglycans that are expressed at the epithelial-mesenchymal interface, by biosynthetic or enzymatic means, results in the branching dysmorphogenesis of various organ systems (23). Besides the structural proteins, some of the mesenchymal secretory proteins that have enzymatic activities, e.g., metalloproteinases, also play a role in renal development by complexing or interacting with those expressed in the ureteric bud epithelia (27, 40). In view of these considerations, it is imperative to search for the molecule(s) that can interact with MEST so that its role in metanephric development can be defined. One possible candidate would be the H19 gene, which is imprinted and developmentally regulated and is expressed in various embryonic epithelia (19). Studies to investigate the interactions between these genes would certainly yield some answers as to their role in metanephric development. Although H19 may not have its translated protein, it is conceivable that these two molecules may modulate renal development via domain-specific DNA-protein, rather than protein-protein, interactions, similar to those postulated for the IGF2-H19 domain (51). Such interactions should be considered speculative in view of the lack of any experimental data, and thus further work is needed to arrive at definitive conclusions.

Intriguingly, the body weight of mice with a targeted mutation of the MEST is 25% of the body weight of their wild-type counterparts. However, they appear to have a normal phenotype; therefore, one may deduce that MEST may have no definitive role in metanephric development, although the status of the urogenital system was not described (34). However, one can argue that the lack of a markedly abnormal phenotype may be due to the compensation by the activity of other imprinted Peg-1-like genes, e.g., Peg-1-Peg-10. A similar scenario has been reported for one of the structural proteins, such as tenascin, where tenascin-deficient mice were shown to have no abnormal phenotype (42). Nevertheless, the in vitro experiments revealed a decreased expression of tenascin when tubulogenesis was inhibited (3). Similarly, the role of several other molecules that are expressed at the epithelial-mesenchymal interface, e.g., ganglioside-3, in metanephric development could be delineated only in in vitro organ culture systems (43). To elucidate the role of MEST in vitro, gene disruption experiments were carried out by inclusion of antisense ODN in the organ culture medium. Antisense technology has been employed in studying various developmental processes (6, 49) and is a reliable method, provided that appropriate controls are included in a given experiment. Also, the specificity of the antisense ODN can be maintained if they are used at relatively low concentrations, as described elsewhere (24, 26, 27, 49). A dose-dependent reduction in the size of explants and a decrease in the population of tubules and glomeruli were observed with the antisense treatment (Fig. 5). The effects were considered to be specific, since RT-PCR analysis revealed a selective decrease in MEST mRNA expression, and no change in the beta -actin expression was observed (Fig. 6). Under basal conditions, that is, in untreated explants, the MEST expression remained constant throughout the period of organ culture (Fig. 4), indicating that the antisense-induced decreased expression was not related to the various stages of the culture of the metanephroi. The major question was whether the decrease in MEST mRNA was the result of altered morphology in the explants or vice versa. To address this issue, in situ hybridization studies were performed at the midpoint of organ culture, and the results revealed a notable decrease of the mRNA expression in the mesenchyme before any change in the morphology of the explants (Fig. 7). The fact that the MEST deficiency preceded the morphological change suggests that it most likely plays a role in in vitro metanephric development.

One can speculate a number of mechanisms by which MEST deficiency resulted in the loss of nascent nephrons and growth retardation of the metanephros. First, there may be interference in the functions of growth factor receptors that are expressed in the metanephric mesenchyme that resulted in the growth retardation. Several growth factors, including platelet-derived growth factor, influence the biology of the metanephric mesenchyme (2, 18). Another interesting possibility that one can entertain would be the "dropout" of the cells from the explants by apoptosis. Apoptosis is an integral process of embryonic development in which the cells drop out and are replaced by other cells to maintain a balance in the induction and proliferation of more differentiated cells (23). Apoptosis is normally seen at basal levels in the developing metanephros, and it is mainly confined to the uninduced mesenchyme (32). Apoptosis was seen to a mild degree in the explants that had marked alterations in the morphology at day 5 of culture (Fig. 5), whereas fulminant apoptosis was observed at day 3 (Fig. 8). This would suggest that the loss of cellular elements or nephrons observed at day 5 was related to dropout of cells during the course of culture with the treatment of MEST antisense ODN that specifically targets the uninduced mesenchyme. Also, a decreased [3H]thymidine incorporation observed with the antisense treatment would further support this contention.

In summary, we have described a paternally imprinted gene exclusively expressed in the embryonic metanephric mesenchyme, the mRNA expression of which abruptly decreases during the neonatal period due to DNA methylation and could conceivably play a role in metanephric development. It is uncertain whether MEST is involved in the cell-cell interactions. However, the fact that its hydropathic profile suggests it to be a transmembrane protein should motivate study of the cell-cell or epithelial-mesenchymal interactions between various imprinted genes that are developmentally regulated and modulate mammalian metanephrogenesis.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-28492 and DK-60635 and Japanese Ministry of Education Grants 10770199 and 11470218. S. Chugh is the recipient of National Institute of Diabetes and Digestive and Kidney Diseases Clinician Scientist Development Award DK-61275.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. S. Kanwar, Dept. of Pathology, Northwestern Univ. Medical School, 303 E. Chicago Ave., Chicago, IL 60611 (E-mail: y-kanwar{at}northwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 4, 2001;10.1152/ajprenal.00200.2001

Received 2 July 2001; accepted in final form 28 November 2001.


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DISCUSSION
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