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
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ABSTRACT |
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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INTRODUCTION |
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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MATERIALS AND METHODS |
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
-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
-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
-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
-actin, was used as the competitive "mutant" DNA template
with an expected 224-bp size of the PCR product (25). The
-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
-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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RESULTS |
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
-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 -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).
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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.
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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.
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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
-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 -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
-actin, no significant
differences in the linearity relationship in the range of logarithmic
dilutions of the competitive DNA between the two groups (
-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 -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 -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.
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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).
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|
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.
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|
 |
DISCUSSION |
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
/
-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., 
and
4
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-1
1 chain with nidogen,
laminin
1-
1-
1 chains with
6
1-integrin receptor, or laminin
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
-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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