*Max Delbrück Center for Molecular Medicine,
Humboldt University of Berlin, Berlin-Buch, Germany. Franz Volhard Clinic, Humboldt University of Berlin, Berlin-Buch,
Germany.
Correspondence to Dr. Andreas Schedl, Max
Delbrück Center for Molecular Medicine,
Robert-Rössle Str. 10, D-13092 Berlin-Buch,
Germany. Phone: 49-30-9406-2337; Fax: 49-30-9406-2110; E-mail:
aschedl{at}mdc-berlin.de
Abstract. Normal development of the kidney is a highly complex
processthat requires precise orchestration of proliferation, differentiation,
andapoptosis. In the past few years, a number of genes that regulatethese
processes, and hence play pivotal roles in kidney development,have been
identified. The Wilms' tumor suppressor gene WT1 hasbeen shown to be
one of these essential regulators of kidneydevelopment, and mutations in this
gene result in the formationof tumors and developmental abnormalities such as
the Denys-Drashand Frasier syndromes. A fascinating aspect of the
WT1 geneis the multitude of isoforms produced from its genomic
locus.In this review, our current understanding of the structuralfeatures of
WT1, how they modulate the transcriptional and post-transcriptional
activitiesof the protein, and how mutations affecting individual isoformscan
lead to diseased kidneys is summarized. In addition, resultsfrom transgenic
experiments, which have yielded important findingsregarding the function of
WT1 in vivo, are discussed. Finally,data on the unusual feature of
RNA editing of WT1 transcriptsare presented, and the relevance of
RNA editing for the normalfunctioning of the WT1 protein in the kidney is
discussed.
The kidney is crucial for survival. The molecular mechanismsunderlying the
development of this organ, however, are onlybeginning to be elucidated. Three
sets of kidneys develop duringembryogenesis, i.e., the pronephros,
the mesonephros, and themetanephros
(1). The precursors of the
permanent kidney in themetanephros result from reciprocal interactions
between theepithelial ureteric bud and the pluripotent metanephric
mesenchyme.The mesenchyme induces the bud to grow and branch, thus forming
theureter, renal pelvis, and collecting ducts. The ureteric budinduces the
mesenchyme to undergo an epithelial transformation,to condense and form (via
the comma- and S-shaped bodies) themature nephron with the glomerulus,
proximal convoluted tubule,loop of Henle, and distal convoluted tubule. In
the past decade,several genes have been identified that demonstrate spatially
andtemporally distinct expression patterns during kidney developmentand lead
to abnormal organ development when disrupted by genetargetingexperiments (for
review, see reference (2). One
of these genesis the Wilms' tumor suppressor gene WT1. This review
focuseson structural features of and how they relate to WT1 function
duringkidney development. This is particularly relevant because wewant to
understand the basis of WT1 mutations related to humanrenal
diseases, such as the Denys-Drash syndrome (DDS) and theFrasier syndrome
(FS).
The human WT1 gene spans approximately 50 kb and includes 10
exons,which generate a 3-kb mRNA
(3,4).
Four zinc finger motifs inthe carboxyl-terminal portion form the DNA-binding
domain andshare homology with the early growth response gene 1 family,
suggestinga role for the WT1 protein as a transcription factor
(Figure 1A).Up to 24 different
isoforms may result from a combinationof alternative translational start
sites, alternative RNA splicing,and RNA editing. One alternative
translational start site (CTG)is located 204 bp upstream of the major ATG
site, creating anisoform with 68 additional amino acids at its amino terminus
(5).Recently, shorter versions
of WT1, resulting from translationinitiation at the second in-frame ATG, have
also been detected(6). Two
alternatively spliced exons are known, one insertingor excluding exon 5,
which encodes 17 amino acids, and the otheraffecting exon 9 via insertion or
exclusion of three amino acids[lysine-threonine-serine (KTS)] between the
third and forthzinc fingers. The ratio of splice variants is highly conserved
innormal fetal kidney and is maintained throughout development
(7).Finally, an RNA-editing
site has been identified in transcriptsof rat and human WT1 genes,
leading to the replacement of anamino acid in exon 6
(8). A more detailed
description of RNAediting is provided below.
Figure 1. (A) Structural features of the Wilms' tumor suppressor gene WT1.
The schematic drawing depicts the main features of the WT1 protein and its
various isoforms (numbers in parentheses refer to references). (B) Frasier
syndrome (FS). The schematic drawing indicates the most common intronic
mutations affecting lysine-threonine-serine (KTS) splicing. aa, amino acid;
PU, proteinuria; NS, nephrotic syndrome; ESRF, end-stage renal failure.
It is now well established that WT1 can bind, via its zinc fingers,to the
promoter regions of >20 putative downstream targetgenes (for review, see
reference (9). WT1 effects on
transcriptioncan be either repressing or activating, depending on the cell
typeand the target gene with which WT1 interacts. Most target geneshave been
identified in transfection assays, and data shouldbe interpreted with care,
because the effects of chromatin orother interacting proteins are usually
neglected in vitro. However,in vitro experiments have been
corroborated by in vivo datafor an increasing number of target genes
that are either activatedor repressed by WT1. These genes include those for
the cellsurface receptor epidermal growth factor receptor
(10), thetranscription factor
paired-box-containing gene 2 (PAX2)
(11),and the growth factor
insulin-like growth factor-2
(12) as targetsdownregulated
by WT1. Amphiregulin, a member of the epidermalgrowth factor family, has been
shown to be a target that canbe activated by WT1
(13).
Because WT1 is expressed in so many different isoforms, whichare highly
conserved throughout evolution
(14,15),
what propertiesdo the different isoforms have? The addition of a 68-amino
acidamino-terminal region from an alternative translational startsite has
been demonstrated to have little effect on the transcriptionalactivity of the
protein (5). In contrast,
amino-terminally shorterversions seem to lead to reduced repression but
increased activationpotential
(6). How important these minor
products are for WT1function in vivo remains unclear. However,
alternatively splicedexon 5 has been suggested to increase the repressing
effectof WT1 on some promoters
(16). Interestingly, the
presence ofthe alternative exon 5 has been found only in mammalian species
(14,15),
indicatingthat it has been adopted by WT1 at a later stage in
evolution.
Increasing amounts of compelling data suggest a role for WT1not only as a
transcription factor but also as a potent modulatorat the
post-transcriptional level. WT1 is able to bind to RNAof the insulin-like
growth factor-2 gene (17). How
the WT1 proteininteracts with RNA is still under discussion. Although one
studyreported that the zinc finger region (in particular, zinc finger1) is
necessary for these protein-RNA interactions
(17), computermodeling
suggests that an RNA recognition motif in the amino-terminalregion is
important (18). These two
regions may be equally important,with the amino-terminal region serving as a
nonspecific bindingdomain and the zinc finger region providing sequence
specificity.Interestingly, the distinct isoforms of WT1 seem to behave
differently,in terms of their nucleic acid-binding capacity. Whereas variants
lackingthe KTS sequence (-KTS) bind to DNA sequences and can act aspotent
transcriptional regulators, KTS-containing isoforms (+KTS)demonstrate higher
affinities for RNA. The actual function ofthe interaction of WT1 with RNA is
still unknown; it could stabilizeor destabilize certain RNA forms or could be
involved in post-transcriptionalmodifications such as RNA editing or
splicing. Evidence forthe latter is derived from immunocolocalization and
immunoprecipitationexperiments. In transfection studies, the +KTS isoforms
seemto preferentially colocalize with splicing factors, whereas-KTS forms
exhibit more diffuse nuclear localization similarto that of transcription
factors (19). Furthermore,
direct interactionof WT1 with the splicing machinery of the nucleus and
particularlythe splicing factor U2AF65 has recently been demonstrated by
two-hybridanalysis and biochemical studies
(20,21).
Despite these excitingand provocative data, the function of these isoforms
in vivois still unclear.
WT1 Mutations and Implications for Human Renal Disease and
Tumorigenesis
The identification of WT1 mutations in human patients has provided
uswith important clues regarding the cellular and developmentalfunctions of
this gene. A variety of WT1 mutations, which eitheraffect
development or induce tumor formation, have been identified.Developmental
defects include the dominant Wilms' tumor/aniridia/genitalanomaly/mental
retardation syndrome, DDS, and FS, whereas tumorsinclude nephroblastoma
(Wilms' tumor), mesothelioma, breastcancer, aggressive desmoplastic small
round cell tumor, andleukemia. For reasons of space, we restrict our
discussion todiseases affecting the urogenital system.
Denys-Drash Syndrome
The DDS is a rare congenital childhood syndrome that includesdiffuse
mesangial sclerosis, severe hypertension, a steroid-resistantnephrotic
syndrome that rapidly progresses to end-stage renaldisease before the age of
5 yr, male pseudoherm-aphroditism,and a high risk of developing Wilms' tumor
(22,23).
In additionto these symptoms, incomplete forms with renal involvement and
eithervarying genital anomalies or Wilms' tumor have been described
(24).Podocytes exhibit
decreased or absent WT1 expression but persistentPAX2 expression
(25). Genital anomalies in
46,XY patients withDDS vary from the total absence of epithelial structures,
with"streak" gonads, to the presence of both the wolffian (male)
andmullerian (female) ducts or female gonads and genitalia. Kidney
transplantation(recurrence of the glomerular phenotype has not yet been
observed)and early bilateral nephrectomy before the development of Wilms'
tumorare life-saving treatments. However, a late manifestation ofWilms'
tumor after renal transplantation in a child with retrospectivelysuspected
DDS has been observed
(26).
More than 60 germline mutations (both familial and de novo)have
been described, which essentially leave the WT1 proteinintact but affect the
DNA-binding zinc finger domain
(27) (forreview, see
reference (28). Most of these
mutations are missensemutations within exons 8 and 9, which code for zinger
fingerdomains 2 and 3, respectively. A mutation hot spot seems tobe present
at nucleotide 1180 in codon 394, leading to the replacementof arginine with
tryptophan (R394W). Only a few deletions, insertions,and nonsense mutations
result in truncated proteins. All mutations,however, alter the structure of
the DNA-binding domain, thuschanging its ability to bind to both DNA and RNA.
The severephenotype produced by only one mutated allele raises several
questions.Does the mutation act in a dominant way with new devastating
effects?Or does the mutated protein act in a dominant-negative manner,
activelysuppressing and inactivating the influence of the wild-typeallele?
Evidence for the latter hypothesis has been obtainedfrom both in
vitro and in vivo analyses. First, WT1 is ableto homodimerize,
and a nonfunctional allele may thus rendersome functional WT1 products (from
the wild-type allele) nonfunctional
(29).This could explain the
much more severe defects observed forpatients with DDS, compared with
patients with heterozygousdeletions of WT1. Second, gene-targeting
experiments with atruncation of WT1 in the zinc finger region
demonstrated a phenotypewith some of the characteristics of DDS
(30). Interestingly,
expressionfrom the mutated allele accounted for only 5% of the total amount
ofWT1 protein, suggesting that even minor amounts of mutant proteincan have
devastating effects on urogenital development.
Frasier Syndrome
FS includes a slowly progressing nephrotic syndrome, attributableto
minimal glomerular changes or focal segmental glomerulosclerosis,and complete
male to female gender reversal in 46,XY patientsbut no signs of Wilms' tumor
(31). Gonadal development in
XXfemale patients is normal. In contrast to DDS, end-stage renaldisease
develops more slowly and at a later stage in life (forreview, see reference
(28). The late onset of renal
diseasein some patients with FS suggests that WT1 is also importantfor
normal functioning of the kidney after development has beencompleted.
Four similar point mutations downstream of the second splicedonor site in
intron 9 have been detected in FS
(32)
(Figure 1B).In vitro
experiments have shown that the intronic mutationsinterfere with recognition
of the second splice donor site andresult in loss of the +KTS isoform from
the mutated allele.Interestingly, FS mutations are dominant, and the wildtype
allelestill produces both the +KTS and -KTS isoforms. Therefore, amere
change of the ratio of the two isoforms must be responsiblefor the severe
developmental defects, emphasizing the importanceof the +KTS isoform for
urogenital development.
Recently, a mutation in exon 9 that did not alter the isoformratio in two
patients classified as having FS was described
(33).In addition, intronic
mutations characteristic of FS have beenobserved in patients with DDS.
Furthermore, a patient with theclassic FS mutation but an unexpected Wilms'
tumor has beendescribed (34).
These recent data fuelled a controversy regardingthe correct classification
of DDS and FS. The majority of publisheddata clearly distinguish between the
two syndromes, on bothclinical and molecular biologic grounds. However, FS
could alsobe an atypical subtype of DDS, because of the variety of incomplete
syndromesand overlapping mutational characteristics. The very similar
phenotypesin FS and DDS, together with the fact that patients with FSdo not
produce abnormal WT1 proteins, support the hypothesisthat DDS is attributable
to dominant-negative mutations, ratherthan a new function of the mutated
protein. Classification ofthe two syndromes should therefore be performed at
the molecularlevel, rather than on the basis of the observed phenotype.
Isolated Diffuse Mesangial Sclerosis (IDMS) and Idiopathic Persistent
Nephrotic Syndrome
In rare familial cases, only the renal phenotype of either DDS(diffuse
mesangial sclerosis) or FS (focal segmental glomerulosclerosis)is observed.
Additional symptoms of the diseases, includingabnormalities of the gonads,
are not found (35). IDMS has
beenshown to be associated with de novo mutations of both exon 8and
exon 9 (24), whereas the most
frequent DDS mutation (R394W)has not been detected. In addition, some
patients with IDMSdo not present with WT1 mutations at all
(36). Although thedescription
of FS has been restricted to XY female patients,the FS mutation can also be
detected in 46,XX patients. Thesefemale patients exhibit normal development
of the genital system,whereas kidney biopsies reveal segmental glomerular
sclerosis
(37,38).
Froma nephrologic point of view, one should search for both intronicand
exonic mutations of WT1, to assess the risk of inheriteddiseases of
the kidney or gonads among the children of patients.This is particularly
important for female patients with focalsegmental glomerulosclerosis or
karyotypic 46,XY male patientswith a family history of therapy-resistant
nephrotic syndrome.The classification of these renal diseases is difficult,
becauseof the heterogeneity of observed phenotypes and the different
mutationsof the WT1 gene.
Wilms' Tumor
Wilms' tumor (nephroblastoma) is a childhood tumor of the kidney(1:10,000
live births) originating from the metanephric blastema.Nephrogenic rests,
caused by the developmental arrest of nephrogenesis,are thought to be
precursors of Wilms' tumor formation
(39).The WT1 gene
has been proven to play an essential role in thepathogenesis of this tumor.
Heterozygous deletions on chromosome11p13 in human subjects are associated
with the congenital Wilms'tumor/aniridia/genital anomaly/mental retardation
syndrome,which consists of a high risk of developing Wilms' tumor anda
combination of developmental abnormalities, including aniridia,genital
anomalies, and mental retardation
(40,41).
Althoughabnormalities of the urogenital system can be attributed tothe
deletion of WT1, other abnormalities are attributable tothe deletion
of additional genes mapping to this region, includingPAX6 and
reticulocalbindin
(42,43).
A 25-bp intragenic deletionin a sporadic Wilms' tumor
(44) and a germline intragenic
deletionin a patient with bilateral nephroblastoma
(45) provided thefirst solid
evidence that WT1 is indeed the long-sought tumorsuppressor gene.
WT1 mutations, including deletions, truncations,translocations, and
missense mutations, were observed in approximately20% of Wilms' tumors. The
cause in the other 80% of cases isunclear but may involve mutations in genes
acting upstream ordownstream of WT1.
The expression pattern of WT1 during embryogenesis is highly
complex
(46,47).
Theinitial differentiation of the metanephric mesenchyme seemsto be
independent of WT1
(48), and the gene is only
weakly expressedin the uncondensed metanephric blastema
(49). Expression dramatically
increasesduring the mesenchyme-to-epithelium conversion in the condensing
mesenchyme,when the renal vesicles and commashaped bodies are formed
(Figure 2, A and B).Similar
mesenchyme-epithelium interactions havebeen observed in a variety of
different developing organs
(50).Gene expression is
highest in the proximal part of the S-shapedbody, which flattens to form the
glomerular podocytes. It hasbeen reported that WT1 activity is
essential for the switchingof cells between mesenchymal and epithelial cell
states. Terminallydifferentiated cells, such as epithelial tubular cells,
exhibitno WT1 expression, whereas cells with epithelial/mesenchymalswitching
potential, such as podocytes, continue to produceWT1
(51,52).
In the fully developed kidney, WT1 expression persistsin the
podocytes and, at a much lower level, in epithelial cellsof the Bowman's
capsule, suggesting that the gene may play arole in more than the initial
stages of kidney development.In addition to the kidney, high levels of
WT1 expression arefound in the spleen
(53), the mesothelium, and the
genital ridgesthat develop into testes or ovaries, depending on the presence
orabsence of the Y chromosome
(54).
Figure 2. Immunohistochemical analysis of Wilms' tumor suppressor gene 1 (WT1) (A and
B) and paired-box-containing gene 2 (PAX2) (C and D) (red) and synaptopodin (A
to D) (green) during kidney development (embryonic day 17.5). (A) Evidence
that WT1 is expressed in the condensing blastema (arrowhead), the proximal
part of S-shaped bodies (arrows), and the podocyte layer of functional
glomeruli (asterisks). (B) High-power view of a glomerulus, showing nuclear
staining for WT1 in podocytes and cytoplasmic staining for podocyte-specific
synaptopodin. (C) Evidence of PAX2 expression in the collecting ducts (arrows)
and condensing blastema (arrowheads) but not in mature glomeruli (asterisks).
(D) High-power view of the reciprocal interactions between the branching
ureteric bud (UB) and the condensing metanephric blastema (arrowhead), both of
which express PAX2. Sections in A, C, and D were counterstained with
diaminophenylindole (blue). Magnifications: x200 in A and C; x630
in B and D.
The complex pattern of WT1 expression suggests that the functionof WT1 is
required at multiple stages during kidney development,and data obtained using
genetically modified mice are beginningto support this hypothesis. In
knockout mice, the ureteric budfails to grow out and the metanephric blastema
undergoes apoptosis(55).
Interestingly, apoptosis occurs even when blastema fromknockout animals is
recombined with ureteric buds from wild-typeanimals in organ culture
experiments. These data suggest thatWT1 has at least two functions
during this first stage of kidneydevelopment. First, it may be required for
the inductive signalingthat induces the outgrowth of the ureter from the
mesonephros.Second, it seems to be involved in either survival or the
receptionof the survival signal from the ureteric bud.
Recent transgenic experiments from our own laboratory have characterizeda
second phase of WT1 action
(56). We examined whether the
humanWT1 locus is able to complement the knockout mutation in mice,
whichare known to die at embryonic day 13.5 because of multiple organ
defects,with renal and gonadal agenesis. To ensure proper expressionof all
isoforms as well as correct transcriptional regulationof the WT1
gene, we decided to introduce the entire genomiclocus on a 280-kb yeast
artificial chromosome (YAC). Interestingly,the transgene only partially
rescued the knockout phenotypewhen it was crossed into the WT1 knockout
background. The transgenecomplementation, however, led to unexpected
postnatal death,within 48 h, because of kidney failure. Histologic analyses
revealedvarying degrees of complementation, i.e., a complete absence
ofureteric budding, outgrowth of the ureter without further branching,or
normal development of the nephrogenic zone with nephrogenesisto the stage of
comma-shaped bodies. Mature glomeruli, however,were never formed. Because the
experiments were not performedwith a defined genetic background, the varying
degrees of kidneydevelopment may be attributable to modifier genes. The
defectsin nephron formation led to increased apoptosis and overallreduced
kidney size (Figure 3).
Interestingly, after the uretericbud invasion, condensation of the
metanephric blastema seemedto occur normally
(Figure 3E), suggesting that
WT1 is not requiredfor this first step of nephron formation. Taken together,
thesedata indicate a continuous requirement for WT1 during nephrogenesis.
Figure 3. Complementation analysis of the WT1 knockout phenotype. WT1 knockout mice
complemented with a human, 280-kb, yeast artificial chromosome (C to E) have
smaller kidneys, with increased amounts of stroma (S), compared with kidneys
of wild-type animals (A and B). Branching of the ureteric bud (UB) and
condensation (arrowheads) occur normally (E), but no functional glomeruli
(asterisk; compare with B) are observed. Magnifications: x40 in A and C;
x200 in B and D; x400 in E.
As illustrated in our rescue experiments, a multitude of positiveand
negative feedback mechanisms operate during the formationof an organ such as
the kidney. For a deeper understanding ofdevelopment, it is important to
examine these interactions ona molecular level. With respect to WT1,
the transcription factorPAX2 is particularly interesting.
PAX2 is an evolutionarilyhighly conserved member of the paired box
gene family (57) thatplays a
pivotal role during the development of the kidney, centralnervous system, and
sensory organs. Ectopic expression of PAX2is associated with
deregulated proliferation during nephrogenesis
(58),whereas PAX2
null mutants lack kidneys, ureters, and gonads
(59,60).
PAX2is down-regulated in precursor cells of the visceral glomerular
epithelium(prepodocytes) and mature glomeruli
(Figure 2C), simultaneously
withincreased WT1 expression, suggesting that PAX2 may be a target
ofWT1. Indeed, binding assays and cotransfection studies
demonstratedthat WT1 can repress PAX2 transcription by binding to
regulatorysequences in the promoter region of PAX2
(61). Interestingly,the
converse, i.e., regulation of the WT1 promoter by
PAX2,also seems to occur. Two PAX2-binding sites have been
identifiedin the upstream region of WT1
(62). Indeed, transfection
experimentsresult in high levels of transcriptional activation of
WT1 byPAX2
(63). Taken together, these
data suggest that the two genesare targets of each other. It is presently
unclear whether thedown-regulation of PAX2 is necessary for proper
kidney developmentand is exclusively mediated by WT1. From our
rescue experiments,however, it seems unlikely that PAX2 is the only
target of WT1during nephron formation, because a lack of
PAX2 repression(resulting in ectopic expression) is unlikely to
cause the observedcomplete absence of nephrons.
The incomplete kidney development in our rescued animals issomewhat
difficult to understand but could be explained by insufficientlevels of WT1
expression. Indeed, RNA analysis indicated three-to fivefold lower levels of
transgene expression, compared withthe endogenous WT1 gene. In
addition, deregulation of post-transcriptionalmodifications may contribute to
the insufficient action of theWT1 transgene. The ratio of the four
different mRNA splice forms,however, was identical to the wild-type
situation. This promptedus to investigate whether the partial rescue of the
urogenitalsystem in WT1 null mice with the WT1 YAC
transgenic lines couldbe attributed to deregulated RNA editing.
RNA editing is a post-transcriptional processing event thatgenerates a new
transcript with nucleotides that do not matchthe bases present in the
original genomic sequence. In mammals,direct nucleotide modifications via
nucleotide deamination (cytidine-to-uridineor adenosine-to-inosine
conversion) are the most frequentlyobserved events (for review, see reference
(64). A differentediting
mechanism has been described for WT1 RNA, in which auridine is
converted to a cytidine at nucleotide 839, resultingin the replacement of
leucine by proline (leucine/proline dimorphism).This process is thought to be
developmentally regulated andoccurs in adults rather than neonates
(8). On a functional level,the
proline-containing edited isoform seems to be a less repressive
transcriptionalmolecule for growth-promoting genes, compared with the
noneditedisoform. The physiologic consequences, however, have not yetbeen
clarified.
Because RNA editing has not yet been described in mice, we firstattempted
to detect edited forms of WT1 mRNA in wild-type mice,using tissues from rats
and a human patient as positive controlsamples. We used single-strand
conformation polymorphism analysisto detect the edited RNA, which should
alter the mobility ofsingle-stranded DNA. In the case of editing, the PCR
productwould contain two slowly moving ±-strands of noneditedT/A839
DNA and two strands of the faster moving edited C/G839DNA (see the legend to
Figure 4 for experimental
details). Surprisingly,kidneys from mice, rats, and a human patient revealed
only thenonedited RNA form and not the four strands expected for editedRNA
samples (Figure 4) (data for
Sprague-Dawley, Wistar-Kyoto,and spontaneously hypertensive rats are not
shown). The resultsof our analysis were in marked contrast to those of
earlierstudies, in which RNA editing was observed in 30% (adult ortumorous
kidney) to 90% (Rat 2 cell line) of mRNA
(8). Couldit be that our
experimental design overlooks a small but relevantamount of edited RNA? To
exclude this possibility, we changedour strategy to a PCR-based subcloning
method. As shown in Figure 5,most colonies harbored the nonedited form, with three bandsafter
MnlI digestion (Figure 5, A and
B). Only 0.4% of the clonesfrom adult mice (1 of 220 colonies)
and 0.7% of those from rats(1 of 126 colonies) exhibited the edited form in
the kidney(Figure 5C). In
addition, we examined 30 colonies from fourdifferent adult Sprague-Dawley
rats (kidney for all four rats,liver for two rats, and lung for one rat). In
rat lung, we detectededited RNA in 2 of 30 clones, whereas all other organs
exhibitedonly nonedited forms (Figure
5C).
Figure 4. Single-strand conformation polymorphism analysis to detect RNA editing.
Total RNA was isolated from snap-frozen kidneys [kidneys from adult female
(MK) and fetal (f MK) (embryonic day 9.5) NMRI mice and a human cadaveric
kidney from a hypertensive adult patient (HK)]. cDNA was prepared using 1
µg of RNA, 25 units of avian myeloblastosis virus reverse transcriptase
(Boehringer Mannheim) in 10x reverse transcription buffer, 20 units of
RNAsin, 100 µM dNTP, and 100 µM random hexanucleotide primers, in a
total volume of 20 µl. The reaction was performed at 42°C for r 1 h and
at 95°C for 7 min. The forward primer for PCR was placed at the end of
exon 5 (5'-CCACGGTATAGGGTACGAGA-3'). We chose a reverse primer
located at the beginning of exon 7 (5'-CAGATACACGCCGCACATC-3').
Although identical in length (116 bp), the PCR products varied at single
nucleotides between the different species, resulting in differently moving
bands in single-strand conformation polymorphism analyses. The PCR products
were diluted (1:1) with stop solution (deionized 95% formamide, 0.05%
bromphenol blue, 20 mM ethylenediaminetetraacetate), denatured at 95°C for
5 min, kept on ice for 3 min, and immediately loaded on a 0.5x
Hydrolink-MDE polyacrylamide gel (BioWhittaker Molecular Application,
Rockland, ME), at 3-W constant wattage, for 12 h at 4°C. The gel was
stained using a silver staining kit. Only two bands of the nonedited form were
detected in all samples.
Figure 5. Colony PCR and MnlI digestion of PCR products. The PCR product of
the reverse-transcribed RNA (Figure
4) was subcloned (TOPO TA cloning kit; Invitrogen, San Diego, CA).
(A) The 116-bp PCR product contains either two MnlI restriction sites
(nonedited CTC form, three resulting fragments) or one MnlI site
(edited CCC form, two fragments) because of the T839/C839 conversion,
resulting in loss of one restriction site. (B) MnlI digestion of the
PCR product from the majority of colonies revealed the nonedited form. (C) The
prevalence of RNA editing in a mouse and four diferent rats (Sprague-Dawley
strain; organ numbers refer to the same animals) is shown.
As expected from the sequence conservation among different species,we were
able to demonstrate RNA editing also in the kidney ofmice, suggesting that
RNA editing is relevant for biologic processes.However, the low prevalence in
adult mice, as well as in "control-species"rats and hypertensive
patients, is in striking contrast to thepublished data. The biologic effects
of RNA editing of the WT1gene might be more variable than expected.
RNA editing has notbeen observed in 15 primary Wilms' tumors
(65) or in the partialrescue
of the kidney phenotype with the WT1-YAC transgenic linein
WT1 knockout mice. To explain these conflicting results,subtle
differences in the genetic background, nutritional andgender influences, or
circadian effects (day/night course) mightbe considered.
Research in the past decade has proven that WT1 has a criticalrole in
proper kidney development. Disrupted gene expressioncan lead to developmental
abnormalities, as well as tumor induction.Target genes for -KTS isoforms have
been identified, but whatis the role of other WT1 variants? The functions of
the varioussplice variants, the regulation of transcription, and splicingof
the gene or RNA editing are far from being understood. Itwill be fascinating
to generate mouse models for DDS by insertingthe most common point mutation
or to mimic the intronic KTSsplice mutation observed in FS. This will enable
us to investigatethe effects of altered isoform ratios or the developmental
andphysiologic functions of individual isoforms, by crossing animalswith the
heterozygous mutation to homozygosity. This will allowus to study the
association of individual isoforms with componentsof the nuclear machinery,
such as transcription and splicingfactors, in a natural environment. Is an
altered WT1 isoformratio responsible for gender determination, and could
decreasedgene expression be associated with abnormal genital development?
Doesa DDS mutation in exon 8 affect genital development more severelythan
does a mutation in exon 9, as recently suggested
(24)?More insight into
molecular mechanisms will certainly permitbetter classification of the
different syndromes.
WT1 has been shown to control cellular proliferation and
mesenchyme-to-epitheliumtransitions. It is tempting to speculate on whether
WT1 makescells of mesodermal origin susceptible to inductive signals.It will
be exciting to further investigate the function of deregulatedWT1 activity by
ectopically expressing the gene under the controlof a renal tissue-specific
promoter. What will happen if WT1is constantly expressed in cells that
normally do not exhibitgene activity after differentiation? Will these cells
returnto their origins, undergo apoptosis, or proliferate into tumorcells?
What is the possible role of WT1 in fully differentiatedcells, such as
Sertoli or granulosa cells? Expression of PAX2under the control of WT1 will
allow us to gain insight intothe regulation and function of podocytes. It is
fascinatingto speculate on whether PAX2 overexpression can inactivate WT1
functionor may be associated with the loss of WT1 activity and cell
dedifferentiationat certain stages of nephrogenesis. These questions are
awaitinganswers from studies with transgenic animal models, as expectedsoon.
Such studies can ultimately help us determine the appropriateconditions for
gene therapy for patients with deregulated WT1gene function.
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Abstract
Introduction
