Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland
¶ Author for correspondence (e-mail: seppo.vainio{at}oulu.fi)
Accepted 31 March 2004
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SUMMARY |
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Key words: Sprouty, Kidney morphogenesis, Fgf, Ureteric branching, Wnt11, Ret, Gdnf, Metanephric mesenchyme, Mouse
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Introduction |
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Organogenesis of the kidney is initiated when the ureteric bud forms as an
outgrowth from the Wolffian duct. The bud invades the adjacent metanephric
blastema, which has obtained a bias by this stage when induced initially by
signals from the ureter to generate nephrons
(Itaranta et al., 2002). As in
many other developing organs, the epithelial signals lead first to
morphological condensation of the mesenchymal cells in the kidney. A selected
pool of such cells then go on to transform into the epithelium in order to
assemble the nephrons and into its associated terminally differentiated cell
types (Vainio and Lin, 2002
;
Vize et al., 2003
). Alongside
this ureteric branching, each of the ureteric tips acts inductively by
secreting as yet unknown factors that lead to the initiation of nephrogenesis
in the mesenchymal cells that flank the ureter bud
(Saxen, 1987
). The number of
tubules assembled during organogenesis depends on the degree of ureteric
branching. Hence nephrogenesis and ureteric branching both contribute to the
secretory and excretory capacities of the mature kidney
(Brenner and Milford,
1993
).
A member of the transforming growth factor ß (Tgfß) superfamily
of secreted signals, the glial cell line-derived neurotrophic factor (Gdnf),
and its receptor, Ret, are crucial for the initiation of kidney organogenesis,
in that they regulate ureteric bud development
(Airaksinen and Saarma, 2002;
Davies and Bard, 1998
;
Kuure et al., 2000
).
Gdnf is expressed in mesenchymal cells that are adjacent to the
ureteric bud, which expresses Ret and a co-receptor for Gdnf,
Gfra1 (Durbec et al.,
1996
; Pichel et al.,
1996
). Ectopic Gdnf signalling can induce bud formation
from the Wolffian duct (Brophy et al.,
2001
; Pepicelli et al.,
1997
; Sainio et al.,
1997
) and knockout of the ligand or receptors for it will perturb
kidney development by inhibition of ureteric branching, indicating a crucial
organogenetic role (Tang et al.,
2002
; Vainio and Lin,
2002
).
Besides Gdnf/Ret, other classes of secreted signals such as the
Wnt genes and bone morphogenetic proteins (Bmps) regulate kidney development
(Dudley et al., 1999;
Dudley and Robertson, 1997
;
Dunn et al., 1997
;
Miyazaki et al., 2000
;
Moore et al., 1996
). Genes
from these families are expressed in epithelial and mesenchymal tissues and
are also essential for their development. Wnt4, for example, is
expressed in mesenchymal pretubular aggregates, and nephrogenesis fails in the
event of its deficiency. Wnt6, Wnt7b and Wnt11 are expressed
in the ureteric bud, and Wnt11, at least, is functional in ureteric
bud branching in vivo (Itaranta et al.,
2002
; Kispert et al.,
1996
; Stark et al.,
1994
; Vainio et al.,
1999
). The pattern of early bud branching is defective in
Wnt11-deficient kidneys, and this is associated with deregulated
expression of the Gdnf genes.
Ret signalling is also necessary to maintain Wnt11 gene
expression in the ureteric bud. These findings suggest that coordination of
the Wnt11/Gndf/Ret pathways is associated with epithelial branching
during kidney development (Majumdar et
al., 2003).
The Bmps that mediate secondary inductive epithelial-mesenchymal
interaction during organogenesis (Vainio
et al., 1993) are also important secreted signals for kidney
development. Bmp4, for example, is expressed in the kidney mesenchyme
and may in turn antagonize ureteric bud development
(Dunn et al., 1997
;
Miyazaki et al., 2000
). Like
Wnt11, Bmp4 also contributes to organogenesis by controlling the
expression of Gdnf
(Raatikainen-Ahokas et al.,
2000
).
In addition to the epithelial and mesenchymal signals, it has become
evident that the renal stroma is a source of inducers that contribute to
organogenesis. This suggestion is based on the fact that disruption of a
winged helix transcription factor, the Foxd1 (previously BF-2) gene,
which is expressed by cells of the renal stroma, impairs kidney development
via reduced ureteric branching and associated nephrogenesis
(Hatini et al., 1996). The
stromal signals remain elusive, however, even though fibroblast growth factor
(Fgf) Fgf7 and retinoic acid appear to fulfil some of the criteria
(Barasch et al., 1997
;
Batourina et al., 2001
;
Mason et al., 1994
;
Qiao et al., 1999
).
Sprouty was identified in an essential gene in the branching process of the
Drosophila tracheal system that apparently antagonizes both the Fgf
and epidermal growth factor (Egf) signalling pathways
(Casci et al., 1999;
Hacohen et al., 1998
;
Wong et al., 2002
). Four mouse
Sprouty homologues have been identified to date and shown to be
expressed during embryogenesis (Mailleux
et al., 2001
; Zhang et al.,
2001
). In the kidney, the Wilms tumor suppressor protein 1 gene
that encodes a transcription factor crucial for nephrogenesis
(Kreidberg et al., 1993
)
regulates Spry1 as one target gene
(Gross et al., 2003
). This
finding suggests a morphogenetic role for the Spry proteins in the developing
kidney in vivo.
We have previously determined the expression pattern of the mouse
Spry1, Spry2 and Spry4 genes in the developing kidney
(Zhang et al., 2001). As Spry
gene expression is prominent in the ureteric bud, this raised the possibility
that the above factors might contribute to kidney organogenesis by controlling
the inductive signalling pathways. This was addressed by expressing human
SPRY2 in the bud in vivo, which led to severe kidney defects and
included either complete unilateral agenesis of the kidney, a reduction in its
size or a division into separate lobes with an ectopic ureteric bud.
Spry2 signalling contributes to kidney development by coordinating
Wnt11/Gdnf/Fgf7 signalling, as expression of these genes was reduced
in mutant cases and ectopic Gdnf/Fgf7 signalling rescued ureteric bud
branching in vitro. We propose that Spry2 controls ureteric bud
branching during kidney assembly by regulating the reciprocal cooperation
between Wnt11/Gdnf-mediated epithelial-mesenchymal and stromal
Fgf7 signalling.
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Materials and methods |
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Histological analysis of phenotypes
Kidneys were isolated from E11.5, E12.5, E15.5 and E17.5 embryos and
newborn mice, and samples were fixed in Bouin's solution, embedded in paraffin
wax and cut by standard methods. Serially sectioned slices were stained with
Haematoxylin and Eosin. The number of glomeruli was defined as described
previously (Bertram et al.,
1992).
Organ culture and the use of growth factors
Culture conditions for the isolated embryonic kidneys were as reported
elsewhere (Lin et al., 2001a;
Lin et al., 2001b
;
Vainio et al., 1993
). Fgf2,
Fgf7, Gdnf (Pepro Tech) and Fgf10 (R&D Systems) were used in the organ
cultures: Fgf2, 50-250 ng/ml of growth factor in the media
(Qiao et al., 2001
); Fgf7, 100
ng/ml (Qiao et al., 1999
);
Fgf10, 500 ng/ml (Qiao et al.,
2001
); and Gdnf, 100 ng/ml
(Sainio et al., 1997
). The
same amounts of bovine serum albumin (BSA, Sigma) served as controls. The drug
PD98059 was applied to the medium to a final concentration of 20 µM
(Fisher et al., 2001
), and the
kidneys were subcultured as indicated in the Results section. The numbers of
ureteric bud tips in the experimentally manipulated explants were evaluated
using Student's t-test.
Fgf-soaked beads were prepared and used as reported earlier
(Lin et al., 2001a;
Sainio et al., 1997
;
Vainio et al., 1993
). The
Affi-gel blue agarose beads (100-200 mesh, 75-150 nm in diameter, 100
beads/unit volume, BioRad Laboratories Hercules, CA) were incubated in pools
separately with human recombinant Fgf2 (50 µg/ml)
(Minowada et al., 1999
), Fgf7
(200 µg/ml) (Lin et al.,
2001b
), Fgf8 (50-100 µg/ml)
(Minowada et al., 1999
) or
Fgf10 (100 µg/ml) (Mailleux et al.,
2001
), washed and combined individually with isolated kidneys and
co-cultured for 24 hours. BSA beads served as controls and were treated and
used identically to those soaked with growth factor.
Localization of diagnostic markers
The Troma-I antibody against cytokeratin EndoA used to monitor ureteric
branching was from the Developmental Studies Hybridoma Bank (USA), while the
antibody against the brush-border antigen used for visualizing induced tubules
was from Dr Aaro Miettinen (University of Helsinki, Finland)
(Ekblom, 1981).
FITC-conjugated donkey anti-rabbit IgG and TRITC-conjugated donkey anti-rat
IgG (Jackson ImmunoResearch Laboratories) were used as secondary antibodies.
The immunoassay was performed on a whole mount basis as described previously
(Lin et al., 2001a
;
Lin et al., 2001b
;
Sainio et al., 1997
).
The whole-mount and non-radioactive section in situ hybridization were
performed as described previously (Zhang
et al., 2001), as was radioactive in situ hybridization
(Parr et al., 1993
;
Kispert et al., 1998
).
Wild-type and transgenic kidneys were processed in the same test tube (whole
mounts) or at the same time (histological sections) to allow the comparison of
staining intensities. A minimum of five hybridizations/marker gene/stage were
performed in order to evaluate changes in gene expression.
The entire SPRY2 cDNA was used to synthesize a riboprobe for
detecting transgene expression in embryonic kidneys. The other probes have
been described earlier and were obtained as gifts: Fgfr1
(Yamaguchi et al., 1994),
Fgfr2, Fgfr3, Fgfr4 and Fgfr7
(Rosenquist and Martin, 1996
),
Fgf2 (Wilkinson et al.,
1989
), Fgf8 (Crossley
and Martin, 1995
), Fgf9
(Colvin et al., 1996
;
Colvin et al., 1999
;
Santos-Ocampo et al., 1996
),
Fgf10 (Bellusci et al.,
1997
), Bmp4 (Bellusci
et al., 1996
), Ret and Gdnf
(Sainio et al., 1997
),
Wnt11 (Kispert et al.,
1996
), Pax2 (Dressler
et al., 1990
), Foxd1
(Hatini et al., 1996
), and
Egf and Egfr (Miettinen
et al., 1995
).
Analysis of changes in cell proliferation and apoptosis
BrdU, an analogue of thymidine that is incorporated into DNA during the
S-phase of the cell cycle (Dolbeare,
1995), served as an indicator of cell entry into mitosis. The cell
proliferation kit (RPN20, Amersham Biosciences, UK) was used as suggested by
the manufacturer. Briefly, pregnant mice were injected intraperitoneally a
solution containing with bromodeoxyuridine (BrdU, Sigma B-5002) at a dose of
50 mg/kg/body weight. The mice were sacrificed at two hours post-injection and
the kidneys were dissected in ice-cold PBS and fixed with 4% paraformaldehyde
(PFA) in phosphate-buffered saline (PBS) overnight at 4°C, dehydrated and
embedded in paraffin wax. Sections (5 µm) were cut and the BrdU
incorporated was detected with the specific antibody provided in the kit.
The terminal deoxynucleotidyl transferase-mediated dUTP nickend labelling (TUNEL) method was used to monitor apoptosis. The paraffin wax was removed from the 6 µm sections with xylene and the tissues were rehydrated and incubated with 20 µg/ml of Proteinase K for 15 minutes at 37°C. Unspecific binding was reduced with a hydrogen peroxide/methanol solution. Fragmented DNA was labelled using a reaction mixture, according to the manufacturer's instructions (Roche). Bound probes were detected using 3, 3'-diaminiobenzidine (DAB) as a substrate (Vectastain ABC kit, Vector Laboratories).
Photography and image analysis
Processed samples were photographed on Ektachrome Tungsten 64 Collagenor
slide film (Kodak, USA), or with a digital camera (DC100 Leica) connected to
an Olympus SZH10 stereo microscope. The composites were assembled with the
Adobe Photoshop v5.0 and Corel Draw programs. All the cultured kidneys, with
or without growth factors, were analysed and photographed for expression of
SPRY2 and GFP at selected time points with a Leica DMLB fluorescence
microscope and an Olympus DP50 digital camera connected to a Leica MZFLIII
stereo microscope.
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Results |
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Expression of the human SPRY2 gene leads to postnatal mortality
Crossing of transgenic mice with another carrier or with wild-type
background mice was performed in order to assay the consequences of human
SPRY2 expression for survival. Genotyping of the litters from such
crosses indicated a gradual loss of those individuals that expressed the human
SPRY2. Approximately 10% of the transgenic mice were recovered two
weeks post partum (Table 1). We
concluded that normal Spry protein signalling is crucial for postnatal
survival. Subsequent studies were performed on the survivors and their
litters. As human SPRY2 was targeted to the ureteric bud of the
embryonic kidney, the likely reason for the transgene-induced death was
deregulated kidney development.
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Expression of the genes encoding Fgf and Egf proteins, their receptors and mouse Spry1 in response to human SPRY2 expression
Spry proteins have been implicated in the control of Fgf and Egf signalling
and organogenesis (Mailleux et al.,
2001; Minowada et al.,
1999
). As Fgf protein activity may similarly regulate kidney
development (Qiao et al.,
1999
), the involvement of human SPRY2 in
Fgf-mediated signalling was also analysed. Of the Fgf proteins,
Fgf2 is normally expressed in the ureteric bud and nephrogenic
mesenchyme during their morphogenesis (Fig.
3A) (Drummond et al.,
1998
), while its expression is substantially reduced in the
presence of human SPRY2 expression
(Fig. 3, compare A with
A' and A'').
|
It was of interest to consider whether human SPRY2 would similarly
influence the expression of Fgf/Egf receptors, the prime candidate targets of
Spry protein-mediated antagonism. Fgfr1 was present in kidney
mesenchymal cells regardless of their genetic status, as reported earlier
(Dudley et al., 1999;
Ford et al., 1997
;
Walshe and Mason, 2000
), but
its expression was lost in the more mature nephrons in the presence of human
SPRY2, unlike the situation in the controls
(Fig. 3, compare E with
E',E''), suggesting that human SPRY2 expression has an
influence on the maturation of the nephron, apparently owing to deregulated
ureteric bud inductive signalling. The expression of Fgf receptors
2-4 appeared to be normal in all the samples analysed
(Fig. 3F-H''), as was the
case for Egfr (data not shown), even though the latter is implicated in the
epithelial branching process in the embryonic lung, for example
(Miettinen et al., 1995
).
Another Spry gene, Spry1 is expressed in the ureteric bud and may be associated with generation of the ectopic human SPRY2 phenotypes. Spry1 expression was normal regardless of the presence of human SPRY2 (Fig. 4F,F'). Hence, besides controlling Fgfr/Egfr, human SPRY2 signalling may also regulate other pathways in the kidney.
|
As Gdnf/Ret signalling is critical for the regulation of ureteric
bud growth in the early stages of kidney organogenesis
(Sainio et al., 1997), and has
recently shown to be coordinated by Wnt11
(Majumdar et al., 2003
), these
genes were candidates for being influenced by human SPRY2. Gdnf is
expressed in kidney mesenchymal cells at E12.5 and E17.5
(Fig. 4A and
Fig. 5A), and human
SPRY2 expression led to a notable reduction at these stages
(Fig. 4A') relative to
controls at E12.5 and E15.5 (Fig.
5, compare A to A',A''). Irrespective of the reduction
in Gdnf expression, expression of Ret, which encodes one of
the identified receptors for Gdnf, was unchanged at the corresponding
developmental stages (Fig. 4, compare B with B'; Fig.
5, compare B with B',B''). Consistent with the
diminished Gdnf expression, Wnt11, a target gene for
Gdnf signalling (Majumdar et al.,
2003
; Pepicelli et al.,
1997
), was also expressed in lesser amounts in the ureteric bud at
E12.5 and E17.5 because of human SPRY2 expression
(Fig. 4, compare C with
C'; Fig. 5, compare C
with C',C'').
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Human SPRY2 expression in the ureteric bud leads to changes in the stromal component of the embryonic kidney
The winged helix transcription factor Foxd1 is required for
ureteric bud development and nephrogenesis via signals that are secreted by
the stromal cells (Hatini et al.,
1996). Human SPRY2 signalling evidently influenced the
stromal component, as Foxd1, a marker of these cells, was severely
reduced at E12.5 and E15.5 in the transgenic kidneys relative to controls
(Fig. 4, compare E with
E'; Fig. 5, compare F
with F',F'').
Induction of Spry2 gene expression by Fgf7
The Spry genes are induced in response to Fgf proteins, and are thought to
antagonize Fgf signalling in order to control the cellular response to these
factors (Mailleux et al.,
2001). As the expression of three Fgf proteins in the kidney was
altered because of human SPRY2 expression, we tested whether these
Fgf proteins also regulated Spry2 gene expression. Agarose beads
soaked with Fgf proteins were combined with kidneys isolated from E11.5
embryos derived from transgenic matings. The tissue was subcultured for 24
hours and changes in Spry2 expression were monitored. Although
control beads soaked with BSA, Fgf8 or Fgf10 had no notable effect, beads
releasing Fgf7 induced Spry2 expression in the nearby cells
(Fig. 6A-D). Hence,
Fgf7 expression is not only dependent on Spry2 function, but
reciprocally, Fgf7 is also sufficient to coordinate Spry2
expression.
|
We set out next to test whether the reduction in expression of Fgf and Gdnf resulting from human SPRY2 expression was causally associated with the phenotypes. For this purpose, the potential of these factors to rescue the defect caused by human SPRY2 was tested by exposing kidneys to them in organ culture. Fgf7, Fgf10 and GDNF all stimulated ureteric branching in the human SPRY2 kidneys, whereas Fgf2 had the opposite effect on this process relative to untreated kidneys also expressing the human SPRY2 gene (Fig. 6H-L). The quantification of the response to growth factors indicated in Fig. 6M is based on counting the number of epithelial tips. Surprisingly, the Wolffian duct of the human SPRY2 embryonic kidney appeared to be sensitized to Fgf7 and GDNF signalling, as these factors induced supernumerary bud formation from the rest of the Wolffian duct. Such an effect was not obtained in normal control kidneys when similarly exposed to these factors in the media (Fig. 6H,K,L, arrows and data not shown). The ectopic buds developed as a response to the growth factors during overnight culture of E11.5 kidneys expressing human SPRY2, and were documented with Fgf7 in 7/9 cases (77.8%) and with GDNF in 9/10 cases (90%).
Sprouty2 is thought to antagonize Fgfr and Egfr signalling upstream of the
ERK-MAP kinase pathway (Hanafusa et al.,
2002). As an initial attempt to characterize the mechanism of
signal transduction controlled by human SPRY2 in these primary kidney
rudiments, the inhibitor of the ERK1/2 pathway, PD98059, was used to
specifically inhibit normal ureteric development
(Fisher et al., 2001
), thus
demonstrating a phenotype that is related to that induced by human
SPRY2 expression. However the drug had an additive effect on the
human SPRY2-mediated defect in ureteric branching in all of the cases
analysed. The bud was even further reduced in development and it generated a
longer ureteric branch than in the untreated transgenic or wild-type control
mice, as demonstrated also in skeletonized diagrams of the ureteric buds
(Fig. 6N-Q). We conclude that
besides Spry proteins other signal transduction pathways are also likely to be
mediated via the ERK-MAP kinase pathway to contribute to ureteric bud
development in the kidney.
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Discussion |
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Human SPRY2-induced defects were manifested in complete agenesis, reduced size, lobularization or cystogenesis of the ureteric bud and an ectopic organ with a second complete ureteric bud was also seen to be formed because of human SPRY2-mediated signalling. Human SPRY2 expression also induced prenatal death, but the reason for this remains unknown at present. Degree of the severity of the kidney phenotypes are likely to be induced by the level of human SPRY2, as the crossing of two human SPRY2 carriers lead to an additive effect to the penetration of the kidney associated phenotypes. A higher level of human SPRY2 is expected to antagonize the associated inductive signal transduction pathway more efficiently, pointing to dose-dependent functioning of the pathway in the process. This point can be analysed once the tools become available. We conclude that the phenotypes caused by human SPRY2 expression suggest that Spry protein signalling is important in coordination of ureteric bud development during kidney organogenesis and that the mouse line serves as a useful model for studying the mechanisms of ureteric bud signalling and pattern formation during organogenesis.
Spry proteins have been implicated in the control of RTKs such as Fgf/Egf
receptors in other developmental systems, and as a target of Fgf signalling
(Hacohen et al., 1998;
Mailleux et al., 2001
).
Ectopic human SPRY2 was also found here to be associated with reduced
Fgf2 expression in the ureteric bud and reduced Fgfr1
expression in the mesenchyme. In addition to these findings, stromally
expressed Fgf7 also induced Spry2 gene expression. However, even
though changed as a response to human SPRY2 expression, these factors
are apparently not the primary targets of the human SPRY2 antagonism
that is expected to take places in the ureteric bud. Deficiency in
Fgf2 does not lead to any overt kidney phenotypes either
(Dono et al., 1998
). The fact
that Fgf signalling induced Spry2 gene expression is consistent with
the findings in the embryonic lung
(Mailleux et al., 2001
;
Tefft et al., 2002
) and in the
zebrafish (Furthauer et al.,
2001
), for example, pointing that Fgf protein signalling controls
Spry gene expression in these systems. However, in the kidney, the target of
human SPRY2-mediated antagonism may include also other then the Fgf
pathway.
Coordination of Gdnf/Wnt11 signalling by human SPRY2 during early organogenesis
A deviation of kidney development from normal in response to human
SPRY2 expression was already noted at E12.5, being manifested as a
reduction in the number of ureteric tips relative to controls at the same
developmental stage. This phenotype was associated with a reduction in the
expression of Gdnf and Wnt11 genes. Wnt11 has
recently been shown to be a functional signal around the same time as the
phenotype of the human SPRY2 transgenic embryos becomes noticeable
(Majumdar et al., 2003). In
the case of Wnt11 deficiency, Gndf expression is also
reduced, suggesting that Wnt11 may regulate Gdnf expression
in kidney mesenchyme, either directly or via another signal
(Fig. 7). The Wnt11
gene, in turn, is also regulated by Ret signalling, as Wnt11
expression is reduced in the kidneys of Ret-deficient embryos
(Majumdar et al., 2003
)
(Fig. 7). The downregulated
expression of Wnt11 and Gdnf genes in kidneys expressing
human SPRY2 and the finding that the ureteric bud defect in the
transgenic kidneys was substantially reversed via stimulated Gdnf
signalling in vitro, support the hypothesis that human SPRY2
contributes to cooperation between the Wnt11/Gdnf/Ret pathways during
early kidney development perhaps by directly antagonising Ret
signalling (Fig. 7).
|
Human SPRY2 expression and the role of stromal cells in kidney development
We noted that the expression of the stromal genes Foxd1 and
Fgf7 was altered because of ectopic human SPRY2 expression
in the ureteric bud. Fgf7, which is normally expressed in the stromal
cells, regulates kidney development via its receptors, which are thought to be
located in the ureteric bud site of the targeted human SPRY2
expression. Moreover, Fgf7 knockout leads similarly to a reduction in
the overall number of nephrons and the size of the kidney
(Qiao et al., 1999), as
attributed here to human SPRY2 expression. In further support of the
speculation that ureteric bud signalling may also influence the stromal cells,
human SPRY2 expression leads to a notable reduction in stromally
located Foxd1 and Fgf7 expression
(Fig. 7), and that the ectopic
Fgf7 signalling that is normally localized to the stromal cells in
the kidney (Qiao et al., 1999
)
also has an rescue effect to the deficiency in ureteric bud branching in
kidneys expressing human SPRY2 in vitro. Batourina et al.
(Batourina et al., 2001
)
proposed that a signalling loop from the stromal cells and ureteric bud
regulates kidney development. Our data are consistent with a model that
implies that, in addition to the stromal-ureteric bud interactions, the
signalling is reciprocal, so that the ureteric bud also coordinates the
stromal cells and involves activities of Spry2
(Fig. 7).
Induction of supernumerary bud formation and ectopic organogenesis due to human SPRY2 expression
In addition to the similarity between the phenotypes of human
SPRY2 expression and Fgf7 deficiency, the consequences of
the lack of function of other genes, the Bmp4 and Foxc genes, relate
to the phenotypes observed in the presence of human SPRY2 expression.
Like embryos expressing human SPRY2, the kidneys of
Bmp4-deficient embryos are hypo/dysplastic and have hydroureter and
double collecting ducts. Such phenotypes are thought to be related to human
congenital anomalies of the kidney and urinary tract (CAKUT)
(Miyazaki et al., 2000), and
to be associated with reduction in the expression of Gdnf and
Wnt11, which are also properties of ectopic human SPRY2.
Like Bmp4 deficiency, human SPRY2 expression induces a
complete second collecting duct system in vivo and in vitro. Moreover, in our
case, exposure of the Wolffian duct to Fgf7 and Gdnf generated supernumerary
epithelial buds not found in the non-transgenic kidneys and is the likely
reason for the formation of the complete ectopic ureteric bud induced by human
SPRY2 signalling. Hence, kidneys expressing human SPRY2
appear to be sensitised to generate ectopic buds from the Wolffian duct in
response to Fgf7 and Gdnf signalling. Even though
Bmp4 has been implicated in the budding process, we did not record
any significant changes in its expression during early kidney development
resulting from human SPRY2 expression. Hence, Spry protein signalling
appears to be involved in the patterning mechanism that defines those cells
that form the ureteric bud from the Wolffian duct. Our data suggest that this
process may be distinct from the one caused by Bmp4. We conclude that
Spry protein-regulated signalling is involved in coordination of the
reciprocal epithelial, mesenchymal and stromal signalling via Wnt11,
Gdnf and Fgf7 that controls the ureteric bud initiation and
subsequent branching process during kidney development.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Department of Medicine, Harvard Medical School, Renal
Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129,
USA
Present address: Department of Medicine, Beth Israel Deaconess Medical
Center, 330 Brookline Avenue, Boston, MA 02215, USA
Present address: Department of Pediatrics and Biocenter Oulu, University of
Oulu, FIN-90014 Oulu, Finland
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Airaksinen, M. S. and Saarma, M. (2002). The GDNF family: signalling, biological functions and therapeutic value. Nat. Rev. Neurosci. 3,383 -394.[CrossRef][Medline]
Barasch, J., Qiao, J., McWilliams, G., Chen, D., Oliver, J. A. and Herzlinger, D. (1997). Ureteric bud cells secrete multiple factors, including bFGF, which rescue renal progenitors from apoptosis. Am. J. Physiol. 273,F757 -F767.[Medline]
Batourina, E., Gim, S., Bello, N., Shy, M., Clagett-Dame, M., Srinivas, S., Costantini, F. and Mendelsohn, C. (2001). Vitamin A controls epithelial/mesenchymal interactions through Ret expression. Nat. Genet. 27, 74-78.[CrossRef][Medline]
Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan,
B. L. (1996). Evidence from normal expression and targeted
misexpression that bone morphogenetic protein (BMP-4) plays a role in mouse
embryonic lung morphogenesis. Development
122,1693
-1702.
Bellusci, S., Grindley, J., Emoto, H., Itoh, N. and Hogan, B.
L. (1997). Fibroblast growth factor 10 (FGF10) and branching
morphogenesis in the embryonic mouse lung. Development
124,4867
-4878.
Bertram, J. F., Soosaipillai, M. C., Ricardo, S. D. and Ryan, G. B. (1992). Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney. Cell Tissue Res. 270,37 -45.[Medline]
Brenner, B. M. and Milford, E. L. (1993). Nephron underdosing: a programmed cause of chronic renal allograft failure. Am. J. Kidney Dis. 21,66 -72.[Medline]
Brophy, P. D., Ostrom, L., Lang, K. M. and Dressler, G. R.
(2001). Regulation of ureteric bud outgrowth by Pax2-dependent
activation of the glial derived neurotrophic factor gene.
Development 128,4747
-4756.
Casci, T., Vinos, J. and Freeman, M. (1999). Sprouty, an intracellular inhibitor of Ras signaling. Cell 96,655 -665.[Medline]
Colvin, J. S., Bohne, B. A., Harding, G. W., McEwen, D. G. and Ornitz, D. M. (1996). Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat. Genet. 12,390 -397.[Medline]
Colvin, J. S., Feldman, B., Nadeau, J. H., Goldfarb, M. and Ornitz, D. M. (1999). Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev. Dyn. 216,72 -88.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The
mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions
that direct outgrowth and patterning in the developing embryo.
Development 121,439
-451.
Davies, J. A. and Bard, J. B. (1998). The development of the kidney. Curr. Top. Dev. Biol. 39,245 -301.[Medline]
DePamphilis, M. L., Herman, S. A., Martinez-Salas, E., Chalifour, L. E., Wirak, D. O., Cupo, D. Y. and Miranda, M. (1988). Microinjecting DNA into mouse ova to study DNA replication and gene expression and to produce transgenic animals. Biotechniques 6,662 -680.[Medline]
Dolbeare, F. (1995). Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part II: Oncology, chemotherapy and carcinogenesis. Histochem. J. 27,923 -964.[Medline]
Dono, R., Texido, G., Dussel, R., Ehmke, H. and Zeller, R.
(1998). Impaired cerebral cortex development and blood pressure
regulation in FGF-2-deficient mice. EMBO J.
17,4213
-4225.
Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O. and Gruss, P. (1990). Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109,787 -795.[Abstract]
Drummond, I. A., Mukhopadhyay, D. and Sukhatme, V. P. (1998). Expression of fetal kidney growth factors in a kidney tumor line: role of FGF2 in kidney development. Exp. Nephrol. 6,522 -533.[CrossRef][Medline]
Dudley, A. T. and Robertson, E. J. (1997). Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient embryos. Dev. Dyn. 208,349 -362.[CrossRef][Medline]
Dudley, A. T., Godin, R. E. and Robertson, E. J.
(1999). Interaction between FGF and BMP signaling pathways
regulates development of metanephric mesenchyme. Genes
Dev. 13,1601
-1613.
Dunn, N. R., Winnier, G. E., Hargett, L. K., Schrick, J. J., Fogo, A. B. and Hogan, B. L. (1997). Haploinsufficient phenotypes in BMP4 heterozygous null mice and modification by mutations in Gli3 and Alx4. Dev. Biol. 188,235 -247.[CrossRef][Medline]
Durbec, P., Marcos-Gutierrez, C. V., Kilkenny, C., Grigoriou, M., Wartiowaara, K., Suvanto, P., Smith, D., Ponder, B., Costantini, F., Saarma, M. et al. (1996). GDNF signalling through the Ret receptor tyrosine kinase. Nature 381,789 -793.[CrossRef][Medline]
Ekblom, P. (1981). Formation of basement
membranes in the embryonic kidney: an immunohistological study. J.
Cell Biol. 91,1
-10.
Finch, P. W., Cunha, G. R., Rubin, J. S., Wong, J. and Ron, D. (1995). Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal-epithelial interactions. Dev. Dyn. 203,223 -240.[Medline]
Fisher, C. E., Michael, L., Barnett, M. W. and Davies, J. A.
(2001). Erk MAP kinase regulates branching morphogenesis in the
developing mouse kidney. Development
128,4329
-4338.
Ford, M. D., Cauchi, J., Greferath, U. and Bertram, J. F. (1997). Expression of fibroblast growth factors and their receptors in rat glomeruli. Kidney Int. 51,1729 -1738.[Medline]
Furthauer, M., Reifers, F., Brand, M., Thisse, B. and Thisse, C. (2001). sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128,2175 -2186.[Medline]
Gross, I., Morrison, D. J., Hyink, D. P., Georgas, K., English,
M. A., Mericskay, M., Hosono, S., Sassoon, D., Wilson, P. D., Little,
M. et al. (2003). The receptor tyrosine kinase regulator
Sprouty1 is a target of the tumor suppressor WT1 and important for kidney
development. J. Biol. Chem.
278,41420
-41430.
Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. and Krasnow, M. A. (1998). sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92,253 -263.[Medline]
Hall, A. B., Jura, N., DaSilva, J., Jang, Y. J., Gong, D. and Bar-Sagi, D. (2003). hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl. Curr. Biol. 13,308 -314.[CrossRef][Medline]
Hanafusa, H., Torii, S., Yasunaga, T. and Nishida, E. (2002). Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat. Cell Biol. 4, 850-858.[CrossRef][Medline]
Hatini, V., Huh, S. O., Herzlinger, D., Soares, V. C. and Lai, E. (1996). Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev. 10,1467 -1478.[Abstract]
Itaranta, P., Lin, Y., Perasaari, J., Roel, G., Destree, O. and Vainio, S. (2002). Wnt-6 is expressed in the ureter bud and induces kidney tubule development in vitro. Genesis 32,259 -268.[CrossRef][Medline]
Kispert, A., Vainio, S. and McMahon, A. P.
(1998). Wnt-4 is a mesenchymal signal for epithelial
transformation of metanephric mesenchyme in the developing kidney.
Development 125,4225
-4234.
Kispert, A., Vainio, S., Shen, L., Rowitch, D. H. and McMahon,
A. P. (1996). Proteoglycans are required for maintenance of
Wnt-11 expression in the ureter tips. Development
122,3627
-3637.
Kreidberg, J. A., Sariola, H., Loring, J. M., Maeda, M., Pelletier, J., Housman, D. and Jaenisch, R. (1993). WT-1 is required for early kidney development. Cell 74,679 -691.[Medline]
Kuschert, S., Rowitch, D. H., Haenig, B., McMahon, A. P. and Kispert, A. (2001). Characterization of Pax-2 regulatory sequences that direct transgene expression in the Wolffian duct and its derivatives. Dev. Biol. 229,128 -140.[CrossRef][Medline]
Kuure, S., Vuolteenaho, R. and Vainio, S. (2000). Kidney morphogenesis: cellular and molecular regulation. Mech. Dev. 92,31 -45.[CrossRef][Medline]
Lin, Y., Liu, A., Zhang, S., Ruusunen, T., Kreidberg, J. A., Peltoketo, H., Drummond, I. and Vainio, S. (2001a). Induction of ureter branching as a response to Wnt-2b signaling during early kidney organogenesis. Dev. Dyn. 222, 26-39.[CrossRef][Medline]
Lin, Y., Zhang, S., Rehn, M., Itaranta, P., Tuukkanen, J.,
Heljasvaara, R., Peltoketo, H., Pihlajaniemi, T. and Vainio, S.
(2001b). Induced repatterning of type XVIII collagen expression
in ureter bud from kidney to lung type: association with sonic hedgehog and
ectopic surfactant protein C. Development
128,1573
-1585.
Mailleux, A. A., Tefft, D., Ndiaye, D., Itoh, N., Thiery, J. P., Warburton, D. and Bellusci, S. (2001). Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis. Mech. Dev. 102, 81-94.[CrossRef][Medline]
Majumdar, A., Vainio, S., Kispert, A., McMahon, J. and McMahon,
A. P. (2003). Wnt11 and Ret/Gdnf pathways cooperate in
regulating ureteric branching during metanephric kidney development.
Development 130,3175
-3185.
Mason, I. J., Fuller-Pace, F., Smith, R. and Dickson, C. (1994). FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions. Mech. Dev. 45, 15-30.[CrossRef][Medline]
Miettinen, P. J., Berger, J. E., Meneses, J., Phung, Y., Pedersen, R. A., Werb, Z. and Derynck, R. (1995). Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376,337 -341.[CrossRef][Medline]
Minowada, G., Jarvis, L. A., Chi, C. L., Neubuser, A., Sun, X.,
Hacohen, N., Krasnow, M. A. and Martin, G. R. (1999).
Vertebrate Sprouty genes are induced by FGF signaling and can cause
chondrodysplasia when overexpressed. Development
126,4465
-4475.
Miyazaki, Y., Oshima, K., Fogo, A., Hogan, B. L. and Ichikawa,
I. (2000). Bone morphogenetic protein 4 regulates the budding
site and elongation of the mouse ureter. J. Clin.
Invest. 105,863
-873.
Moore, M. W., Klein, R. D., Farinas, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L. F., Ryan, A. M., Carver-Moore, K. and Rosenthal, A. (1996). Renal and neuronal abnormalities in mice lacking GDNF. Nature 382, 76-79.[CrossRef][Medline]
Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. (2003). Manipulating the Mouse Embryo: A Laboratory Manual. New York: Cold Spring Harbor Laboratory.
Parr, B. A., Shea, M. J., Vassileva, G. and McMahon, A. P.
(1993). Mouse Wnt genes exhibit discrete domains of expression in
the early embryonic CNS and limb buds. Development
119,247
-261.
Pepicelli, C. V., Kispert, A., Rowitch, D. H. and McMahon, A. P. (1997). GDNF induces branching and increased cell proliferation in the ureter of the mouse. Dev. Biol. 192,193 -198.[CrossRef][Medline]
Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A. C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B. J. et al. (1996). GDNF is required for kidney development and enteric innervation. Cold Spring Harb. Symp. Quant. Biol. 61,445 -457.[Medline]
Qiao, J., Uzzo, R., Obara-Ishihara, T., Degenstein, L., Fuchs,
E. and Herzlinger, D. (1999). FGF-7 modulates ureteric
bud growth and nephron number in the developing kidney.
Development 126,547
-554.
Qiao, J., Bush, K. T., Steer, D. L., Stuart, R. O., Sakurai, H., Wachsman, W. and Nigam, S. K. (2001). Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis. Mech. Dev. 109,123 -135.[CrossRef][Medline]
Raatikainen-Ahokas, A., Hytonen, M., Tenhunen, A., Sainio, K. and Sariola, H. (2000). BMP-4 affects the differentiation of metanephric mesenchyme and reveals an early anterior-posterior axis of the embryonic kidney. Dev. Dyn. 217,146 -158.[CrossRef][Medline]
Rosenquist, T. A. and Martin, G. R. (1996). Fibroblast growth factor signalling in the hair growth cycle: expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle. Dev. Dyn. 205,379 -386.[CrossRef][Medline]
Ryan, G., Steele-Perkins, V., Morris, J. F., Rauscher, F. J.,
3rd and Dressler, G. R. (1995). Repression of Pax-2 by
WT1 during normal kidney development. Development
121,867
-875.
Sainio, K., Suvanto, P., Davies, J., Wartiovaara, J.,
Wartiovaara, K., Saarma, M., Arumae, U., Meng, X., Lindahl, M.,
Pachnis, V. et al. (1997). Glial-cell-line-derived
neurotrophic factor is required for bud initiation from ureteric epithelium.
Development 124,4077
-4087.
Santos-Ocampo, S., Colvin, J. S., Chellaiah, A. and Ornitz, D.
M. (1996). Expression and biological activity of mouse
fibroblast growth factor-9. J. Biol. Chem.
271,1726
-1731.
Sasaki, H. and Hogan, B. L. (1994). HNF-3 beta as a regulator of floor plate development. Cell 76,103 -115.[Medline]
Saxen, L. (1987). Organogenesis of Kidney. Cambridge: Cambridge University Press.
Stark, K., Vainio, S., Vassileva, G. and McMahon, A. P. (1994). Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature 372,679 -683.[CrossRef][Medline]
Tang, M. J., Cai, Y., Tsai, S. J., Wang, Y. K. and Dressler, G. R. (2002). Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase. Dev. Biol. 243,128 -136.[CrossRef][Medline]
Tefft, D., Lee, M., Smith, S., Crowe, D. L., Bellusci, S. and
Warburton, D. (2002). mSprouty2 inhibits FGF10-activated MAP
kinase by differentially binding to upstream target proteins. Am.
J. Physiol. Lung Cell Mol. Physiol.
283,L700
-L706.
Torres, M., Gomez-Pardo, E., Dressler, G. R. and Gruss, P.
(1995). Pax-2 controls multiple steps of urogenital development.
Development 121,4057
-4065.
Vainio, S. and Lin, Y. (2002). Coordinating early kidney development: lessons from gene targeting. Nat. Rev. Genet. 3,533 -543.[CrossRef][Medline]
Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I. (1993). Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75,45 -58.[Medline]
Vainio, S. J., Itaranta, P. V., Perasaari, J. P. and Uusitalo, M. S. (1999). Wnts as kidney tubule inducing factors. Int. J. Dev. Biol. 43,419 -423.[Medline]
Vize, P. D., Woolf, A. S. and Bard, J. B. L. (2003). The Kidney: From Normal Development to Congenital Disease. USA: Elsevier.
Walshe, J. and Mason, I. (2000). Expression of FGFR1, FGFR2 and FGFR3 during early neural development in the chick embryo. Mech. Dev. 90,103 -110.[CrossRef][Medline]
Wilkinson, D. G., Bhatt, S. and McMahon, A. P. (1989). Expression pattern of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal development. Development 105,131 -136.[Abstract]
Wong, E. S., Fong, C. W., Lim, J., Yusoff, P., Low, B. C.,
Langdon, W. Y. and Guy, G. R. (2002). Sprouty2 attenuates
epidermal growth factor receptor ubiquitylation and endocytosis, and
consequently enhances Ras/ERK signalling. EMBO J.
21,4796
-4808.
Yamaguchi, F., Saya, H., Bruner, J. M. and Morrison, R. S. (1994). Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas. Proc. Natl. Acad. Sci. USA 91,484 -488.[Abstract]
Zhang, S., Lin, Y., Itaranta, P., Yagi, A. and Vainio, S. (2001). Expression of Sprouty genes 1, 2 and 4 during mouse organogenesis. Mech. Dev. 109,367 -370.[CrossRef][Medline]