1 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity
Avenue, Cambridge, MA 02138, USA
2 Department of Pathology, Brigham and Women's Hospital, 75 Francis Street,
Boston, MA 02115, USA
3 Department of Pediatrics, Tufts-New England Medical Center, 750 Washington
Street, Boston, MA 02111, USA
* Author for correspondence (e-mail: Elizabeth.Robertson{at}well.ox.ac.uk)
Accepted 23 June 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: BMP, Kidney development, Lineage analysis, Nephrogenic mesenchyme, Smad4, Stroma, TGFß
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Development of the kidney
The Wolffian duct (WD) differentiates from mesoderm within the nephrogenic
cord at approximately E9.0 in the mouse, and induces pro- and mesonephric
tubules as it extends caudally toward the cloaca. At approximately E10.5, the
ureteric bud (UB) appears as a thickening of the WD at the level of the
hindlimb. The UB grows out in a cranial direction and invades the metanephric
blastema, a population of cells at the caudal end of the nephrogenic cord.
Upon induction by the metanephric mesenchyme, the UB extends and branches
within the mesenchyme, forming the collecting duct (CD) system. In turn,
metanephric mesenchyme is induced to condense and form nephrogenic mesenchyme
(NM), which gives rise to the nephron. This cycle of induction initiates a
program of reciprocal interactions between the mesenchyme and the CD
epithelium. The molecular signals governing these processes are only partially
understood: glial cell-derived neurotrophic factor (Gdnf), produced by
mesenchyme, and signaling through the receptor tyrosine kinase Ret (c-ret) in
CD epithelium, is required for directional growth and branching of collecting
ducts (Sanchez et al., 1996;
Schuchardt et al., 1994
;
Vega et al., 1996
). The
inductive signals to the mesenchyme remain unknown.
TGFß superfamily signal transduction
The TGFß superfamily is composed of a collection of structurally
related ligands with diverse functions in development
(Miyazono et al., 2001). Two
distinct families of receptors elicit diverse responses to ligand binding;
TGFß, nodal and activin result in phosphorylation of receptor-associated
Smads (R-Smads) 2 and 3, whereas the BMPs elicit phosphorylation of R-Smads 1,
5 and 8. The GDF group comprises factors that signal via both R-Smads 2 and 3,
and R-Smads 1, 5 and 8. Phosphorylated R-Smads associate with Smad4, which
contains a nuclear translocation signal. In the nucleus, the R-Smad:Smad4
complex associates with a variety of cofactors that determine the outcome of
the transcriptional response
(Massagué, 2000
). Smad4
is thus an integral component of the signal transduction machinery employed by
both the TGFß and BMP pathways.
The role of TGFß superfamily signaling in metanephric kidney development
A role for Gdf11 in induction of the metanephros has recently been
shown. Mice deficient for Gdf11 display uni- or bilateral kidney
agenesis. Normally, Gdf11 from either the ureteric bud or the metanephric
mesenchyme activates Gdnf expression in mesenchyme cells, which
initiates the reciprocal inductive program leading to formation of the kidney
(Esquela and Lee, 2003).
Tgfb2 has also been implicated in induction of the metanephros, as
loss of Tgfb2 gives rise to incompletely penetrant kidney agenesis in
females (Sanford et al.,
1997
). In addition, Tgfß2 has been identified as an active
component of rat ureteric bud conditioned medium with inductive capacity
(Plisov et al., 2001
).
Genetic studies have shown roles for Bmp4 and Bmp7 in
organogenesis of the kidney. Loss of Bmp7 leads to premature
termination of kidney development, with a depletion of nephrogenic mesenchyme
cells (Dudley et al., 1995).
Explant experiments indicate that Bmp7 acts as a survival factor for
nephrogenic progenitor cells, suggesting that Bmp7 is required in vivo for
replenishment of the progenitor cell pool
(Dudley et al., 1999
).
Bmp4 expressed in mesenchyme surrounding the Wolffian duct inhibits
ectopic budding of the ureteric bud
(Miyazaki et al., 2000
).
Additionally, in embryos that form a single ureteric bud there is a paucity of
epithelial structures in the E14.5 kidney
(Miyazaki et al., 2003
). The
reduction in nephron number and the premature arrest of kidney development
indicate that Bmp4 may also act as a survival signal for nephrogenic
mesenchyme.
In this study we have used cell-type specific inactivation of Smad4 to further define the role of TGFß superfamily signals in kidney development. Surprisingly, removal of Smad4 in the epithelium of the Wolffian duct, the ureteric bud and the collecting duct system does not impair the development of the metanephros. However, loss of Smad4 in the metanephric mesenchyme leads to ectopic mesenchymal cell death and premature depletion of nephrogenic mesenchyme. Strikingly, there is a marked expansion of the peripheral stromal layer and impaired condensation of nephrogenic mesenchyme, implying a role for TGFß signaling in the morphogenesis of the mesenchyme at the earliest stages of nephrogenesis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The HoxB7cre transgene was generated by cloning Cre
recombinase cDNA fused to a nuclear localization signal
(Gu et al., 1993) into a
cassette containing the HoxB7 enhancer/promoter element, a polylinker
and 3' sequence from the human ß-globin gene with a splice
donor, acceptor and polyadenylation site
(Srinivas et al., 1999
).
PmlI linearized DNA was microinjected into fertilized oocytes using
standard procedures (Hogan et al.,
1994
), and offspring were genotyped by Cre PCR. Positive
males were intercrossed with ROSA26R reporter females
(Soriano, 1999
), and embryos
were dissected and stained at E14.5 to verify Cre activity in the collecting
duct system of the kidney. Of six positive males, two showed reliable excision
in collecting ducts. Both strains were used in this study.
The Smad4 conditional strain carries loxP sites flanking the first coding
exon of Smad4, which results in removal of the DNA-binding domain and nuclear
localization signal upon recombination (Chu
et al., 2004).
Sample preparation
For Hematoxylin and Eosin staining, in situ hybridization and
immunohistochemistry, tissues were fixed in 4% paraformaldehyde in PBS,
dehydrated, embedded in paraffin wax, and sectioned at 6 µm. Hematoxylin
and Eosin staining was performed using standard procedures. For X-Gal
staining, whole tissues were fixed for 90 minutes in 0.5% glutaraldehyde, 1%
paraformaldehyde in PBS, and staining was performed as described previously
(Michael et al., 1999).
In situ hybridization
Sections were rehydrated and in situ hybridization was performed using
standard procedures (Mendelsohn et al.,
1999). Whole mount in situ hybridization was performed as
described previously (Wilkinson,
1992
). Probes used in this study were: Wt1
(Kreidberg et al., 1993
), Pax2
(Dressler et al., 1990
), Ret
(Pachnis et al., 1993
), Raldh2
(Batourina et al., 2001
), Lhx1
(Barnes et al., 1994
) and Gdnf
(Hellmich et al., 1996
).
Immunohistochemistry
Immunohistochemistry was performed as described
(Oxburgh and Robertson, 2002),
with 1:200 dilutions of affinity-purified rabbit antisera specific for
phosphorylated Smad1 and phosphorylated Smad2 (a kind gift of Peter ten
Dijke). Pax2 staining with antigen unmasking was performed as described
(Schnabel et al., 2001
). PCNA
staining was performed using the PC10 mouse monoclonal antibody (Santa Cruz
Biotechnology) and the Mouse-on-Mouse kit (Vector Laboratories), according to
the manufacturer's instructions.
RT-PCR assays
E11.5 kidneys and E17.5 whole embryos were dissected into Trizol
(Invitrogen), and RNA was extracted according to the manufacturer's
instructions. cDNA synthesis and PCR were performed as previously described
(Oxburgh and Robertson, 2002).
The oligonucleotide combinations used to amplify transcripts for members of
the TGFß superfamily are listed in the supplementary table (see Table S1
at
http://dev.biologists.org/cgi/content/full/131/18/4593/DC1).
Kidney explant culture
Mesenchymes were separated from ureteric buds of E11.5 kidneys, as
previously described (Godin et al.,
1998), and cultured on filter rafts with and without 15 mM LiCl in
the culture medium. After 72 hours, cultures were either processed for
sectioning, or stained for laminin as described
(Bard et al., 2001
) and viewed
using a Zeiss LSM510 confocal microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Bmp7cre/+ activated the ROSA26R reporter in components of the kidney derived from both the ureteric bud and the metanephric mesenchyme. From the earliest stages of metanephric development, recombination occurs in the ureteric bud, and subsequently labeled cells are seen throughout the collecting duct system. At E11.5, there is mosaic recombination throughout the metanephric mesenchyme (Fig. 1B), and by E12.0, labeling is evident within the four to five cell layers of nephrogenic mesenchyme around collecting duct tips, with a few unlabeled cells surrounding collecting duct trunks and the periphery of the kidney (Fig. 1C). At E13.5, labeled cells are seen in nephrogenic mesenchyme and epithelial structures such as renal vesicles, comma-shaped bodies, Bowman's capsule and podocytes (Fig. 1D,F,G). Interestingly, labeling is not seen in the peripheral stroma. The medullary stroma contains a few labeled cells, which we attribute to migration of Bmp7-expressing cells of neural origin. This general pattern of labeling is maintained up to E17.5 (data not shown).
We next compared Bmp7lacZ/+ reporter expression
(Godin et al., 1998) with the
domain of Rosa26R activation by Bmp7cre/+.
Bmp7lacZ/+ is expressed throughout the collecting ducts, but
is restricted in the mesenchyme (Fig.
1E). Expression is robust in nephrogenic mesenchyme, but is
maintained in only a few cells of the renal vesicle. Expression can be seen
only at the distal end of the developing tubule and in the podocytes. By
contrast, Bmp7cre/+ labeling is seen throughout the
developing nephron, from the renal vesicle stage onward, demonstrating that
the entire nephron is derived from Bmp7-expressing cells. As active
Bmp7 expression is limited to the distal tubule and podocytes, and
HoxB7cre labeling shows that collecting duct cells are strictly
confined to collecting ducts, we conclude that the renal vesicle and nascent
epithelial structures are derived from Bmp7cre/+-marked
nephrogenic mesenchyme. This experiment conclusively demonstrates that the
nephrogenic mesenchyme is the exclusive progenitor population of the
nephron.
Domains of TGFß superfamily signaling in the developing kidney
We previously described Smad protein distribution in the developing kidney
(Oxburgh and Robertson, 2002).
At E11.5, Smads 1, 2 and 4 are expressed only in the mesenchyme. Upon contact
with the ureteric bud epithelium, expression of these Smads is downregulated
in the nephrogenic mesenchyme, but remains high in uninduced mesenchyme.
Subsequently, expression is limited to the peripheral and medullary stroma,
mesangial cells and the proximal tubules of mature nephrons. Smad4 is
expressed in the collecting duct system at low levels from approximately
E11.75 onwards.
To ascertain which cells are actively transducing BMP and TGFß
signals, we performed immunohistochemistry on paraffin sections of E11.5-E13.5
kidneys using antisera detecting phosphorylated forms of Smad1 and Smad2
(Persson et al., 1998).
Surprisingly, we find that the domains of activation of the two pathways are
superimposable. At E11.5, signaling is seen almost exclusively in scattered
cells throughout the metanephric blastema
(Fig. 2A,B). By E12.5, the
domains of signaling are more organized, with high levels of activation within
the peripheral and mature stromal compartments. Some activation is seen in
collecting ducts, but very little within nephrogenic mesenchyme
(Fig. 2C,D). At E13.5, a
similar pattern is observed. In addition, signaling is active in the tips and
trunks of collecting ducts (Fig.
2E,F). To verify that the paucity of nuclear phospho-Smad staining
in the nephrogenic mesenchyme was not due to our staining method, we performed
immunohistochemistry using an antigen unmasking protocol
(Schnabel et al., 2001
). No
difference could be seen in phospho-Smad staining using this protocol, despite
homogenous nephrogenic mesenchyme staining using Pax2 antiserum as a control
on adjacent sections (data not shown).
|
Inactivation of Smad4 in the collecting ducts
HoxB7cre was introduced into the Smad4 conditional
background to generate HoxB7cre;Smad4/CA mice that
were born at approximately Mendelian frequencies (21%, n=42). Mutant
mice were phenotypically normal at 6 weeks of age, but by 8 weeks, 3 out of 8
mutant mice had died. Kidneys from these mice did not display overt
morphological defects (data not shown), and
HoxB7cre;Smad4/CA mice are to be assessed for
kidney function. These results will be reported elsewhere. At E16.5, no
difference between mutant and wild-type kidneys was documented, either
macroscopically or histologically (Fig.
3B). Loss of Smad4 in the collecting ducts thus has no effect on
kidney development up to E16.5.
|
Close examination of collecting ducts at E16.5 revealed that the tips are surrounded by variable amounts of nephrogenic mesenchyme, with approximately half of the tips entirely devoid of these cells. This feature of the phenotype is also apparent at E14.5, where a prominent thickening of the cell layer between the nephrogenic mesenchyme and the kidney capsule is seen (Fig. 3E). In wild-type kidneys, this single layer of peripheral stromal cells is located between the nephrogenic mesenchyme and the kidney capsule, and populates the clefts between aggregates of nephrogenic mesenchyme. At E12.5, the lack of nephrogenic mesenchyme in mutants is less obvious, but a marked thickening of the cortical cell layer is evident compared with wild type. Extensive cell death can be seen in cortical regions between aggregates of nephrogenic mesenchyme (Fig. 7A-F). A reduction in the number of collecting ducts can be seen in the mutant, with the average number of peripheral collecting duct tips counted in sagittal sections from the center of three individual kidneys being: 7(±1) in wild type and 6(±1) in mutants at E12.5; 20(±2) in wild type and 8(±1) in mutants at E14.5; and 28(±2) in wild type and 9(±2) in mutants at E16.5.
|
|
Nephrogenic mesenchyme and stroma are incompletely segregated in Bmp7cre/+;Smad4/CA kidneys
In order to understand whether the distribution of stroma and nephrogenic
mesenchyme is disturbed from the outset of metanephric development, we
analyzed gene expression in E11.5 and E12.5 mutant kidneys. At E11.5, the
metanephric mesenchyme of mutant kidneys expresses both Wt1 and
Pax2, indicating normal patterning
(Fig. 5A-D). Expression of
Raldh2 is seen throughout the mesenchyme, and is excluded from
condensates surrounding ureteric bud tips, an expression pattern
indistinguishable from that of wild type
(Fig. 5E,F). Strong expression
of Lhx1 in foci within the mesenchyme indicates the formation of
epithelial structures at comparable times in wild-type and mutant kidneys
(Fig. 5G,H). We therefore
conclude that the distribution of cell types in E11.5 mutant kidneys is
normal.
|
In order to ascertain whether the reduced number of collecting duct branches seen in mutant kidneys could be caused by a lack of expression of Gdnf, in situ hybridization for this gene was carried out at E13.0, a time point at which the depletion of nephrogenic mesenchyme is not yet pronounced. Mutant kidneys showed Gdnf expression similar to wild type (Fig. 5Q,R), indicating that this is not the case.
Lineage analysis of Bmp7cre;Smad4/CA kidneys
To determine the ontogeny of cells within the expanded cortical layer of
mutant kidneys, we next introduced the ROSA26R conditional
reporter into either the
Bmp7cre/+;Smad4/CA or the
Bmp7cre/+ backgrounds (hereafter referred to as mutant and
wild-type, respectively). Kidneys were analyzed at E11.5, E12.0, E14.5 and
E16.5 for lacZ activity. In the mutant, cells derived from
Smad4-deficient progenitors are labeled by lacZ activity. In
both wild-type and mutant kidneys, as expected, the collecting ducts are
homogenously stained at all time-points analyzed. However, comparison of the
mesenchymal and stromal components of wild-type and mutant kidneys reveals
significant differences. At E11.5, mutant mesenchyme is indistinguishable from
wild type, labeled cells being distributed through the blastema but primarily
localized to the area immediately surrounding ureteric bud tips
(Fig. 6B). By E12.0 in
wild-type kidneys, labeled cells are closely associated with collecting duct
tips and display the morphological characteristics of nephrogenic mesenchyme
(Fig. 6C,E). By contrast, there
is significant mixing of labelled, and thus Smad4-deficient, cells
with unlabeled cells of other lineages around the collecting duct tips of the
mutant (Fig. 6D,F). Also,
labeled cells are distributed through the mesenchyme and do not appear to
condense tightly in the vicinity of duct tips. In E14.5 wild-type kidneys,
labeled cells surround the tips of collecting ducts, comprising the
nephrogenic mesenchyme lineage (Fig.
6G). No labeled cells are seen within the peripheral stromal layer
separating the nephrogenic mesenchyme and the kidney capsule. However, the
mutant displays a reduced overall volume of nephrogenic mesenchyme, and
labeled Smad4-deficient cells are ectopically located within the
expanded cortical layer (Fig.
6H). However, this localization is transient because marked cells
are largely missing from the cortical layer of mutants at E16.5
(Fig. 6J).
|
Bmp7cre/+;Smad4/CA kidneys display increased peripheral cell death
To understand the role of cell death in the mutant phenotype, we performed
a careful histological analysis for pyknotic nuclei on serial sections of
wild-type and mutant kidneys. At E12.0, E14.5 and E16.5, clusters of dead
cells can be seen in the thickened areas of the mesenchyme at the periphery of
mutant kidneys (Fig. 7B,D,F). These clusters of dead cells localize to regions distant from collecting duct
tips. This contrasts with the patterns of cell death observed in wild-type
kidneys (Fig. 7A,C,E), in which
only sporadic pyknotic nuclei can be seen at the periphery. In areas of mutant
kidneys in which recognizable nephrogenic mesenchyme is organized around
collecting duct tips, pyknotic nuclei could be seen at a frequency comparable
with that of wild type, indicating that the depletion of nephrogenic
mesenchyme does not occur specifically through cell death.
To assess the degree of proliferation, we immunostained sections for the presence of proliferating cell nuclear antigen (PCNA). Proliferation in the cortical region of the wild-type kidney is distributed evenly throughout the collecting ducts, nephrogenic mesenchyme and peripheral stroma (Fig. 7G,I). This pattern of proliferation is maintained in the mutant. Importantly, collecting ducts display an even distribution of proliferating cells, indicating that the primary cause for the paucity of collecting ducts is not an innate failure of the collecting duct epithelium to proliferate.
Bmp7cre/+;Smad4/CA metanephric mesenchyme responds to induction in vitro
To explore whether the disturbed aggregation of nephrogenic mesenchyme is
due to a defect in the induction of mutant mesenchyme, we used an in vitro
culture system to compare the inducibility of wild-type and mutant mesenchyme
at E11.5, using LiCl as an inducing agent
(Davies and Garrod, 1995)
(Fig. 8). After enzymatic
separation, mesenchyme was cultured on filter rafts in the presence or absence
of LiCl for 72 hours. Mutant mesenchyme was induced, but attained consistently
smaller volumes than wild type at the end of the culture period
(n=10). Histological analysis of induced mesenchyme revealed that
epithelial structures formed both in wild-type and mutant mesenchyme, with
wild-type mesenchyme developing slightly more complex tubular epithelial
structures (Fig. 8E,F).
Immunostaining of mesenchyme revealed an appropriate deposition of laminin
(Fig. 8G,H). This result was
reproduced upon co-culture of mesenchyme with the heterologous inducer spinal
cord (data not shown). We conclude from this assay that
Bmp7cre/+;Smad4/CA mesenchyme is fully
competent to respond to inducing signals.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lineage relationships in the developing kidney
As the Cre-loxP system induces heritable labeling of cells in the
ROSA26R conditional reporter strain, it provides a
convenient tool for lineage analysis. The predicted domains of expression for
the Cre-expressing lines used in this study are: (1) the ureteric bud and
collecting duct system for HoxB7cre
(Srinivas et al., 1999); and
(2) the ureteric bud, collecting duct system and nephrogenic mesenchyme for
Bmp7cre/+ (Godin et
al., 1998
). Because the expression domains of HoxB7 and
Bmp7 overlap exactly in the ureteric bud and collecting ducts, it is
possible to deduce the fate of the nephrogenic mesenchyme by comparison of the
two.
Two main points regarding lineage relationships between cell populations
within the metanephros emerge from this study. The first is that cells of
ureteric bud origin do not contribute in significant numbers to the
mesenchyme, as has previously been suggested
(Herzlinger et al., 1993). The
few lacZ-expressing cells that can be seen outside the collecting
ducts in HoxB7cre;R26R kidneys are confined to the
medullary stroma, and are never seen within the nephrogenic mesenchyme or
mesenchymal structures such as renal vesicles or nephric tubules. A possible
reason for the discrepancy between our findings and those of Herzlinger et al.
(Herzlinger et al., 1993
) is
the fact that the latter study was performed in organ culture using cell
labeling, which is a less accurate technique than in vivo fate mapping. Also,
it cannot be excluded that cell lineages are less strictly segregated in
cultured organs, perhaps owing to the removal of surrounding tissue. The
second finding is that cells of the nephrogenic mesenchyme labeled by
Bmp7cre/+ are restricted to the nephrogenic lineage; only
a scattering of labeled cells can be found within the medullary stroma. The
same scattering of labeled cells was seen with HoxB7cre, suggesting
that these represent cells that have migrated into the kidney. Commitment to
either stromal or nephrogenic lineages thus occurs prior to induction and the
condensation around collecting duct tips, if indeed cells of these lineages
are derived from the same progenitor. As labeled cells are seen in mosaic
fashion in the early metanephric mesenchyme and subsequently consolidate
around collecting duct tips, we conclude that segregation of nephrogenic and
stromal progenitors occurs at this early time point. Future studies using Cre
transgenes that label the stromal cell population will test whether cells of
stromal origin incorporate into the nephrogenic mesenchyme and will clarify
whether these lineages derive from a common precursor.
The role of Smad4 in collecting duct epithelium
In the ureteric bud, onset of BMP and TGFß pathway activation is
relatively late, with appreciable levels of phosphorylated Smads being
detectable first at E12.5 in collecting duct trunks. Removal of Smad4
within this tissue has no effect on kidney development up to E16.5. This is a
surprising result, as several studies have shown that BMP and TGFß
ligands applied to E11.5 kidney explants inhibit growth and branching of the
collecting duct system, and it has therefore been assumed that these pathways
are involved in collecting duct morphogenesis
(Bush et al., 2004;
Clark et al., 2001
;
Piscione et al., 1997
;
Ritvos et al., 1995
;
Rogers et al., 1993
).
Interestingly, a study in which constitutively activated Alk3 was expressed in
collecting ducts using the HoxB7 enhancer-promoter revealed an
inhibitory effect of BMP pathway activation on collecting ducts, confirming
these in vitro findings (Hu et al.,
2003
). However, it was found that phosphorylated Smad1 complexes
with ß-catenin leading to activation of the Wnt pathway, presumably
without the participation of Smad4. It thus seems likely that TGFß
superfamily signals are transduced through Smad4-independent pathways in the
collecting duct. Further studies using inhibitors of alternative pathways that
have been shown in other organ systems to be activated by TGFß
superfamily ligands, such as the MAP kinase cascade, may shed additional light
on this mechanism of signal transduction.
The role of Smad4 in metanephric mesenchyme
The earliest co-localization of Bmp7cre/+ and
Smad4 expression in kidney development is limited to a dispersed
population of cells within the metanephric blastema at E11.0-E11.5. Upon
induction of the blastema by the ureteric bud, the population of
Bmp7cre/+ labeled cells coalesces around the ureteric bud
to form the nephrogenic mesenchyme, and from this point onward
Bmp7cre/+ labeled cells are confined to the nephrogenic
mesenchyme and its derivatives. Nephrogenic mesenchyme expresses very little
Smad4, and shows limited pathway activation
(Oxburgh and Robertson, 2002).
We therefore conclude that the phenotype seen upon loss of Smad4 in
dispersed cells of the metanephric blastema occurs prior to formation of the
nephrogenic mesenchyme. However, it cannot be excluded that the very small
number of nephrogenic mesenchyme cells displaying Smad4 expression at
later points in development contribute to the phenotype.
The profound effects of Bmp7cre/+ inactivation of
Smad4 can be divided into two categories: those that phenocopy
previously described TGFß superfamily pathway mutants, and novel effects.
Like the Bmp7 homozygous null mutant, the
Bmp7cre/+;Smad4/CA mutant displays
ectopic cell death at the periphery of the kidney at E12.5, and premature
depletion of nephrogenic mesenchyme. The Bmp7 mutant phenotype
originates from a lack of Bmp7 signaling to the progenitor cell
population of the developing kidney
(Dudley et al., 1999). That
this phenotype can be recapitulated by the inactivation of Smad4 in
Bmp7-expressing cells implies that these progenitors reside within
the nephrogenic mesenchyme. Bmp7 thus appears to act in an autocrine manner to
promote progenitor survival. The early and mosaic appearance of the
Bmp7-expressing cell population suggests that the metanephric
blastema is composed of distinct cell types, and that the first inductive
contact with the ureteric bud serves as a signal to organize nephrogenic
progenitors around the inducer.
The expansion of the cortical layer of
Bmp7cre/+;Smad4/CA mutant kidneys is
previously undescribed in any TGFß superfamily mutation. Our analysis of
Wt1 and Pax2 expression in wild-type kidneys reveals that
these markers are expressed in partially overlapping cell populations in the
mesenchyme. At E11.5, Wt1 is expressed throughout the metanephric
blastema, whereas Pax2 expression is localized mainly to mesenchyme
surrounding ureteric bud tips. At E12.5, Wt1 expression is still seen
in the majority of the mesenchyme, and expression is intensified in
nephrogenic mesenchyme surrounding collecting duct tips. Pax2
expression is limited to nephrogenic mesenchyme and maturing epithelial
structures. The Wt1-expressing but Pax2-negative cell
population at the periphery of the kidney overlaps with cells expressing
Raldh2, a stromal cell marker. This cell population, with
characteristics of both nephrogenic mesenchyme and stroma, is greatly expanded
in Bmp7cre/+;Smad4/CA mutant kidneys
from E12.5 to E14.5. We suggest that this immature precursor of both
nephrogenic mesenchyme and peripheral stroma remains uninduced in the mutant
kidney, and is gradually recruited into the nephrogenic and stromal lineages.
Induction by collecting duct tips recruits mesenchyme into the nephrogenic
lineage, thus leaving the cortical region of the mutant kidney devoid of
Wt1-expressing cells by E16.5. Alternatively, co-expression of
Wt1 and Raldh2 in cells of the thickened cortical layer
could indicate the presence of an immature population of cells committed to
the nephrogenic lineage but expressing certain stromal cell markers that are
downregulated upon induction into the nephrogenic mesenchyme. Interestingly,
focal cell death can be seen in areas of the expanded peripheral cell layer
distant from collecting duct tips, indicating that this cell population is
dependent on signals from collecting ducts for its survival. The cortical
expansion of a population of cells with both nephrogenic and stromal
characteristics is also seen in compound mutants of the retinoic acid
receptors and ß2 (Mendelsohn
et al., 1999
). This phenotype can be rescued by transgenic
overexpression of Ret in collecting ducts, conclusively showing that
the mesenchyme phenotype is secondary to a collecting duct defect. The
similarity of this primarily collecting duct phenotype to the mesenchymal
Bmp7cre/+;Smad4/CA phenotype supports
our hypothesis, as one would expect a defect in inductive capacity of the
collecting ducts, or a poor capacity of mesenchyme to be induced, to result in
insufficient recruitment of nephrogenic mesenchyme and a persistent peripheral
population of primitive mesenchyme. Analysis of a ureteric bud cell line
supernatant with inductive capacity has identified Tgfß2 as a component
of the inducer (Plisov et al.,
2001
). Considering the many functions that TGFßs display in
the control of cell-matrix interactions and the deposition of extracellular
matrix (Verrecchia et al.,
2001
), it is tempting to speculate that the phenotype seen in
Bmp7cre/+;Smad4/CA mutant kidneys is due
to an inability of mesenchyme to respond to a TGFß signal that determines
a change in extracellular matrix composition allowing aggregation or
compaction of these cells.
In conclusion, the data presented here provide the first evidence that a TGFß superfamily signal mediated through Smad4 is required to recruit mesenchyme cells from a primitive state in which they display both nephrogenic and stromal characteristics into the nephrogenic mesenchyme. The surprising finding that deletion of Smad4 in the ureteric bud and collecting ducts does not result in an appreciable phenotype is most readily explained by alternative pathway activation by TGFß superfamily ligands and receptors, possibly through ß-catenin and the Wnt pathway. Further genetic lineage analysis of the early metanephros will clarify our understanding of the sequence of events leading from specification of the metanephric blastema to the segregation of the various cell-types required for morphogenesis.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bard, J. B., Gordon, A., Sharp, L. and Sellers, W. I. (2001). Early nephron formation in the developing mouse kidney. J. Anat. 199,385 -392.[CrossRef][Medline]
Barnes, J. D., Crosby, J. L., Jones, C. M., Wright, C. V. and Hogan, B. L. (1994). Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis. Dev. Biol. 161,168 -178.[CrossRef][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]
Bosukonda, D., Shih, M. S., Sampath, K. T. and Vukicevic, S. (2000). Characterization of receptors for osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7) in rat kidneys. Kidney Int. 58,1902 -1911.[Medline]
Bush, K. T., Sakurai, H., Steer, D. L., Leonard, M. O., Sampogna, R. V., Meyer, T. N., Schwesinger, C., Qiao, J. and Nigam, S. K. (2004). TGF-[beta] superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud. Dev. Biol. 266,285 -298.[CrossRef][Medline]
Chu, G. C., Dunn, N. R., Anderson, D. C., Oxburgh, L. and
Robertson, E. J. (2004). Differential requirement for
Smad4 in TGFb-dependent patterning of the early mouse embryo.
Development 131,3501
-3512.
Clark, A. T., Young, R. J. and Bertram, J. F. (2001). In vitro studies on the roles of transforming growth factor-beta 1 in rat metanephric development. Kidney Int. 59,1641 -1653.[CrossRef][Medline]
Davies, J. A. and Garrod, D. R. (1995). Induction of early stages of kidney tubule differentiation by lithium ions. Dev. Biol. 167,50 -60.[CrossRef][Medline]
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]
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., Lyons, K. M. and Robertson, E. J. (1995). A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9,2795 -2807.[Abstract]
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.
Dymecki, S. M. (1996). Flp recombinase promotes
site-specific DNA recombination in embryonic stem cells and transgenic mice.
Proc. Natl. Acad. Sci. USA
93,6191
-6196.
Esquela, A. F. and Lee, S. E.-J. (2003). Regulation of metanephric kidney development by growth/differentiation factor 11. Dev. Biol. 257,356 -370.[CrossRef][Medline]
Godin, R. E., Takaesu, N. T., Robertson, E. J. and Dudley, A.
T. (1998). Regulation of BMP7 expression during kidney
development. Development
125,3473
-3482.
Gu, H., Zou, Y. R. and Rajewsky, K. (1993). Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73,1155 -1164.[Medline]
Hellmich, H. L., Kos, L., Cho, E. S., Mahon, K. A. and Zimmer, A. (1996). Embryonic expression of glial cell-line derived neurotrophic factor (GDNF) suggests multiple developmental roles in neural differentiation and epithelial-mesenchymal interactions. Mech. Dev. 54,95 -105.[CrossRef][Medline]
Herzlinger, D., Abramson, R. and Cohen, D. (1993). Phenotypic conversions in renal development. J. Cell Sci. 17,61 -64.
Hogan, B. L., Beddington, R. S., Costantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hu, M. C., Piscione, T. D. and Rosenblum, N. D.
(2003). Elevated SMAD1/{beta}-catenin molecular complexes and
renal medullary cystic dysplasia in ALK3 transgenic mice.
Development 130,2753
-2766.
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]
Lagna, G., Hata, A., Hemmati-Brivanlou, A. and Massagué, J. (1996). Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 383,832 -836.[CrossRef][Medline]
Massagué, J. (2000). How cells read TGF-beta signals. Nat. Rev. Mol. Cell Biol. 1, 169-178.[CrossRef][Medline]
Mendelsohn, C., Batourina, E., Fung, S., Gilbert, T. and Dodd,
J. (1999). Stromal cells mediate retinoid-dependent functions
essential for renal development. Development
126,1139
-1148.
Michael, S. K., Brennan, J. and Robertson, E. J. (1999). Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci. Mech. Dev. 85, 35-47.[CrossRef][Medline]
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.
Miyazaki, Y., Oshima, K., Fogo, A. and Ichikawa, I. (2003). Evidence that bone morphogenetic protein 4 has multiple biological functions during kidney and urinary tract development. Kidney Int. 63,835 -844.[CrossRef][Medline]
Miyazono, K., Kusanagi, K. and Inoue, H. (2001). Divergence and convergence of TGF-beta/BMP signaling. J. Cell Physiol. 187,265 -276.[CrossRef][Medline]
Oxburgh, L. and Robertson, E. J. (2002). Dynamic regulation of Smad expression during mesenchyme to epithelium transition in the metanephric kidney. Mech. Dev. 112,207 -211.[CrossRef][Medline]
Pachnis, V., Mankoo, B. and Costantini, F.
(1993). Expression of the c-ret proto-oncogene during mouse
embryogenesis. Development
119,1005
-1017.
Pelton, R. W., Saxena, B., Jones, M., Moses, H. L. and Gold, L. I. (1991). Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J. Cell Biol. 115,1091 -1105.[Abstract]
Persson, U., Izumi, H., Souchelnytskyi, S., Itoh, S., Grimsby, S., Engstrom, U., Heldin, C. H., Funa, K. and ten Dijke, P. (1998). The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation. FEBS Lett. 434,83 -87.[CrossRef][Medline]
Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L. and Rosenblum, N. D. (1997). BMP-2 and OP-1 exert direct and opposite effects on renal branching morphogenesis. Am. J. Physiol. 273,F961 -F975.[Medline]
Plisov, S. Y., Yoshino, K., Dove, L. F., Higinbotham, K. G.,
Rubin, J. S. and Perantoni, A. O. (2001). TGFß2, LIF and
FGF2 cooperate to induce nephrogenesis. Development
128,1045
-1057.
Ritvos, O., Tuuri, T., Eramaa, M., Sainio, K., Hilden, K., Saxen, L. and Gilbert, S. F. (1995). Activin disrupts epithelial branching morphogenesis in developing glandular organs of the mouse. Mech. Dev. 50,229 -245.[CrossRef][Medline]
Rogers, S. A., Ryan, G., Purchio, A. F. and Hammerman, M. R. (1993). Metanephric transforming growth factor-beta 1 regulates nephrogenesis in vitro. Am. J. Physiol. 264,F996 -F1002.[Medline]
Sanchez, M. P., Silos-Santiago, I., Frisen, J., He, B., Lira, S. A. and Barbacid, M. (1996). Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382,70 -73.[CrossRef][Medline]
Sanford, L. P., Ormsby, I., Gittenberger-de Groot, A. C.,
Sariola, H., Friedman, R., Boivin, G. P., Cardell, E. L. and
Doetschman, T. (1997). TGFbeta2 knockout mice have multiple
developmental defects that are non-overlapping with other TGFbeta knockout
phenotypes. Development
124,2659
-2670.
Saxén, L. (1987). Organogenesis of the kidney. Cambridge, UK: Cambridge University Press.
Schnabel, C. A., Selleri, L., Jacobs, Y., Warnke, R. and Cleary, M. L. (2001). Expression of Pbx1b during mammalian organogenesis. Mech. Dev. 100,131 -135.[CrossRef][Medline]
Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V. (1994). Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367,380 -383.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Srinivas, S., Goldberg, M. R., Watanabe, T., D'Agati, V., al-Awqati, Q. and Costantini, F. (1999). Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis. Dev. Genet. 24,241 -251.[CrossRef][Medline]
Vega, Q. C., Worby, C. A., Lechner, M. S., Dixon, J. E. and
Dressler, G. R. (1996). Glial cell line-derived
neurotrophic factor activates the receptor tyrosine kinase RET and promotes
kidney morphogenesis. Proc. Natl. Acad. Sci. USA
93,10657
-10661.
Verrecchia, F., Chu, M. L. and Mauviel, A.
(2001). Identification of novel TGF-beta /Smad gene targets in
dermal fibroblasts using a combined cDNA microarray/promoter transactivation
approach. J. Biol. Chem.
276,17058
-17062.
Vukicevic, S., Latin, V., Chen, P., Batorsky, R., Reddi, A. H. and Sampath, T. K. (1994). Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes. Biochem. Biophys. Res. Commun. 198,693 -700.[CrossRef][Medline]
Wilkinson, D. G. (1992). Whole-mount in situ hybridization of vertebrate embryos. Oxford, UK: IRL Press.
Xiao, Z., Latek, R. and Lodish, H. F. (2003). An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity. Oncogene 22,1057 -1069.[CrossRef][Medline]