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First published online 30 November 2005
doi: 10.1242/dev.02174
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1 Division of Stem Cell Regulation, The Institute of Medical Science, The
University of Tokyo, Tokyo 108-8639, Japan.
2 Department of Life Sciences (Biology), Graduate School of Arts and Sciences,
The University of Tokyo, Tokyo 153-8902, Japan.
3 ICORP, JST, Saitama 332-0012, Japan.
4 Division of Integrative Cell Biology, Institute of Molecular Embryology and
Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
5 Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625
Hannover, Germany.
6 PRESTO, JST, Saitama 332-0012, Japan.
* Author for correspondence (e-mail: ryuichi{at}kaiju.medic.kumamoto-u.ac.jp)
Accepted 24 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Progenitor, Kidney, Colony-forming assay, Sall1, Wnt, PCP, JNK, Rho, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Inductive
signals have been vigorously investigated, and several factors have been
elucidated that trigger epithelialization of metanephric mesenchyme in explant
culture system; the members of Wnt family
(Herzlinger et al., 1994;
Kispert et al., 1998),
leukemia inhibitory factor (LIF) (Barasch
et al., 1999; Plisov et al.,
2001), and transforming growth factor ß2 (TGFß2)
(Plisov et al., 2001). These
studies have also suggested the presence of clonal cells in mesenchymal
rudiments, which sequentially form renal condensation, comma (C)- and S-shaped
bodies, and terminally epithelia of glomeruli and renal tubules, and the
existence of single epithelial precursors responding to LIF was demonstrated
in mesenchyme (Barasch et al.,
1999). One previous report suggested retrospectively the presence
of multipotent cells in embryonic kidneys, demonstrating that cells in several
portions of nephron were derived from a single stem cell using lacZ
gene transduction with retrovirus into a single cell of mesenchyme
(Herzlinger et al., 1992).
However, none has isolated prospectively the renal progenitor cells with a
multilineage differentiation potential from the embryonic kidney, and none has
examined their differentiation mechanisms in a single cell culture. There has
been a lack of assay systems that specifically identify renal progenitors, as
in cases of the neurosphere method for neural stem cells
(Reynolds et al., 1992) and
the colony assay for hematopoietic progenitors
(Pluznik and Sachs, 1965;
Bradley and Metcalf, 1966).
We previously generated mice in which the green fluorescence protein gene
(GFP) was knocked into the locus of Sall1
(Sall1-GFP mice), a zinc finger nuclear factor that is expressed in
the metanephric mesenchyme and that is essential for kidney development
(Nishinakamura et al., 2001;
Takasato et al., 2004).
Sall1 is also expressed in the subventricular zone of the central
nervous system and progress zones of limb buds, where neural and mesenchymal
stem cells reside, respectively, leading to speculation that Sall1
might have some association with stem cells in several organs, including the
kidney.
Targeted disruption of Wnt4 results in kidney agenesis and impairs
mesenchymal-to-epithelial transformation
(Stark et al., 1994), and
co-culture with 3T3Wnt4 induces tubulogenesis in the mesenchyme rudiment in
organ culture (Kispert et al.,
1998), suggesting both essential and sufficient roles of Wnt4 for
epithelial differentiation of metanephric mesenchyme. Recently, Wnt9b
expressed in the ureteric bud was shown to function upstream of Wnt4
(Carroll et al., 2005). Thus,
we attempted to set up assay systems that can identify and characterize the
progenitor cells with multipotent differentiation potential from uninduced
metanephric mesenchyme using Wnt4 signal. Wnt genes are known to regulate
multiple cellular functions using at least three intracellular signaling
branches: the ß-catenin pathway (canonical pathway), in which stabilized
ß-catenin interacts with members of the lymphoid enhancer factor/T cell
factor (LEF/TCF) family of transcription factors and activates gene expression
in the nucleus (Wodarz and Nusse,
1998; Miller et al.,
1999); the planar cell polarity (PCP) pathway, which involves Jun
N-terminal kinase (JNK) and the Rho family of small guanosine triphosphatases
(GTPases) and which directs cytoskeletal rearrangements, coordinated
polarization within the plane of epithelial sheets, and morphogenetic
movements during development (Veeman et
al., 2003; Wallingford et al.,
2002); and the Wnt/Ca2+ pathway, which leads to release
of intracellular calcium and is implicated in Xenopus ventralization
and in the regulation of embryonic cell movements
(Miller et al., 1999;
Veeman et al., 2003;
Wallingford et al., 2002).
Mechanisms by which Wnt pathways mediate cellular effects in kidney
development are poorly understood.
In this study, we established a novel colony-forming assay system using 3T3-expressing Wnt4 to identify renal progenitors in the metanephric mesenchyme. Combining our colony-forming assay with flow cytometry, we found that these progenitors could be enriched by using Sall1 as a marker. We also examined the effects of Wnt downstream branches on the renal progenitors.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were
mitotically inactivated with mitomycin C before use. Single mesenchymal cells
were sorted by FACS Vantage (Becton Dickinson) and plated onto these feeder
cells at a low density (5x103 cells/well of 6-well plates),
then cultured in DMEM/F12 with 5% knockout serum replacement (Invitrogen), 10
µg/ml insulin, 6.7 µg/l sodium selenite, 5.5 µg/ml transferrin,
1x10-7 mol/l dexamethasone, 10 mmol/l nicotinamide, 2 mmol/l
L-glutamine, 50 µmol/l ß-mercaptoethanol, 5 mmol/l HEPES
and penicillin/streptomycin.
RT-PCR
Primers used for PCR were as follows:
8, 5'-GGCGAAAGTGCAGTCCTAAA-3' and
5'-GAAGGAGACATTCGGAGTGG-3'; 3, 5'-CGGCCTGTCATCAATATCCT-3' and
5'-CGAACATTGTCCATCAGCAG-3'; -actinin-4, 5'-TGGTGCAACTCTCATCTTCG-3' and
5'-CCGCAGCTTGTCATACTCAA-3'; PCR cycles were as follows: Gapdh, initial denaturation at 94°C for 2.5 minutes, followed by 22 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and final extension at 72°C for 10 minutes; other genes, initial denaturation at 94°C for 2.5 minutes, followed by 28-33 cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 30 seconds, and final extension at 72°C for 10 minutes.
Organ culture
In order to examine the in vitro differentiating potential of cell
populations included in the metanephric mesenchyme, each cell population was
separated by flow cytometry and was pelleted down by low-speed centrifugation
(380 g). The resultant cell pellet (1x104
cells per pellet) was cultured on 3T3Wnt4 cells at air-fluid interface on a
polycarbonate filter (0.4 µm, Nucleopore) supplied with DMEM plus 10% fetal
calf serum at 37°C, 5% carbon dioxide. 3T3Wnt4 cells (50,000 cells in 50
µl medium) were seeded on the filter 24 hours before the experiments, as
described (Kispert et al.,
1998). To examine the influence of reagents on tubulogenesis, two
metanephroi or mesenchyme rudiments from E11.5 embryos were cultured on a
polycarbonate filter. For the culture of mesenchyme rudiments, 3T3Wnt4 cells
were used as described above.
Retroviral infection
The cDNA clones of the active mutant form of ß-catenin
(pUC-EF-1-ß-cateninSA-3HA)
(Miyagishi et al., 2000), the
full length of rat axin (pBSKS-rAxin)
(Ikeda et al., 1998), and both
constitutively-active and dominant-negative mutant forms of human
Rac1 and RhoA with N-terminus flag tag [pCAGIP-flag-Rac1
(Val), pCAGIP-flag-Rac1 (Asn), pCAGIP-flag-RhoA (Val), pCAGIP-flag-RhoA
(Asn)] were subcloned into retroviral vector pMY-IRES-EGFP
(Kitamura et al., 2003). To
produce recombinant retrovirus, these plasmid vectors were transfected into
the virus packaging cell line PLAT-E
(Morita et al., 2000) using
FuGENE (Roche), and supernatant from the transfected cells was collected to
infect cells of the metanephric mesenchyme. The viral supernatant was
centrifuged at 20,000 g overnight at 4°C to concentrate
the virus. To infect mesenchymal cells with the retrovirus, dissociated
mesenchymal cells were resuspended into the concentrated virus supernatant
with adding polybrene. The suspension was centrifuged 1400 g
for 4 hours at room temperature. After washing with PBS, mesenchymal cells
were plated onto 3T3 feeder cells.
Immunocytochemistry and lectin staining
The colonies formed on 3T3Wnt4 feeder were fixed with 4% paraformaldehyde
in PBS for 20 minutes at 4°C. After washing with PBS, PBS containing 2%
skimmed milk and 0.1% Triton-X was incubated as a blocking solution for 1 hour
at room temperature. The fixed dishes were incubated with primary antibodies
overnight at 4°C followed by incubating with secondary antibodies for 1
hour at room temperature. The following antibodies were used: rabbit anti-Pax2
(Babco), rabbit anti-WT1 (Santa Cruz), mouse anti-E-cadherin (Becton
Dickinson), rabbit anti-AQP1 (Chemicon), and rabbit anti-phosphorylated JNK1
and 2 (Biosource). Rhodamine-conjugated anti-rabbit IgG (H+L) and anti-mouse
IgG (Chemicon) were used as secondary antibodies. To examine the expression of
a proximal renal tubule-specific marker, fluorescein isothiocyanate
(FITC)-conjugated Lotus Tetragonobulus lectin (LTL; Vector Labs) was
used. After each step, the cultured cells were washed three times with PBS
containing 0.1% Triton-X. For detection of Sall1, mesenchymal cells derived
from Sall1-GFP heterozygote embryos were cultured on 3T3 feeder and
subjected to GFP immunostaining procedure using rabbit anti-GFP (Molecular
Probes). Rhodamine-conjugated peanut agglutinin (PNA; Vector Labs) staining
was done as described (Gilbert et al.,
1994). Organ culture tissues were fixed with 4% paraformaldehyde
in PBS for 1 hour at 4°C and incubated in PBS including 0.1% saponin
(Sigma) for 1 hour at 37°C, then the same staining procedure was carried
out. Staining with rabbit anti-secreted frizzled-related protein 2 (sFRP2;
Santa Cruz) and FITC-conjugated Dolichos biflorus agglutinin (DBA;
Vector Labs) were also used on sections of paraffin-embedded explants to
examine the effect of reagents on tubule formation and branching,
respectively.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was used to distinguish mesenchyme-derived cells from feeder
cells, and single cells sorted by flow cytometry were cultured at a low cell
density on 3T3Wnt4. This culture condition resulted in the formation of
sheet-like colonies not formed on 3T3lacZ
(Fig. 1A, upper panels), while
scattered fibroblast-like cells were observed in both conditions
(Fig. 1A, lower panels,
arrows). Colonies were not formed in the presence of frizzled (Fz)-Fc chimeric
protein, a Wnt inhibitor, thus confirming an essential role of Wnt4 for colony
formation (Fig. 1B). Colonies
were not formed by culturing in the conditioned medium from 3T3Wnt4 without
feeder cells (data not shown). Colonies were also formed on 3T3Wnt3a, but not
in feeder-free conditions using a purified recombinant Wnt3a protein (data not
shown). These data suggested the requirement of other signals from 3T3 cells,
in addition to the Wnt signals for the colony formation. In the presence of
serum, colonies were not formed even on 3T3Wnt4, and some factors in the serum
might prevent colony formation (data not shown). When colonies on 3T3Wnt4 were
dissociated and plated onto fresh feeder cells at day 10 of culture, few
colonies were obtained, and maintenance of these colonies could not be
achieved (data not shown). When we tried colony-formation by using
polycarbonate filters, which separate mesenchymal cells from the feeder layer,
colonies were formed but the number of colonies formed was much smaller than
that formed by directly culturing on feeder cells (data not shown).
To characterize the molecular profiles of the colonies, genes expressed in
the metanephric mesenchyme were examined by RT-PCR using RNA from the colonies
together with 3T3Wnt4 (Fig.
1C). All the mesenchymal genes examined (Pax2, Lim1, Eya1,
Sall1, WT1, Hoxa11, Gdnf, integrin 8, integrin 3,
Ncam, E-cadherin and K-cadherin) were expressed, and the expression
continued to day 20 (Fig. 1C,
lanes 3-5). By contrast, when cultured on 3T3lacZ, the expression of
these genes was below the detection level (lanes 7-9). The expression of
ureteric bud markers (Ret and Hoxb7) were not detected in
mesenchyme separated from ureteric bud, suggesting that the separation was
successful (lane 1). To determine the potential for differentiation within the
colonies, markers for terminally differentiated epithelia in glomeruli
(podocyte), proximal or distal tubules, and the loop of Henle were also
examined (glomeruli: -actinin-4, CD2-AP, P-cadherin,
podoplanin and podocalyxin; proximal tubule: Aqp1, Clc5, cubilin,
megalin and Sglt1; Henle's loop: Brn1 and Nkcc2;
Henle's loop or distal tubule: Clck2, polycystin 2, and
Romk2; distal tubule: ENaC, Na/Ca exchanger and polycystin
1. These markers encode: (1) cytoskeletal or structural proteins:
-actinin-4, CD2-AP, P-cadherin, podoplanin and podocalyxin;
(2) transcription factor: Brn1; (3) water or ion channels: Aqp1,
Clc5, Clck2, Romk2, ENaC, and polycystin1 and 2; and (4) transporters:
cubilin, megalin, Sglt1, Nkcc2 and Na/Ca exchanger. As shown in
Fig. 1C, almost all the genes
examined were expressed at day 20 on 3T3Wnt4 (lane 5), while these markers
were not expressed on 3T3lacZ (lanes 7-9). To ascertain that these
genes were expressed by the colony-forming cells, colonies were formed from
GFP transgenic mesenchyme, and cells expressing GFP were separated from feeder
layers by using flow cytometry sorting. RT-PCR using RNA from these cells
suggested that the marker genes examined were indeed expressed by
colony-forming cells (lane 10). Furthermore, we made use of
immunocytochemistry and found that Pax2
(Fig. 1D-F), E-cadherin
(Fig. 1G-I), Sall1
(Fig. 1J,K), and Aqp1
(Fig. 1L,M) were expressed on
colonies. The expression of Pax2 and E-cadherin was not detected on
immunocytochemistry at day 3, and was subsequently upregulated by day 10,
which was consistent with the result of RT-PCR
(Fig. 1D,E,G,H). These data
suggest that dissociated cells from the metanephric mesenchyme form colonies
on 3T3Wnt4 feeder cells in serum-free conditions, and that these colonies
contain differentiated epithelia expressing marker genes for epithelia in
glomeruli (podocyte), proximal or distal tubules, and the loop of Henle.
Colonies are derived from a single multipotent renal progenitor
To confirm that these colonies were derived from a single cell, each single
cell sorted from the EGFP transgenic mesenchyme was cultured in an individual
well of 96-well plates coated with 3T3Wnt4. The sheet-like colony was found in
166 wells out of a total of 1632 (10.2%) from three independent experiments
(Fig. 2A).
To examine the multilineage differentiation of single cell-derived colonies, RT-PCR was done for 22 independent wells containing a colony at day 20. The representative data from three colonies are shown in Fig. 2B (lanes 1-3). Although variation existed between colonies, all the colonies expressed markers for each of the three segments: glomerular podocytes, proximal tubules and Henle's loop or distal tubules. Double staining using PNA and LTL, specific to glomerular podocytes and the proximal renal tubule, respectively, showed that adult kidney (8 weeks old) contained three kinds of cells; single-positive for PNA (those in the glomerulus); single-positive for LTL (those in the proximal renal tubule); and double-negative for LTL or PNA (Fig. 2C, left panel). Similarly, a single cell-derived colony at day 20 contained these three kinds of cells (Fig. 2C, right panel). With a combination of LTL and E-cadherin, at least three cell types were observed in adult kidney (Fig. 2D, left panel) and in a single cell-derived colony (right panel): cells strongly expressing only E-cadherin characteristic of distal renal tubules (Fig. 2D, arrows), and LTL-positive or -negative cells, with a faint expression of E-cadherin in the cell boundary. These results suggest that a colony was derived from a single progenitor, with multipotent differentiating capacity into epithelial cells in glomeruli, proximal and distal tubule, and the loop of Henle.
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). As
Sall1 is expressed in mesenchyme-derived tissues, GFP was detected in
the mesenchyme around the ureteric bud at E11.5 in the Sall1-GFP
heterozygous mouse (Fig. 3A,
arrows). At E17.5, GFP-expressing cells were observed in the mesenchyme near
the surface, as well as in C- or S-shaped bodies, and parts of renal tubules
(Fig. 3B). By flow-cytometrical
analysis, three subpopulations were fractionated based on the expression of
Sall1-GFP: Sall1-GFPhigh, Sall1-GFPlow and
Sall1-GFPnegative (Fig.
3C), and cells in these subpopulations were separated by flow
cytometry sorting to be characterized using RT-PCR. As shown in
Fig. 3D,
Sall1-GFPhigh cells expressed Sall1 and Pax2.
Sall1-GFPlow cells expressed markers of stroma (Foxd1,
previously known as BF2), endothelia (Flk1 and VE-cadherin),
and blood cell (Cd45), in addition to Sall1 and
Pax2. Sall1-GFPnegative cells expressed Flk1 and
Cd45. The markers of fully differentiated renal epithelia were not
expressed in these three populations. These data suggested that cells of
stromal lineage were included in cell populations weakly expressing
Sall1 and that those of hemangiogenic lineage were included in both
Sall1-GFPlow and Sall1-GFPnegative populations. Then,
the numbers of the colony-forming progenitors in each subpopulation were
examined using the low-density culture on 3T3Wnt4
(Fig. 3E). At E11.5, colonies
were formed exclusively from the Sall1-GFPhigh population, and not
from Sall1-GFPlow or Sall1-GFPnegative populations. At
E14.5 and 17.5, colonies were also formed only from Sall1-GFP high
subpopulations, but the frequency of colony-forming progenitors decreased as
gestation proceeded. These results indicate that renal progenitors with
multipotent differentiating capacity are included in cell populations strongly
expressing Sall1 throughout gestation periods.
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;
Kispert et al., 1998).
Sall1-GFPhigh, Sall1-GFPlow and
Sall1-GFPnegative cells were separated by flow cytometry,
aggregated to form a cell pellet by centrifugation and cultured on 3T3Wnt4
feeder cells in an organ culture setting. Starting from day 3 in culture,
tubulogenesis was observed only in the aggregate of the
Sall1-GFPhigh population (Fig.
4A, upper panels), while that from Sall1-GFPlow or
Sall1-GFPnegative did not differentiate and disappeared by day 7
(Fig. 4A, lower panels; data of
Sall1-GFPnegative, not shown). In sections of the
Sall1-GFPhigh aggregate (Fig.
4B), many tubule- (t) and glomerulus-like structures (g) were
observed, and the expression of markers for glomerular podocyte (Wt1,
Fig. 1C, red) and proximal
tubule (LTL, green) was confirmed by confocal microscopy. These data suggest
that only Sall1-GFPhigh cells differentiate into renal epithelia in
vitro in a three-dimensional setting, in addition to forming colonies.
Colony size is affected by the absence of Sall1
To investigate the role of Sall1 in colony formation, mesenchymal
cells from Sall1+/+, Sall1+/- and
Sall1-/- embryos at E11.5, which were obtained from
intercrosses of Sall1-GFP mice, were plated on 3T3Wnt4 feeder cells
at a low density. Ten days after culture, double immunostaining using anti-GFP
and anti-E-cadherin antibodies was done to strengthen the green fluorescence
and to examine the expression of E-cadherin, respectively
(Fig. 5). The numbers of
colonies formed were not significantly different among wild-type, heterozygous
and homozygous mesenchyme, suggesting that colony-forming progenitors do exist
and are not decreased in the absence of Sall1 (data not shown).
Colonies derived from Sall1+/+ wild-type mesenchyme were
not stained with GFP (Fig. 5A),
while Sall1+/- and
Sall1-/- colonies were positive for GFP
(Fig. 5C,E, green), indicating
that Sall1 itself is not required for Sall1 promoter
activity. Colonies from all three groups
(Sall1+/+,
Sall1+/- and
Sall1-/-) were also positive for E-cadherin
(Fig. 5B,D,F), suggesting that
differentiation (mesenchymal-to-epithelial transformation) may not be impaired
in the absence of Sall1. Indeed, marker gene expression for
terminally differentiated epithelia in glomeruli and renal tubules was not
changed among Sall1+/+,
Sall1+/- and
Sall1-/- colonies on RT-PCR analyses (data not
shown). By contrast, the size of Sall1-/-
colonies (Fig. 5E,F) was
significantly smaller than Sall1+/+ and
Sall1+/- colonies
(Fig. 5B-D), and this was
confirmed statistically (Table
1). Thus, Sall1 is not required for generation or
differentiation of renal progenitors, but the colony size is affected by
Sall1 absence. This is consistent with our previous report that
Sall1-deficient mesenchyme is competent with respect to epithelial
differentiation tested by spinal cord recombination
(Nishinakamura et al., 2001).
In the spinal cord recombination experiments, Sall1-deficient
mesenchyme was consistently smaller than wild-type mesenchyme, but this could
be due to differences in the initial size of the mesenchyme. Using the
colony-forming assay starting from a single cell, we now show that
Sall1 is indeed required for the colony from the mesenchyme to
develop into a normal size.
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;
Kitamura et al., 2003), we
observed EGFP expression in 12.9% of colony-forming progenitor cells (116
colonies expressing green fluorescence per total of 896 colonies formed from
three independent experiments). Thus, the colony-forming assay in this study
enables us to investigate direct effects of reagents and gene transduction on
colony-forming progenitor cells, allowing us to examine the roles of Wnt and
its downstream branches in kidney development. Positive immunostaining of the
colonies for activated JNK1 and 2 indicated that the JNK branch of the PCP
pathways (Boutros et al., 1998)
may be activated downstream of Wnt4 (Fig.
6A). Indeed, the addition of two kinds of JNK inhibitor (JNKI1 and
JNKI2) (Bonny et al., 2001;
Bennett et al., 2001) gave rise
to smaller colonies than did the control without reagents
(Fig. 6B,C, colonies from EGFP
transgenic mesenchyme, Table
2). The result of control experiments using the HIV-TAT peptide
excluded the possibility that the effects of JNKI1 were derived from
non-specific toxicity of the peptide constituting the inhibitor
(Fig. 6B). We then investigated
effects of both activation and inactivation of Rac1, one of the Rho family
GTPases implicated in PCP pathways (Habas
et al., 2003), on colony formation. Cells from wild-type E11.5
mesenchyme were transduced with both constitutively active (CA) and
dominant-negative (DN) forms of Rac1 using the retroviral vector
pMY-IRES-EGFP. Colonies consisting of cells expressing both EGFP and
CA-Rac1 were larger than those transduced with pMY-IRES-EGFP controls
(Fig. 6D,
Table 3), suggesting positive
effects on colony size. By contrast, the transduction of DN-Rac1 gave
rise to smaller colonies than did the controls
(Fig. 6D,
Table 3). The numbers of
colonies formed were not significantly changed either with the addition of
inhibitors or with gene transduction (data not shown). These data indicate
that Rac and JNK pathways positively regulate colony size.
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; Winter et al.,
2001; Habas et al.,
2001; Habas et al.,
2003), with the addition of ROCK inhibitor, Y27,632
(Uehata et al., 1997)
(Fig. 6E, colonies from EGFP
transgenic mesenchyme, Table
2), or the transduction of DN-RhoA, increased the colony
size (Fig. 6F, Table 3), while the activation
with CA-RhoA decreased it (Fig.
6F, Table 3).
Activation of ß-catenin signaling both with the addition of two kinds of
glycogen synthetase kinase (GSK)-3 inhibitors, lithium chloride (LiCl)
(Klein and Melton, 1996)
(Fig. 6G, colonies from EGFP
transgenic mesenchyme, Table 2) and (2'Z, 3'E)-6-Bromoindirubin-3'-oxime (BIO)
(Sato et al., 2004)
(Table 2) and with the
transduction of the active form of ß-catenin
(Fig. 6H,
Table 3) gave rise to smaller
colonies. However, inactivation of the ß-catenin pathway with the
addition of recombinant dickkopf homolog 1 (Dkk-1), a specific inhibitor of
the ß-catenin pathway (Glinka et al.,
1998) and with the transduction of axin
(Zeng et al., 1997) exerted no
significant effects on colony formation (Tables
2,
3). These data suggest
inhibitory roles of Rho/ROCK and ß-catenin pathways in regulating colony
size.
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), and
DBA (Fig. 7K,L, green),
respectively. While some tubules expressing sFRP2 were found in explants
treated with control HIV-TAT peptide (Fig.
7K,M, arrows), they were lost with JNK inhibitor 1 both in whole
metanephroi (Fig. 7L) and in
mesenchyme explants (Fig. 7N), suggesting the involvement of JNK pathways in mesenchymal-to-epithelial
transformation in organ culture. By contrast, branching of ureteric bud was
proportional to the size of explants, and there were no specific effects of
the reagents observed on the ureteric bud itself (data not shown). These
findings were consistent with the results observed in the colony-forming assay
(Fig. 6,
Table 2). Thus our
colony-forming assay, which enables analysis at a single cell level, could be
used for examining mechanisms of three-dimensional kidney development.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The cell line expresses marker genes of endothelia and
smooth muscle cells with treatment of TGFß1, in addition to gene
expression of mesenchyme and renal epithelia. It has not been known, however,
whether they normally resided in the fetal kidney or accidentally emerged by
the influence of the process with immortalization. In our colony-forming
system, gene expression of endothelia and smooth muscle cells was not
observed, and expression of Foxd1 (BF2), a marker gene
specific to stroma, a third cell population included in metanephros was not
found (data not shown). Thus, it remains to be elucidated whether embryonic
kidney contains stem cells that can differentiate into endothelium, smooth
muscle or stroma in addition to epithelia of glomerulus and renal tubules. The renal progenitors defined by our colony-forming assay are included in cell populations strongly expressing Sall1 throughout gestation periods, and they might continue to reside in the outer layer of embryonic kidney, where undifferentiated metanephric mesenchyme resides and strongly expresses Sall1 (Fig. 3B). As shown in Table 5, the total cell numbers of metanephros increased and the frequency of colony-forming Sall1-GFP high cells decreased as gestation proceeded. Interestingly, the calculated numbers of the colony-forming cells remained almost constant throughout gestation periods (400-800 cells/embryonic kidney). The amplification of these progenitors might not occur in the embryonic kidney. One interesting question is whether they continue to remain in the adult kidney. From 8-week-old mice, however, colonies were not formed under the same culture conditions (data not shown). Renal progenitors defined by our colony-forming assay might be lost by the time kidney development is complete.
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Mice lacking the constituent genes involved in downstream branches of Wnt
signaling pathways often show early embryonic lethality, such as Rac1
(Sugihara et al., 1998),
Jnk1 and Jnk2 (Kuan et
al., 1999), ß-catenin
(Haegel et al., 1995), axin
(Zeng et al., 1997), and their
functions in kidney morphogenesis remain largely unknown. Using our culture
system, functions of these genes in metanephros development were elucidated.
Furthermore, experiments for colony formation from mesenchyme of
Sall1-mutant embryos demonstrated the roles of Sall1 for the
colony size. Thus, the colony-assay system set up in this study can also be
applied to the analysis of genetic mouse models.
Roles of PCP pathway in kidney development
Among downstream branches of Wnt4 signal, we found that Rac- and
JNK-dependent PCP pathways positively regulated the colony size and the
differentiation of colony-forming cells. This result is compatible with
several previous reports (Du et al.,
1995; Ungar et al.,
1995; Maretto et al.,
2003). In frogs and fish, the Wnt4 family does not strongly
activate the ß-catenin pathway, and affects convergent extension, which
is polarized movement during embryonic development regulated by the PCP
pathway (Du et al., 1995;
Ungar et al., 1995).
Activation of the ß-catenin signaling was not detected at various stages
of differentiation of the metanephric mesenchyme, which was examined using
transgenic mice expressing the lacZ reporter genes under the control
of ß-catenin/TCF responsive elements
(Maretto et al., 2003).
Furthermore, activation of the ß-catenin pathway is implicated in
epithelial-to-mesenchymal transition during mesoderm formation in embryonic
development and tumorigenesis (Polakis,
2000; Bienz and Clevers,
2000), which is opposite to the process we examined in this study:
mesenchymal-to-epithelial transformation. Thus it may be possible that PCP
pathways, not the ß-catenin pathway, play central roles as downstream
branches of Wnt4 for epithelial differentiation of metanephric mesenchyme.
We demonstrated that Rac1 and RhoA play positive and negative roles for the
regulation of colony size, respectively. The Rho family of small GTPases is
known to be implicated in cell proliferation by the regulation of cell cycle
progression, in addition to its effects on the cytoskeleton
(Etienne-Manneville and Hall,
2002). Antagonism, or the opposing activities, between two Rho
GTPases have been noted in some cell types
(Luo, 2000;
Gu et al., 2005). For
instance, a hematopoietic-specific Rho GTPase, RhoH, negatively regulates both
growth and actin-based function of hematopoietic progenitors via suppression
of Rac-mediated signaling (Gu et al.,
2005). Similarly, our data suggested the possibility that Rac1 and
RhoA might antagonistically regulate the growth of progenitors in kidney
development. Recently the roles of the JNK pathway in epithelial morphogenesis
have been noted both in Drosophila and in mice
(Xia and Karin, 2004). Our
data also suggested the essential roles of JNK pathways in epithelialization,
as well as in regulation of colony size. Common mechanisms regulating
epithelial morphogenesis might underlie these processes. The PCP pathways,
including the Rho family of small GTPases and JNK, control several
developmental processes, mainly by regulating cell cytoskeletons, such as the
polarity of hairs on the epidermal cells of Drosophila wings, the
arrangement of ommatidial cells of Drosophila eyes, the polarity of
stereocilia in the inner ears of mammals, and convergent extension in
Xenopus and zebrafish (Veeman et
al., 2003; Wallingford et al.,
2002). In addition to these processes, we provide a novel
hypothesis of the involvement of the PCP pathways in kidney development.
In summary, we set up a novel colony-forming assay by which we demonstrated the presence and the frequency of multipotent progenitor cells in embryonic kidneys. This assay would serve as a useful tool for analyzing differentiation mechanisms in the kidney at a single cell level, taking advantage of the facility of gene transfer.
|
ACKNOWLEDGMENTS |
|---|
-ß-cateninSA-3HA, Dr H. Koide for
pCAGIP-flag-Rac1 and RhoA, Dr T. Kitamura for
pMY-IRES-EGFP and PLAT-E, Y. Morita for technical support for FACS,
and Dr C. Kobayashi for critically reading the manuscript. This work was
partly supported by the Ministry of Health, Labor, and Welfare of Japan. |
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