2330 RESEARCH ARTICLE
Development 139, 2330-2339 (2012) doi:10.1242/dev.076299
© 2012. Published by The Company of Biologists Ltd
COUP-TFII is essential for metanephric mesenchyme
formation and kidney precursor cell survival
Cheng-Tai Yu1, Ke Tang1, Jae Mi Suh1,*, Rulang Jiang3, Sophia Y. Tsai1,2,‡ and Ming-Jer Tsai1,2,‡
SUMMARY
Development of the metanephric kidney in mammals requires complex reciprocal tissue interactions between the ureteric
epithelium and the mesenchyme. It is believed that Gdnf, produced in the metanephric mesenchyme, activates Ret signaling in
the Wolffian duct to initiate the formation of the metanephros. However, the molecular mechanism for induction of Gdnf in the
metanephric mesenchyme is not completely defined. Previous studies demonstrated that during the early stages of kidney
development, loss of Osr1, Eya1, Pax2 or Wt1 gene function in the metanephric mesenchyme compromises the formation of the
kidney. Moreover, it has been shown that the Hox11-Eya1-Pax2 complex activates the expression of Six2 and Gdnf in the
metanephric mesenchyme to drive nephrogenesis. Here, we demonstrate that the orphan nuclear receptor chicken ovalbumin
upstream promoter transcription factor II (COUP-TFII, also known as Nr2f2) is required for the specification of the metanephric
mesenchyme. Deletion of COUP-TFII at E7.5 results in improper differentiation of the metanephric mesenchyme and absence of
essential developmental regulators, such as Eya1, Six2, Pax2 and Gdnf. Importantly, we show that COUP-TFII directly regulates the
expression of both Eya1 and Wt1 in the metanephric mesenchyme. Our findings reveal, for the first time, that COUP-TFII plays a
central role in the specification of metanephric fate and in the maintenance of metanephric mesenchyme proliferation and
survival by acting as a crucial regulator of Eya1 and Wt1 expression.
INTRODUCTION
The mammalian kidney originates from the intermediate mesoderm
and develops through three distinct stages: pronephros,
mesonephros and metanephros. Organogenesis of the permanent
kidney occurs through reciprocal interactions between the Wolffian
(mesonephric) duct and the metanephric mesenchyme. At
embryonic day (E) 10.5 in mouse, the metanephric mesenchyme
secretes glial cell line-derived neurotrophic factor (Gdnf), which
induces the Wolffian duct to invade the metanephric mesenchyme
and induce adjacent mesenchymal cells to condense. These
condensed metanephric mesenchyme cells then induce ureteric bud
outgrowth and branching, leading to the formation of nephrons
(Dressler, 2006; Saxén, 1987). Specifically, Gdnf promotes ureteric
bud outgrowth by binding to its receptor tyrosine kinase (Ret) and
co-receptor Gdnf family receptor alpha-1 (Gfr1) on the surface of
the ureteric bud (Durbec et al., 1996; Moore et al., 1996; Sainio et
al., 1997; Vainio and Lin, 2002).
Although it is well established that the Gdnf-Ret signal
transduction pathway initiates metanephric induction, the detailed
mechanism of this signal pathway is still not well understood.
During the metanephric mesenchyme condensation, many marker
genes, including Osr1 (James et al., 2006), Pax2 (Torres et al.,
1995), Eya1 (Sajithlal et al., 2005), Wt1 (Kreidberg et al., 1993),
Six1 (Xu et al., 2003), Six2 (Self et al., 2006) and the Hox11
1
Department of Molecular and Cellular Biology, 2Program in Developmental
Biological, Baylor College of Medicine, Houston, TX 77030, USA. 3Division of
Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati,
OH 45229, USA.
*Present address: Department of Neurobiology and Anatomy, University of Texas
Medical School at Houston, Houston, TX 77030, USA
‡
Authors for correspondence ([email protected]; [email protected])
Accepted 17 April 2012
paralogous group (Hoxa11, Hoxc11 and Hoxd11) (Wellik et al.,
2002), are expressed in the metanephric mesenchyme. Ablation of
any of these genes in mice leads to renal agenesis, indicating that
these genes are essential for the proper formation of the kidney.
Osr1 and Eya1, the two earliest genes expressed in kidney
precursor cells, sit atop the signal cascade and activate expression
of other marker genes. Osr1 is expressed first. Mice lacking Osr1
do not form metanephric mesenchyme and do not express Eya1,
Six2, Pax2, Sall1 and Gdnf. Eya1 is expressed downstream of Osr1
and is required for renal genesis (Xu et al., 1999). Eya proteins
have no intrinsic DNA-binding domain but can localize to the
nucleus and function as transcriptional co-activators (Ohto et al.,
1999). Eya1 not only works with Six1 and Pax2 to regulate Gdnf
expression, but also complexes with Hox11 paralogous proteins
(including Hoxa11, Hoxc11 and Hoxd11) and Pax2. The Hox11Eya1-Pax2 complex binds to the Six2 enhancer to induce the
expression of Six2, which, in turn, mediates Six2 and Gdnf
activation (Gong et al., 2007). Thus, Eya1 acts as a key regulator
of Gdnf expression and, hence, determination of metanephric fate
within the intermediate mesoderm (Sajithlal et al., 2005).
Chicken ovalbumin upstream promoter-transcription factor I and
II (COUP-TFI and COUP-TFII; Nr2f1 and Nr2f2, respectively –
Mouse Genome Informatics) are members of the nuclear receptor
superfamily (Qiu et al., 1994). Although biochemical studies indicate
that these two factors have similar DNA-binding and transcriptional
activity in vitro, the patterns of COUP-TFI and COUP-TFII
expression are distinct from each other. COUP-TFII is expressed in
the mesenchyme of developing organs and is shown to play a key
role in their organogenesis, cell fate determination, cell
differentiation, angiogenesis and metabolic homeostasis (Kim et al.,
2009; Kurihara et al., 2007; Li et al., 2009; Lin et al., 2010; Qin et
al., 2010; Qin et al., 2008; Tang et al., 2010; Xie et al., 2011; You et
al., 2005a; You et al., 2005b). During kidney development, COUPTFII expression is detectable in the condensed mesenchyme and
DEVELOPMENT
KEY WORDS: COUP-TFII, Metanephric mesenchyme, Eya1, Wt1, Mouse
COUP-TFII in early nephrogenesis
experiments were carried out as described (Lin et al., 2010). The primer
sequences are listed in supplementary material Table S2. Means for mRNA
levels in control and knockdown cells were compared using Student’s ttest.
Chromatin immunoprecipitation (ChIP) and PCR
ChIP assays were carried out with an EZ ChIP Chromatin
Immunoprecipitation Kit (Millipore) by following the manufacturer’s
protocol. Primers are shown in supplementary material Table S3.
Luciferase assays
The human EYA1 and WT1 promoter regions, which contained the putative
Sp1-binding sites, were amplified from human BAC clone RP11-160C13
(Children’s Hospital Oakland Research Institute, CA, USA) and CTD2083A15 (Invitrogen) separately. The PCR primers for amplifying the
promoter region or for generating the Sp1-binding site mutations are as
shown in supplementary material Table S4. The amplified promoter
fragments were cloned into the pGL2-basic-luc vector (Promega). HEK
293 cells were transfected with pGL2-Eya1-basic-luc or pGL2-Wt1-luc
using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
instructions.
MATERIALS AND METHODS
Statistics
Animals
Statistical analysis was carried out by Student’s t-test. P values of less than
0.05 were considered significant.
Generation of the floxed COUP-TFII mice and COUP-TFII-lacZ knockin mice was as described previously (Takamoto et al., 2005). The Rosa26Cre-ERT2/+ knock-in mouse strain was provided by T. Ludwig (Columbia
University, New York City, USA) (de Luca et al., 2005). For inducible
deletions during embryonic stages, 2 mg tamoxifen (Tam), dissolved in
corn oil (Sigma-Aldrich; 10 mg/ml), was injected intraperitoneally into
pregnant females at E7.5 or E8.5. Embryos from littermates were collected
at indicated stages. All mouse strains were maintained in a mixed genetic
background and received standard rodent chow.
X-gal staining
Whole-mount X-gal staining of embryos (up to E10.5) was performed
according to published methods (Takamoto et al., 2005).
Histology, immunofluorescence and in situ hybridization
All the histology and immunofluorescence staining of paraffin-embedded
slides were performed as described (You et al., 2005a). Antibodies used
were: anti-COUP-TFII (R&D, 1:2000), anti-Pax2 (Covance, 1:500), antiKi67 (BD Biosciences, 1:400), anti--galactosidase (Biogenesis, 1:1500),
anti-Wt1 (Santa Cruz, 1:500), anti-Gdnf (Santa Cruz, 1:200) and anti-Six2
(Proteintech group, 1:400). Non-radioactive in situ hybridization was
carried out as described previously (Bramblett et al., 2004). The RNA
probe for mouse Osr1 was provided by Thomas M. Schultheiss (Harvard
University, Boston, USA) and the mouse Eya1 probe was as described (Xu
et al., 1999). Cell apoptosis was detected by cleaved Caspase 3 (Cell
Signaling, 1:500) staining or by TUNEL assay (Roche, In Situ Cell Death
Detection Kit) according to the manufacturer’s instructions.
Kidney organ culture and staining
The metanephric mesenchymes containing the Wolffian duct from 10.5
days post-coitum (dpc) mouse embryos were cultured on transwell filters
as described (Shakya et al., 2005). The Wolffian duct and ureteric bud were
visualized by staining with anti-E-Cadherin (BD Biosciences, 1:200).
Cell lines and transfection
Human embryonic kidney 293 (HEK 293) cells were transfected with
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.
The rat inducible metanephric mesenchyme (RIMM-18) cell line was
provided by Alan O. Perantoni (National Institutes of Health, NCI,
Bethesda, MD, USA) and the culture conditions were as described
(Levashova et al., 2003).
RNA interference and quantitative real-time RT-PCR assay
The siRNA oligonucleotides were purchased from Thermo Fisher
Scientific or Applied Biosystems/Ambion. The target sequences for rat
siRNA are described in supplementary material Table S1. RNA interference
RESULTS
COUP-TFII is expressed specifically in kidney
precursor cells
We showed previously that COUP-TFII is expressed in the
metanephric blastema at E11.5 and later in the developing nephron,
nephrogenic cortex and stromal cells. In the adult kidney, high
COUP-TFII expression is also detectable in the distal tubules,
podocytes and epithelial cells of Bowman’s capsule (Suh et al.,
2006). Nonetheless, the expression pattern of COUP-TFII in
kidney precursor cells has not been determined. Here, we
employed a COUP-TFII-specific antibody to investigate its
expression pattern in the nephrogenic mesoderm from E9.5 to E10
(Fig. 1A-C). At E9.5, COUP-TFII staining was observed in the
intermediate mesoderm and other mesenchyme cells surrounding
the Wolffian duct (Fig. 1A). Using Pax2 to mark the Wolffian duct,
we demonstrated that COUP-TFII is only expressed in the
intermediate mesoderm and not in the duct (Fig. 1B). Half a day
later, at E10, COUP-TFII expression was observed in the
nephrogenic mesenchyme region surrounding the Wolffian duct
and in the urogenital ridge (Fig. 1C). These results indicate that
COUP-TFII is expressed in kidney precursor cells.
Inducible deletion of COUP-TFII in kidney
precursor cells
In order to bypass early embryonic lethality of COUP-TFII
homozygous mutant at E10.5, we conditionally inactivated COUPTFII by crossing the COUP-TFII floxed mouse strain (COUPTFIIf/f) (Takamoto et al., 2005) with Rosa26-Cre-ERT2/+, a strain
that harbors a tamoxifen-inducible Cre recombinase under the
control of the ubiquitously active ROSA26 promoter (de Luca et al.,
2005). Rosa26-Cre-ERT2/+;COUP-TFIIf/f males (hereafter referred
to as COUP-TFIId/d) were intercrossed with COUP-TFIIf/f females,
and a single dosage of 2 mg Tam was injected intraperitoneally into
pregnant dams to induce COUP-TFII deletion. Specifically, we
performed Tam injection at E7.5. In our mouse model, a lacZ gene
inserted into the COUP-TFII locus is turned on when the COUPTFII gene is deleted. One and half a days after Tam treatment,
COUP-TFII was deleted throughout the whole body of COUP-
DEVELOPMENT
derivates, including pretubular aggregates, comma-shaped bodies
and S-shaped bodies. Finally, in the adult mouse kidney, COUP-TFII
is found in the distal tubules, podocytes and the epithelial cells of the
Bowman’s capsule (Suh et al., 2006). These observations imply that
COUP-TFII might play an important role in the genesis of the kidney
and maintenance of its function. Here, we provide new insights into
the role of COUP-TFII in the formation of the metanephric
mesenchyme. Not only is COUP-TFII expressed in the
undifferentiated kidney precursor cells, it is also indispensable for
the establishment and maintenance of the metanephric mesenchyme.
It acts as a crucial link in determination of metanephric fate through
its recruitment to the promoter of the Eya1 gene and direct regulation
of Eya1 transcription. In addition, COUP-TFII directly regulates the
transcription of Wt1, an essential factor for maintaining metanephric
mesenchyme cell survival (Davies et al., 2004; Kreidberg et al.,
1993). Therefore, our studies clearly indicate that COUP-TFII is a
key early regulator of the formation of metanephric mesenchyme and
the subsequent formation and differentiation of the kidney.
RESEARCH ARTICLE 2331
Fig. 1. Expression pattern and conditional deletion of COUP-TFII
in kidney precursor cells. (A)Transverse section of wild-type E9.5
mouse embryo shows that COUP-TFII is expressed in the intermediate
mesoderm (im), lateral plate mesoderm (lpm) and mesenchyme cells
surrounding the Wolffian duct (wd). (B)High magnification view
indicates that COUP-TFII is not expressed in the Wolffian ducts, which
are stained positively with Pax2. (C)Transverse section of an E10
embryo shows that COUP-TFII is expressed in the nephrogenic
mesenchyme (nm) and urogenital ridge (ur), but not in the Wolffian
duct (wd). (D-I)Tamoxifen was administered at E7.5 to induce COUPTFII deletion and analyzed at E9. The whole-mount X-gal staining
indicates that COUP-TFII is deleted throughout the whole mutant
embryo, including the nephrogenic mesenchyme (nm) (G). The
transverse section also shows deletion of COUP-TFII in the intermediate
mesoderm and lateral plate mesoderm of the mutant embryo (E,H). At
E10.5, COUP-TFII expression is detected in the urogenital ridge and
metanephric mesenchyme (mm) (F). COUP-TFII expression is totally lost
in the mutant embryo three days after Tam administration (I). DAPI
counterstain for nuclei (blue) was applied for A, C, F and I. Scale bars:
50m.
TFIId/d mutant embryos, including the nephrogenic mesenchyme
(Fig. 1D,G). Further examination of X-gal staining of transverse
sections at the nephrogenic mesenchyme indicated that deletion of
COUP-TFII occurred in the intermediate mesoderm and lateral
plate mesoderm of COUP-TFIId/d mutant embryos (Fig. 1E,H).
Using the COUP-TFII-specific antibody, we showed that COUPTFII is highly expressed in the urogenital ridge and the developing
condensed nephrogenic mesenchyme, but not in the Wolffian duct
of the COUP-TFIIf/f control at E10.5 (Fig. 1F). In COUP-TFII
mutants, expression was undetectable (Fig. 1I). These data indicate
the Tam-inducible deletion system effectively deletes COUP-TFII
in kidney progenitor cells. Unfortunately, deletion of COUP-TFII
at E7.5 led to embryonic lethality at ~E11, which limits our study
to embryos before E11 (data not shown).
COUP-TFII is essential for metanephric
mesenchyme formation but not for the formation
of mesonephros
During the formation of the metanephros, the metanephric
mesenchyme appears morphologically as an aggregate of the
nephrogenic mesenchyme cells at the caudal end of the nephrogenic
cord at E10.5 (Fig. 2A,C). This structure is largely missing in
COUP-TFIId/d mutant embryos (Fig. 2B,D). In addition, serial
sections through the entire metanephric mesenchyme region were
Development 139 (13)
Fig. 2. COUP-TFII is essential for the metanephric mesenchyme
formation. Tamoxifen was administered at E7.5 to delete COUP-TFII.
(A-D)Transverse sections of metanephric mesenchyme (mm) from these
embryos were stained with hematoxylin and eosin at E10.5. The
Wolffian duct (wd) and condensed metanephric mesenchyme (mm) are
seen in the COUP-TFIIf/f control (A,C). The metanephric mesenchyme is
completely absent in the COUP-TFIId/d mutant embryo (B,D). (E-J)At
E10.5, the metanephric mesenchyme of COUP-TFIIf/f control embryos
expresses Pax2, Six2 and Gdnf (E,G,I). By contrast, these three gene
products are undetectable in the metanephric mesenchyme region of
COUP-TFIId/d null mutants (F,H,J,). However, the Pax2 expression in the
Wolffian duct remains (F). lacZ staining indicates that COUP-TFII is
deleted in the mesenchyme cells (F). Boxed areas in I and J are shown in
insets. (K,L)Kidney organ cultures from the metanephric mesenchyme
of COUP-TFIIf/f control (K) and COUP-TFIId/d mutant (L) embryos
indicated that the ureteric bud (ub) outgrowth, visualized by E-cadherin
staining, was detected from the Wolffian duct of the COUP-TFIIf/f
control (K), but not from the COUP-TFIId/d mutant embryo (L). DAPI
counterstain for nuclei (blue) was applied for E-J. Scale bars: 50m.
examined with littermates of the same somite number. The results
clearly show that the metanephric mesenchyme is not formed in the
COUP-TFIId/d mutant (supplementary material Fig. S1). As the
paired box-containing transcription factor Pax2 is a key regulator of
kidney development (Torres et al., 1995), we investigated whether
the expression of this gene is compromised. In the mouse kidney, the
expression of Pax2 is initiated in the intermediate mesoderm and
maintained throughout the development of the pronephros and
mesonephros (Bouchard et al., 2000). Pax2 is expressed in the
induced metanephric mesenchyme and in the Wolffian duct of the
metanephros in the controls (Fig. 2E). By contrast, Pax2 expression
DEVELOPMENT
2332 RESEARCH ARTICLE
is totally lost in the nephrogenic mesenchyme area, whereas it is
intact in the Wolffian duct of the COUP-TFIId/d mutants (Fig. 2F).
lacZ-positive cells, which denote COUP-TFII ‘expressing cells’ in
the COUP-TFIId/d mutants, indicated that some ‘COUP-TFII
expressing’ metanephric mesenchyme cells surrounding the Wolffian
duct are still present (Fig. 2F).
In addition to Pax2, the homeobox gene Six2 is also specifically
expressed in the metanephric mesenchyme before ureteric bud
outgrowth (Xu et al., 1999) and is required to activate Gdnf
expression by directly binding to its promoter during kidney
development (Brodbeck et al., 2004). Therefore, we examined Six2
expression in COUP-TFII mutants. As indicated in Fig. 2G,H, Six2
expression is not detectable in the mutant metanephric mesenchyme.
As both Pax2 and Six2 lie upstream of Gdnf, which is essential for
promoting the ureteric bud outgrowth (Costantini and Shakya, 2006),
we investigated whether Gdnf expression in the nephrogenic
mesenchyme cells is also compromised in the mutants. Gdnf is
detectable in the cytoplasm of the metanephric mesenchyme in
control animals (Fig. 2I), but is not detected in the COUP-TFIId/d
mutants (Fig. 2J). Together, these results strongly implied that
COUP-TFII is likely to regulate the expression of genes essential for
metanephric mesenchyme formation and kidney development.
In order to bypass the lethality of COUP-TFII mutant embryo at
E11, we injected Tam at E7.5 to delete COUP-TFII and collected
the embryos at E10.5. We then dissected the metanephric
mesenchyme tissue with the Wolffian duct and cultured it for 36
hours. The results obtained from organ cultured of three litters of
mice indicate that the ureteric bud was induced to outgrow from
the Wolffian duct and started to branch from all ten COUP-TFIIf/f
controls (Fig. 2K). By contrast, we found that majority of COUPTFIId/d mutants (six out of eight) did not display any ureteric bud
outgrowth (Fig. 2L). Therefore, the inability of the mutant
metanephric mesenchyme to induce ureteric bud outgrowth and
branching suggests that the mature kidney will not be able to form
in the COUP-TFII mutants at later stages.
During this stage, the early mesonephros structure also formed.
The mesonephros develops when the Wolffian duct reaches the
prospective mesonephric mesenchyme and induces adjacent
mesenchymal cells to condense and form mesonephric tubules
(Saxén, 1987). First, we investigated whether COUP-TFII is also
expressed in the mesonephros (also called the mesonephric tubule).
COUP-TFII staining in E10 embryos indicated that COUP-TFII is
expressed in the mesonephros but not in the Wolffian duct
(supplementary material Fig. S2A). Pax2 is expressed in the
Wolffian duct and mesonephros. By using Pax2 as a mesonephric
tubule marker, we found COUP-TFII colocalizes with Pax2 in the
mesonephros but not in the Wolffian duct (supplementary material
Fig. S2B). We examined the E9.5 embryos and found that the
mesonephros is still intact in the COUP-TFII–/– mutants in which
COUP-TFII is deleted very early in the germ cells, compared with
COUP-TFII+/+ controls (supplementary material Fig. S2C,D).
Therefore, even though COUP-TFII is expressed in the
mesonephros, it is not essential for mesonephros formation.
COUP-TFII specifically regulates Eya1, Wt1 and
Six2 in the metanephric mesenchyme
Although the metanephric mesenchyme markers, such as Pax2 or
Six2 expression, are not detected in the COUP-TFIId/d mutant
embryos (Fig. 3F,H), the presence of lacZ-positive cells indicates
that it is still possible that the lack of marker gene expression could
be due to the loss of metanephric mesenchyme cells. To demonstrate
that COUP-TFII does indeed regulate metanephric mesenchyme
RESEARCH ARTICLE 2333
Fig. 3. COUP-TFII specifically regulates the metanephric
mesenchyme gene expression. Tamoxifen was administered at E8.5
to delete COUP-TFII. Transverse sections of the metanephric
mesenchyme region from the embryos were stained with Hematoxylin
and Eosin at E10.5. (A,B)The condensed metanephric mesenchyme
(mm) was seen in both the COUP-TFIIf/f control (A) and the COUP-TFIId/d
mutant (B) embryos. (C-H)At E10.5, the metanephric mesenchyme of
COUP-TFIId/d mutant embryos expresses Eya1 (D), Wt1 (F) and Six2 (H);
however, the expression levels per cell of all of these three factors are
lower than control (C,E,G). Eya1 expression level was measured by in
situ hybridization (C,D). The DAPI counterstain for nuclei (blue) was
applied in E-H. (I-L)lacZ staining indicates that COUP-TFII is deleted in
the mesenchyme cells and is specifically colocalized with Six2 in that
area of COUP-TFIId/d mutant (I,J). J shows a high magnification of the
dashed box in I. Arrows indicate that cells that express higher Six2 levels
have lower lacZ expression (L) and arrowheads indicate that cells that
express higher lacZ have lower Six2 expression (K). (M)The Six2
expression level of Six2-positive cells was measured by the fluorescence
density divided by area (densitometric mean) from COUP-TFIIf/f control
and COUP-TFIId/d mutant embryos. Four metanephric mesenchyme
samples were measured and the results indicate that COUP-TFII deletion
in the metanephric mesenchyme significantly reduces Six2 expression.
**P<0.005. ur, urogenital ridge; wd, Wolffian duct. Scale bars: 50m.
marker gene expression, we deleted COUP-TFII one day later by
injecting Tam at E8.5 and collecting embryos at E10.5. We reasoned
that by deleting COUP-TFII one day later, we would have
metanephric mesenchyme formation, but in some cells COUP-TFII
would be completely deleted, in some one allele would be deleted,
DEVELOPMENT
COUP-TFII in early nephrogenesis
and in some it would not be deleted at all. In this scenario, a decrease
in overall expression of these marker genes per cell would be
observed in the formed metanephric mesenchyme. In addition, those
cells with COUP-TFII deletion would have lower levels of target
gene expression and those cells with no COUP-TFII deletion would
have higher levels of target gene expression. As expected, we found
that the metanephric mesenchyme was formed in both COUP-TFIIf/f
control (Fig. 3A) and COUP-TFIId/d mutant (Fig. 3B) embryos, even
though the mutant metanephric mesenchyme is smaller and less
condensed compared with the control. Using in situ hybridization to
examine Eya1 expression, we found that both the number of cells
expressing Eya1 and the expression level per cell are decreased in
the COUP-TFIId/d mutant (Fig. 3C,D). Similarly, Wt1 and Six2
expression levels in the metanephric mesenchyme are significantly
decreased in the COUP-TFIId/d mutant (Fig. 3F,H) compared with
the controls (Fig. 3E,G). To define further whether COUP-TFII
specifically regulated Six2 expression in the metanephric
mesenchyme, we double labeled Six2 with lacZ and found that Six2
colocalizes with lacZ expression in the metanephric mesenchyme of
COUP-TFIId/d mutant (Fig. 3I). Under high magnification, we
examined the Six2-lacZ-positive cells (Fig. 3J) and found that those
cells with a higher lacZ signal (i.e. in which COUP-TFII is deleted)
have lower Six2 expression (Fig. 3K,L, arrowheads). By contrast, if
lacZ expression is low (i.e. COUP-TFII is not deleted), Six2
expression is higher (Fig. 3K,L, arrows).
In order to quantify the results, we measured the fluorescence
intensity of Six2-positive cells (metanephric mesenchyme cells) in
the COUP-TFIIf/f controls (n131 cells) and COUP-TFIId/d mutants
(n65 cells) shown in Fig. 3G,H and divided by the cell area to
obtain densitometric levels of Six2 expression. The result shows that
deletion of COUP-TFII significantly decreases Six2 expression in
the COUP-TFIId/d mutant (Fig. 3M). These data clearly indicate that
COUP-TFII does indeed regulate the expression of the metanephric
mesenchyme markers Six2, Eya1 and Wt1.
Deletion of COUP-TFII abolishes Wt1 expression
and increases metanephric mesenchyme cell
apoptosis
We observed a reduction in cell number (Fig. 4A,B) in the
nephrogenic mesenchyme region of the COUP-TFIId/d mutant
embryos compared with controls. We investigated whether the loss
of mesenchymal cells in this region is due to decreased cell
proliferation or increased apoptosis. Embryo-wide comparison of
Ki67 staining revealed no obvious differences in overall
proliferation in controls versus COUP-TFIId/d mutants (Fig. 4A,B).
By contrast, the COUP-TFIId/d mutant metanephric mesenchyme
region showed a significant decrease in Ki67-positive cell numbers
(Fig. 4C,D, red circled regions), indicating a specific decrease in
proliferation of the metanephric mesenchyme. Furthermore, we
observed an increase in the number of apoptotic cells in the
metanephric mesenchyme of mutant mice as detected by both
cleaved Caspase 3 and tunnel assays (Fig. 4E-H).
The Wilms tumor suppressor gene (Wt1) is expressed in the
metanephric mesenchyme and has been shown to be essential for
the survival of metanephric mesenchymal cells (Kreidberg et al.,
1993; Kuure et al., 2000; Moore et al., 1999). Therefore, we
investigated whether Wt1 expression is compromised in COUPTFIId/d mutants. First, we determined whether COUP-TFII and
Wt1 colocalize with each other. As shown in supplementary
material Fig. S3A-C, we found that COUP-TFII colocalizes with
Wt1 in the metanephric mesenchyme and in the urogenital ridge.
Next, we investigated whether Wt1 expression level is altered with
Development 139 (13)
Fig. 4. COUP-TFII deletion abolishes Wt1 expression and
increases apoptosis of metanephric mesenchyme cells. (A-F)Tam
was administered at E8.5 and embryos were examined at E10.5.
Proliferation was determined by Ki67 immunostaining which appeared
to show similar Ki67-positive cells for both mutant (B) and control (A).
However, upon close examination, the metanephric mesenchyme (C,D
red circles) had fewer Ki-67-positive cells in the mutant. Apoptosis
analysis results indicated that there are many more cleaved Caspase 3
(C-Casp3)-positive cells located in the metanephric mesenchyme region
of the COUP-TFIId/d mutant embryos compared with control embryo
(E,F). (G-J)Tam was administered one day earlier at E7.5 and embryos
were examined at E10.5. TUNEL assay shows a significantly increase in
apoptosis in the metanephric mesenchyme region of the COUP-TFII
knockout embryo (G,H). Wt1 is expressed in the urogenital ridge and
metanephric mesenchyme in the COUP-TFIIf/f control embryo (I) and the
expression is lost in the COUP-TFIId/d embryo (J). lacZ staining indicates
that COUP-TFII-deleted cells are still present. The slides are
counterstained with DAPI for nuclei (blue). Scale bar: 50m.
COUP-TFII deletion. Indeed, we found Wt1 expression to be
significantly decreased in the COUP-TFII mutant urogenital ridge
and metanephric mesenchyme (deletion cells shown as the lacZ
positive cells) (Fig. 3E,F and Fig. 4I,J). These results indicate Wt1
lies downstream of COUP-TFII and is regulated by COUP-TFII.
Owing to the importance of Wt1 in metanephric mesenchyme cell
survival, our findings suggested that loss of Wt1 expression due to
COUP-TFII deletion is the likely reason for the increased
metanephric mesenchyme apoptosis in COUP-TFIId/d mutants.
COUP-TFs regulate the expression of Eya1, Six2,
Wt1 and Gdnf in metanephric mesenchyme cells
In order to study the detailed molecular mechanism by which
COUP-TFII controls regulatory genes in the metanephric
mesenchyme, we employed the conditionally immortalized rat
inducible metanephric mesenchyme cell line (RIMM-18). RIMM-18
cells were generated from rat mesenchymal cells by transfection with
a vector encoding an estradiol-dependent E1A-ER fusion protein and
DEVELOPMENT
2334 RESEARCH ARTICLE
COUP-TFII in early nephrogenesis
RESEARCH ARTICLE 2335
Fig. 5. COUP-TFs regulate Gdnf, Eya1, Six2 and Wt1 expression in
the metanephric mesenchyme in vitro. (A-F)Quantitative RT-PCR
analysis of COUP-TFI (A), COUP-TFII (B), Gdnf (C), Eya1 (D), Six2 (E) and
Wt1 (F) in rat metanephric mesenchyme-derived cells (RIMM-18), after
knockdown of both COUP-TFI and COUP-TFII using specific siRNAs. All
mRNA measurements were normalized to 18S rRNA. Error bars indicate
s.d. **P<0.005.
these cells maintain its metanephric mesenchyme characteristics
(Levashova et al., 2003). Using RT-PCR, we found that the
expression levels of both COUP-TFI and COUP-TFII are high in
RIMM-18 cells. Despite this, in the E10.5 mouse embryos, only
COUP-TFII is expressed in the metanephric mesenchyme, whereas
COUP-TFI is located in the stroma mesenchyme. Our laboratory has
reported that COUP-TFI and COUP-TFII are highly homologous
and also functionally redundant (Tsai and Tsai, 1997; Wu et al.,
2010). Therefore, during in vitro experiments, we used siRNAs to
knock down both COUP-TFI and COUP-TFII to study the
underlying mechanism of COUP-TF regulation of transcription
factors important for kidney development. Knockdown of COUPTFI and COUP-TFII significantly decreases Gdnf, Eya1, Six2 and
Wt1 (Fig. 5C-F) expression. In order to confirm whether COUP-TFII
alone can regulate these transcription factors in RIMM-18 cells, we
also used siRNA to knock down COUP-TFII specifically. We found
that knockdown of COUP-TFII alone is enough to significantly
decrease Eya1 and Wt1 (supplementary material Fig. S4B,C)
expression. These results indicate that COUP-TFs can indeed
positively regulate genes expressed early in metanephric
mesenchyme development.
COUP-TFII signaling is independent of Osr1 in the
nephrogenic mesenchyme
Similar to COUP-TFII, Odd-skipped related 1 (Osr1) is also required
for the formation of the metanephric mesenchyme and is upstream
of Eya1. In order to define the relationship between Osr1 and COUPTFII in the regulatory cascade, we first investigated whether COUPTFII is upstream of Osr1 by examining the expression of Osr1 in
COUP-TFII mutants by in situ hybridization. Osr1 is broadly
expressed in the nephrogenic mesenchyme regions in the control
mice (supplementary material Fig. S5A). Despite the fact that the
COUP-TFII directly regulates Eya1 gene expression
by interacting with Sp1
Eya1 has been shown to specify the metanephric mesenchyme fate
and is indispensable for metanephric mesenchyme formation. In
order to determine whether Eya1 expression is regulated by COUPTFII at the transcriptional level, we carried out in situ hybridization
to see whether Eya1 is regulated by COUP-TFII at the mRNA level.
Indeed, Eya1 mRNA is detected in the metanephric mesenchyme of
COUP-TFIIf/f controls but is absent in COUP-TFIId/d mutants (Fig.
6A,B). This indicates that Eya1 is a COUP-TFII target and is likely
to be regulated by COUP-TFII at the transcriptional level.
To support this conclusion further, we carried out chromatin
immunoprecipitation (ChIP) assays to determine whether COUPTFII is recruited to the promoter/enhancer region of the Eya1 gene
locus. Our laboratories have previously demonstrated that COUPTFII acts as a positive regulator, enhancing target gene expression
by interacting with Sp1 at Sp1-binding sites (Kim et al., 2009; Lin
et al., 2010; Pipaon et al., 1999; Qin et al., 2010). To assess
whether COUP-TFII regulates Eya1 expression through its
interaction with Sp1, we searched for the evolutionarily conserved
Sp1-binding sites in human, mouse and rat sequences on and
surrounding the Eya1 gene and found three conserved sites
upstream of the transcription start site (Fig. 6C, black boxes). We
then performed ChIP assays to evaluate whether endogenous
COUP-TFII is recruited to the Sp1 sites of the Eya1 promoter.
Indeed, we found COUP-TFII is preferentially recruited to the
promoter regions containing Sp1-binding sites but not recruited to
the region lacking Sp1-binding sites (Fig. 6C, Sp1). In parallel, Sp1
was also specifically recruited to the same region as COUP-TFII,
but not to the region without Sp1-binding sites (Fig. 6C). To
substantiate that COUP-TFII was recruited by Sp1 to the Eya1
promoter, we knocked down endogenous Sp1 expression with Sp1specific siRNA (si-Sp1). Recruitment of COUP-TFII and Sp1 to
the Eya1 promoter was, in fact, significantly reduced in Sp1knockdown cells compared with controls (Fig. 6D,E).
To test further whether COUP-TFII binding to the Eya1 promoter
leads to activation of transcription, we performed luciferase reporter
assays in HEK293 cells using a 1.8-kb (–731 bp to 1079 bp) human
EYA1 promoter fragment that included the three conserved Sp1binding sites. Luciferase reporter activity was significantly increased
DEVELOPMENT
COUP-TFIId/d mutant has fewer mesenchyme cells, the Osr1
expression level in the mesenchyme is equivalent to that of the
control (supplementary material Fig. S5B). Next, we examined
COUP-TFII expression in Osr1 knockout mice to determine whether
COUP-TFII is downstream of Osr1. The COUP-TFII expression
level is similar in the urogenital ridge of Osr1 knockout and wildtype mice (supplementary material Fig. S5C-F). Although Osr1
knockouts lack the metanephric mesenchyme, COUP-TFII
expression in the nephrogenic mesenchyme surrounding the Wolffian
duct is similar compared with wild-type controls (supplementary
material Fig. S5C-F). These results indicate that Osr1 and COUPTFII act in parallel with one another and are both required for Eya1
activation to control metanephric mesenchyme differentiation and
nephrogenesis. To support this conclusion further, we employed
RIMM-18 cell culture experiments and found that simultaneous
knockdown of COUP-TFI and COUP-TFII by siRNA does not affect
expression of Osr1 (supplementary material Fig. S5G-I). Similarly,
knockdown of Osr1 had no significant effect on COUP-TFI or
COUP-TFII expression (supplementary material Fig. S5J-L). These
results support the conclusion that COUP-TFII and Osr1 act in
parallel to regulate Eya1 expression.
2336 RESEARCH ARTICLE
Development 139 (13)
when COUP-TFII was expressed compared with control cells (Fig.
5F). Next, we mutated these three conserved Sp1-binding sites as
depicted in supplementary material Fig. S4A. We generated reporters
with three single Sp1-binding sites or with all three sites mutated
(supplementary material Fig. S6A; pGL2-Eya1-M1, pGL2-Eya1M2, pGL2-M3 and the triple-mutation pGL2-Eya1-M123). In the
presence of COUP-TFII expression plasmid, activation of all four
mutant luciferase reporters was significantly diminished versus the
intact reporter (Fig. 6F). This result indicates that COUP-TFII works
through Sp1 on the Sp1-binding sites to activate Eya1 expression.
Collectively, these results substantiate a model in which COUP-TFII
is recruited to the Eya1 promoter in an Sp1-dependent manner to
directly activate Eya1 transcription.
COUP-TFII interacts synergistically with Sp1 to
regulate Wt1 expression directly
Next, we investigated whether COUP-TFII also directly regulates the
transcription of Wt1 through its interactions with Sp1. We identified
two evolutionarily conserved Sp1-binding sites surrounding the Wt1
promoter (Fig. 7A, black boxes). ChIP analysis showed that COUPTFII is indeed preferentially recruited to Sp1-binding sites at the Wt1
promoter but is not recruited to the region lacking Sp1-binding sites
(Fig. 7A). Sp1 was recruited to the same regions as COUP-TFII. To
determine whether recruitment of COUP-TFII to the Wt1 promoter
is Sp1-dependent, we knocked down endogenous Sp1 by siRNA.
With knockdown, COUP-TFII recruitment to the Wt1 promoter was
significantly reduced (Fig. 7B,C).
To confirm whether COUP-TFII binding to the Wt1 promoter
leads to transcription activation, we performed luciferase reporter
assays using a 1.1-kb (–736 bp to 377 bp) human WT1 promoter
fragment that contains the two conserved Sp1-binding sites. As
shown in Fig. 7D, Wt1-luciferase reporter (pGL2-Wt1) activity was
significantly increased by COUP-TFII. In addition, when Sp1binding sites were mutated in the Wt1-luciferase reporter
(supplementary material Fig. S6B; pGL2-Wt1-M1, pGL2-Wt1-M2
and the double-mutation pGL2-Wt1-M12), COUP-TFII-dependent
luciferase activity decreased dramatically (Fig. 7D). Taken together,
these results strengthen our proposed model that recruitment of
COUP-TFII to the Wt1 promoter is Sp1-dependent, and that COUPTFII directly activates Wt1 transcription.
DISCUSSION
COUP-TFII directly regulates transcription of Eya1
to modulate Gdnf expression during metanephric
induction
COUP-TFII is expressed in the mesenchyme of developing organs
and has been shown to play a key role in their organogenesis. In
this study, we found that COUP-TFII is expressed in the kidney
DEVELOPMENT
Fig. 6. COUP-TFII directly regulates
Eya1 gene transcription by tethering to
Sp1 at Sp1-binding sites. (A,B)Tam was
administered at E7.5 and embryos were
examined at E10.5 for Eya1 expression in
the metanephric mesenchyme by in situ
hybridization. Eya1 RNA is detected in the
metanephric mesenchyme (mm) of the
COUP-TFIIf/f control embryo (A), but is
absent in the COUP-TFIId/d mutant embryo
(B). Scale bars: 50m. (C)ChIP analysis
showed that COUP-TFII and Sp1 are
recruited to the conserved Sp1-binding
sites of the Eya1 promoter in RIMM-18
cells (C). (D,E)RT-PCR analysis of ChIP assay
of COUP-TFII (CII; D) and Sp1 binding (E) to
the conserved Sp1-binding sites of the rat
Eya1 promoter control siRNA- (siCtrl, 25
nM) or Sp1- (siSp1, 25 nM) specific siRNAtreated RIMM-18 cells. All values were
normalized to the total input and error bars
indicate s.d. *P<0.05, **P<0.005.
(F)Relative luciferase activities of Eya1
promoter-driven reporter pGL2-Eya1 and
the Sp1-binding site mutant reporters
pGL2-Eya1-M1, pGL2-Eya1-M2, pGL2Eya1-M3 and pGL2-Eya1-M123 (250 ng
each) were measured after co-transfection
with or without COUP-TFII expression
plasmid (50 ng) into HEK 293 cells.
COUP-TFII in early nephrogenesis
RESEARCH ARTICLE 2337
precursor intermediate mesoderm and later expressed in the
metanephric mesenchyme. Knockout of COUP-TFII at an early
developmental stage (E7.5) results in the loss of metanephric
mesenchyme in the mutant embryo at E10.5. In addition, ablation
of COUP-TFII results in the loss of expression of many key
factors, including Six2, Pax2, Wt1, Eya1 and Gdnf, which are
essential for metanephric mesenchyme formation. These results
place COUP-TFII as an upstream regulator of these important
regulatory factors.
Our in situ hybridization data showed that Eya1 expression is
significantly decreased in the COUP-TFII mutant (Fig. 3C,D),
suggesting that COUP-TFII directly regulates Eya1 expression in
the metanephric mesenchyme. Indeed, ChIP assays showed that
COUP-TFII is recruited to the conserved Sp1-binding site of the
Eya1 promoter by tethering to Sp1 for direct regulation of Eya1
expression. Furthermore, expression of COUP-TFII can enhance
Eya1 promoter-driven reporter activity. Collectively, these results
clearly indicate that COUP-TFII promotes Gdnf signaling cascade
by direct regulation of Eya1 expression and, thus, induces
metanephric mesenchyme fate in kidney progenitor cells. It should
be noted that in Eya1 knockout mice, ureteric buds fail to grow out
into the metanephric mesenchyme (Sajithlal et al., 2005). Similar
to Eya1, we observed no ureteric bud outgrowth in six out of eight
COUP-TFII mutants using the kidney organ cultures (Fig. 2L). It
is not clear, however, whether the remaining two COUP-TFII
mutants have ureteric bud outgrowth (supplementary material
S7A,B, arrowheads). In any event, if they indeed have ureteric bud
outgrowth, it is possible that COUP-TFII was not deleted early
enough in these two particular mutants, resulting in partially
committed metanephric mesenchyme and subsequent induction of
ureteric bud outgrowth. As the majority of mutants do not have
ureteric buds outgrowth, it indicates that COUP-TFII is required
for the proper formation of metanephric mesenchyme and
subsequent induction of ureteric bud outgrowth.
COUP-TFII acts in parallel with Osr1 to regulate
Eya1 expression during metanephric induction
Osr1 is known to be the earliest marker for kidney development in the
intermediate mesoderm. Mice lacking Osr1 do not form metanephric
mesenchyme and do not express many factors essential for
metanephric mesenchyme formation (James et al., 2006; Wang et al.,
2005). These phenotypes bear strong resemblance to that of the
COUP-TFII knockout mice. To address whether these two genes work
in the same pathway, we examined whether Osr1 expression is
affected in the COUP-TFII knockout. Our results clearly show that
Osr1 expression in the nephrogenic mesenchyme region is not altered
in the COUP-TFII mutant. Similarly, COUP-TFII expression in the
metanephric mesenchyme region remains the same in the Osr1
knockout embryo. Together, these results indicate that COUP-TFII
and Osr1 act independently to regulate kidney morphogenesis. This
notion is further supported by in vitro cell culture experiments, in
which we showed that COUP-TFII and Osr1 do not regulate each
other’s expression. Interestingly, whereas Wt1 is expressed in the
metanephric mesenchyme of Osr1 null mice (James et al., 2006), Wt1
expression in the COUP-TFII knockout mutant is totally lost. This
result indicates that although both COUP-TFII and Osr1 are expressed
in the intermediate mesoderm and both regulate Eya1, Pax2 and Six2
expression, they do not regulate each other to control the expression
of those key factors important for metanephric mesenchyme
formation. Based on all these results, our working model is that
COUP-TFII and Osr1 act in parallel to regulate Eya1 and its
downstream target Gdnf to specify metanephric mesenchyme (Fig. 8).
COUP-TFII is essential for survival of the
metanephric mesenchyme and for nephron
differentiation
Wt1 mutant mesenchyme cannot be induced to form nephric
tubules (Kreidberg et al., 1993), suggesting that Wt1 is cellautonomously required for nephron differentiation. Subsequently,
DEVELOPMENT
Fig. 7. COUP-TFII synergistically
interacts with Sp1 to regulate Wt1
expression. (A)ChIP analysis showed
that COUP-TFII and Sp1 are recruited
to the conserved Sp1-binding sites of
the Wt1 promoter in RIMM-18 cells.
(B,C)RT-PCR analysis of ChIP assay of
COUP-TFII (CII; B) and Sp1 (C) binding
to the conserved Sp1-binding sites of
the rat Wt1 promoter in control
siRNA- (si-Ctrl, 25 nM) or Sp1- (si-Sp1,
25 nM) specific siRNA-treated RIMM18 cells. All values were normalized to
the total input and error bars indicate
s.d. **P<0.005. (D)Relative luciferase
activity of wild-type promoter-driven
reporter (pGL2-Wt1, 250 ng) and Sp1binding site mutant reporters (pGL2Wt1-M1, pGL2-Wt1-M2 and pGL2Wt1-M12, 250 ng each) were
measured after co-transfection with or
without COUP-TFII expression plasmid
(50 ng) in HEK 293 cells.
2338 RESEARCH ARTICLE
Development 139 (13)
Funding
This work was supported by grants from the National Institutes of Health
[DK62434 and DK59820 to S.Y.T. and M.-J.T., HL76448 to S.Y.T., DK45641
and HD17379 to M.-J.T. and the Diabetes Endocrinology Research Center P30
DK079638 to M.-J.T.]. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Fig. 8. COUP-TFII directly regulates Eya1 and Wt1 expression to
specify metanephric mesenchyme cell fate and maintain
metanephric mesenchyme precursor cell survival. COUP-TFII acts in
parallel with Osr1 to regulate Eya1 transcription. COUP-TFII in the
intermediate mesoderm kidney precursor cells specifies metanephric
mesenchyme fate by directly regulating Eya1 expression. Eya1 regulates
Pax2 expression, forms a complex with Pax2 and Hox11 paralogous
proteins (including Hoxa11, Hoxc11 and Hoxd11) and binds directly to
the Six2 enhancer to regulate the expression of Six2 and its
downstream target Gdnf. Gdnf, a crucial factor for the specification of
the metanephric mesenchyme, will then induce ureteric bud outgrowth
from the Wolffian duct and initiate kidney organogenesis. In addition,
COUP-TFII is essential for the survival of the metanephric mesenchyme
precursor cells through its direct regulation of Wt1 gene expression.
Wt1 plays an anti-apoptotic role to maintain metanephric mesenchyme
cell survival. MM, metanephric mesenchyme; UB, ureteric bud.
it was shown that kidney precursor cells undergo apoptosis in Wt1deficient mutant mice, consistent with the notion that Wt1 is
essential for the early stage of kidney development (Davies et al.,
2004; Kreidberg et al., 1993). The colocalization of COUP-TFII
and Wt1 in the metanephric mesenchyme and urogenital ridge and
the similar phenotypes exhibited by COUP-TFII and Wt1 mutants
in terms of decreased cell numbers in the metanephric mesenchyme
strongly implicate that these two factors function in the same
pathway. As the expression of Wt1 is drastically decreased in
COUP-TFII mutant mice in the metanephric mesenchyme and
urogenital ridge (Fig. 3E,F and Fig. 4I,J), it suggests that COUPTFII is upstream of Wt1 in the signaling cascade. This notion is
supported by ChIP analysis, which showed that COUP-TFII is
recruited by Sp1 to the conserved Sp1-binding site in the Wt1
promoter to regulate Wt1 transcription directly. Therefore, Wt1
mediates COUP-TFII function to maintain metanephric
mesenchyme cell differentiation and survival (Fig. 8).
In summary, we have shown that COUP-TFII has two major
roles during metanephric mesenchyme formation. First, COUPTFII directly regulates Eya1 transcription to specify the kidney
precursor cell differentiation into the mature metanephric
mesenchyme. Second, COUP-TFII directly regulates Wt1
expression to maintain metanephric mesenchyme survival and
differentiation into the mature nephron.
Acknowledgements
We thank Dr Thomas Ludwig for providing the ROSA26CRE-ERT2 mice, Dr
Thomas M. Schultheiss for the mouse Osr1 probe, Dr Pin-Xian Xu for the Eya1
mouse probe and Dr Alan O. Perantoni for sharing RIMM-18 cells. We
appreciate technical help from Ms Wei Qian, Xuefei Tong and Grace Wen
Chen. We would like to express gratitude to Eric Buras for the English editing.
References
Bouchard, M., Pfeffer, P. and Busslinger, M. (2000). Functional equivalence of
the transcription factors Pax2 and Pax5 in mouse development. Development
127, 3703-3713.
Bramblett, D. E., Pennesi, M. E., Wu, S. M. and Tsai, M. J. (2004). The
transcription factor Bhlhb4 is required for rod bipolar cell maturation. Neuron
43, 779-793.
Brodbeck, S., Besenbeck, B. and Englert, C. (2004). The transcription factor Six2
activates expression of the Gdnf gene as well as its own promoter. Mech. Dev.
121, 1211-1222.
Costantini, F. and Shakya, R. (2006). GDNF/Ret signaling and the development
of the kidney. BioEssays 28, 117-127.
Davies, J. A., Ladomery, M., Hohenstein, P., Michael, L., Shafe, A., Spraggon,
L. and Hastie, N. (2004). Development of an siRNA-based method for
repressing specific genes in renal organ culture and its use to show that the Wt1
tumour suppressor is required for nephron differentiation. Hum. Mol. Genet. 13,
235-246.
de Luca, C., Kowalski, T. J., Zhang, Y., Elmquist, J. K., Lee, C., Kilimann, M.
W., Ludwig, T., Liu, S. M. and Chua, S. C., Jr (2005). Complete rescue of
obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B
transgenes. J. Clin. Invest. 115, 3484-3493.
Dressler, G. R. (2006). The cellular basis of kidney development. Annu. Rev. Cell
Dev. Biol. 22, 509-529.
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.
Gong, K. Q., Yallowitz, A. R., Sun, H., Dressler, G. R. and Wellik, D. M.
(2007). A Hox-Eya-Pax complex regulates early kidney developmental gene
expression. Mol. Cell. Biol. 27, 7661-7668.
James, R. G., Kamei, C. N., Wang, Q., Jiang, R. and Schultheiss, T. M. (2006).
Odd-skipped related 1 is required for development of the metanephric kidney
and regulates formation and differentiation of kidney precursor cells.
Development 133, 2995-3004.
Kim, B. J., Takamoto, N., Yan, J., Tsai, S. Y. and Tsai, M. J. (2009). Chicken
ovalbumin upstream promoter-transcription factor II (COUP-TFII) regulates
growth and patterning of the postnatal mouse cerebellum. Dev. Biol. 326, 378391.
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.
Kurihara, I., Lee, D. K., Petit, F. G., Jeong, J., Lee, K., Lydon, J. P., DeMayo, F.
J., Tsai, M. J. and Tsai, S. Y. (2007). COUP-TFII mediates progesterone
regulation of uterine implantation by controlling ER activity. PLoS Genet. 3,
e102.
Kuure, S., Vuolteenaho, R. and Vainio, S. (2000). Kidney morphogenesis:
cellular and molecular regulation. Mech. Dev. 92, 31-45.
Levashova, Z. B., Plisov, S. Y. and Perantoni, A. O. (2003). Conditionally
immortalized cell line of inducible metanephric mesenchyme. Kidney Int. 63,
2075-2087.
Li, L., Xie, X., Qin, J., Jeha, G. S., Saha, P. K., Yan, J., Haueter, C. M., Chan, L.,
Tsai, S. Y. and Tsai, M. J. (2009). The nuclear orphan receptor COUP-TFII plays
an essential role in adipogenesis, glucose homeostasis, and energy metabolism.
Cell Metab. 9, 77-87.
Lin, F. J., Chen, X., Qin, J., Hong, Y. K., Tsai, M. J. and Tsai, S. Y. (2010). Direct
transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps
in murine lymphatic vessel development. J. Clin. Invest. 120, 1694-1707.
Moore, A. W., McInnes, L., Kreidberg, J., Hastie, N. D. and Schedl, A. (1999).
YAC complementation shows a requirement for Wt1 in the development of
epicardium, adrenal gland and throughout nephrogenesis. Development 126,
1845-1857.
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.
DEVELOPMENT
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.076299/-/DC1
Ohto, H., Kamada, S., Tago, K., Tominaga, S. I., Ozaki, H., Sato, S. and
Kawakami, K. (1999). Cooperation of six and eya in activation of their target
genes through nuclear translocation of Eya. Mol. Cell. Biol. 19, 6815-6824.
Pipaon, C., Tsai, S. Y. and Tsai, M. J. (1999). COUP-TF upregulates NGFI-A gene
expression through an Sp1 binding site. Mol. Cell. Biol. 19, 2734-2745.
Qin, J., Tsai, M. J. and Tsai, S. Y. (2008). Essential roles of COUP-TFII in Leydig cell
differentiation and male fertility. PLoS ONE 3, e3285.
Qin, J., Chen, X., Xie, X., Tsai, M. J. and Tsai, S. Y. (2010). COUP-TFII regulates
tumor growth and metastasis by modulating tumor angiogenesis. Proc. Natl.
Acad. Sci. USA 107, 3687-3692.
Qiu, Y., Cooney, A. J., Kuratani, S., DeMayo, F. J., Tsai, S. Y. and Tsai, M. J.
(1994). Spatiotemporal expression patterns of chicken ovalbumin upstream
promoter-transcription factors in the developing mouse central nervous system:
evidence for a role in segmental patterning of the diencephalon. Proc. Natl.
Acad. Sci. USA 91, 4451-4455.
Sainio, K., Suvanto, P., Davies, J., Wartiovaara, J., Wartiovaara, K., Saarma,
M., Arumae, U., Meng, X., Lindahl, M., Pachnis, V. et al. (1997). Glial-cellline-derived neurotrophic factor is required for bud initiation from ureteric
epithelium. Development 124, 4077-4087.
Sajithlal, G., Zou, D., Silvius, D. and Xu, P. X. (2005). Eya 1 acts as a critical
regulator for specifying the metanephric mesenchyme. Dev. Biol. 284, 323-336.
Saxén, L. (1987). Organogenesis Of The Kidney. London: Cambridge University
Press.
Self, M., Lagutin, O. V., Bowling, B., Hendrix, J., Cai, Y., Dressler, G. R. and
Oliver, G. (2006). Six2 is required for suppression of nephrogenesis and
progenitor renewal in the developing kidney. EMBO J. 25, 5214-5228.
Shakya, R., Watanabe, T. and Costantini, F. (2005). The role of GDNF/Ret
signaling in ureteric bud cell fate and branching morphogenesis. Dev. Cell 8, 6574.
Suh, J. M., Yu, C. T., Tang, K., Tanaka, T., Kodama, T., Tsai, M. J. and Tsai, S. Y.
(2006). The expression profiles of nuclear receptors in the developing and adult
kidney. Mol. Endocrinol. 20, 3412-3420.
Takamoto, N., You, L. R., Moses, K., Chiang, C., Zimmer, W. E., Schwartz, R.
J., DeMayo, F. J., Tsai, M. J. and Tsai, S. Y. (2005). COUP-TFII is essential for
radial and anteroposterior patterning of the stomach. Development 132, 21792189.
RESEARCH ARTICLE 2339
Tang, K., Xie, X., Park, J. I., Jamrich, M., Tsai, S. and Tsai, M. J. (2010). COUPTFs regulate eye development by controlling factors essential for optic vesicle
morphogenesis. Development 137, 725-734.
Torres, M., Gomez-Pardo, E., Dressler, G. R. and Gruss, P. (1995). Pax-2
controls multiple steps of urogenital development. Development 121, 40574065.
Tsai, S. Y. and Tsai, M. J. (1997). Chick ovalbumin upstream promotertranscription factors (COUP-TFs): coming of age. Endocr. Rev. 18, 229-240.
Vainio, S. and Lin, Y. (2002). Coordinating early kidney development: lessons
from gene targeting. Nat. Rev. Genet. 3, 533-543.
Wang, Q., Lan, Y., Cho, E. S., Maltby, K. M. and Jiang, R. (2005). Odd-skipped
related 1 (Odd 1) is an essential regulator of heart and urogenital development.
Dev. Biol. 288, 582-594.
Wellik, D. M., Hawkes, P. J. and Capecchi, M. R. (2002). Hox11 paralogous
genes are essential for metanephric kidney induction. Genes Dev. 16, 14231432.
Wu, S. P., Lee, D. K., Demayo, F. J., Tsai, S. Y. and Tsai, M. J. (2010). Generation
of ES cells for conditional expression of nuclear receptors and coregulators in
vivo. Mol Endocrinol. 6, 1297-1304.
Xie, X., Qin, J., Lin, S. H., Tsai, S. Y. and Tsai, M. J. (2011). Nuclear receptor
chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII)
modulates mesenchymal cell commitment and differentiation. Proc. Natl. Acad.
Sci. USA 108, 14843-14848.
Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S. and Maas, R. (1999).
Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of
organ primordia. Nat. Genet. 23, 113-117.
Xu, P. X., Zheng, W., Huang, L., Maire, P., Laclef, C. and Silvius, D. (2003). Six1
is required for the early organogenesis of mammalian kidney. Development 130,
3085-3094.
You, L. R., Lin, F. J., Lee, C. T., DeMayo, F. J., Tsai, M. J. and Tsai, S. Y. (2005a).
Suppression of Notch signalling by the COUP-TFII transcription factor regulates
vein identity. Nature 435, 98-104.
You, L. R., Takamoto, N., Yu, C. T., Tanaka, T., Kodama, T., Demayo, F. J., Tsai,
S. Y. and Tsai, M. J. (2005b). Mouse lacking COUP-TFII as an animal model of
Bochdalek-type congenital diaphragmatic hernia. Proc. Natl. Acad. Sci. USA 102,
16351-16356.
DEVELOPMENT
COUP-TFII in early nephrogenesis