sign in sign up
<<
Home , Intermediate mesoderm, Mesonephric duct

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 formation and precursor 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 requires complex reciprocal tissue interactions between the ureteric 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 , 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.

KEY WORDS: COUP-TFII, Metanephric mesenchyme, Eya1, Wt1, Mouse

INTRODUCTION paralogous group (Hoxa11, Hoxc11 and Hoxd11) (Wellik et al., The mammalian kidney originates from the intermediate 2002), are expressed in the metanephric mesenchyme. Ablation of and develops through three distinct stages: , any of these genes in mice leads to renal agenesis, indicating that and metanephros. of the permanent these genes are essential for the proper formation of the kidney. kidney occurs through reciprocal interactions between the Wolffian Osr1 and Eya1, the two earliest genes expressed in kidney (mesonephric) duct and the metanephric mesenchyme. At precursor cells, sit atop the signal cascade and activate expression embryonic day (E) 10.5 in mouse, the metanephric mesenchyme of other marker genes. Osr1 is expressed first. Mice lacking Osr1 secretes glial cell line-derived neurotrophic factor (Gdnf), which do not form metanephric mesenchyme and do not express Eya1, induces the Wolffian duct to invade the metanephric mesenchyme Six2, Pax2, Sall1 and Gdnf. Eya1 is expressed downstream of Osr1 and induce adjacent mesenchymal cells to condense. These and is required for renal genesis (Xu et al., 1999). Eya proteins condensed metanephric mesenchyme cells then induce have no intrinsic DNA-binding domain but can localize to the outgrowth and branching, leading to the formation of nucleus and function as transcriptional co-activators (Ohto et al., (Dressler, 2006; Saxén, 1987). Specifically, Gdnf promotes ureteric 1999). Eya1 not only works with Six1 and Pax2 to regulate Gdnf bud outgrowth by binding to its receptor tyrosine kinase (Ret) and expression, but also complexes with Hox11 paralogous proteins co-receptor Gdnf family receptor alpha-1 (Gfr1) on the surface of (including Hoxa11, Hoxc11 and Hoxd11) and Pax2. The Hox11- the ureteric bud (Durbec et al., 1996; Moore et al., 1996; Sainio et Eya1-Pax2 complex binds to the Six2 enhancer to induce the al., 1997; Vainio and Lin, 2002). expression of Six2, which, in turn, mediates Six2 and Gdnf Although it is well established that the Gdnf-Ret signal activation (Gong et al., 2007). Thus, Eya1 acts as a key regulator transduction pathway initiates metanephric induction, the detailed of Gdnf expression and, hence, determination of metanephric fate mechanism of this signal pathway is still not well understood. within the (Sajithlal et al., 2005). During the metanephric mesenchyme condensation, many marker Chicken ovalbumin upstream promoter-transcription factor I and genes, including Osr1 (James et al., 2006), Pax2 (Torres et al., II (COUP-TFI and COUP-TFII; Nr2f1 and Nr2f2, respectively – 1995), Eya1 (Sajithlal et al., 2005), Wt1 (Kreidberg et al., 1993), Mouse Genome Informatics) are members of the nuclear receptor Six1 (Xu et al., 2003), Six2 (Self et al., 2006) and the Hox11 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 1Department of Molecular and Cellular Biology, 2Program in Developmental expression are distinct from each other. COUP-TFII is expressed in Biological, Baylor College of Medicine, Houston, TX 77030, USA. 3Division of the mesenchyme of developing organs and is shown to play a key , Cincinnati Children’s Hospital Medical Center, Cincinnati, role in their organogenesis, cell fate determination, cell OH 45229, USA. differentiation, angiogenesis and metabolic homeostasis (Kim et al., *Present address: Department of Neurobiology and Anatomy, University of Texas 2009; Kurihara et al., 2007; Li et al., 2009; Lin et al., 2010; Qin et Medical School at Houston, Houston, TX 77030, USA al., 2010; Qin et al., 2008; Tang et al., 2010; Xie et al., 2011; You et ‡Authors for correspondence ([email protected]; [email protected]) al., 2005a; You et al., 2005b). During kidney development, COUP-

Accepted 17 April 2012 TFII expression is detectable in the condensed mesenchyme and DEVELOPMENT COUP-TFII in early nephrogenesis RESEARCH ARTICLE 2331 derivates, including pretubular aggregates, comma-shaped bodies experiments were carried out as described (Lin et al., 2010). The primer and S-shaped bodies. Finally, in the adult mouse kidney, COUP-TFII sequences are listed in supplementary material Table S2. Means for mRNA is found in the distal , and the epithelial cells of the levels in control and knockdown cells were compared using Student’s t- Bowman’s capsule (Suh et al., 2006). These observations imply that test. COUP-TFII might play an important role in the genesis of the kidney Chromatin immunoprecipitation (ChIP) and PCR and maintenance of its function. Here, we provide new insights into ChIP assays were carried out with an EZ ChIP Chromatin the role of COUP-TFII in the formation of the metanephric Immunoprecipitation Kit (Millipore) by following the manufacturer’s mesenchyme. Not only is COUP-TFII expressed in the protocol. Primers are shown in supplementary material Table S3. undifferentiated kidney precursor cells, it is also indispensable for Luciferase assays the establishment and maintenance of the metanephric mesenchyme. The EYA1 and WT1 promoter regions, which contained the putative It acts as a crucial link in determination of metanephric fate through Sp1-binding sites, were amplified from human BAC clone RP11-160C13 its recruitment to the promoter of the Eya1 gene and direct regulation (Children’s Hospital Oakland Research Institute, CA, USA) and CTD- of Eya1 transcription. In addition, COUP-TFII directly regulates the 2083A15 (Invitrogen) separately. The PCR primers for amplifying the transcription of Wt1, an essential factor for maintaining metanephric promoter region or for generating the Sp1-binding site are as mesenchyme cell survival (Davies et al., 2004; Kreidberg et al., shown in supplementary material Table S4. The amplified promoter 1993). Therefore, our studies clearly indicate that COUP-TFII is a fragments were cloned into the pGL2-basic-luc vector (Promega). HEK key early regulator of the formation of metanephric mesenchyme and 293 cells were transfected with pGL2-Eya1-basic-luc or pGL2-Wt1-luc using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s the subsequent formation and differentiation of the kidney. instructions.

MATERIALS AND METHODS Statistics Animals Statistical analysis was carried out by Student’s t-test. P values of less than Generation of the floxed COUP-TFII mice and COUP-TFII-lacZ knock- 0.05 were considered significant. in mice was as described previously (Takamoto et al., 2005). The Rosa26- Cre-ERT2/+ knock-in mouse strain was provided by T. Ludwig (Columbia University, New York City, USA) (de Luca et al., 2005). For inducible RESULTS deletions during embryonic stages, 2 mg tamoxifen (Tam), dissolved in COUP-TFII is expressed specifically in kidney corn oil (Sigma-Aldrich; 10 mg/ml), was injected intraperitoneally into precursor cells pregnant at E7.5 or E8.5. from littermates were collected at indicated stages. All mouse strains were maintained in a mixed genetic We showed previously that COUP-TFII is expressed in the background and received standard rodent chow. metanephric blastema at E11.5 and later in the developing , nephrogenic cortex and stromal cells. In the adult kidney, high X-gal staining COUP-TFII expression is also detectable in the distal tubules, Whole-mount X-gal staining of embryos (up to E10.5) was performed podocytes and epithelial cells of Bowman’s capsule (Suh et al., according to published methods (Takamoto et al., 2005). 2006). Nonetheless, the expression pattern of COUP-TFII in Histology, immunofluorescence and in situ hybridization kidney precursor cells has not been determined. Here, we All the histology and immunofluorescence staining of paraffin-embedded employed a COUP-TFII-specific antibody to investigate its slides were performed as described (You et al., 2005a). Antibodies used expression pattern in the nephrogenic mesoderm from E9.5 to E10 were: anti-COUP-TFII (R&D, 1:2000), anti-Pax2 (Covance, 1:500), anti- (Fig. 1A-C). At E9.5, COUP-TFII staining was observed in the Ki67 (BD Biosciences, 1:400), anti--galactosidase (Biogenesis, 1:1500), intermediate mesoderm and other mesenchyme cells surrounding anti-Wt1 (Santa Cruz, 1:500), anti-Gdnf (Santa Cruz, 1:200) and anti-Six2 the Wolffian duct (Fig. 1A). Using Pax2 to mark the Wolffian duct, (Proteintech group, 1:400). Non-radioactive in situ hybridization was we demonstrated that COUP-TFII is only expressed in the carried out as described previously (Bramblett et al., 2004). The RNA intermediate mesoderm and not in the duct (Fig. 1B). Half a day probe for mouse Osr1 was provided by Thomas M. Schultheiss (Harvard later, at E10, COUP-TFII expression was observed in the University, Boston, USA) and the mouse Eya1 probe was as described (Xu nephrogenic mesenchyme region surrounding the Wolffian duct et al., 1999). Cell was detected by cleaved Caspase 3 (, 1:500) staining or by TUNEL assay (Roche, In Situ Cell Death and in the urogenital ridge (Fig. 1C). These results indicate that Detection Kit) according to the manufacturer’s instructions. COUP-TFII is expressed in kidney precursor cells. Kidney culture and staining Inducible deletion of COUP-TFII in kidney The metanephric mesenchymes containing the Wolffian duct from 10.5 precursor cells days post-coitum (dpc) mouse embryos were cultured on transwell filters In order to bypass early embryonic lethality of COUP-TFII as described (Shakya et al., 2005). The Wolffian duct and ureteric bud were homozygous mutant at E10.5, we conditionally inactivated COUP- visualized by staining with anti-E-Cadherin (BD Biosciences, 1:200). TFII by crossing the COUP-TFII floxed mouse strain (COUP- f/f T2/+ Cell lines and transfection TFII ) (Takamoto et al., 2005) with Rosa26-Cre-ER , a strain Human embryonic kidney 293 (HEK 293) cells were transfected with that harbors a tamoxifen-inducible Cre recombinase under the Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. control of the ubiquitously active ROSA26 promoter (de Luca et al., The rat inducible metanephric mesenchyme (RIMM-18) cell line was 2005). Rosa26-Cre-ERT2/+;COUP-TFIIf/f males (hereafter referred provided by Alan O. Perantoni (National Institutes of Health, NCI, to as COUP-TFIId/d) were intercrossed with COUP-TFIIf/f females, Bethesda, MD, USA) and the culture conditions were as described and a single dosage of 2 mg Tam was injected intraperitoneally into (Levashova et al., 2003). pregnant dams to induce COUP-TFII deletion. Specifically, we RNA interference and quantitative real-time RT-PCR assay performed Tam injection at E7.5. In our mouse model, a lacZ gene The siRNA oligonucleotides were purchased from Thermo Fisher inserted into the COUP-TFII locus is turned on when the COUP- Scientific or Applied Biosystems/Ambion. The target sequences for rat TFII gene is deleted. One and half a days after Tam treatment,

siRNA are described in supplementary material Table S1. RNA interference COUP-TFII was deleted throughout the whole body of COUP- DEVELOPMENT 2332 RESEARCH ARTICLE Development 139 (13)

Fig. 1. Expression pattern and conditional deletion of COUP-TFII in kidney precursor cells. (A)Transverse section of wild-type E9.5 mouse shows that COUP-TFII is expressed in the intermediate mesoderm (im), (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 COUP- TFII 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 Fig. 2. COUP-TFII is essential for the metanephric mesenchyme metanephric mesenchyme (mm) (F). COUP-TFII expression is totally lost formation. Tamoxifen was administered at E7.5 to delete COUP-TFII. in the mutant embryo three days after Tam administration (I). DAPI (A-D)Transverse sections of metanephric mesenchyme (mm) from these counterstain for nuclei (blue) was applied for A, C, F and I. Scale bars: embryos were stained with hematoxylin and eosin at E10.5. The 50m. 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 TFIId/d mutant embryos, including the nephrogenic mesenchyme E10.5, the metanephric mesenchyme of COUP-TFIIf/f control embryos (Fig. 1D,G). Further examination of X-gal staining of transverse expresses Pax2, Six2 and Gdnf (E,G,I). By contrast, these three gene products are undetectable in the metanephric mesenchyme region of sections at the nephrogenic mesenchyme indicated that deletion of d/d COUP-TFII occurred in the intermediate mesoderm and lateral COUP-TFII null mutants (F,H,J,). However, the Pax2 expression in the d/d Wolffian duct remains (F). lacZ staining indicates that COUP-TFII is plate mesoderm of COUP-TFII mutant embryos (Fig. 1E,H). deleted in the mesenchyme cells (F). Boxed areas in I and J are shown in Using the COUP-TFII-specific antibody, we showed that COUP- insets. (K,L)Kidney organ cultures from the metanephric mesenchyme TFII is highly expressed in the urogenital ridge and the developing of COUP-TFIIf/f control (K) and COUP-TFIId/d mutant (L) embryos condensed nephrogenic mesenchyme, but not in the Wolffian duct indicated that the ureteric bud (ub) outgrowth, visualized by E-cadherin of the COUP-TFIIf/f control at E10.5 (Fig. 1F). In COUP-TFII staining, was detected from the Wolffian duct of the COUP-TFIIf/f mutants, expression was undetectable (Fig. 1I). These data indicate control (K), but not from the COUP-TFIId/d mutant embryo (L). DAPI the Tam-inducible deletion system effectively deletes COUP-TFII counterstain for nuclei (blue) was applied for E-J. Scale bars: 50m. 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). examined with littermates of the same number. The results clearly show that the metanephric mesenchyme is not formed in the COUP-TFII is essential for metanephric COUP-TFIId/d mutant (supplementary material Fig. S1). As the mesenchyme formation but not for the formation paired box-containing transcription factor Pax2 is a key regulator of of mesonephros kidney development (Torres et al., 1995), we investigated whether During the formation of the metanephros, the metanephric the expression of this gene is compromised. In the mouse kidney, the mesenchyme appears morphologically as an aggregate of the expression of Pax2 is initiated in the intermediate mesoderm and nephrogenic mesenchyme cells at the caudal end of the nephrogenic maintained throughout the development of the pronephros and cord at E10.5 (Fig. 2A,C). This structure is largely missing in mesonephros (Bouchard et al., 2000). Pax2 is expressed in the COUP-TFIId/d mutant embryos (Fig. 2B,D). In addition, serial induced metanephric mesenchyme and in the Wolffian duct of the

sections through the entire metanephric mesenchyme region were metanephros in the controls (Fig. 2E). By contrast, Pax2 expression DEVELOPMENT COUP-TFII in early nephrogenesis RESEARCH ARTICLE 2333 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 COUP- TFIId/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. Fig. 3. COUP-TFII specifically regulates the metanephric mesenchyme gene expression. Tamoxifen was administered at E8.5 The mesonephros develops when the Wolffian duct reaches the to delete COUP-TFII. Transverse sections of the metanephric prospective mesonephric mesenchyme and induces adjacent mesenchyme region from the embryos were stained with Hematoxylin mesenchymal cells to condense and form and Eosin at E10.5. (A,B)The condensed metanephric mesenchyme (Saxén, 1987). First, we investigated whether COUP-TFII is also (mm) was seen in both the COUP-TFIIf/f control (A) and the COUP-TFIId/d expressed in the mesonephros (also called the mesonephric ). mutant (B) embryos. (C-H)At E10.5, the metanephric mesenchyme of COUP-TFII staining in E10 embryos indicated that COUP-TFII is COUP-TFIId/d mutant embryos expresses Eya1 (D), Wt1 (F) and Six2 (H); expressed in the mesonephros but not in the Wolffian duct however, the expression levels per cell of all of these three factors are (supplementary material Fig. S2A). Pax2 is expressed in the lower than control (C,E,G). Eya1 expression level was measured by in Wolffian duct and mesonephros. By using Pax2 as a mesonephric situ hybridization (C,D). The DAPI counterstain for nuclei (blue) was tubule marker, we found COUP-TFII colocalizes with Pax2 in the 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 mesonephros but not in the Wolffian duct (supplementary material area of COUP-TFIId/d mutant (I,J). J shows a high magnification of the Fig. S2B). We examined the E9.5 embryos and found that the dashed box in I. Arrows indicate that cells that express higher Six2 levels –/– mesonephros is still intact in the COUP-TFII mutants in which have lower lacZ expression (L) and arrowheads indicate that cells that COUP-TFII is deleted very early in the germ cells, compared with express higher lacZ have lower Six2 expression (K). (M)The Six2 COUP-TFII+/+ controls (supplementary material Fig. S2C,D). expression level of Six2-positive cells was measured by the fluorescence Therefore, even though COUP-TFII is expressed in the density divided by area (densitometric mean) from COUP-TFIIf/f control mesonephros, it is not essential for mesonephros formation. and COUP-TFIId/d mutant embryos. Four metanephric mesenchyme samples were measured and the results indicate that COUP-TFII deletion COUP-TFII specifically regulates Eya1, Wt1 and in the metanephric mesenchyme significantly reduces Six2 expression. Six2 in the metanephric mesenchyme **P<0.005. ur, urogenital ridge; wd, Wolffian duct. Scale bars: 50m. Although the metanephric mesenchyme markers, such as Pax2 or Six2 expression, are not detected in the COUP-TFIId/d mutant marker gene expression, we deleted COUP-TFII one day later by embryos (Fig. 3F,H), the presence of lacZ-positive cells indicates injecting Tam at E8.5 and collecting embryos at E10.5. We reasoned that it is still possible that the lack of marker gene expression could that by deleting COUP-TFII one day later, we would have be due to the loss of metanephric mesenchyme cells. To demonstrate metanephric mesenchyme formation, but in some cells COUP-TFII

that COUP-TFII does indeed regulate metanephric mesenchyme would be completely deleted, in some one allele would be deleted, DEVELOPMENT 2334 RESEARCH ARTICLE Development 139 (13) 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 Fig. 4. COUP-TFII deletion abolishes Wt1 expression and intensity of Six2-positive cells (metanephric mesenchyme cells) in increases apoptosis of metanephric mesenchyme cells. (A-F) Tam f/f d/d  the COUP-TFII controls (n131 cells) and COUP-TFII mutants was administered at E8.5 and embryos were examined at E10.5. (n65 cells) shown in Fig. 3G,H and divided by the cell area to Proliferation was determined by Ki67 immunostaining which appeared obtain densitometric levels of Six2 expression. The result shows that to show similar Ki67-positive cells for both mutant (B) and control (A). deletion of COUP-TFII significantly decreases Six2 expression in However, upon close examination, the metanephric mesenchyme (C,D the COUP-TFIId/d mutant (Fig. 3M). These data clearly indicate that red circles) had fewer Ki-67-positive cells in the mutant. Apoptosis COUP-TFII does indeed regulate the expression of the metanephric analysis results indicated that there are many more cleaved Caspase 3 mesenchyme markers Six2, Eya1 and Wt1. (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 Deletion of COUP-TFII abolishes Wt1 expression were examined at E10.5. TUNEL assay shows a significantly increase in and increases metanephric mesenchyme cell apoptosis in the metanephric mesenchyme region of the COUP-TFII apoptosis knockout embryo (G,H). Wt1 is expressed in the urogenital ridge and We observed a reduction in cell number (Fig. 4A,B) in the metanephric mesenchyme in the COUP-TFIIf/f control embryo (I) and the nephrogenic mesenchyme region of the COUP-TFIId/d mutant expression is lost in the COUP-TFIId/d embryo (J). lacZ staining indicates embryos compared with controls. We investigated whether the loss that COUP-TFII-deleted cells are still present. The slides are of mesenchymal cells in this region is due to decreased cell counterstained with DAPI for nuclei (blue). Scale bar: 50m. 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). COUP-TFII deletion. Indeed, we found Wt1 expression to be By contrast, the COUP-TFIId/d mutant metanephric mesenchyme significantly decreased in the COUP-TFII mutant urogenital ridge region showed a significant decrease in Ki67-positive cell numbers and metanephric mesenchyme (deletion cells shown as the lacZ (Fig. 4C,D, red circled regions), indicating a specific decrease in positive cells) (Fig. 3E,F and Fig. 4I,J). These results indicate Wt1 proliferation of the metanephric mesenchyme. Furthermore, we lies downstream of COUP-TFII and is regulated by COUP-TFII. observed an increase in the number of apoptotic cells in the Owing to the importance of Wt1 in metanephric mesenchyme cell metanephric mesenchyme of mutant mice as detected by both survival, our findings suggested that loss of Wt1 expression due to cleaved Caspase 3 and tunnel assays (Fig. 4E-H). COUP-TFII deletion is the likely reason for the increased The Wilms tumor suppressor gene (Wt1) is expressed in the metanephric mesenchyme apoptosis in COUP-TFIId/d mutants. metanephric mesenchyme and has been shown to be essential for the survival of metanephric mesenchymal cells (Kreidberg et al., COUP-TFs regulate the expression of Eya1, Six2, 1993; Kuure et al., 2000; Moore et al., 1999). Therefore, we Wt1 and Gdnf in metanephric mesenchyme cells investigated whether Wt1 expression is compromised in COUP- In order to study the detailed molecular mechanism by which TFIId/d mutants. First, we determined whether COUP-TFII and COUP-TFII controls regulatory genes in the metanephric Wt1 colocalize with each other. As shown in supplementary mesenchyme, we employed the conditionally immortalized rat material Fig. S3A-C, we found that COUP-TFII colocalizes with inducible metanephric mesenchyme cell line (RIMM-18). RIMM-18 Wt1 in the metanephric mesenchyme and in the urogenital ridge. cells were generated from rat mesenchymal cells by transfection with

Next, we investigated whether Wt1 expression level is altered with a vector encoding an estradiol-dependent E1A-ER fusion protein and DEVELOPMENT COUP-TFII in early nephrogenesis RESEARCH ARTICLE 2335

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 wild- type 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 COUP- TFII 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. Gdnf Eya1 Six2 Wt1 Fig. 5. COUP-TFs regulate , , and expression in COUP-TFII directly regulates Eya1 gene expression the metanephric mesenchyme in vitro. (A-F)Quantitative RT-PCR by interacting with Sp1 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 Eya1 has been shown to specify the metanephric mesenchyme fate knockdown of both COUP-TFI and COUP-TFII using specific siRNAs. All and is indispensable for metanephric mesenchyme formation. In mRNA measurements were normalized to 18S rRNA. Error bars indicate order to determine whether Eya1 expression is regulated by COUP- s.d. **P<0.005. TFII 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 these cells maintain its metanephric mesenchyme characteristics COUP-TFIIf/f controls but is absent in COUP-TFIId/d mutants (Fig. (Levashova et al., 2003). Using RT-PCR, we found that the 6A,B). This indicates that Eya1 is a COUP-TFII target and is likely expression levels of both COUP-TFI and COUP-TFII are high in to be regulated by COUP-TFII at the transcriptional level. RIMM-18 cells. Despite this, in the E10.5 mouse embryos, only To support this conclusion further, we carried out chromatin COUP-TFII is expressed in the metanephric mesenchyme, whereas immunoprecipitation (ChIP) assays to determine whether COUP- COUP-TFI is located in the stroma mesenchyme. Our laboratory has TFII is recruited to the promoter/enhancer region of the Eya1 gene reported that COUP-TFI and COUP-TFII are highly homologous locus. Our laboratories have previously demonstrated that COUP- and also functionally redundant (Tsai and Tsai, 1997; Wu et al., TFII acts as a positive regulator, enhancing target gene expression 2010). Therefore, during in vitro experiments, we used siRNAs to by interacting with Sp1 at Sp1-binding sites (Kim et al., 2009; Lin knock down both COUP-TFI and COUP-TFII to study the et al., 2010; Pipaon et al., 1999; Qin et al., 2010). To assess underlying mechanism of COUP-TF regulation of transcription whether COUP-TFII regulates Eya1 expression through its factors important for kidney development. Knockdown of COUP- interaction with Sp1, we searched for the evolutionarily conserved TFI and COUP-TFII significantly decreases Gdnf, Eya1, Six2 and Sp1-binding sites in human, mouse and rat sequences on and Wt1 (Fig. 5C-F) expression. In order to confirm whether COUP-TFII surrounding the Eya1 gene and found three conserved sites alone can regulate these transcription factors in RIMM-18 cells, we upstream of the transcription start site (Fig. 6C, black boxes). We also used siRNA to knock down COUP-TFII specifically. We found then performed ChIP assays to evaluate whether endogenous that knockdown of COUP-TFII alone is enough to significantly COUP-TFII is recruited to the Sp1 sites of the Eya1 promoter. decrease Eya1 and Wt1 (supplementary material Fig. S4B,C) Indeed, we found COUP-TFII is preferentially recruited to the expression. These results indicate that COUP-TFs can indeed promoter regions containing Sp1-binding sites but not recruited to positively regulate genes expressed early in metanephric the region lacking Sp1-binding sites (Fig. 6C, Sp1). In parallel, Sp1 mesenchyme development. was also specifically recruited to the same region as COUP-TFII, but not to the region without Sp1-binding sites (Fig. 6C). To COUP-TFII signaling is independent of Osr1 in the substantiate that COUP-TFII was recruited by Sp1 to the Eya1 nephrogenic mesenchyme promoter, we knocked down endogenous Sp1 expression with Sp1- Similar to COUP-TFII, Odd-skipped related 1 (Osr1) is also required specific siRNA (si-Sp1). Recruitment of COUP-TFII and Sp1 to for the formation of the metanephric mesenchyme and is upstream the Eya1 promoter was, in fact, significantly reduced in Sp1- of Eya1. In order to define the relationship between Osr1 and COUP- knockdown cells compared with controls (Fig. 6D,E). TFII in the regulatory cascade, we first investigated whether COUP- To test further whether COUP-TFII binding to the Eya1 promoter TFII is upstream of Osr1 by examining the expression of Osr1 in leads to activation of transcription, we performed luciferase reporter COUP-TFII mutants by in situ hybridization. Osr1 is broadly assays in HEK293 cells using a 1.8-kb (–731 bp to 1079 bp) human expressed in the nephrogenic mesenchyme regions in the control EYA1 promoter fragment that included the three conserved Sp1-

mice (supplementary material Fig. S5A). Despite the fact that the binding sites. Luciferase reporter activity was significantly increased DEVELOPMENT 2336 RESEARCH ARTICLE Development 139 (13)

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 siRNA- treated 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, pGL2- Eya1-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.

when COUP-TFII was expressed compared with control cells (Fig. is Sp1-dependent, we knocked down endogenous Sp1 by siRNA. 5F). Next, we mutated these three conserved Sp1-binding sites as With knockdown, COUP-TFII recruitment to the Wt1 promoter was depicted in supplementary material Fig. S4A. We generated reporters significantly reduced (Fig. 7B,C). with three single Sp1-binding sites or with all three sites mutated To confirm whether COUP-TFII binding to the Wt1 promoter (supplementary material Fig. S6A; pGL2-Eya1-M1, pGL2-Eya1- leads to transcription activation, we performed luciferase reporter M2, pGL2-M3 and the triple- pGL2-Eya1-M123). In the assays using a 1.1-kb (–736 bp to 377 bp) human WT1 promoter presence of COUP-TFII expression plasmid, activation of all four fragment that contains the two conserved Sp1-binding sites. As mutant luciferase reporters was significantly diminished versus the shown in Fig. 7D, Wt1-luciferase reporter (pGL2-Wt1) activity was intact reporter (Fig. 6F). This result indicates that COUP-TFII works significantly increased by COUP-TFII. In addition, when Sp1- through Sp1 on the Sp1-binding sites to activate Eya1 expression. binding sites were mutated in the Wt1-luciferase reporter Collectively, these results substantiate a model in which COUP-TFII (supplementary material Fig. S6B; pGL2-Wt1-M1, pGL2-Wt1-M2 is recruited to the Eya1 promoter in an Sp1-dependent manner to and the double-mutation pGL2-Wt1-M12), COUP-TFII-dependent directly activate Eya1 transcription. luciferase activity decreased dramatically (Fig. 7D). Taken together, these results strengthen our proposed model that recruitment of COUP-TFII interacts synergistically with Sp1 to COUP-TFII to the Wt1 promoter is Sp1-dependent, and that COUP- regulate Wt1 expression directly TFII directly activates Wt1 transcription. Next, we investigated whether COUP-TFII also directly regulates the transcription of Wt1 through its interactions with Sp1. We identified DISCUSSION two evolutionarily conserved Sp1-binding sites surrounding the Wt1 COUP-TFII directly regulates transcription of Eya1 promoter (Fig. 7A, black boxes). ChIP analysis showed that COUP- to modulate Gdnf expression during metanephric TFII is indeed preferentially recruited to Sp1-binding sites at the Wt1 induction promoter but is not recruited to the region lacking Sp1-binding sites COUP-TFII is expressed in the mesenchyme of developing organs (Fig. 7A). Sp1 was recruited to the same regions as COUP-TFII. To and has been shown to play a key role in their organogenesis. In

determine whether recruitment of COUP-TFII to the Wt1 promoter this study, we found that COUP-TFII is expressed in the kidney DEVELOPMENT COUP-TFII in early nephrogenesis RESEARCH ARTICLE 2337

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 RIMM- 18 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 Sp1- binding site mutant reporters (pGL2- Wt1-M1, pGL2-Wt1-M2 and pGL2- Wt1-M12, 250 ng each) were measured after co-transfection with or without COUP-TFII expression plasmid (50 ng) in HEK 293 cells.

precursor intermediate mesoderm and later expressed in the COUP-TFII acts in parallel with Osr1 to regulate metanephric mesenchyme. Knockout of COUP-TFII at an early Eya1 expression during metanephric induction developmental stage (E7.5) results in the loss of metanephric Osr1 is known to be the earliest marker for kidney development in the mesenchyme in the mutant embryo at E10.5. In addition, ablation intermediate mesoderm. Mice lacking Osr1 do not form metanephric of COUP-TFII results in the loss of expression of many key mesenchyme and do not express many factors essential for factors, including Six2, Pax2, Wt1, Eya1 and Gdnf, which are metanephric mesenchyme formation (James et al., 2006; Wang et al., essential for metanephric mesenchyme formation. These results 2005). These phenotypes bear strong resemblance to that of the place COUP-TFII as an upstream regulator of these important COUP-TFII knockout mice. To address whether these two genes work regulatory factors. in the same pathway, we examined whether Osr1 expression is Our in situ hybridization data showed that Eya1 expression is affected in the COUP-TFII knockout. Our results clearly show that significantly decreased in the COUP-TFII mutant (Fig. 3C,D), Osr1 expression in the nephrogenic mesenchyme region is not altered suggesting that COUP-TFII directly regulates Eya1 expression in in the COUP-TFII mutant. Similarly, COUP-TFII expression in the the metanephric mesenchyme. Indeed, ChIP assays showed that metanephric mesenchyme region remains the same in the Osr1 COUP-TFII is recruited to the conserved Sp1-binding site of the knockout embryo. Together, these results indicate that COUP-TFII Eya1 promoter by tethering to Sp1 for direct regulation of Eya1 and Osr1 act independently to regulate kidney morphogenesis. This expression. Furthermore, expression of COUP-TFII can enhance notion is further supported by in vitro cell culture experiments, in Eya1 promoter-driven reporter activity. Collectively, these results which we showed that COUP-TFII and Osr1 do not regulate each clearly indicate that COUP-TFII promotes Gdnf signaling cascade other’s expression. Interestingly, whereas Wt1 is expressed in the by direct regulation of Eya1 expression and, thus, induces metanephric mesenchyme of Osr1 null mice (James et al., 2006), Wt1 metanephric mesenchyme fate in kidney progenitor cells. It should expression in the COUP-TFII knockout mutant is totally lost. This be noted that in Eya1 knockout mice, ureteric buds fail to grow out result indicates that although both COUP-TFII and Osr1 are expressed into the metanephric mesenchyme (Sajithlal et al., 2005). Similar in the intermediate mesoderm and both regulate Eya1, Pax2 and Six2 to Eya1, we observed no ureteric bud outgrowth in six out of eight expression, they do not regulate each other to control the expression COUP-TFII mutants using the kidney organ cultures (Fig. 2L). It of those key factors important for metanephric mesenchyme is not clear, however, whether the remaining two COUP-TFII formation. Based on all these results, our working model is that mutants have ureteric bud outgrowth (supplementary material COUP-TFII and Osr1 act in parallel to regulate Eya1 and its S7A,B, arrowheads). In any event, if they indeed have ureteric bud downstream target Gdnf to specify metanephric mesenchyme (Fig. 8). outgrowth, it is possible that COUP-TFII was not deleted early enough in these two particular mutants, resulting in partially COUP-TFII is essential for survival of the committed metanephric mesenchyme and subsequent induction of metanephric mesenchyme and for nephron ureteric bud outgrowth. As the majority of mutants do not have differentiation ureteric buds outgrowth, it indicates that COUP-TFII is required Wt1 mutant mesenchyme cannot be induced to form nephric for the proper formation of metanephric mesenchyme and tubules (Kreidberg et al., 1993), suggesting that Wt1 is cell-

subsequent induction of ureteric bud outgrowth. autonomously required for nephron differentiation. Subsequently, DEVELOPMENT 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.

Supplementary material Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.076299/-/DC1

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 Fig. 8. COUP-TFII directly regulates Eya1 and Wt1 expression to 43, 779-793. specify metanephric mesenchyme cell fate and maintain Brodbeck, S., Besenbeck, B. and Englert, C. (2004). The transcription factor Six2 metanephric mesenchyme precursor cell survival. COUP-TFII acts in activates expression of the Gdnf gene as well as its own promoter. Mech. Dev. 121, 1211-1222. parallel with Osr1 to regulate Eya1 transcription. COUP-TFII in the Costantini, F. and Shakya, R. (2006). GDNF/Ret signaling and the development intermediate mesoderm kidney precursor cells specifies metanephric of the kidney. BioEssays 28, 117-127. mesenchyme fate by directly regulating Eya1 expression. Eya1 regulates Davies, J. A., Ladomery, M., Hohenstein, P., Michael, L., Shafe, A., Spraggon, Pax2 expression, forms a complex with Pax2 and Hox11 paralogous L. and Hastie, N. (2004). Development of an siRNA-based method for proteins (including Hoxa11, Hoxc11 and Hoxd11) and binds directly to repressing specific genes in renal organ culture and its use to show that the Wt1 the Six2 enhancer to regulate the expression of Six2 and its tumour suppressor is required for nephron differentiation. Hum. Mol. Genet. 13, downstream target Gdnf. Gdnf, a crucial factor for the specification of 235-246. de Luca, C., Kowalski, T. J., Zhang, Y., Elmquist, J. K., Lee, C., Kilimann, M. the metanephric mesenchyme, will then induce ureteric bud outgrowth W., Ludwig, T., Liu, S. M. and Chua, S. C., Jr (2005). Complete rescue of from the Wolffian duct and initiate kidney organogenesis. In addition, obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B COUP-TFII is essential for the survival of the metanephric mesenchyme transgenes. J. Clin. Invest. 115, 3484-3493. precursor cells through its direct regulation of Wt1 gene expression. Dressler, G. R. (2006). The cellular basis of kidney development. Annu. Rev. Cell Wt1 plays an anti-apoptotic role to maintain metanephric mesenchyme Dev. Biol. 22, 509-529. cell survival. MM, metanephric mesenchyme; UB, ureteric bud. 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. it was shown that kidney precursor cells undergo apoptosis in Wt1- Gong, K. Q., Yallowitz, A. R., Sun, H., Dressler, G. R. and Wellik, D. M. deficient mutant mice, consistent with the notion that Wt1 is (2007). A Hox-Eya-Pax complex regulates early kidney developmental gene essential for the early stage of kidney development (Davies et al., expression. Mol. Cell. Biol. 27, 7661-7668. 2004; Kreidberg et al., 1993). The colocalization of COUP-TFII 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 Wt1 in the metanephric mesenchyme and urogenital ridge and and regulates formation and differentiation of kidney precursor cells. the similar phenotypes exhibited by COUP-TFII and Wt1 mutants Development 133, 2995-3004. in terms of decreased cell numbers in the metanephric mesenchyme Kim, B. J., Takamoto, N., Yan, J., Tsai, S. Y. and Tsai, M. J. (2009). Chicken strongly implicate that these two factors function in the same ovalbumin upstream promoter-transcription factor II (COUP-TFII) regulates growth and patterning of the postnatal mouse cerebellum. Dev. Biol. 326, 378- pathway. As the expression of Wt1 is drastically decreased in 391. COUP-TFII mutant mice in the metanephric mesenchyme and Kreidberg, J. A., Sariola, H., Loring, J. M., Maeda, M., Pelletier, J., Housman, urogenital ridge (Fig. 3E,F and Fig. 4I,J), it suggests that COUP- D. and Jaenisch, R. (1993). WT-1 is required for early kidney development. Cell TFII is upstream of Wt1 in the signaling cascade. This notion is 74, 679-691. Kurihara, I., Lee, D. K., Petit, F. G., Jeong, J., Lee, K., Lydon, J. P., DeMayo, F. supported by ChIP analysis, which showed that COUP-TFII is J., Tsai, M. J. and Tsai, S. Y. (2007). COUP-TFII mediates progesterone recruited by Sp1 to the conserved Sp1-binding site in the Wt1 regulation of uterine implantation by controlling ER activity. PLoS Genet. 3, promoter to regulate Wt1 transcription directly. Therefore, Wt1 e102. Kuure, S., Vuolteenaho, R. and Vainio, S. (2000). Kidney morphogenesis: mediates COUP-TFII function to maintain metanephric cellular and molecular regulation. Mech. Dev. 92, 31-45. mesenchyme cell differentiation and survival (Fig. 8). Levashova, Z. B., Plisov, S. Y. and Perantoni, A. O. (2003). Conditionally In summary, we have shown that COUP-TFII has two major immortalized cell line of inducible metanephric mesenchyme. Kidney Int. 63, roles during metanephric mesenchyme formation. First, COUP- 2075-2087. Li, L., Xie, X., Qin, J., Jeha, G. S., Saha, P. K., Yan, J., Haueter, C. M., Chan, L., TFII directly regulates Eya1 transcription to specify the kidney Tsai, S. Y. and Tsai, M. J. (2009). The nuclear orphan receptor COUP-TFII plays precursor cell differentiation into the mature metanephric an essential role in adipogenesis, glucose homeostasis, and energy metabolism. mesenchyme. Second, COUP-TFII directly regulates Wt1 Cell Metab. 9, 77-87. expression to maintain metanephric mesenchyme survival and 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 differentiation into the mature nephron. 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). Acknowledgements YAC complementation shows a requirement for Wt1 in the development of We thank Dr Thomas Ludwig for providing the ROSA26CRE-ERT2 mice, Dr epicardium, and throughout nephrogenesis. Development 126, Thomas M. Schultheiss for the mouse Osr1 probe, Dr Pin-Xian Xu for the Eya1 1845-1857. mouse probe and Dr Alan O. Perantoni for sharing RIMM-18 cells. We Moore, M. W., Klein, R. D., Farinas, I., Sauer, H., Armanini, M., Phillips, H., appreciate technical help from Ms Wei Qian, Xuefei Tong and Grace Wen Reichardt, L. F., Ryan, A. M., Carver-Moore, K. and Rosenthal, A. (1996).

Chen. We would like to express gratitude to Eric Buras for the English editing. Renal and neuronal abnormalities in mice lacking GDNF. Nature 382, 76-79. DEVELOPMENT COUP-TFII in early nephrogenesis RESEARCH ARTICLE 2339

Ohto, H., Kamada, S., Tago, K., Tominaga, S. I., Ozaki, H., Sato, S. and Tang, K., Xie, X., Park, J. I., Jamrich, M., Tsai, S. and Tsai, M. J. (2010). COUP- Kawakami, K. (1999). Cooperation of six and eya in activation of their target TFs regulate eye development by controlling factors essential for optic vesicle genes through nuclear translocation of Eya. Mol. Cell. Biol. 19, 6815-6824. morphogenesis. Development 137, 725-734. Pipaon, C., Tsai, S. Y. and Tsai, M. J. (1999). COUP-TF upregulates NGFI-A gene Torres, M., Gomez-Pardo, E., Dressler, G. R. and Gruss, P. (1995). Pax-2 expression through an Sp1 binding site. Mol. Cell. Biol. 19, 2734-2745. controls multiple steps of urogenital development. Development 121, 4057- Qin, J., Tsai, M. J. and Tsai, S. Y. (2008). Essential roles of COUP-TFII in Leydig cell 4065. differentiation and male fertility. PLoS ONE 3, e3285. Tsai, S. Y. and Tsai, M. J. (1997). Chick ovalbumin upstream promoter- Qin, J., Chen, X., Xie, X., Tsai, M. J. and Tsai, S. Y. (2010). COUP-TFII regulates transcription factors (COUP-TFs): coming of age. Endocr. Rev. 18, 229-240. tumor growth and metastasis by modulating tumor angiogenesis. Proc. Natl. Vainio, S. and Lin, Y. (2002). Coordinating early kidney development: lessons Acad. Sci. USA 107, 3687-3692. from gene targeting. Nat. Rev. Genet. 3, 533-543. Qiu, Y., Cooney, A. J., Kuratani, S., DeMayo, F. J., Tsai, S. Y. and Tsai, M. J. Wang, Q., Lan, Y., Cho, E. S., Maltby, K. M. and Jiang, R. (2005). Odd-skipped (1994). Spatiotemporal expression patterns of chicken ovalbumin upstream related 1 (Odd 1) is an essential regulator of heart and urogenital development. promoter-transcription factors in the developing mouse central nervous system: Dev. Biol. 288, 582-594. evidence for a role in segmental patterning of the diencephalon. Proc. Natl. Wellik, D. M., Hawkes, P. J. and Capecchi, M. R. (2002). Hox11 paralogous Acad. Sci. USA 91, 4451-4455. genes are essential for metanephric kidney induction. Genes Dev. 16, 1423- Sainio, K., Suvanto, P., Davies, J., Wartiovaara, J., Wartiovaara, K., Saarma, 1432. M., Arumae, U., Meng, X., Lindahl, M., Pachnis, V. et al. (1997). Glial-cell- Wu, S. P., Lee, D. K., Demayo, F. J., Tsai, S. Y. and Tsai, M. J. (2010). Generation line-derived neurotrophic factor is required for bud initiation from ureteric of ES cells for conditional expression of nuclear receptors and coregulators in epithelium. Development 124, 4077-4087. vivo. Mol Endocrinol. 6, 1297-1304. Sajithlal, G., Zou, D., Silvius, D. and Xu, P. X. (2005). Eya 1 acts as a critical Xie, X., Qin, J., Lin, S. H., Tsai, S. Y. and Tsai, M. J. (2011). Nuclear receptor regulator for specifying the metanephric mesenchyme. Dev. Biol. 284, 323-336. chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) Saxén, L. (1987). Organogenesis Of The Kidney. London: Cambridge University modulates mesenchymal cell commitment and differentiation. Proc. Natl. Acad. Press. Sci. USA 108, 14843-14848. Self, M., Lagutin, O. V., Bowling, B., Hendrix, J., Cai, Y., Dressler, G. R. and Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S. and Maas, R. (1999). Oliver, G. (2006). Six2 is required for suppression of nephrogenesis and Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of progenitor renewal in the developing kidney. EMBO J. 25, 5214-5228. organ primordia. Nat. Genet. 23, 113-117. Shakya, R., Watanabe, T. and Costantini, F. (2005). The role of GDNF/Ret Xu, P. X., Zheng, W., Huang, L., Maire, P., Laclef, C. and Silvius, D. (2003). Six1 signaling in ureteric bud cell fate and branching morphogenesis. Dev. Cell 8, 65- is required for the early organogenesis of mammalian kidney. Development 130, 74. 3085-3094. Suh, J. M., Yu, C. T., Tang, K., Tanaka, T., Kodama, T., Tsai, M. J. and Tsai, S. Y. You, L. R., Lin, F. J., Lee, C. T., DeMayo, F. J., Tsai, M. J. and Tsai, S. Y. (2005a). (2006). The expression profiles of nuclear receptors in the developing and adult Suppression of Notch signalling by the COUP-TFII transcription factor regulates kidney. Mol. Endocrinol. 20, 3412-3420. vein identity. Nature 435, 98-104. Takamoto, N., You, L. R., Moses, K., Chiang, C., Zimmer, W. E., Schwartz, R. You, L. R., Takamoto, N., Yu, C. T., Tanaka, T., Kodama, T., Demayo, F. J., Tsai, J., DeMayo, F. J., Tsai, M. J. and Tsai, S. Y. (2005). COUP-TFII is essential for S. Y. and Tsai, M. J. (2005b). Mouse lacking COUP-TFII as an animal model of radial and anteroposterior patterning of the stomach. Development 132, 2179- Bochdalek-type congenital diaphragmatic hernia. Proc. Natl. Acad. Sci. USA 102, 2189. 16351-16356. DEVELOPMENT