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Department of Molecular and Cellular Biology (J.M.S., C.-T.Y., K.T., M.-J.T., S.Y.T) and Program of Development Biology (M.-J.T., S.Y.T.), Baylor College of Medicine, Houston, Texas 77030; and Laboratory for Systems Biology and Medicine (T.T., T.K.), Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan
Address all correspondence and requests for reprints to: Sophia Y. Tsai, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: stsai{at}bcm.tmc.edu.
ABSTRACT
Nuclear receptors are transcriptional regulators that play important roles in embryonic development and organogenesis. To study the potential roles of nuclear receptors in kidney development, we examined the expression patterns of a subset of nuclear receptors in which specific antibodies are available for profiling using immunohistochemistry. As a prototype for our analysis, we investigated the expression patterns of chicken ovalbumin upstream promoter transcription factor (COUP-TF) -I and -II in more details during embryonic development and in the adult by immunohistochemistry. We showed that COUP-TFI is expressed in the stroma and mesenchymal cells at embryonic d 11.5 (E11.5) and expression persists throughout embryonic development. In the adult kidney, only mesangial cells show meaningful COUP-TFI expression. In contrast, COUP-TFII expression is detected as early as E9.5 and high expression is seen in the mesenchymal-derived epithelial cells but not in the ureteric buds through E12.5. At E13.5, COUP-TFII expression becomes regionalized with higher expression in the region that gives rise to the distal tubule. The proximal part of the S-shaped body that will become the glomerulus after endothelial cell migration shows COUP-TFII expression in podocyte precursor cells and epithelial cells of the Bowman’s capsule. In the adult mouse kidney, COUP-TFII is detected in distal tubules, podocytes, and the epithelial cells of the Bowman’s capsule. In addition to COUP-TFs, we also examined the expression profiles of eight other nuclear receptors (farnesoid X receptor, vitamin D receptor, hepatocyte nuclear factor 4
, retinoid X receptor , mineralocorticoid receptor, steroidogenic factor 1, liver receptor homolog-1, and germ cell nuclear factor). Our results suggest that these nuclear receptors are likely to play important physiological roles in the kidney development.
CHICKEN OVALBUMIN UPSTREAM promoter transcription factor I (COUP-TFI) and II (COUP-TFII) show more than 97% amino acid identity in the DNA binding domain and the putative ligand binding domain (1). Molecular biological studies indicate that they have similar DNA binding and transcriptional activity; however, COUP-TFI and COUP-TFII expression patterns are distinct from each other. COUP-TFI is highly expressed in the neural system, whereas COUP-TFII is expressed in the mesenchyme of developing organs (2). Consistent with these distinct expression patterns, gene knockout studies indicate that COUP-TFI and COUP-TFII have different physiological functions. COUP-TFI is known to be important in neurogenesis and neural crest cell differentiation during embryonic development (3, 4, 5, 6), whereas COUP-TFII is shown to be a major regulator of angiogenesis and vein identity (7, 8); in addition, it plays an important role in development of organs such as stomach, limb, heart, and diaphragm (9, 10, 11).
The kidney is a complex organ, and its development has been well studied. The actual kidney, metanephros, begins to form when the ureteric bud invades the metanephric blastema around embryonic d 10.5 (E10.5)–E11, and the formation is completed around 2 wk after birth. The ureteric bud is one of the early epithelial tissues of metanephros extended from Wollfian ducts (12). After invasion, the ureteric buds induce metanephric mesenchymal cells to condense around them, and the condensed mesenchymal cells help ureteric buds to branch. The condensed mesenchyme then undergoes a mesenchyme-to-epithelial transition and forms renal vesicle/pretubular aggregates. After multiple developmental stages, including formation of comma-shaped bodies and S-shaped bodies, as well as migration of endothelial cells, embryonic kidney tubules develop into nephrons. Nephrons are the excretory units of the kidney, which consist of the glomerulus, the proximal tubule, the loop of Henle, and the distal tubule. In the adult kidney, millions of glomeruli are present in the nephrogenic cortex and each glomerulus contains mesangial cells, endothelial cells, podocytes, and epithelial cells of Bowman’s capsule. The proximal tubule and the loop of Henle are developed from the middle part of the S-shaped body and are responsible for reabsorption of most essential molecules and salts from urine. The distal tubule fuses to the collecting duct and participates in the regulation of blood pressure and the levels of potassium, sodium, and calcium and the pH. The branched ureteric buds become collecting ducts. In the meantime, the stroma develops in the region surrounding the mesenchyme and in between ureteric buds (12).
Loss-of-function studies in COUP-TFI and -II demonstrated the importance of COUP-TFI and -II in embryonic development and organogenesis. To investigate the potential roles of COUP-TFI and -II in kidney organogenesis, we used immunohistochemistry to examine the expression patterns of COUP-TFI and -II in the embryonic (E9.5, E11.5, E12.5, and E14.5) and adult mouse kidney. The expression profiles of COUP-TFI and -II suggest that these two orphan receptors might play key roles in kidney development. In addition to COUP-TFI and -II, we also examined the expression patterns of eight other nuclear receptors during mouse embryonic kidney development and in the adulthood.
RESULTS
COUP-TFI and -II Antibodies Are Specific
To validate the specificity of the employed antibodies, we carried out immunohistochemistry on sections from littermates of both wild-type and knockout mouse. As shown in Fig. 1
, intense COUP-TFI antibody positively stained cells are shown in a wild-type embryonic brain section at E13.5 (Fig. 1A), whereas virtually no signal was detected in the mutant brain section (Fig. 1B).
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). In contrast, intense staining was observed in the comparable stomach section of the wild-type littermate (Fig. 1C). Therefore, both COUP-TFI and COUP-TFII antibodies used are very specific in immunostaining and are suitable for profiling the expression of these receptors during mouse development.
The Expression of COUP-TFI in the Developing and Adult Kidney
COUP-TFI expression is detected in the mesenchyme of the developing kidney at E11.5 (Fig. 2A). In the metanephric blastema, COUP-TFI is expressed mostly in the stroma but not in the ureteric bud or the nephrogenic mesenchyme (Fig. 2B). As expected, neural cell adhesion molecule (NCAM)-positive mesenchyme derivatives are negative for COUP-TFI, indicating that the mesenchyme derivatives do not show any expression of COUP-TFI (Fig. 2C). At E14.5, COUP-TFI expression persists in the stroma (Fig. 2, D and E). In the adult kidney, COUP-TFI expression is observed inside the glomerulus and cells in the proximity of the glomerular vascular pole (Fig. 2F). However, COUP-TFI expression from the inside of the glomerulus is not colocalized with podocalyxin, a marker for podocytes; thus, the cells with COUP-TFI expression are most likely the mesangial cells or pericytes. In the vascular pole of the glomerulus, a bunch of mesangial cells, called extraglomerular mesangial cells (EGM), are positively stained with COUP-TFI. Taken together, we conclude that strong COUP-TFI expression is detected in the EGM cells adjacent to the glomerulus, the mesangial cells inside the glomerulus, and pericytes surrounding the glomerular capillary (Fig. 2, G and H).
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). As development proceeds, COUP-TFII is detected in the metanephric blastema at E11.5 (Fig. 3B); particularly, strong expression of COUP-TFII is seen in the condensed mesenchymal cells (Fig. 3C). To examine whether COUP-TFII is expressed in the epithelial cells derived from the mesenchyme-to-epithelial transition, we used NCAM as a marker for mesenchyme-derived epithelial cells. As shown in Fig. 3D, high COUP-TFII expression was observed in the mesenchymal derivatives, colocalized with NCAM-positive cells. To further examine the detailed expression patterns of COUP-TFII during kidney development, we performed immunohistochemistry at various stages during embryonic kidney development. At E12.5, COUP-TFII expression was detected in the developing nephron, the nephrogenic cortex, and stromal cells but not in the ureteric buds (Fig. 3E). The mesenchymal-derived epithelial cells of the developing nephrons such as the condensed mesenchyme, the renal vesicle/pretubular aggregate, and the comma-shaped body also showed very strong expression of COUP-TFII (Fig. 3E). At E13.5, COUP-TFII expression started to decrease in the S-shaped body (Fig. 3F). The distal part of the S-shaped body near the ureteric bud, which will develop into the distal tubule, still showed the expression (narrow arrow in Fig. 3G), but the middle part, which gives rise to the proximal tubule, no longer shows COUP-TFII expression (star in Fig. 3G; see also panel H). The proximal part of the S-shaped body, which will become the glomerulus after endothelial cell migration, showed COUP-TFII expression in podocyte precursor cells (triangle in Fig. 3G) and epithelial cells of the Bowman’s capsule (bold arrow in Fig. 3G). However, other regions of the embryonic kidney still showed strong COUP-TFII expression (Fig. 3, F–H). At E13.5, cells in the middle part of the S-shaped body already completely lacked COUP-TFII expression and, by E14.5, most COUP-TFII expression disappeared in the S-shaped body (Figs. 3, I and J). However, low levels of COUP-TFII expression remained in the distal and proximal parts of the S-shaped body (bold arrowheads in Fig. 3J).
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) or a collecting duct marker, aquaporin 3 (14, 15) (Fig. 4B). The major structural components of the kidney cortex consist of glomeruli, proximal tubules, distal tubules, and collecting ducts. Because the tubules with strong COUP-TFII expression are not proximal tubules or collecting ducts of the kidney cortex, these are presumed to be distal tubules (Fig. 4, A and B). To support this notion, we used E-cadherin, which is expressed in proximal tubules, distal tubules, and collecting ducts in kidney (16), to ask whether COUP-TFII is coexpressed with some of the E-cadherin-expressed cells. In coimmunostaining with E-cadherin (Fig. 4D), we observed that some E-cadherin-positive tubules are also positive for COUP-TFII, suggesting that COUP-TFII is expressed in the distal tubules. This conclusion is consistent with the expression pattern of COUP-TFII in the distal end of S-shape bodies adjacent to the ureteric buds, which later develop into distal tubules (Fig. 3, G–J). In addition to distal tubules, the macula densa cells in a tubule lining next to a glomerulus showed strong COUP-TFII expression (Fig. 4C). In the kidney, macula densa lies next to a renal vascular pole, and a group of epithelial cells in the distal tubule is a component of the juxtaglomerular apparatus involved in the release of rennin. The star in Fig. 4C indicates the vascular pole of the glomerulus filled with COUP-TFI-positive mesangial cells or pericytes. Taken together, these results strongly indicate that COUP-TFII is highly expressed in the distal tubules including macula densa but not in the collecting ducts of the adult mouse kidney. However, in the proximal tubules, we observed an extremely low positive signal of COUP-TFII, but the signal was almost the same as a background. Therefore, we regard the weak signal as a background.
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). In addition to podocytes, there are other cells with relatively low levels of COUP-TFII expression. These cells are either endothelial cells or mesangial cells. Furthermore, we also detected the expression of COUP-TFII in the epithelial cells of the Bowman’s capsule (Fig. 4D). In conclusion, in the adult kidney, COUP-TFII expression is detected in distal tubules, podocytes, and epithelial cells of the Bowman’s capsule.
The Expression Profiles of Other Nuclear Receptors in the Developing and Adult Kidney
In addition to COUP-TFI and -II, we examined the expression patterns of eight other nuclear receptors during mouse embryonic kidney development and in adulthood. Their expression patterns in different cell types are presented in the supplemental figures, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org, and are summarized in Tables 1 (embryonic development) and 2 (adult). Here we will only briefly summarize their expression patterns. The specificities of the antibodies were validated with either knockout mouse (farnesoid X receptor, FXR) or small interfering RNA knockdown cells (data not shown). Retinoid X receptor expression was detected in the ureteric buds and stromal cells of embryonic kidney and proximal tubules and collecting ducts (see supplemental data), at low levels in the glomeruli, and at very low levels in the distal tubules of the adult kidney. Hepatocyte nuclear factor 4 (HNF4) was expressed only in the developing proximal tubules during kidney development, and this expression persisted through the adulthood (see supplemental data). FXR, vitamin D receptor (VDR), mineralocorticoid receptor (MR), liver receptor homolog-1 (LRH-1), and germ cell nuclear factor (GCNF) were expressed only in the adult kidney. FXR was strongly detected in the proximal tubules; VDR was expressed in the distal tubules and collecting ducts; MR was expressed in the collecting ducts and distal tubules at a level slightly above that of a background, and both LRH-1 and GCNF were expressed in both proximal and distal tubules. Finally, steroidogenic factor 1 (SF-1) was not expressed in any cell type during kidney development or adult although it is expressed in the adrenal gland. Therefore, many receptors are expressed during kidney development and in the adult.
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DISCUSSION
The specific expression patterns of COUP-TFI and -II and other nuclear receptors in different cell types during kidney development and in adulthood imply that these various receptors might play a role in the formation of kidney and the maintenance of kidney function in the adulthood. The high expression of COUP-TFII in the condensed mesenchyme and renal vesicle as well as in S- and comma-shaped bodies during kidney development indicates its likely role during kidney development. Also, the apparent nonoverlapping expression pattern between COUP-TFI and COUP-TFII in the kidney suggests that these two closely related orphan receptors may have different functional roles. Finally, the persistent expression of these two receptors in adult suggests that they may play a role in the physiological function of the adult kidney.
Sugawara et al. (17) have reported that RXR is expressed only in the proximal and distal tubules and is functionally important for vitamin D signaling in the regulation of calcium homeostasis by formation of a heterodimer with VDR. As reported, we also observed RXR expression in the proximal and distal tubules, but, interestingly, we also observed RXR expression in glomeruli which, suggests an additional role of RXR in kidney function. VDR was reported by Liu et al. (18) to be expressed in only distal tubules of rat kidney, but Sugawara et al. (17) and Kumar et al. (19) reported its expression in both proximal and distal tubules in rat and human, respectively. However, we observed VDR expression only in the distal tubules and collecting ducts (Table 2). The discrepancy between observations from different groups might arrive from the threshold of the staining, which is considered positive by one laboratory but not by the others, as suggested by Sugawara et al. (17). MR is known to be important in the stimulation of electrolyte and water transfer in the kidney. In accordance with its function, MR was shown to be highly expressed in collecting ducts but was not highly expressed in distal tubules (Table 2). This result is consistent with a recent study by Gomez-Sanchez et al. (20) who showed MR expression in the distal tubules and collecting ducts. In agreement with our observation, FXR (21) and HNF4 (22) expressions were previously shown in proximal tubules.
In conclusion, the expression profile reported here will serve as a platform for future exploration of the critical function of these nuclear receptors in kidney development and will provide new avenues for potential treatment of kidney diseases once their function is defined. It is anticipated that future conditional ablation of a specific receptor in kidney might reveal its precise function in the developing and the adult kidney.
MATERIALS AND METHODS
The mouse embryos and the adult mouse kidney at postnatal 21 d were fixed in 4% paraformaldehyde and embedded in paraffin. Samples were sectioned in a cross or sagittal manner in 3- to 7-µm thickness depending on the experimental purpose. Immunohistochemistry was performed with COUP-TFI, -TFII, and the nuclear receptor antibodies, which were provided by PPMX (Perseus Proteomics Inc., Tokyo, Japan; except LRH-1 and GCNF, which were provided by Austin J. Cooney, Baylor College of Medicine). For double immunostaining, TSA system (nos. 22 and 25; Invitrogen, Carlsbad, CA) was applied and processed according to protocol furnished by the manufacturer. Markers used were purchased from Chemicon (Temecula, CA; NCAM, AB5032; aquaporin 3, AB3276), Zymed (E-cadherin, 13–1900), R&D Systems (Minneapolis, MN; podocalyxin, MAB1556), and Sigma (St. Louis, MO; fluorescein- LTL, FL-1321). 4',6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. siCON and small interfering RNAs of RXR, MR, VDR, HNF4 were purchased from Ambion (Austin, TX).
ACKNOWLEDGMENTS
We thank PPMX (Perseus Proteomics Inc.) for providing nuclear receptor antibodies including COUP-TFI and II and Austin J. Cooney for providing LRH-1 and GCNF antibodies. Also, we thank Dr. David Moore for FXR knockout mouse sections and Wei Qian and Grace Chen for their technical assistance.
FOOTNOTES
This work is supported by The Nuclear Receptor Signaling Atlas Grant U19DK 62434 to S.Y.T and M.-J.T. Also, this study was supported by the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, by the Focus 21 project of New Energy and Industrial Technology development Organization, and by the Special Coordination Fund for Science and Technology from Ministry of Education, Culture, Sports, Science and Technology to T.T. and T.K.
Disclosure statement: The authors have nothing to disclose.
First Published Online September 14, 2006
Abbreviations: COUP-TF, Chicken ovalbumin upstream promoter transcription factor; DAPI, 4',6-diamidino-2-phenylindole; EGM, extraglomerular mesangial cells; FXR, farnesoid X receptor; GCNF, germ cell nuclear factor; HNF4, hepatocyte nuclear factor 4; LRH-1, liver receptor homolog-1; LTL, lotus tetragonolobus lectin; MR, mineralocorticoid receptor; NCAM, neural cell adhesion molecule; SF-1, steroidogenic factor 1; VDR, vitamin D receptor.
Received for publication July 31, 2006. Accepted for publication September 1, 2006.
REFERENCES
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