Deregulated Expression of the Homeobox Gene Cux-1 in Transgenic Mice Results in Downregulation of p27^kip1 Expression during Nephrogenesis, Glomerular Abnormalities, and Multiorgan Hyperplasia

Abstract

Cux-1 is a murine homeobox gene that is highly expressed in the developing kidney with expression restricted to the nephrogenic zone. Cux-1 is highly expressed in cyst epithelium of polycystic kidneys from C57BL/6J-cpk/cpk mice, but not in kidneys isolated from age-matched phenotypically normal littermates. To further elucidate the role of Cux-1 in renal development, we generated transgenic mice expressing Cux-1 under the control of the CMV immediate early gene promoter. Mice constitutively expressing Cux-1 developed multiorgan hyperplasia and organomegaly, but not an overall increase in body size. Transgenic kidneys were enlarged 50% by 6 weeks of age, with the increased growth primarily restricted to the cortex. Proliferating cells were found in proximal and distal tubule epithelium throughout the cortex, and the squamous epithelium that normally lines Bowman's capsule was replaced with proximal tubule epithelium. However, the total number of nephrons was not increased. In the developing kidneys of transgenic mice, Cux-1 was ectopically expressed in more highly differentiated tubules and glomeruli, and this was associated with reduced expression of the cyclin kinase inhibitor, p27. Transient transfection experiments revealed that Cux-1 is an inhibitor of p27 promoter activity. These results suggest that Cux-1 regulates cell proliferation during early nephrogenesis by inhibiting expression of p27.

INTRODUCTION

The metanephros, the definitive kidney of mammals, is derived from a complex series of inductive and morphogenetic events involving the branching ureteric bud and induction of the metanephric mesenchyme (Grobstein, 1955; Saxen, 1987). During metanephric kidney development, the mesenchyme induces the growth and branching of the ureteric bud which differentiates into the collecting ducts, calyces, pelvis, and ureter of the mature kidney. Conversely, each new branch of the ureteric bud induces the cells of the metanephric mesenchyme to condense into a proliferating epithelial renal vesicle, which differentiates into the glomerulus, proximal tubule, loop of Henle, and distal tubule of a mature nephron. Nephrogenesis involves many developmental processes, including cell proliferation, induction, cell fate specification, epithelial and stromal differentiation, and apoptosis (Lechner and Dressler, 1997; Burrow, 2000). The analysis of animal models and the use of organ culture experiments have identified a number of regulatory proteins involved in these processes (Lechner and Dressler, 1997). Many of these proteins are members of signal transduction pathways that are well-established in vertebrate and invertebrate development. These include the Wnts (Stark et al., 1994; Kispert et al., 1996), bone morphogenetic proteins (Dudley et al., 1995; Luo et al., 1995), TGFβ (Stuart and Nigam , 1995), and RET/GDNF (Schuchardt et al., 1994; Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996). Recent evidence suggests that RET/GDNF signaling is responsible for the centripetal growth of the ureteric bud, which ultimately determines the histoarchitecture of the adult kidney (Srinivas et al., 1999). A number of transcription factors are also essential for the normal patterning and differentiation of the kidney, as determined by gene knockout studies (Lechner and Dressler, 1997; Burrow, 2000). These include BF-2 (Hatini et al., 1996), Pax-2 (Torres et al., 1995), WT-1 (Kreidberg et al., 1993), HNF-1α (Pontoglio et al., 1996), and Pod-1 (Quaggin et al., 1999).

Homeobox genes encode transcription factors that regulate developmental gene expression in many organisms (Krumlauf, 1994). Cux-1 is a murine homeobox gene that is related to the Drosophila cut gene (Valarche et al., 1993; Vanden Heuvel et al., 1996a). During mouse embryogenesis, Cux-1 is expressed in the developing brain, limb, lung, and kidney. In the developing metanephros, Cux-1 is highly expressed at 13.5 days postcoitus (d.p.c.), shortly after metanephric induction, and expression remains high until the completion of nephrogenesis ~1 week postpartum (Vanden Heuvel et al., 1996a). Thereafter, expression of Cux-1 decreases such that only low levels of expression are detected in the adult kidney. Studies using in situ hybridization have shown that Cux-1 is highly expressed in the nephrogenic zone in uninduced and condensed mesenchymal cells and in developing epithelial structures (ureteric buds, renal vesicles, S-shaped bodies). During later stages of nephrogenesis, Cux-1 is downregulated such that expression is undetectable in mature tubules or glomeruli (Vanden Heuvel et al., 1996a). In polycystic kidneys, Cux-1 is highly expressed in cyst epithelium, but is minimally expressed in phenotypically normal littermates. This temporally and spatially restricted pattern of expression suggests that Cux-1 plays a role in kidney development.

In addition to Cux-1, mammalian Cut homologues have been identified in human [CCAAT displacement protein (CDP)] (Neufeld et al., 1992), mouse (Cux) (Valarche et al., 1993), dog (Clox) (Andres et al., 1992), and rat (CDP-2) (Yoon and Chikaraishi, 1994). While these homologues all contain a cut homeodomain and three cut repeats, several truncated Cut proteins have been identified, including testis Cux-1 (Vanden Heuvel et al., 1996b) and CASP (Lievens et al., 1997). Mammalian Cut homologues function as transcriptional repressors of many different genes (Superti-Furga et al., 1989; Skalnik et al., 1991; Andres et al., 1992; Valarche et al., 1993; Dufort and Nepveu, 1994; Banan et al., 1997; Higgy et al., 1997; Liu et al., 1997; Coqueret et al., 1998). The binding of Cut proteins to the promoters of these genes appears to be limited to tissues or developmental stages where the target genes are not expressed. Upon terminal differentiation, Cut proteins are downregulated or lose the ability to bind to the promoters, and transcription of the target genes is permitted. Cut proteins function to repress transcription by two different mechanisms: (1) competition for CCAAT or Sp1 binding site occupancy, preventing activation by the corresponding transcription factors, or (2) active repression via a carboxy-terminal repression domain following binding at a distance from the transcription start site (Mailly et al., 1996). Targets of repression by Cut homologues include γ-globin (Superti-Furga et al., 1989), c-Myc (Dufort and Nepveu, 1994), myosin heavy chain (Andres et al., 1992), NCAM (Valarche et al., 1993), CD8a (Banan et al., 1997), c-mos (Higgy et al., 1997), MMTV long terminal repeats (Liu et al., 1997), and gp91-phox (Skalnik et al., 1991). As well, a role for mammalian Cut proteins in repressing the expression of the cyclin kinase inhibitor, p21, during G ₁-S phase has been described (Coqueret et al., 1998). In addition to their repressor function, mammalian Cut proteins have been implicated as transcriptional activators of histone gene expression, independent of E2F during G ₁-S transition (van Wijnen et al., 1996). Because of the precise downregulation of Cux-1 during nephrogenesis and the persistent expression of Cux-1 in the cystic epithelium of polycystic kidneys, we postulated that persistent expression of Cux-1 could lead to developmental abnormalities. To test this hypothesis, Cux-1 expression was deregulated in transgenic mice.

MATERIALS AND METHODS

Transgene Construct

To obtain the entire coding region for Cux-1, we employed two PCR-based strategies. A 3.1-kb cDNA, corresponding to nt 605-3734 of the published murine Cux sequence (Valarche et al., 1993), was amplified by standard PCR using gene-specific primers 5′-ATAGAGGTGCTGACCCGATCCA-3′ and 5′-TTCGAGCTGAAGGTGAGTCGCT-3′. Conditions for PCR were as described previously (Igarashi et al., 1995). The template was 10 ng of first-strand cDNA that was synthesized from neonatal mouse kidney poly(A)⁺ RNA by using reverse transcriptase. Thirty five cycles of PCR were performed, each consisting of incubation at 94°C for 30 s, 55°C for 30 s, and 72°C for 4 min. Reaction products were resolved on low-melting agarose gels, and products of the desired size were cloned into pCRII (Invitrogen). Because the published murine Cux sequence diverged from all other mammalian Cut homologues at the amino-terminal end (Valarche et al., 1993), we used ligation-anchored PCR to obtain the murine 5′ sequence. Conditions for ligation-anchored PCR were as described previously. The template was 10 ng of first-strand cDNA that was synthesized from neonatal mouse kidney poly(A)⁺ RNA by using a gene-specific primer (5′-TTCGAGCTGAAGGTGAGTCGCT-3′). An anchor oligonucleotide (Clontech) was added by using T4 RNA ligase according to manufacturer's directions. A 1.1-kb cDNA corresponding to the 5′ end of Cux-1 was amplified by 5′ RACE using an anchor-specific primer (Clontech) and a gene-specific primer (5′-GGCCGATGAGAGCTGTTCCCTTA-3′). Five microliters of a 1:50 dilution of the PCR products was then reamplified by using nested PCR with a second anchor-specific primer (Clontech) and a second gene-specific primer (5′-GTGAGATCTGGCTGGCGGAAT-3′). The 3.1-kb cDNA and 1.1-kb cDNA were sequenced and ligated with an existing cDNA (10F2) containing the 3′ end of Cux-1 in pBluescript (Stratagene) (Vanden Heuvel et al., 1996). The full-length composite sequence was 6564 bp and contained 27 bp of 5′ untranslated region, 4544 of coding region, and 1993 of 3′ untranslated region. The inferred protein contained 110 amino acids at the amino terminus, not present in the published murine Cux sequence (Valarche et al., 1993), but found in human CDP (Neufeld et al., 1992). The full-length cDNA was subcloned into pcDNA 3.1 (Invitrogen) and sequenced across the ligation sites to verify the integrity of the coding region. The Cux-1 cDNA, corresponding to the entire coding region, was subcloned into pCEP4 (Invitrogen) to create the CMV/Cux-1 construct for the transgenic experiments. Subsequent digestion with SalI, which does not cut within the Cux-1 cDNA, released a linear fragment containing only the CMV promoter, Cux-1 coding region, and SV40 small t intron and polyadenylation signal without additional vector sequences.

Production of Transgenic Mice

A total of 4.3 μg of the SalI fragment was separated on an agarose gel and purified by using the Elutrap electroelution system (Schleicher & Schuell) according to manufacturer's directions. The DNA was resuspended in 40 μl of injection buffer (10 mM Tris, pH 7.5, 5 mM NaCl, 0.2 mM EDTA) and used for pronuclear injection into mouse eggs as described by Hogan et al. (1994). Next, 200–500 copies of the DNA fragment were microinjected into pronuclei of fertilized mouse oocytes (C57Bl/6 × C3H). The oocytes were then implanted into pseudopregnant female mice. This resulted in 68 mice. Genomic DNA was isolated from tail biopsies of the 68 mice and digested with EcoRI. Southern blot analysis using a CMV-specific riboprobe revealed that 5 of the 68 mice carried the transgene. The positive founder transgenics were bred with nontransgenic littermates to generate permanent transgenic lines. We have successfully established three transgenic lines that we have named 2235, 2200, and 2189. We were unable to generate transgenic offspring from the other two positive founders, suggesting that the transgene had not been transmitted to the germline in these animals. To obtain embryos at defined developmental stages, mice were randomly mated, and the morning of detection of copulatory plugs was designated 0.5 d.p.c. At selected times, pregnant mice were euthanized by cervical dislocation, and the embryos were removed. Genotype was determined by Southern blot analysis of placental DNA. Gestational age was verified according to the criteria of Theiler (1972).

Southern and Northern Blot Analyses

For Southern blot analysis, genomic DNA was isolated from tail biopsies and digested with EcoRI, to cut once within the transgene. Following electrophoresis, DNA was transferred to nylon membranes (Hybond-N), then hybridized with a CMV ³²P-riboprobe generated from a 532-bp HincII CMV fragment excised from pCEP4 and subcloned into pBluescript. Hybridization was performed at 55°C in 5× Denhardt's, 50% formamide, 200 μg/ml salmon sperm DNA, 200 μg/ml yeast tRNA, 200 μg/ml SDS, 2 × 10⁶ cpm/ml radiolabeled probe. Southern blots were washed twice in 0.1× SSC, 0.1% SDS at 68°C then exposed to Hyperfilm (Amersham) with a single intensifying screen.

Poly(A)⁺ RNA was isolated from metanephroi, electrophoresed, and transferred to nylon membranes as described previously (Igarashi et al., 1995). Northern blots were hybridized with a full-length p27 antisense ³²P-labeled riboprobe. Hybridization was performed at 60°C in 5× Denhardt's, 50% formamide, 200 μg/ml salmon sperm DNA, 200 μ/ml yeast tRNA, 200 μg/ml SDS, 2 × 10⁶ cpm/ml radiolabeled probe. Northern blots were washed twice in 0.1× SSC, 0.1% SDS at 68°C then exposed to Hyperfilm (Amersham) with a single intensifying screen. Northern blots were stripped and rehybridized with a 450-bp cDNA fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (Igarashi et al., 1995).

Anatomical and Histological Analysis

Organs were isolated and weighed from 6-week-, 3-month-, and 6-month-old wild-type and transgenic mice (3 males and 3 females for each genotype and time point). For histological analysis, tissues were fixed in freshly prepared 4% paraformaldehyde in PBS, dehydrated with graded ethanols, cleared in xylene, and embedded in paraffin. Slides prepared with 5-μM sections were stained with hematoxylin and eosin. Images from midsagittal sections of adult kidneys were captured from a Wild 400 macroscope. The cross-sectional area of 6-week-old kidneys was estimated from midsagittal sections by measuring their length (rostrocaudal aspect) and width (mediolateral aspect). NIH Image (Scion Corp.) was used to estimate cortex, outer medulla, and inner medulla areas from midsagittal sections. The boundaries of cortex, outer medulla, or inner medulla were outlined and the relative area was determined. Glomeruli in wild-type or transgenic adult kidneys were counted in midsagittal sections from 18 animals. The numbers of glomeruli in wild-type or transgenic fields were compared by using Student's t test for paired samples. Cell counts were obtained from toluidine blue-stained, 1-μM sections of kidney cortex from 4 transgenic (2 male and 2 female) and 4 sex-matched wild-type mice. Photomicrographs were taken at 400×, and the number of renal epithelial nuclei in 5 high-power fields were counted.

Western Blot Analysis

Nuclear extracts (30 μg), prepared as previously described (Vanden Heuvel et al., 1996b), were solubilized in SDS–PAGE sample buffer and electrophoresed on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose filters as described previously (Biemesderfer et al., 1993). The immunoblot was blocked in TTBS (100 mM Tris, pH 7.5, 0.9% NaCl, 0.1% Tween) for 1 h at room temperature (RT). Goat anti-CDP (Cux-1) (Santa Cruz #sc-6327) antibody was added at a dilution of 1:250. After overnight incubation at 4°C, filters were washed three times at RT with TBS, then incubated at RT for 1 h with biotin-conjugated donkey anti-goat IgG (1:1000 dilution; Santa Cruz), washed three times with TBS, and incubated for 1 h at RT with avidin-biotin-peroxidase complex (ABC staining system; Santa Cruz). Following three additional washes in TBS, bound antibody was detected by incubation with 3,3′-diaminobenzidine (DAB). In some experiments, whole 3-day-old kidney lysates (50 μg) were solubilized in SDS–PAGE sample buffer, electrophoresed on 15% polyacrylamide gels, and transferred to nitrocellulose filters. Rabbit anti-p27 (Santa Cruz #sc-528) was added at a dilution of 1:100, and bound antibody was detected as described above.

Immunostaining

Isolated metanephroi or whole embryos were immersion fixed in 4% paraformaldehyde and blocked in paraffin. Then, 5-μM-thick tissue sections were deparaffinized with xylene and rehydrated with graded ethanols. Endogenous peroxidase was blocked with 1% hydrogen peroxide for 10 min and the samples were then rinsed in PBS. To obtain adequate signal, the slides were treated with antigen-unmasking solution (Vector) according to manufacturer's protocol, or slides were treated for 10 min with trypsin (1 mg/ml) at 37°C. To reduce background, the sections were blocked for 1 h at RT in 10% normal serum from the species the secondary antibody was made in. Commercial reagents used were: goat anti-CDP (Cux-1) (Santa Cruz #sc-6327); goat anti-p27 (Santa Cruz #sc-528-G); rabbit anti-p27 (Santa Cruz #sc-528); goat anti-Pax-2 (Santa Cruz #sc-7747); rabbit anti-WT-1 (Santa Cruz #sc-192); goat anti-PECAM (Santa Cruz #sc-1506); rabbit anti-desm in (Sigma # D-8281); monoclonal anti-PCNA (Sigma #p-8825); biotinconjugated Lotus Tetragonolobus (LTA, Vector # B-1325); biotin-conjugated Dolichos Biflorus (DB, Vector # B-1035). Antibody dilutions were 1:100 for CDP Ab, 1:100 for WT-1 Ab, 1:100 for Pax-2 Ab, 1:100 for p27 AB, 1:3000 for PCNA Ab, 1:20 for PECAM Ab, in 2% blocking serum in PBS. Biotin-conjugated Lotus Tetragonolobus was used at 50 μg/ml, and biotin-conjugated Dolichos Biflorus was used at 5 μg/ml. Slides were incubated at room temperature with 100 μl of antibody in a humid chamber and then washed four times in PBS. Biotin-conjugated secondary antibodies (Vector) were diluted 1:400 in PBS containing 2% serum from the species in which the secondary antibody was generated. After incubation for 1 h at room temperature, slides were washed four times in PBS, then incubated with avidin-biotin-peroxidase complex (ABC-Elite; Vector) and then DAB. In some experiments, nickel chloride was added to the DAB to give a black reaction product. The tissue sections were then dehydrated with graded ethanols, mounted with Permount (Fisher), and covered with glass coverslips. Lectin binding was detected by ABC and DAB as described for antibody detection.

TUNEL Assay

Terminal deoxynucleotidal transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) was used to detect cells containing fragmented DNA indicative of apoptosis in 10-μM sections from transgenic and wild-type embryos as previously described (Oberhaus et al., 1997).

Transient Reporter Assay

Human embryonic kidney (293T) cells were obtained from the American Type Culture Collection. Exponentially growing 293T cells were cultured in DMEM with 4.5 g/L glucose and 10% heat-inactivated FBS. Transient transfections were performed by the calcium phosphate precipitation method, using 1 μg luciferase reporter plasmid containing either p27 promoter sequence (–609 to +178) or Nkcc2 promoter sequence (–469 to +1) along with 0.3 μg β-galactosidase-expressing plasmid (to correct for transfection efficiencies), pCMV/Cux-1 at the concentrations indicated, and 2.5 μg pCEP4 (to control for nonspecific vector effects). After 48 h, the cells were lysed, and luciferase and β-galactosidase activities were determined by enzyme assay kits. Luciferase activity was normalized to β-galactosidase activity as an internal transfection control.

Materials

Restriction endonucleases were obtained from New England Biolabs, Promega, or Boehringer Mannheim. T3 and T7 RNA polymerases were from Stratagene. Radionucleotides and nylon filters were from Amersham. Other reagents were of molecular biological grade from Sigma, Boehringer-Mannheim, Promega, or U.S. Biochemicals.

RESULTS

Generation of Transgenic Mice Constitutively Expressing Cux-1

To ask whether expression of Cux-1 throughout the kidney, instead of only in the nephrogenic zone, arrests renal development or causes cyst form ation, we expressed a wild-type Cux-1 cDNA under the control of the CMV promoter (Fig. 1A). The CMV promoter was used previously to ectopically express Pax-2 in the developing kidneys of transgenic mice (Dressler et al., 1993). Three transgenic mouse lines were generated containing the CMV/Cux-1 construct. Figure 1B shows an autoradiogram of EcoRI-digested DNA in which adult mice from lines 2189, 2200, and 2235 are transgenic as indicated by hybridization to a ³²P-radiolabeled CMV-specific riboprobe. The presence of a hybridizing band at 8 kb, which would be expected from concatamers of the transgene, indicated that transgenic mice from lines 2235 and 2189 carried multiple copies of the transgene. Importantly, Southern analysis also demonstrated that the transgenes were integrated into different genomic sites for each line, indicating that the phenotype was not due to position effects. Absolute quantitation of transgene copy number for lines 2189 and 2235 was not performed. All three transgenic mouse lines expressed the transgenic Cux-1 mRNA in the adult kidney (data not shown). The two lines with the highest expression in the kidney (2189 and 2200) were used for further studies and proved to have identical phenotypes. However, the third line (2235) was not different from wild-type mice and was not used for further analysis.

FIG. 1.

CMV/Cux-1 transgene construct and Southern blot analysis of transgenic lines 2189, 2200, and 2235. (A) Schematic diagram of transgene construct. The 6.5-kb Cux-1 cDNA was subcloned into pCEP4, and the CMV/Cux-1 construct was excised by digestion with SalI. The bar below the CMV promoter corresponds to the probe used to identify transgenic mice by Southern blot analysis. (B) Genomic DNA was isolated from CMV/Cux-1 transgenic mice and digested with EcoRI. DNA from transgenic lines 2189 (lane 1), 2200 (lane 2), and 2235 (lane 3) are shown. Molecular weight standards (in kb) are shown on the left. Hybridization to the 8-kb fragment in lanes 1 and 3 indicates concatamers of the transgene. Line 2200 (lane 2) carries a single copy of the transgene.

To determine whether the CMV/Cux-1 transgene produced a protein, immunoblot analysis was performed. For these experiments, we used a polyclonal antibody generated against a 19-amino-acid peptide from the amino-terminal end of murine Cux-1. Figure 2 shows results of immunoblot analysis in which Cux-1 protein can be detected in nuclear extracts isolated from adult transgenic and normal tissues. In the normal tissues, we detected the predicted 180- to 190-kDa Cux-1 protein at low levels in the kidney and liver, but not in spleen or testis. In transgenic tissues, however, we detected abundant Cux-1 protein in the kidney, liver, spleen, and testis. Since the antibody used was directed against the amino terminus of Cux-1, we did not detect the truncated testis-specific form of Cux-1 (Vanden Heuvel et al., 1996b).

FIG. 2.

Expression of Cux-1 protein in kidneys, livers, spleens, and testes from adult CMV/Cux-1 transgenic mice. Expression of Cux-1 protein in kidney (lanes 1 and 5), liver (lanes 2 and 6), spleen (lanes 3 and 7), and testis (lanes 4 and 8) isolated from adult CMV/Cux-1 transgenic (lanes 1–4) and wild-type (lanes 5–8) mice. Thirty micrograms of nuclear extract prepared from transgenic and wild-type adult mouse tissues was subjected to SDS–PAGE and transferred to nitrocellulose membranes. The presence of Cux-1 protein was detected by using a polyclonal antibody to CDP. The Cux-1 protein is 180 kDa (arrow) and was detected at high levels in all transgenic tissues, but at low levels or not at all in nontransgenic tissues. Positions of molecular weight standards (X kDa) are shown on left.

CMV/Cux-1 Mice Exhibit Organomegaly

At birth, wild-type and transgenic mice were indistinguishable. Transgenic mice survived to adulthood and appeared identical to wild-type mice. Adult CMV/Cux-1 mice exhibited enlargement of multiple visceral organs, but the average body weight of the transgenic mice was not increased (Fig. 3). By 6 weeks of age, the kidney, liver, and spleen were 50–100% larger than in wild-type mice. At 3 and 6 months of age, the kidney, liver, and heart were still proportionately larger in the transgenic mice, but the transgenic spleen was comparable in size to that in the wild-type mice. At all three ages, the testes were slightly larger in the transgenic mice, but as the mice got older, this difference in size was less significant. We also observed an increased size of the seminal vesicles in the CMV/Cux-1 mice, as compared with those in the wild-type mice.

FIG. 3.

CMV/Cux-1 mice exhibit organomegaly. (A) Increased organ weights in transgenic mice relative to wild-type littermates. Three male and three female CMV/Cux-1 mice (transgenic line 2200) and wild-type littermates were weighed at 6 weeks (open bars), 3 months (black bars), and 6 months (lined bars). Heart, kidney, liver, spleen, seminal vesicle, and testes from these mice were isolated and weighed. The data are plotted as the average percent increase of body and organ weights of the CMV/Cux-1 mice compared with the wild-type mice. (B) Gross appearance of heart, kidney, testis, spleen, seminal vesicle, and liver from CMV/Cux-1 transgenic and wild-type mice.

CMV/Cux-1 Transgene Causes Renal Hyperplasia and Glomerular Abnormalities

Kidneys isolated from adult transgenic mice ranging in age from 6 weeks to 6 months were compared with kidneys from age-matched nontransgenic littermates. Transgenic kidneys were larger than normal kidneys both by weight and by cross-sectional area (Figs. 3, 4A, and 4B). While the overall organization of the cortex and medulla in the transgenic kidneys appeared normal, the increased growth was primarily restricted to the cortex (Fig. 5). Histological analysis of transgenic renal cortices revealed hyperplasia in tubules and glomeruli (Figs. 4C–4F). To determine the increase in cell number in transgenic kidneys, the number of nuclei in 1-μm kidney sections from transgenic and wild-type kidneys were counted. This showed a significant increase in the number of cells in the transgenic kidneys as compared with the wild-type kidneys (Table 1). To determine whether the total number of nephrons was changed, the total number of glomeruli was counted in midsagittal sections from wild-type and transgenic 6-week-old kidneys. These results indicated that there was no significant change in nephron number between wild-type and transgenic kidneys (Fig. 5).

FIG. 4.

Renal hyperplasia and glomerular abnormalities in CMV/Cux-1 mice. Light micrographs of wild-type (A, C, E, G) and transgenic (B, D, F, H) adult kidneys. (A, B) Sagittal sections of adult kidneys demonstrating an increase in cross-sectional area of transgenic kidneys (B) as compared with wild-type kidneys (A). Hematoxylin and eosin stain. (C–F) Hematoxylin and eosin-stained sections showing replacement of normal squamous epithelium of Bowman's capsule with tubule-like epithelial cells (arrow) in transgenic mice. These cells were continuous with the proximal tubule epithelium (arrowheads), and were observed in all glomeruli examined from transgenic lines 2189 and 2200. Transgenic kidneys also exhibited glomerular (g) and tubular (t) hypercellularity. (G, H) Tetragonolobus lotus lectin specifically stains proximal tubules (arrowhead) in wild-type (G) and transgenic (H) kidneys. Cells lining Bowman's capsule in transgenic (H) kidneys also stain with Tetragonolobus lotus lectin (arrows). Original magnification: ×5 (A, B), ×100 (C, D), ×200 (E–H).

FIG. 5.

Increased cortex in CMV/Cux-1 transgenic kidneys. (A) The cross-sectional area of the cortex, outer medulla, and inner medulla were measured from midsagittal sections of wild-type and transgenic six-week-old kidneys using NIH Image (Scion Corp.). The area in mm² for wild-type (open bars) and transgenic (filled bars) are shown. (B) The number of glomeruli per midsagittal section was counted on 10 wild-type and 10 transgenic six-week-old kidneys. Standard deviation is indicated.

TABLE 1.

Kidney Cell Counts

	Wild-type	Transgenic	Relative increase (%)
Kidney cortex	278 (5.5)	454 (3.1)	63
Glomerulus	22 (0.55)	27 (1.4)	23

Note. Cell counts were obtained from toluidine blue-stained, 1-μ sections of kidney cortex from four adult transgenic (2 male and 2 female) and four sex-matched wild-type mice. Photomicrographs were taken at 400×, and the number of total or glomerular nuclei in 5 high-power fields from each was counted. Data are expressed as group means of the number of cells per field or per glomerulus. Standard error is indicated in parentheses.

Within all glomeruli examined, tubule-like epithelial cells were observed completely lining Bowman's capsule, instead of the normal squamous cells making up the parietal epithelium (Figs. 4C–4F). Moreover, these cells had a brush border and appeared to be continuous with the proximal tubule epithelium (Figs. 4D and 4F). Furthermore, staining with Tetragonolobus lotus, a lectin that binds to the brush border of proximal tubules (D’Agati and Trudel, 1992), demonstrated that these cells had characteristics common to proximal tubule epithelium (Figs. 4G and 4H).

To determine the basis of the increase in cell number in the transgenic kidneys, we stained wild-type and transgenic kidney sections for the presence of proliferating cell nuclear antigen (PCNA) (Fig. 6). In contrast to wild-type kidney, transgenic adult kidney sections exhibited numerous PCNA-positive cells, including cells lining Bowman's capsule (Fig. 7D).

FIG. 6.

Increased proliferation in transgenic kidneys. (A, B) The proportion of proliferating cells is increased in kidney cortex from CMV/Cux-1 mice (B). Kidney sections from 6-week-old mice were labeled with anti-PCNA antibody. Sections from wild-type (A, C) or CMV/Cux-1 transgenic (B, D) are shown. (D) Inset shows PCNA-positive cells in epithelium lining Bowman's capsule. Original magnification: (A, B) 100× (Bar, 100 μm). (C, D) 400× (Bar, 50 μm).

FIG. 7.

Expression of Cux-1 in capillary loop-staged glomeruli. Serial sections of wild-type (A, B) or transgenic (C, D) E15.5 kidneys were stained for Cux-1 (A, C) or WT-1 (B, D) protein. Capillary loop-staged glomeruli are defined morphologically by capping of podocytes (stained for WT-1) around underlying mesangial and endothelial precursors. Cux-1 is expressed in podocytes, but not in underlying cells in normal kidney (A). In transgenic kidneys, Cux-1 is expressed both in podocytes and in underlying cells (C). Original magnification: 400× (Bar, 25 μm).

Cux-1 Is Ectopically Expressed in Developing Kidneys of Transgenic Mice

To examine the developmental basis of the renal abnormalities in CMV/Cux-1 mice, embryonic and newborn kidneys were dissected and subjected to histological analysis. During nephrogenesis, maturing nephrons proceed through an orderly sequence of developmental stages that can be distinguished morphologically. These stages are described as renal vesicle (stage I), comma and S-shaped bodies (stage II), developing capillary loop (stage III), and maturing glomerulus (stage IV). In the developing kidney, a centrifugal gradient of nephron development exists in which nephrons at the earliest stages of development (stages I and II) are restricted to the nephrogenic zone immediately below the renal capsule, while progressively more mature nephrons (stages III and IV) are located toward the center of the kidney (Reeves et al., 1978). Thus, a single cross-section of a late gestation embryonic or newborn kidney exhibits a gradient of nephrons in all stages of development.

Previously, we showed that Cux-1 mRNA is highly expressed in the subcapsular nephrogenic zone in stages I and II, but was markedly downregulated after the S-shaped body stage, with no expression detected in the maturing glomerulus stage (Vanden Heuvel et al., 1996). To evaluate expression of Cux-1 protein during nephrogenesis in wild-type and transgenic mice, we performed immunohistochemical studies on embryonic and newborn kidney sections. In embryonic kidneys from transgenic mice, Cux-1 protein was detected at high levels in uninduced and condensing mesenchyme, ureteric buds, and S-shaped bodies in the nephrogenic zone, similar to wild-type kidneys (data not shown). However, at the capillary loop stage, expression of Cux-1 in transgenic mice was seen both in podocytes that form a C-shaped cap, and in the underlying glomerular endothelial and mesangial cells, where Cux-1 is normally not expressed (Fig. 7). And, while Cux-1 protein was not detected in maturing glomeruli or surrounding tubule epithelia of wild-type mice, Cux-1 continued to be highly expressed in maturing glomeruli and tubules of transgenic mice (Figs. 8E and 8F). The ectopic glomerular expression of Cux-1 results in an increase in the number of mesangial cells, but not endothelial cells, and leads to the development of glomerulosclerosis (J.G.B. et al., unpublished observations).

FIG. 8.

Light micrographs of wild-type (A, C, E, G) and transgenic (B, D, F, H) kidneys at P3 (A–F) and at E15.5 (G, H). (A, B) No obvious abnormalities are present in the nephrogenic zone (NZ) of developing transgenic kidneys. Flattened squamous epithelium lining Bowman's capsule (arrowheads) is observed in maturing glomeruli in both wild-type (C) and transgenic (D) kidneys. (E) Cux-1 protein in the developing kidney is highest in the nephrogenic zone (NZ) but is downregulated in maturing glomeruli (arrow), reflecting the previously described mRNA expression pattern (Vanden Heuvel et al., 1996a). (F) In transgenic mice, Cux-1 protein is highly expressed in the NZ, but continues to be expressed at high levels in maturing glomeruli (arrow) and surrounding tubular epithelial cells. (G, H) Sections of E15 kidney were labeled with biotin-12-dUTP using the TUNEL method. Similar numbers of TUNEL-positive cells (arrows) were detected in both wild-type (E) and transgenic (F) kidneys. Original magnification: (A, B, E, F) 100×. (C, D, G, H) 1000×.

Apoptosis occurs during normal kidney development primarily in the nephrogenic zone. Previous studies by Quaggin et al. (1997) indicated that inhibition of Cux-1 expression in metanephroi in organ culture resulted in an abnormal increase in apoptosis during development. One possible explanation for the increased cellularity in the CMV/Cux-1 mice was protection against apoptosis resulting from dysregulated expression of Cux-1. When embryonic day 15 transgenic and normal mouse embryos were subjected to the TUNEL assay, we observed no difference in the number of TUNEL-positive cells (Figs. 8G and 8H).

The similarm orphology of the condensing mesenchyme, ureteric buds, and S-shaped bodies of transgenic and wild-type metanephroi, together with no change in the overall number of nephrons, suggested that early nephrogenic events were unaffected in the transgenic mice. However, the appearance of proximal tubule epithelium lining Bowman's capsule in adult transgenic kidneys suggested that patterning of the nephron was disrupted. Thus, to evaluate patterning changes in the developing nephrons of transgenic mice, we stained 15 d.p.c. embryo sections with markers for condensing mesenchyme and early nephric structures (Pax-2), and for differentiating glomeruli (WT-1). Both Pax-2 and WT-1 showed identical staining in wild-type and transgenic embryonic kidneys (data not shown). Moreover, morphological analysis of newborn transgenic kidneys, when compared with nontransgenic kidneys, showed no observable changes in nephrogenesis (Figs. 8A and 8B). In particular, the maturing glomeruli of transgenic newborn kidneys exhibited flattened squamous epithelium lining Bowman's capsule, identical to wild type (Figs. 8C and 8D).

The Cyclin Kinase Inhibitor p27^kip1 Is Dow nregulated in Developing Kidneys of CMV/Cux-1 Transgenic Mice

Previous studies by Coqueret et al. (1998) identified a role for Cux-1 in regulating the expression of the cyclin kinase inhibitor (CKI) p21 in G ₁-S transition in cultured cells. These authors demonstrated that Cux-1 repressed the expression of p21 by periodic occupation of the p21 promoter on a region containing the TATA box and an Sp1 binding site. Therefore, we hypothesized that ectopic expression of Cux-1 might alter cell cycle regulation. However, no gross renal abnormalities, including an overall increase in renal size, have been observed in mice lacking p21 (Williams et al., 1999). Another CKI, related to p21, is p27. In contrast to p21, targeted deletion of p27 results in multiorgan hyperplasia, including enlarged kidneys (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). Since the expression of p27 in the developing kidney is inversely proportional to the expression of Cux-1 (Combs et al., 1998), we hypothesized that p27 may be a target of Cux-1 regulation during renal development. To address this possibility, we examined the expression of p27 in newborn wild-type and transgenic kidneys by immunohistochemistry. During normal kidney development, p27 expression is upregulated in the newly differentiated cells of capillary loop staged and maturing glomeruli and tubules, where cell proliferation has ceased (Combs et al., 1998). When we examined p27 protein expression in newborn wild-type kidneys, we observed high levels of p27 expression in the nuclei of maturing glomeruli and surrounding tubules (Fig. 9A). In contrast, in CMV/Cux-1 mice, p27 expression was markedly downregulated in cells of developing glomerular structures, where ectopic Cux-1 protein was the most abundantly expressed (Figs. 9B). To approximate the reduction of p27 in CMV/Cux-1 kidneys, we performed Western blot analysis on whole kidney lysates from newborn wild-type and CMV/Cux-1 kidneys. Figure 9D shows an overall decrease in p27 protein in transgenic kidneys. This was accompanied by a decrease in p27 mRNA in CMV/Cux-1 kidneys as determined by Northern blot analysis (Fig. 9C). To determine whether Cux-1 represses p27 promoter activity, and thus, whether Cux-1 could be regulating p27 expression in the CMV/Cux-1 mice, we performed transient reporter assays. Unsynchronized 293T cells were cotransfected with a p27/luciferase reporter construct and either the empty CMV vector or the CMV/Cux-1 expression construct. In three separate experiments, the luciferase activity was found to be progressively decreased in the presence of increasing amounts of Cux-1 (Fig. 10). Luciferase activity of a control plasmid was unaltered by addition of Cux-1, indicating that the effect of Cux-1 on the p27 promoter is specific.

FIG. 9.

Reduced expression of p27 in kidneys ectopically expressing Cux-1. Light micrographs of wild-type (A) and transgenic (B) kidneys at P3. p27 is highly expressed in developing glomeruli from wild-type kidneys (A), but expression of p27 is reduced in developing glomeruli from transgenic kidneys (B). (C) Northern blot analysis of mRNA isolated from wild-type and transgenic P3 kidneys showing reduced p27 mRNA in transgenic kidneys. Seven micrograms of poly(A)⁺ RNA was used for Northern blotting and hybridized with a murine p27 riboprobe. Equal RNA loading was verified by reprobing with GAPDH. (D) Western blot analysis showing reduced amounts of p27 protein in transgenic kidneys. Fifty micrograms of total kidney lysate isolated from wild-type and transgenic P3 kidneys was subjected to SDS–PAGE and transferred to nitrocellulose membranes. The presence of p27 protein (arrow) was detected using a rabbit polyclonal antibody to p27. Positions of molecular weight standards (X kDa) are shown on right. Original magnification: (A, B) 1000×.

FIG. 10.

p27 promoter-reporter response to increasing amounts of Cux-1. 293T cells were transiently transfected with 1 μg of reporter construct containing p27 upstream sequences from –1609 to +178 (Kwon et al., 1996) fused to the luciferase reporter gene (+), or with 1 μg reporter construct containing Nkcc2 upstream sequences from –469 to +1 (Igarashi et al., 1996) fused to the luciferase reporter gene (+), or with 1 μg of the empty luciferase reporter plasmid (–), together with different concentrations of the Cux-1 expression vector (amounts shown are in μg) or with 2.5 μg of the empty expression vector (–). Promoter activity in 293T cells was plotted as relative light units (RLU) normalized to the expression of a cotransfected β-galactosidase expression construct. Activity is expressed as the mean of three separate experiments performed in triplicate. Error bars indicate standard deviation. Expression of Cux-1 protein in transfected 293T cells was confirmed by immunofluorescent labeling (not shown).

DISCUSSION

The development of the nephron from the ureteric bud epithelium and metanephric mesenchyme requires the phenotypic conversion of the metanephric mesenchyme to epithelium and the orderly progression through a series of developmental stages that pattern the epithelium along a proximal–distal axis. Stage-dependent expression is a characteristic of transcription factors known to be involved in nephrogenesis, such as c-Myc and Pax-2 (Mugrauer and Ekblom, 1991; Dressler et al., 1990). Moreover, appropriate downregulation of transcription factors transiently expressed during nephrogenesis appears to be required for normal kidney development. Prevention of c-Myc or Pax-2 downregulation by constitutive expression in transgenic mice results in severe renal abnormalities (Trudel et al., 1991; Dressler et al., 1993). Previous studies demonstrated that Cux-1 was highly and transiently expressed during metanephric kidney development, consistent with a role in regulation of gene expression during nephrogenesis. Furthermore, Cux-1 was highly expressed in cyst epithelial cells in polycystic kidneys, but was minimally expressed in normal kidneys from age-matched littermates. These results support a model in which Cux-1 downregulation is necessary for normal kidney development. To test this hypothesis and further elucidate the role of Cux-1 in renal development, we generated transgenic mice expressing Cux-1 under the control of the CMV immediate early gene promoter.

Mice constitutively expressing Cux-1 developed renal hyperplasia and organomegaly, indicating a broad role of Cux-1 in growth control. Consistent with this notion is the observation that the Cux-1 gene is normally expressed in many cell types and tissues (Vanden Heuvel et al., 1996b). Despite widespread organomegaly, constitutive expression of Cux-1 was not associated with gross congenital defects, and organs displaying hyperplasia appeared structurally normal. These observations indicate that downregulation of Cux-1 is not essential for renal organomorphogenesis.

In kidneys from adult transgenic mice, tubule-like epithelial cells were observed completely lining Bowman's capsule. Furthermore, these cells had a brush border and bound the lectin Tetragonolobus lotus, characteristic of proximal tubule epithelium. This suggested that patterning of the nephron was disrupted. However, when we examined the expression of two transcription factors involved in nephrogenesis, no changes were observed. Furthermore, we observed flattened squamous epithelium lining Bowman's capsule in maturing glomeruli in newborn transgenic kidneys. Together with the observation that the total number of nephrons was unchanged, these results suggest that nephron induction was unaffected in the transgenic kidneys. One possible explanation for the change in Bowman's capsule is that the proximal tubule cells of already formed nephrons continued to proliferate, growing beyond the glomerular border and displacing the parietal epithelium. This is supported by the presence of proliferating cells found both in proximal tubules and lining Bowman's capsule of mature nephrons. Of interest, there is an increase in the number of proliferating tubule cells following acute renal failure, sometimes leading to proximal tubule epithelium lining Bowman's capsule (Witzgall et al., 1994; Megyesi et al., 1998).

Increased organ size can be caused by an increase in cell number (hyperplasia), an increase in individual cell size (hypertrophy), or a decrease in apoptosis. Previous studies indicated a specific increase in apoptosis in kidney organotypic cultures treated with antisense oligonucleotides directed against Cux-1 (Quaggin et al., 1997). These studies suggest that an increase in Cux-1 might protect against apoptosis. However, in developing CMV/Cux-1 transgenic kidneys, there was no obvious decrease in apoptosis. Instead, the increased renal size correlated with a greater number of proliferating cells as compared with wild-type kidney.

To proliferate, a cell must enter the cell cycle where DNA synthesis and mitosis occur (reviewed in Pardee, 1989). Progression through the cell cycle is controlled by a group of nuclear proteins known as cell cycle regulatory proteins. These proteins can positively (cyclins and cyclin-dependent kinases) or negatively (cyclin kinase inhibitors) regulate cell proliferation. The cyclins form an active complex with a catalytic subunit, the cyclin-dependent kinase (CDK) (Morgan, 1995). Both are located in the nucleus and specific cyclin–CDK complexes are expressed in each phase of the cell cycle. These complexes phosphorylate the protein retinoblastoma (RB), causing the release of the transcription factor E2F, which targets genes that are involved in DNA synthesis. Thus, cyclin–CDKs act through RB to induce DNA synthesis via E2F, driving the progression of the cell cycle (Nevins, 1992). Cyclin kinase inhibitors (CKI) are nuclear proteins that bind to the cyclin–CDK complex, preventing the phosphorylation of RB and other substrates (Sherr and Roberts, 1995; Peter and Herskowitz, 1994). This prevents E2F release and subsequent gene activation dependent on E2F, resulting in cell cycle arrest.

The mechanism underlying the increase in cell number associated with constitutive expression of Cux-1 is not clear at present. One possibility is that deregulated expression of Cux-1 results in a faster division cycle of progenitor or stem cells prior to cell cycle withdrawal in terminal differentiation, as has been described for the targeted deletion of the cyclin kinase inhibitor genes, p18 and p27 (Franklin et al., 1998; Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). Consistent with this idea is the observation that downregulation of Cux-1 is not required for cell cycle withdrawal during terminal differentiation.

Mammalian Cut protein expression or activity appears to be restricted to proliferating cells in many different tissues (Nepveu, 2001). As such, the role of Cut proteins in cell cycle regulation has been examined. Cux-1 was found to be a component of the histone gene transcription factor HiNF-D, contributing to the induction of the histone H4 gene in G ₁-S transition, independent of E2F (van Wijnen et al., 1996). And Coqueret et al. (1998) showed that Cux-1 repressed the cyclin kinase inhibitor p21 in G ₁-S transition in cultured cells. p21 expression is transcriptionally regulated, and characterization of the basal promoter has identified important Sp1 sites (Datto et al., 1995). The specific Cux-1 binding site in the p21 promoter overlapped the TATA box and an Sp1 binding site, suggesting that Cux-1 repressed p21 by competition for binding site occupancy. p21 null mice, however, do not exhibit an increase in renal size (Williams et al., 1999).

We propose an alternate model to explain the renal hyperplasia in CMV/Cux-1 transgenic mice, in which the ectopically expressed Cux-1 negatively regulates the expression of p27. Several observations are consistent with this model. First, in the CMV/Cux-1 mice, ectopic expression of Cux-1 in transgenic kidneys resulted in the down-regulation of p27 mRNA and protein. Second, during normal kidney development, p27 is not expressed in the nephrogenic zone where Cux-1 is highly expressed, but is upregulated in maturing glomeruli and tubules following downregulation of Cux-1 (Combs et al., 1998). It is therefore plausible that Cux-1 represses p27 in the early stages of nephrogenesis prior to terminal differentiation, maintaining a proliferative state for cells in the nephrogenic zone. Third, organs in which the Cux-1 transgene is highly expressed display hyperplasia. This is similar to p27 null mice, which displayed hyperplasia of all the organs studied resulting from an increase in cell proliferation. However, tubulization of Bowman's capsule in p27 null mice has not been described. Fourth, in cotransfection experiments, Cux-1 repressed p27 gene expression. Functional analysis of the p27 promoter revealed regulation by two Sp1 sites (Kwon et al., 1996; Minami et al., 1997; Zhang and Lin, 1997), as well as a nearby CCAAT site, which are both targets of Cux-1 (Mailly et al., 1996). Thus, similar to the p21 promoter, Cux-1 may compete for binding site occupancy with transcriptional activators to negatively regulate p27 gene expression. Because p27 mRNA levels are unchanged during the cell cycle, it has been suggested that p27 is not under transcriptional control (Hengst and Reed, 1996). However, a number of studies have demonstrated upregulation or downregulation of p27 mRNA levels in response to various mitogens or growth factors (Moore et al., 1996; Li and Brooks, 1997; Cordon-Cardo et al., 1998; Johnson et al., 1998; Kortylewski et al., 1999; Lee et al., 1999; Park et al., 1999; Robson et al., 1999), suggesting that the p27 gene is also transcriptionally regulated. While previous studies have characterized the regulation of p21 gene expression by Cux-1 in S phase, further studies will be required to determine how Cux-1 regulates p27 gene expression.

Given the role for mammalian cut homologues as transcriptional repressors of developmentally regulated gene expression, we initially hypothesized that Cux-1 must be downregulated for terminal differentiation of glomeruli and tubules. However, in transgenic kidneys, we observed no signs of delayed or disrupted nephrogenesis. And the total number of nephrons as well as the overall histoarchitecture of adult transgenic kidneys was not changed. Therefore, while our data indicate that downregulation of Cux-1 is not required for terminal differentiation, they suggest a role for Cux-1 in promoting cell proliferation during early stages of nephrogenesis. The ability of Cux-1 to repress p27 promoter activity in 293T cells suggests this is accomplished through repression of p27^kip1 gene expression in developing nephrons.

We also observed a significant increase in the proportion of proliferating cells in adult CMV/Cux-1 transgenic kidneys. One possible explanation for this increase is that Cux-1 is able to induce differentiated cells to proliferate. While there is very little proliferation in a normal adult kidney, glomerular and tubular epithelial cell proliferation increases in certain renal pathologies. Experimentally induced acute renal failure in p21 null mice results in a significant increase in the number of proliferating cells, both in proximal tubules and lining Bowman's capsule, with more severe damage and higher mortality (Megyesi et al., 1998). Moreover, downregulation of p27 is associated with mesangioproliferative glomerulonephritis (Shankland etal.,1996). Thus, aberrant expression of Cux-1 may contribute to the disease process. Future studies will be directed at examining the effects of constitutive Cux-1 expression on renal function and disease. Finally, our studies support a general role for Cux-1 in growth regulation, and provide additional evidence in support of a dual role for mammalian Cut proteins in determining cell type specificity in differentiation, and serving as cell cycle-dependent transcription factors in proliferating cells.