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Human Molecular Genetics Advance Access originally published online on October 17, 2006
Human Molecular Genetics 2006 15(23):3420-3428; doi:10.1093/hmg/ddl418
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Regulation of c-Ret in the developing kidney is responsive to Pax2 gene dosage

Jason C. Clarke1, Sanjeevkumar R. Patel2, Richard M. Raymond, Jr1, Scott Andrew4,5, Bruce G. Robinson5, Gregory R. Dressler3 and Patrick D. Brophy1,*

1 Department of Pediatrics, 2 Department of Internal Medicine and 3 Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA, 4 Department of Pathology, Queens University, Kingston, Ontario, Canada K7L 3N6 and 5 Kolling Institute, Royal North Shore Hospital, St Leonards and University of Sydney, Cancer Genetics Laboratory, New South Wales 2065, Australia.

* To whom correspondence should be addressed at: Department of Pediatrics, University of Michigan, 1150 West Medical Center Drive, MSRB I Rm 4500, Ann Arbor, MI 48109, USA. Tel: +1 7346479922; Fax: +1 7349986416; Email: pbrophy{at}umich.edu

Received August 13, 2006; Revised October 9, 2006; Accepted October 14, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
During kidney development, Pax2 and Pax8 are expressed very early in the mammalian nephric duct and both precede the expression of receptor tyrosine kinase, c-Ret. However, in Pax2–/– mutant mice, expression of c-Ret is lost after embryonic day 10.5. As the Ret/Gdnf pathway is necessary for renal development and there is a temporal and spatial relationship of Pax2 and c-Ret expression in the developing genito-urinary system, we postulate that Pax2 is necessary for c-Ret expression in the developing kidney. In vitro, Pax2 protein is capable of physically interacting with a c-RET promoter, and both Pax2 and Pax8 can activate the expression of a reporter gene driven by the c-RET promoter. Compound heterozygous null mice (Pax2+/–: Ret+/–) display an increased incidence of unilateral and bilateral renal agenesis, and smaller kidneys with fewer nephrons. Furthermore, the expression of Gdnf is reduced 2–3-fold, whereas c-Ret expression is reduced 9–47-fold in Pax2 heterozygous embryonic kidneys as detected by real-time quantitative RT (QRT)–PCR. The data demonstrate that Pax2 plays an integral role in the initiation and maintenance of the Ret/Gdnf pathway by not only activating the ligand of the pathway, but by also enhancing the expression of the pathway receptor Ret. The effects of reduced Pax2 gene dosage are thus amplified resulting in a haploinsufficient phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In humans and in mice, different Pax genes control the development of a variety of structures including the eye, the hindbrain, the vertebral column, the neural crest and the kidney (reviewed in 1). In all cases, development is particularly sensitive to gene dosage resulting in haploinsufficient phenotypes. For example, Pax2 heterozygous (Pax2+/–) mice and humans have smaller kidneys, whereas loss of Pax2 in mice results in complete renal agenesis (2–4). The mammalian metanephric kidney arises from the posterior intermediate mesoderm through a series of reciprocal inductive interactions between the ureteric bud epithelium and the metanephric mesenchyme (MM) (5). The ureteric bud is an outgrowth of the nephric duct, a bilateral epithelial duct that extends along the anteroposterior body axis beginning at approximately somite 12 and ending in the cloaca. Signals from the MM, at the posterior aspect of the intermediate mesoderm, stimulate ureteric bud outgrowth. In the mouse, this process is initiated at approximately embryonic day 10.5 (E10.5). The uninduced MM secretes glial-derived neurotrophic factor (Gdnf) that acts as a chemoattractant for nephric duct epithelial cells (6–8) to promote the invasion of the MM. Gdnf binds to the high affinity receptor Gfr{alpha}-1 and the receptor tyrosine kinase Ret, which are expressed in the nephric duct epithelia. Homozygous null mice for c-Ret (9), Gdnf (10–12) and Gfr{alpha}-1 (13) all exhibit bilateral renal agenesis due to the inhibition of ureteric bud growth and branching morphogenesis.

The spatial and temporal regulation of Gdnf and c-Ret expression is essential for initiation and patterning of metanephric development. The c-Ret gene encodes the receptor tyrosine kinase, Ret, which is expressed within the nephric duct and the advancing tip of the ureteric bud, as well as in the developing peripheral and central nervous systems (9,14). The c-Ret transcripts are first observed at E8.5 in the developing nephric ducts of murine embryos. By E10 to E11, c-Ret is expressed along the entire nephric duct, and by E11 to E11.5, can be seen in the ureteric bud epithelium, but not the MM (14). Expression persists in the tips of the branching ureteric bud system but not in the mature stalks. The c-Ret–/– mice die soon after birth and display a spectrum of renal defects from complete renal agenesis to severe renal hypoplasia, due to the lack of ureteric bud outgrowth or subsequent branching (9,15). The expression of Gdnf is restricted to mesenchymal cells along the medial aspect of the nephric duct, eventually becoming localized in the MM (16). Within the MM, Gdnf expression is positively regulated by Pax2 (17) and Eya1 (18), whereas the anterior boundary of Gdnf expression is negatively regulated by FoxC1/2 (19). However, little is known regarding the regulation of c-Ret expression in the early epithelial derivatives of the intermediate mesoderm. Genetic studies suggest that Pax2 is required for the maintenance of c-Ret expression at the time of metanephric induction (4,17). Prior to this stage, c-Ret expression may be regulated by the related gene Pax8 whose expression overlaps with Pax2 in the nephric duct epithelia. However, a direct role for Pax2/8 regulation of the c-Ret gene has not been established.

Among the earliest markers restricted to the intermediate mesoderm are the transcription factors Pax2 and Pax8. Members of the Pax2/5/8 subfamily possess extensive homology and their protein products can substitute for each other in development presumably because of similar biochemical activities (20,21). In the mouse, Pax2 transcripts are first detectable in the intermediate mesoderm prior to the formation of the nephric duct at E8.5 (22,23) and are subsequently found in the nephric duct epithelia and in the mesenchyme that generates the mesonephric and metanephric tubules. In Pax2–/–, embryos the nephric duct loses the expression of c-Ret by E10.5 and ureteric buds capable of inducing the metanephroi do not form, resulting in a complete lack of kidney development (4). Furthermore, Pax2–/– MM is unable to respond to inductive signals in vitro, suggesting an intrinsic defect in the ability of intermediate mesoderm to form epithelia (17). In humans, the loss of one PAX2 allele can result in renal hypoplasia, vesicouretal reflux and optic nerve colobomas (22,24,25). The hypoplastic renal phenotype is mainly due to reduced calyces and upper part of the ureters, suggesting defects in branching of the nephric duct and/or cell proliferation. These phenotypes are consistent with a potential role for Pax2 in the regulation of the Ret/Gdnf signaling pathway.

Mice lacking Pax8 have no discernible kidney phenotypes, presumably because of the redundant function of Pax2, though they do exhibit thyroid abnormalities (26). However, Pax2/8 double null mutants, Pax2–/–: Pax8–/–, exhibit an earlier and more severe phenotype in the intermediate mesoderm including the absence of the nephric duct at the earliest stages and no expression of c-Ret in the nephric duct (22). Temporally, the expression of Pax2 occurs prior to the expression of c-Ret, but after the expression of Pax8 within the primary nephric duct (22). Owing to the spatial relationship and upstream temporal expression of Pax2 and Pax8 to that of c-Ret in the nephric duct, and the inherent loss of a viable nephric duct and c-Ret expression in the Pax2–/– mice, we postulated that Pax2 and Pax8 can directly regulate c-Ret expression in the developing murine kidney. In this report, we examine the relationship between Pax2 and c-Ret in vitro and in vivo. The Pax2 and Pax8 proteins are able to directly activate the c-RET promoter in cultured cells. Direct binding of the Pax2 paired domain (PD) to the c-RET promoter is consistent with this observation. To test the interaction of Pax2 and c-Ret in vivo, we analyzed the renal phenotypes and expression of c-Ret in wild-type, Pax2+/–, c-Ret+/– and compound Pax2+/–: c-Ret+/– mice. The data demonstrates that Pax2/8 can directly regulate the level of c-Ret expression to affect branching morphogenesis, glomerular density and the frequency of unilateral and bilateral renal agenesis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To examine whether Pax proteins can regulate the c-Ret gene directly, we utilized a 680 bp region of the human c-RET promoter described previously (27). Since both Pax2 and Pax8 are expressed in the intermediate mesoderm at the time of nephric duct growth, we examined activation of a luciferase reporter under the control of the c-RET promoter in response to increasing amounts of Pax2 or Pax8 in transient transfections (Fig. 1A and B). In the absence of Pax proteins, the c-RET promoter construct did not express more than the vector pGL3 alone, consistent with the lack of c-Ret expression in NIH3T3 cells. However, both Pax2 and Pax8 were able to activate luciferase expression 5–6-fold when co-transfected with pGL3-RET680. However, neither a 0.5 µg dosage of either Pax2 or Pax8 expression plasmids were capable of appreciably activating the vector pGL3 lacking the c-RET promoter.


Figure 4181
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  		Figure 1. Activation of the c-RET promoter by Pax2 or Pax8. Mouse NIH 3T3 fibroblast cells were transiently transfected with 0.5 µg of the pGL3 RET680 vector, which contains a 680 bp fragment of the c-RET 5' promoter sequence driving a luciferase reporter, and either increasing concentrations of CMV-Pax2b (A) or CMV-Pax8 (B) cDNA plasmids. Note both Pax2 and Pax8 can drive the pGL3-RET680 with 5–6-fold induction compared with the RET-pGL3 vector alone. (C) Removal of the 167 bp Pax2/8 binding site from the c-RET promoter (black bars) results in a reduction of luciferase activation in the presence of 0.5 µg of either Pax2 or Pax8 when compared with the full length promoter (gray bars). The pGL3-Basic vector was utilized at 0.5 µg as a control (white bars).

 
Given the ability of Pax2/8 to transactivate the c-RET promoter, we examined binding of the Pax2 protein to the c-RET promoter fragment. The full length 680 bp fragment and smaller sub-fragments were used for electrophoretic mobility shift assays with either full length Pax2b from transfected cells or a recombinant Pax2 PD (amino acids 1–170) isolated from E. coli (Fig. 2B and C). The full length Pax2 protein bound to the whole 680 bp fragment and to a sub-fragment from either a DdeI or AluI digests (Fig. 2B). Of note, one of the AluI sub-fragments consisted of a 167 bp region that is highly conserved between mouse and human and contained the specific Pax2 binding site. This sub-fragment was isolated and used in electrophoretic mobility shift assay (EMSA) experiments with just the recombinant Pax2 PD (Fig. 2C) to test for specificity. Binding to this sub-fragment could be easily competed with a molar excess of unlabeled fragment. As a positive control, we also utilized the well characterized H2A Pax2 binding site (28). The 167 bp sub-fragment bound full length Pax2 and exhibited multiple protein/DNA complexes when incubated with the PD alone, although the full length Pax2 protein only showed a single protein/DNA species. These data indicate at least one specific Pax2 binding site is present in this fragment. DNA sequence analysis did not reveal a perfect consensus sequence within the 167 bp region. However, a sequence containing 10/13 identical nucleotides (GCTTGCTCTGCGT) from the WT1 promoter to the Pax2 binding site is present (29). To determine whether Pax-dependent activation required this conserved 167 bp region, we deleted this fragment from the 680 bp c-RET promoter in the luciferase reporter construct pGL3-RET680del167 (Fig. 1C). Using fixed amounts of Pax2 or Pax8, the reporter plasmid with the deletion of the 167 bp conserved region did not activate significantly more than the pGL3 vector alone. Thus, the Pax responsive elements are within the 167 bp region, the same region known to bind the PD. The necessity of the 167 bp region for strong Pax2 binding was confirmed by electrophoretic mobility shift assays utilizing nuclear lysates from HA-epitope tagged Pax2b expressing cells (Fig. 2D). The full length 680 bp c-RET promoter showed a strong protein/DNA complex, using Pax2b-HA containing lysates, which was not detected with 293 lysates. This protein/DNA complex could be supershifted with a monoclonal antibody against the HA tag. Upon deletion of the 167 bp Pax2 binding region, the protein/DNA complex was reduced and could no longer be supershifted with anti-HA.


Figure 4182
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  		Figure 2. Binding of Pax2 to the c-RET promoter. (A) Schematic diagram of the 680 bp c-RET DNA promoter fragment (26). Restriction digest cut sites are denoted for DdeI and AluI. The black box denotes the position of the first exon following the ATG codon. The black bar designates the 167 bp sub-fragment resulting from the Alu1 digest. (B) The 680 bp c-RET 5' promoter fragment from digests with Alu1 (lanes 1–4), DdeI (lanes 5–8) and the non-cutter Sau3A as a positive control (lanes 9–12). These fragments were radiolabeled and incubated with HEK 293 nuclear lysates from either a sham transfection or a transfection containing 1 µg of full length Pax2b. Lanes 2, 6 and 10 are negative controls containing the nuclear lysates from the sham transfected HEK 293 cells. These lysates do not interact with the AluI or DdeI sub-fragments or the full length promoter fragment. Lanes 4, 8 and 12 contain nuclear lysates from HEK 293 cells transfected with full length Pax2b. A specific shift is noted in these lanes (black arrows) in not only the full length promoter fragment but also the DdeI and smaller AluI sub-fragments. Lanes 1, 3, 5, 7, 9 and 11 are controls containing no NL for each respective restriction digest. (C) EMSA analysis using the conserved 167 bp c-RET promoter fragment with recombinant Pax2 PD. The binding to the 167 bp promoter sub-fragment was blocked with an excess of the known Pax2 binding sequence H2A (30) or the 167 bp fragment itself as cold competitors. (D) Super shift assay with the (32)-P-labeled full length c-RET promoter (RET680) and with the 167 bp deletion mutant c-RET promoter (RET680del167). The free probes (lanes 1 and 7) migrate relatively unaffected in the presence of 2 µg of anti-HA (lanes 2 and 8). The addition of nuclear lysates (NL) from HEK 293 cells transfected with Pax2b-HA does shift both the RET680 and RET680del167 probes (lanes 3 and 9) (lower left and right black arrows). However, the RET680 probe exhibits more binding with Pax2-HA NL and is supershifted by anti-HA (lane 4) (top left black arrow). The mutant RET680del167 probe (lane 10) shows weak binding and does not super shift with anti-HA. Control reactions with NL not containing Pax2b-HA protein are capable of shifting both the probes (lanes 5 and 11) but do not super shift (lanes 6 and 12).

 
The above experiments suggest a direct interaction between the Pax2 protein and the c-Ret regulatory region. In vivo, both Pax2 and c-Ret homozygous null mutations have severe renal abnormalities. However, heterozygous c-Ret+/– animals have normal kidneys, whereas Pax2+/– animals show a reduction in kidney size that depends in part on the genetic background. To examine genetic interactions between c-Ret and Pax2, we examined mice heterozygous for both Pax2 and c-Ret null alleles. Matings between Pax2+/– and c-Ret+/– mice generated compound heterozygous offspring, in addition to the expected Pax2+/– and c-Ret+/– individual heterozygous animals. Embryos were analyzed at E11.5 and E18.5 and newborns at 15 days post-natal (dpn). A review of the gross pathology was conducted as well as a gene expression analysis. In the 15 dpn newborns, wild-type and c-Ret+/– mice had similar kidney to body mass ratios (Table 1). However, the Pax2+/– mice had a significantly reduced kidney to body mass ratio, 0.56% (±0.15 s.d.) (t-test P<0.001) compared with the 0.75% kidney to body mass ratio of the wild-type and c-Ret+/– littermates. The compound heterozygous (Pax2+/–: Ret+/–) mice also had a significantly reduced kidney to body mass ratio of 0.56% (±0.15 s.d.) (t-test P<0.001) relative to wild-type and c-Ret+/– mice, but did not differ from their Pax2+/– littermates in this regard.


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  		Table 1. Kidney abnormalities observed at E18.5 and 15 dpn

 
Although the ratio of kidney to body mass is one measure of development, a more precise indicator of Ret activity and branching morphogenesis is the number of glomeruli in the adult kidney, as reduced branching will result in reduced nephron induction. Thus, we determined the mean glomerular density for all genotypes at 15 dpn. Histological sections (n=71) were analyzed by counting the number of glomeruli per unit area. Representative sections are shown in Fig. 3A. The wild-type kidneys had a mean glomerular density of 8.95 (±1.9 s.d.) compared with 7.25 (±1.9 s.d.) for the c-Ret+/– littermates (Fig. 3B). The Pax2+/– kidneys displayed a reduction of glomerular density and overall number of glomeruli with a mean of 5.79 (±1.9 s.d.) glomeruli. The compound heterozygotes displayed an even further reduction in overall glomerular density with a mean of 4.57 (±1.8 s.d.) glomeruli. This is a reduction of ~49, 37 and 22% in the glomerular density of the compound heterozygote kidney sections when compared with those of the wild-type, c-Ret+/– and Pax2+/– kidney sections, respectively. The glomerular densities of the three mutant groups were found to be significantly different when compared with the wild-type kidneys and to each other in all cases.


Figure 4183
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  		Figure 3. Glomerular density analysis of 15 dpn mid-sagittally sectioned kidneys. (A) Representative photographs of the upper poles of kidneys at 20x magnification. Areas of ~255 µm2 were used to count the number of glomeruli at four anatomical reference points on each kidney section. Genotypes are noted on each section. Black scale bar in WT panel is 10 µm. (B) Comparison of the glomerular density revealed that the c-Ret+/– (n=18), Pax2+/– (n=17) and compound heterozygous (n=24) samples were significantly different (t-test; P<0.001) when compared with the wild-type samples (n=12). The Pax2+/– and compound heterozygous samples were significantly different (t-test; P<0.001) when compared with the c-Ret+/– samples. Lastly, the compound heterozygous samples were significantly different (t-test; P<0.001) when compared with the Pax2+/– samples. Also, in comparison with the WT, Pax2+/– and c-Ret+/– there appears to be more interstitial and medullary stromal tissue in the compound heterozygotes. WT, wild-type.

 
The compound heterozygous mice were significantly underrepresented, from the expected Mendelian distribution, in the newborn population at weaning (Table 2, {chi}2; P<0.01) suggesting a partially penetrant lethal phenotype. Given the underrepresentation of the compound heterozygotes at 15 dpn, we examined litters at E18.5 to determine if lethality occurred in utero or post-natally. The wild-type and c-Ret+/– embryos at this time point had a similar kidney to body mass ratio (Table 1), whereas the Pax2+/– embryos had a significantly reduced kidney to body mass ratio, 0.26% (±0.16 s.d.), when compared with the wild-type and Ret+/– embryos (t-test P<0.001). Compound heterozygous embryos were not significantly different from Pax2+/– littermates. While the compound heterozygote kidneys were slightly more affected in terms of renal mass and nephron number, more significant differences were observed in the prevalence of unilateral or bilateral agenesis. At 15 dpn, 4% of the c-Ret+/– and 15% of the compound heterozygous mice exhibited unilateral renal agenesis (Table 1). However, in the E18.5 embryos, a high frequency of unilateral and bilateral renal agenesis was observed in the compound heterozygotes (Table 1). At this analyzed time point, 52% of the compound heterozygous mice displayed unilateral renal agenesis and 13% displayed bilateral renal agenesis. In the observed instances of renal agenesis, the ipsilateral ureters of the agenic kidney(s) were also absent. However, in all instances of renal agenesis, the adrenal glands, bladder and genitals were still present. Thus, the underrepresentation of the compound heterozygotes post-natally is probably due to renal insufficiency after birth in a proportion of the newborns.


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  		Table 2. Observed genotypes at each time point

 
Owing to the high frequency of renal agenesis in the E18.5 compound heterozygotes, we examined embryos at E11.5 just after ureteric bud invasion and MM induction. These E11.5 kidneys were cultured in vitro to determine the pattern of branching morphogenesis over time. In all genotypes examined, ureteric bud invasion had occurred by E11.5 and induction of the MM was evident. After 48 h in culture, both wild-type (n=13) and c-Ret+/– explants (n=17) displayed similar branching with a mean of 5.65 (±2.08 s.d.) and 6.79 (±2.67 s.d.) branch points, respectively (Fig. 4). Explants from Pax2+/– embryos (n=7) did not significantly differ from the wild-type and c-Ret+/– explants, with a mean of 4.89 (±1.98 s.d.). Explants from the compound heterozygotes (n=9) had an obviously stunted pattern of branching morphogenesis with a significantly reduced number of branch points, 3.08 (±1.15 s.d.) (ANOVA; P<0.05) when compared with the wild-type and c-Ret+/– explants. Similar effects on branching morphogenesis were noted in organ cultures of E12.5 littermates (data not shown), as compound heterozygotes again exhibited fewer branch points and abnormal patterning.


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  		Figure 4. Branching morphogenesis analysis of E11.5 explants. Kidney rudiments (n=46) were microdissected from E11.5 embryos, grown in culture for 48 h, and stained with anti-laminin and anti-cytokeratin. A branch point was defined as the location where the ureteric bud epithelium bifurcated to form one or more subsequent branches. Compound heterozygous explants (n=9) had a significant reduction in a stunted pattern of branching morphogenesis with a significantly reduced number of branch points, 3.08 (±1.15 s.d.) (ANOVA; P<0.05) when compared with the wild-type and c-Ret+/– explants. WT, wild-type.

 
To address whether reduced Pax2 gene dosage specifically affected the expression of the c-Ret gene in vivo, real-time QRT–PCR was performed on E11.5 and E18.5 murine kidneys. We compared levels of c-Ret mRNA in wild-type, c-Ret+/– and Pax2+/– kidneys. At E11.5, the Pax2+/– kidney rudiments showed a 9.3 relative fold reduction (±2.5 s.d.) (t-test P<0.001) in c-Ret expression relative to the wild-type kidneys (Fig. 5A). At the E18.5 time point, the Pax2+/– kidneys showed a 47.5 relative fold reduction (±10.05 s.d.) (t-test P<0.002) in c-Ret expression relative to the wild-type kidneys (Fig. 5B). Strikingly, levels of c-Ret mRNA were not significantly different in c-Ret+/– kidneys, consistent with the lack of phenotype and suggesting that there is some mechanism for dosage compensation. We also examined the expression of Gdnf at E18.5 in Pax2 and c-Ret heterozygotes (Fig. 5C). Gdnf expression was examined in Pax2+/– embryos previously, although this was not very quantitative (17). At E18.5, Gdnf mRNA levels were reduced slightly in c-Ret+/– mutants and reduced ~2–3-fold in Pax2+/– mutants. This reduction in Gdnf could further amplify the inhibition of c-Ret expression, through a potential feedback loop. These data support our hypothesis that Pax2 regulates c-Ret in vivo and that the Pax2 haploinsufficient phenotype is due, at least in part, to reduced expression of c-Ret.


Figure 4185
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  		Figure 5. Real-time QRT–PCR of c-Ret and Gdnf expression. (A) Relative c-Ret expression in E11.5 embryonic kidneys: relative to the wild-type kidneys the c-Ret+/– kidneys had an ~1.2-fold reduction (±1.5 s.d.) in c-Ret expression. However, the Pax2+/– kidneys showed a 9.3 relative fold reduction (±2.5 s.d.) (t-test P<0.001) in c-Ret expression compared with wild-types. (B) Relative c-Ret expression in E18.5 embryonic kidneys: relative to the wild-type kidneys the c-Ret+/– kidneys had an ~3.4-fold reduction (±2.1 s.d.) in c-Ret expression and again showed no significant difference when compared with the wild-type kidneys. The Pax2+/– embryonic kidneys demonstrate an ~47.5 relative fold reduction (±10.05 s.d.) (t-test P<0.002) in c-Ret expression when compared with wild-type littermates. (C) Relative Gdnf expression in E18.5 embryonic kidneys: the c-Ret+/– kidneys showed an ~0.2-fold reduction (±1.3 s.d.) and the Pax2+/– kidneys an ~2.0-fold reduction (±0.06 s.d.) in Gdnf expression.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Gene dosage effects, or haploinsufficiency, is a common characteristic of dominant Pax2/PAX2 gene mutations in both mice and humans with renal hypoplasia as the common observed haploinsufficient phenotype. Yet the molecular basis for these effects are unclear. The size of the kidney and the number of nephrons present is dependent upon the activity of the c-Ret receptor tyrosine kinase. The data presented in this report suggest a direct link between Pax2 gene dosage and c-Ret expression. Pax2 can bind to and activate a c-RET promoter in tissue culture cells. Furthermore, Pax2 heterozygotes show significantly reduced levels of c-Ret expression at early stages of kidney development. Genetic analyses suggests that the combination of reduced c-Ret expression in Pax2 heterozygotes and the reduction in c-Ret gene dosage in the compound heterozygotes results in a significant increase in renal agenesis above what is observed in either single heterozygote alone. These results establish a molecular basis for Pax2 gene dosage effects in the developing kidney.

The expression levels and activity of the c-Ret receptor tyrosine kinase is regulated at multiple levels. The secreted ligand, Gdnf, not only activates the kinase activity but also increases expression of c-Ret mRNA, suggesting a positive feedback loop (30). Retinoids activate c-Ret expression and promote branching morphogenesis through a stromal cell-mediated mechanism (31). If retinoic acid receptors in the stromal cells are lost, c-Ret expression in the tips of the branching ureteric buds is also suppressed. Coincidentally, Pax2 is expressed highly in the ureteric bud tips, although it is not clear if Pax2 expression is affected in the retinoic acid receptor mutants. The importance of retinoids and c-Ret expression is underscored in embryos that develop renal hypoplasia in a vitamin-A deficient environment (32).

In vitro, Pax2 binds to the c-RET promoter and activates transcription in a reporter system. Similarly, Pax8 can activate the c-RET promoter. Given the partial overlap in expression patterns between Pax2 and Pax8 and the identical DNA binding PDs, we postulate that Pax2/8 play a role in directly activating the c-Ret gene during formation of the nephric duct within the intermediate mesoderm at the earliest stage of kidney development. In compound Pax2/Pax8 double mutants, the nephric duct is never formed at E9.5 and c-Ret expression is undetectable (22). In Pax2–/– embryos, the nephric duct is formed and c-Ret expression can be detected from E9.5–E10.5. However, c-Ret expression in the posterior nephric duct, at the time of kidney induction (E11.5), has disappeared indicating that Pax2 plays a necessary role in specifically regulating and maintaining c-Ret expression during the phase of branching morphogenesis. This is consistent with a lack of Pax8 expression in the ureteric bud epithelium after E11.5 (33). Thus, either Pax2 or Pax8 are capable of activating c-Ret expression early, but only Pax2 sustains the expression of c-Ret within the nephric duct because Pax8 expression is lost. The biological relevance of this type of Pax gene redundancy has also been demonstrated in the development and maintenance of the zebra fish otic placode (21).

In a wild-type genetic background, the c-Ret+/– mice show no observable renal abnormalities. In fact, quantitative analyses of c-Ret expression show no significant differences in mRNA levels at early and late gestation. That the reduction in c-Ret gene dosage has little effect on gene expression may be due to the ability of Gdnf to positively up-regulate expression of its own receptor (30). Activation of c-Ret expression by Gdnf may be Pax2-dependent or independent. Since Gdnf is expressed in the mesenchyme, where it is regulated by Pax2, Gdnf levels are not likely to be affected by reduced c-Ret gene dosage. However, in Pax2+/– mutants Gdnf, expression levels are affected, with a 2–3-fold reduction. Because Gdnf upregulates c-Ret expression, this feedback loop may further suppress c-Ret in the ureteric bud when Gdnf levels are reduced. Thus, the 9 and 47-fold decrease in c-Ret expression levels in Pax2+/– kidneys at E11.5 and E18.5, respectively, are likely to reflect the decrease in Pax2 gene dosage and the decreased Gdnf-mediated signaling. This model is outlined schematically in Fig. 6 and can explain why renal size is particularly sensitive to Pax2 gene dosage. Since Pax2 can directly regulate both the ligand and the receptor and since receptor activation by the ligand reinforces expression of the receptor, the negative effects of reduced Pax2 gene dosage on c-Ret expression are amplified. Thus, the reduction in renal size and the increased frequency of complete agenesis reflects the inherent decreased activity of the Ret receptor tyrosine kinase. That renal size is determined by Ret activity is best illustrated in the retinoic acid mutants, which also down regulate c-Ret expression and show reduced kidney size, yet the phenotype can be rescued merely by expressing a c-Ret transgene in the ureteric buds (31).


Figure 4186
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  		Figure 6. Model of c-Ret Regulation by Pax2. (A) In wild-type kidneys, Pax2 activates both the Gdnf and c-Ret promoters. Gdnf signaling from the MM provides an additional transcriptional boost, that may be Pax2-independent or dependent, to maintain normal levels of c-Ret expression. (B) In c-Ret+/– kidneys, Gdnf expression is normal and the positive feedback loop of receptor activation may compensate for the reduced c-Ret gene dosage by expressing more c-Ret mRNA from the remaining normal allele. (C) In Pax2+/– kidneys, reduced Pax2 gene dosage lowers Gdnf levels and c-Ret expression levels. Reduced Gdnf signaling further reduces c-Ret expression as positive feedback is diminished. Kidney size is affected due to decreased Ret signaling. (D) Reduction of both Pax2 and c-Ret gene dosage reduces c-Ret expression and Ret signaling to the point where renal agenesis is now frequent.

 
The renal agenesis in compound heterozygous mice is not due to a failure of the ureteric bud to induce the metanephros, as evident in the E11.5 metanephric explants (Fig. 4), but rather a post induction developmental arrest and regression. Based on the expected Mendelian ratios, approximately half of the compound heterozygotes in this study survived to weaning with smaller kidneys. The proportion of unilateral renal agenesis at E18.5 and 15 dpn was 52 and 15%, respectively. This suggests that many compound heterozygous mice presenting with unilateral renal agenesis died postnatally, most likely due to renal hypoplasia and insufficiency. This variable renal phenotype is suggestive that unilateral and bilateral renal agenesis have similar genetic underpinnings.

In summary, Pax2 expression in the kidney can regulate both the expression of Gdnf and its receptor, the Ret tyrosine kinase. Because the Gdnf/Ret signaling pathway also regulates expression of the receptor, the effects of Pax2 gene dosage are amplified. This model can explain why some developing tissues are particularly sensitive to Pax gene dosage. Whether similar receptor/ligand feedback loops are controlled by other Pax genes such as in the eye and neural crest, tissues that are sensitive to Pax6 and Pax3 gene dosage, remains to be determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and expression vectors
The pGL3-RET680 reporter construct contained a 680 bp c-RET promoter fragment as previously described (27). The mutant pGL3-RET680del167 c-RET promoter fragment was designed and constructed by Blue Heron Biotechnology and sequence verified. This resulted in removal of the 167 bp Pax2/8 binding site on the 680 bp c-RET promoter fragment. The Pax2b-CMV HA tagged vector was constructed as previously described by Lechner and Dressler (34) and the Pax2 PD containing amino acids 1–170 was fused to a poly-histidine expression vector (pRSET, Invitrogen). The Pax8-CMV plasmid was a kind gift from D. Plachov and has been previously described (33).

Luciferase assays
NIH 3T3 or HEK 293 cells were plated at 5x105 cells per 60 mM dish, cultured in DMEM+10% FCS and 1% penicillin/streptomycin at 37°C, and transfected the following day with FUGENE (Roche), according to the manufacturer's protocol. For each 60 mm dish, 6 µl of FUGENE was used per 3 µg of DNA, which contained 0.5 µg of reporter plasmid and 0.2 µg CMV-ß-gal for standardization, along with increasing amounts of full-length Pax2b or Pax8 (0.1, 0.25, 0.5 and 1 µg). After a 2 h exposure to the FUGENE/DNA in DMEM (serum-free), DMEM+10% FCS and 1% penicillin/streptomycin were added to each dish to a final volume of 10 ml. Forty-eight hours post-transfection, cells were scraped into PBS, centrifuged and resuspended in 300 µl of 0.25 M Tris (pH 7.6). Cells were lysed, debris was pelleted and 50 µl of lysate was assayed for ß-gal activity. Luminescence was read utilizing a Turner Design TD –20/20 luminometer per manufacturer's protocol.

Generation of Pax2 and c-Ret mice
The murine Pax2 knockout strain is that as described by Torres et al. (4). The murine c-Ret knockout strain is that as described by Schuchardt et al. (15).

Matings
We reciprocally crossed Pax2+/– mice to c-Ret+/– mice to generate individual and compound heterozygous offspring for analysis. The reciprocal mating scheme was done to observe any parent-of-origin effects that may occur in regards to genotype frequency. Females were checked for the presence of a vaginal plug daily, that if observed was recorded as embryonic day 0.5 (E0.5). Embryos and mice were analyzed at E11.5, E18.5 and 15 dpn on the morning of the appropriate time point.

PCR assay
Mice were genotyped by PCR utilizing primers unique to each of the two mutant lines. Pax2 primer sequences are: Pax2 Fwd. 5'-CCCACCGTCCCTTCCTTTTCTCCTCA-3', Pax2 Rev. 5'-GAAAGGCCAGTGTGGCCTCTAGGGTG-3' and PGK-FX3, 5'AGACTGCCTT GGGAAAAGCGC3' (Pax2 primer sequences were a kind gift from R. Behringer). The c-Ret primer sequences are: c-Ret Fwd. 5'-TGATGTCAAAGCAGTCTTCAGAGC-3' and c-Ret Rev. 5'-GTGCCCAGTCATAGCCGAATAG-3'. The Pax2 PCR program was performed with the Touch Down protocol: 94°C for 5 min, followed by 94°C for 30 s, 72°C for 30 s (dropping 0.5°/cycle) and 72°C for 30 s for a total of 35 cycles followed by a 5 min extension at 72°C. The c-Ret PCR program was performed as follows: 94° C for 5 minutes, followed by: 94°C for 1 min, 65°C for 2 min and 72°C for 3 min for a total of 28 cycles followed by a 5 min extension at 72°C.

Whole mount organ culture and nephric branch point analysis
Metanephric explants (n=46) from E11.5 mice were grown in culture for 2 days as described by Dressler and Douglass (35). Antibodies against laminin and cytokeratin were used to visualize the nephric duct and glomerular structures. The branch points of the nephric duct were then counted in a double-blinded study (n=4). A branch point was defined as the location where the ureteric bud epithelia bifurcated to form one or more subsequent branches. Statistical analysis was conducted utilizing a one-way ANOVA with significance at P<0.05.

EMSA and super shift assay
For initial screening, the 680 bp c-RET promoter fragment was digested with Alu1, Dde1 or Sau3A. Fragment products of the restriction enzyme digests were kinase labeled with [{gamma}-32P] ATP. Binding reactions were performed using nuclear lysates from HEK 293 cells that had been either transfected with full-length Pax2b or sham transfected with Bluescript in a total volume of 10 µl for 30 min at room temperature, and contained increasing amounts of the full length Pax2b protein, 100 ng poly(dI-dC) and labeled probe (10 000 dpm). Free DNA and DNA/protein complexes were resolved at room temperature on a 3.5, 4 or 8% neutral Tris–glycine polyacrylamide gel in 0.5x Tris–glycine buffer at 120 V. For specific fragment analysis and competition experiments, the Pax2 PD protein was purified by metal affinity chromatography under denaturing conditions. The denatured protein was dialyzed stepwise in decreasing amounts of urea and finally into Z-buffer (25 mM Hepes pH 7.8, 20% glycerol, 12.5 mM MgCl2, 0.1 M KCl, 1 mM DTT) at a concentration of 100 µg/ml. The purified Pax2 PD, the [{gamma}-32P] ATP labeled 167 bp fragment from the Alu1 digest of the c-RET promoter and unlabeled competitors were used at 50- and 500-fold molar excess. Binding reactions were carried out in a buffer composed of 20 mM Hepes pH 7.9, 1 mM MgCl2, 4% Ficoll, 0.5 mM DTT, 50 mM KCl and 1.5 µg (10%) of poly (dI-dC). The cold competition Pax2 target DNA fragment (H2A) has previously been described (28). For super shift assays, reactions were performed using nuclear lysates from HEK 293 cells that had been either transfected with full-length Pax2b-HA or sham transfected with Bluescript. Either the full length c-RET promoter (RET680) or mutant c-RET promoter (RET680del167) was labeled with [{gamma}-32P] ATP and incubated with the nuclear lysates with and without the addition of 2 µg of monoclonal anti-HA (Sigma). Antibodies were pre-incubated with nuclear lysates in binding buffer for 20 min on ice prior to being incubated with the DNA probes.

Glomerular density analysis
Kidneys from 15 dpn mice were fixed in 4% paraformaldehyde then dehydrated through a graded alcohol series and embedded into paraffin. From each kidney two to three slides were produced with two ribbons of 3 µm thick mid-sagittal serial sections. A 40 µm interval between each serial ribbon reduced the likelihood of counting the same glomeruli repeatedly. Sections were stained with PAS and glomeruli were counted using a bright field microscope with a viewfinder reticle at 20x magnification. The viewfinder box was measured with a micrometer at 20x magnification to be 255 µm2. For each of the sections, the viewfinder box was placed in the cortical region of four anatomical reference points: (i) upper pole, (ii) upper anterior pole, (ii) lower anterior pole and (iv) lower pole, and the glomeruli within the viewfinder were counted by a blinded observer. Statistical analysis was conducted utilizing Student's t-test with significance at P<0.05.

RNA extraction and real-time QRT–PCR
RNA from E11.5 and E18.5 murine kidneys was extracted by homogenization with Trizol as per the manufacturer's instructions (Life Technology). cDNAs were synthesized from 1.2 and 5.0 µg of total RNA using the Superscript II First Strand Synthesis kit (Invitrogen) as per the manufacturer's instructions. Real-time QRT–PCR reactions utilized TaqMan chemistry and contained TaqMan Fast Universal PCR Master Mix (2x) (Applied Biosystems), pre-developed Taqman gene specific probes for either murine GAPDH, c-Ret or Gdnf (Applied Biosystems part numbers 4352932E, 171883 and 770223, respectively) and equal volumes of cDNA products. Reactions were setup as per the manufacturer's instructions (Applied Biosystems). Samples from individual embryos were run in triplicate and standardized to probe specific standard curves. The real-time QRT–PCR assay was repeated a minimum of three times at each time point on a BioRad MyiQ single color real-time PCR machine with respective software. Relative fold change between wild-type and mutant samples was determined using the method previously described by Pflaffl (36).


   ACKNOWLEDGEMENTS
 
The authors would like to thank Nancy Smith (ret.) and Lisa Riggs from the Department of Pathology, University of Michigan for their preparation of histological samples, F. Costantini for the c-Ret mice, R. Behringer for the Pax2 primer sequences and D. Plachov for the Pax8-CMV plasmid. This work was supported in part by NIH grants DK54740 and DK62914 to G.R.D., DK02803 to S.R.P. and DK064333-01 to P.D.B., as well as the Carl W. Gottschalk Award from the American Society of Nephrology and the Elizabeth Kennedy Fund to P.D.B.

Conflict of Interest statement. None declared.


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