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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2011 Jun;22(6):1053–1063. doi: 10.1681/ASN.2010080841

The Pro-Apoptotic Protein Bim Is a MicroRNA Target in Kidney Progenitors

Jacqueline Ho *,†,✉, Priyanka Pandey *,†, Tobias Schatton *,‡, Sunder Sims-Lucas §, Myda Khalid *,†, Markus H Frank *,‡, Sunny Hartwig *,†, Jordan A Kreidberg *,†,✉
PMCID: PMC3103725  PMID: 21546576

Abstract

Understanding the mechanisms that regulate nephron progenitors during kidney development should aid development of therapies for renal failure. MicroRNAs, which modulate gene expression through post-transcriptional repression of specific target mRNAs, contribute to the differentiation of stem cells, but their role in nephrogenesis is incompletely understood. Here, we found that the loss of miRNAs in nephron progenitors results in a premature depletion of this population during kidney development. Increased apoptosis and expression of the pro-apoptotic protein Bim accompanied this depletion. Profiling of miRNA expression during nephrogenesis identified several highly expressed miRNAs (miR-10a, miR-106b, miR-17-5p) in nephron progenitors that are either known or predicted to target Bim. We propose that modulation of apoptosis by miRNAs may determine congenital nephron endowment. Furthermore, our data implicate the pro-apoptotic protein Bim as a miRNA target in nephron progenitors.

Kidney development begins with the outgrowth of the ureteric bud from the Wolffian duct into the metanephric mesenchyme.1,2 The metanephric mesenchyme condenses as a tight “cap” of nephron progenitors around the tip of the ureteric bud and the ureteric bud branches to form the collecting system. Nephron progenitors have the capacity to self-renew to generate the full complement of nephrons and to differentiate into the multiple cell types required to form the nephron. This process continues in an iterative fashion during nephrogenesis such that the most immature cells are present in the subcapsular cortex of the developing kidney, termed the nephrogenic zone.

MicroRNAs (miRNAs) are a group of endogenous, small noncoding RNAs that function by causing the post-transcriptional repression of their respective target mRNAs. The first suggestion that miRNAs are critical in stem cell populations came from the observation that embryos that are null for Dicer, an enzyme required for the production of functional miRNAs, lose expression of Oct4 in the epiblast.3 Furthermore, Dicer null embryonic stem (ES) cells display a profound proliferative defect and fail to differentiate under conditions in which ES cells typically form embryoid bodies.4,5 Since these initial studies, a number of ES cell–specific miRNAs have been implicated in regulating the cell cycle, pluripotency, and differentiation of stem cells.6–8 These observations raise the intriguing question of whether miRNAs are critical in the regulation of other stem or progenitor cell populations.

Recent studies have shown that miRNAs are essential in specific tissue lineages such as the epidermis and hair follicle, lung epithelia, and cardiac, limb, and skeletal muscle, using a conditional approach to knock down Dicer.9–15 In mammals, Dicer is believed to be required predominantly for the processing of mature miRNAs, although it remains possible that a portion of these phenotypes are caused by the loss of other Dicer-dependent small RNAs.16,17 In the kidney, Dicer activity is critical in podocytes for the maintenance of the glomerular filtration barrier and in the juxtaglomerular apparatus.18–21 Interestingly, loss of Dicer activity in the proximal tubule confers resistance to ischemia–reperfusion injury.22

We hypothesized that miRNAs regulate the self-renewal and differentiation of nephron progenitors during kidney development. In this study, we used a conditionally floxed Dicer model to show that the loss of miRNAs in nephron progenitors resulted in a premature depletion of these cells, and as a consequence, a marked decrease in nephron number. This was accompanied by increased apoptosis and elevated expression of the pro-apoptotic protein Bim (also known as Bcl-2L11) in nephron progenitors. Expression profiling in the embryonic kidney identified several miRNAs (mmu-miR-10a, mmu-miR-17-5p, and mmu-miR-106b) that are expressed in nephron progenitors that are known, or are predicted, to target the Bim transcript. Together, these results are consistent with a model in which miRNA-mediated regulation of Bim expression plays an important role in nephron progenitor survival.

RESULTS

Loss of miRNAs in Nephron Progenitors Results in Premature Depletion of Progenitors during Kidney Development

Mesenchymal Six2-expressing cells represent a population of multipotent, self-renewing nephron progenitors during kidney development.23 To define a requirement for miRNAs in progenitors, we generated a mouse model using a floxed Dicer allele11 and a Six2-TGC BAC transgenic allele.23 The Six2-TGC allele expresses a fusion of the green fluorescence protein (GFP) and Cre recombinase behind the Six2 promoter. Cre expression in nephron progenitors was confirmed by simultaneous detection of GFP with immunofluorescence (IF) staining for pan-cytokeratin, which labels the ureteric bud, and for Wt1, a known marker of nephron progenitors (Figure 1, A and B). Excision of the floxed Dicer allele by the Six2-TGC allele would be expected to result in loss of miRNAs within nephron progenitors and their cellular descendants. To verify this prediction, locked nucleic acid in situ hybridization (LNA-ISH) was performed in embryonic day 14.5 (E14.5) kidneys from control Six2-TGCtg/+; DicerFlx/+ and mutant Six2-TGCtg/+; DicerFlx/Flx embryos. Mmu-miR-10a is normally expressed in the ureteric bud and nephron progenitors, and mmu-miR-30a occurs in the early epithelial derivatives of the nephron progenitors, such as the pretubular aggregate (Figure 1, C and E). In Six2-TGCtg/+; DicerFlx/Flx mutant kidneys, there was a loss of mmu-miR-10a in nephron progenitors and mmu-miR-30a in the pretubular aggregate, whereas the mmu-miR-10a expression in the ureteric bud was maintained (Figure 1, D and F). There was also decreased mmu-miR-10a and mmu-miR-30a levels by Northern blot in Six2-TGCtg/+; DicerFlx/Flx kidneys (Supplemental Figure 1).

Figure 1.

Figure 1.

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Loss of mature miRNAs occurs in Six2-TGCtg/+; DicerFlx/Flx kidneys. Visualization of GFP in Six2-TGCtg/+ E14.5 kidneys co-labeled with pan-cytokeratin (A) or Wt1 (B) showed expression of the fusion GFP-cre protein in nephron progenitors. Scale bar, 20 μm. LNA-ISH for mmu-miR-10a or mmu-miR-30a in E14.5 Six2-TGCtg/+; DicerFlx/+ (C and E) and Six2-TGCtg/+; DicerFlx/Flx (D and F) kidneys showed the loss of miRNA expression in nephron progenitors and their derivatives in Six2-TGCtg/+; DicerFlx/Flx mutant kidneys. Scale bar, 50 μm.

Nephron progenitors in the embryonic kidney can be visualized as a “cap” of cells tightly clustered around ureteric bud tips. In mutant Six2-TGCtg/+; DicerFlx/Flx kidneys, nephron progenitors were present at E14.5 (Figure 2, A and B). These cells largely disappeared in mutant kidneys by E16.5, indicating that there is a premature depletion of progenitors (Figure 2, C and D, arrows). There was also a marked decrease in the number of immature nephrons in the mutant kidneys, and the nephrons that do develop have glomeruli that lack capillary loops. The few nephrons that form in Six2-TGCtg/+; DicerFlx/Flx kidneys may represent the cellular descendents of nephron progenitors that escaped Dicer deletion driven by the Six2-TGC allele.

Figure 2.

Figure 2.

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Premature depletion of nephron progenitors in Six2-TGCtg/+; DicerFlx/Flx kidneys during nephrogenesis. Histologic analysis of Six2-TGCtg/+; DicerFlx/+ (A and C) and Six2-TGCtg/+; DicerFlx/Flx (B and D) E14.5 kidney sections and E16.5 showed a marked loss of nephron progenitors in E16.5 mutant kidneys. In each panel, the double-headed arrows delimit the extent of progenitors. Scale bar, 20 μm. Lineage tracing with the Rosa26-lacZ reporter allele showed that β-galactosidase expression remains confined to the nephron lineage in Six2-TGCtg/+; DicerFlx/Flx kidneys (F and H) and is greatly reduced compared with control kidneys (E and G). Scale bar, 50 μm.

To more closely examine the influence of miRNA loss in nephron progenitors, several transcription factors known to be functionally important in nephron progenitors were examined: Six2, Osr1, Eya1, Sall1, Wt1, and Pax2.24–31 The earliest changes in Six2 and Pax2 expression occurred at E14.5, with relative preservation of Wt1 (Supplemental Figure 2). By E16.5, there was a significant decrease in the expression of these transcription factors in nephron progenitors in Six2-TGCtg/+; DicerFlx/Flx kidneys (Figure 3). Together, these results are consistent with the hypothesis that miRNAs are required to maintain nephron progenitors throughout kidney development.

Figure 3.

Figure 3.

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ISH for known transcription factors in nephron progenitors for E16.5 Six2-TGCtg/+; DicerFlx/+ (A, E, I, M, Q, and U) and Six2-TGCtg/+; DicerFlx/Flx (B, F, J, N, R, and V) kidneys reveals decreased expression of Six2, Eya1, Osr1, Wt1, Pax2, and Sall1 in the mutant kidneys. The stromal markers FoxD1 and Pod1 (G–L) remained unchanged in their expression pattern, and a small decrease in Raldh2 expression (C and D) in the cortical stroma of E16.5 Six2-TGCtg/+; DicerFlx/Flx was observed. The expression of the ureteric bud marker, c-ret (O and P), was unchanged. Wnt4 expression was present in the pretubular aggregates of mutant Six2-TGCtg/+; DicerFlx/Flx kidneys (S and T). Scale bar, 50 μm. CM, cap mesenchyme; SM, stromal mesenchyme; UB, ureteric bud; PA, pretubular aggregate.

The interaction between the developing renal stroma and nephron progenitors is critical to the ability of nephron progenitors to differentiate and self-renew. The expression of the cortical stroma marker, FoxD1,32 and the more general stromal marker, Pod1,33 were unchanged in Six2-TGCtg/+; DicerFlx/Flx kidneys (Figure 3). In contrast, Raldh2, another marker of the stroma,34 was slightly decreased in the mutant kidneys. The decreased Raldh2 expression may reflect cross-talk between the nephron progenitor population and the renal stroma. Indeed, loss of retinoic acid signaling in Rara−/−; RarB2−/− mice has previously been shown to result in abnormal stromal patterning.35

The tips of the branching ureteric buds retain c-ret expression, and there is some degree of epithelial differentiation, as measured by the presence of Wnt4 (Figure 3).36 The overall number of Wnt4-positive renal vesicles and the complexity of individual Wnt4-expressing structures is similar between control and mutant at embryonic day 16. However, histologically apparent renal vesicles are greatly reduced in number in mutants by postnatal day 0 (Supplemental Figure 3). This may reflect either a dimunition in the progenitor pool or a specific role for miRNAs in differentiation of the nephron. Resolving this question will require conditional mutation of Dicer in the pretubular aggregate or renal vesicle.

Increased Apoptosis in Nephron Progenitors of Six2-TGCtg/+; DicerFlx/Flx Kidneys

There are several potential explanations for the premature loss of nephron progenitors in Six2-TGCtg/+; DicerFlx/Flx kidneys. First, nephron progenitors may become renal stroma, either through a cell fate switch or a loss of commitment to the nephron lineage in the absence of miRNAs. To test this hypothesis, the Rosa26-lacZ reporter allele37 was crossed into the control and mutant kidneys. Activation of the β-galactosidase reporter would be expected to occur in any cells that had expressed the Cre recombinase and their cellular descendants. Consistent with previously published results,23 β-galactosidase expression was present in nephron progenitors and developing nephrons in control kidneys (Figure 2, E and G). In the Six2-TGCtg/+; DicerFlx/Flx kidneys, β-galactosidase expression continued to be confined to the nephron lineage, indicating that Six2-expressing cells are not lost through a conversion to the stromal lineage (Figure 2, F and H). This experiment also confirms the premature depletion of progenitors in mutant kidneys.

Other explanations for this observation include a failure in the ability of nephron progenitors to self-renew or increased apoptosis. Proliferating cells were identified with bromodeoxyuridine (BrdU) labeling in the developing kidney. There was no obvious gross difference between BrdU+ cells in the nephron progenitor population of control compared with mutant kidneys, indicating that decreased self-renewal of nephron progenitors is not sufficient to explain the phenotype seen in Six2-TGCtg/+; DicerFlx/Flx kidneys (Figure 4, A and B).

Figure 4.

Figure 4.

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Increased apoptosis in the nephrogenic zone occurs in Six2-TGCtg/+; DicerFlx/Flx kidneys. Proliferating cells in E15.5 kidneys were identified by BrdU uptake. The number of BrdU+ nephron progenitors was similar between Six2-TGCtg/+; DicerFlx/+ (A) and Six2-TGCtg/+; DicerFlx/Flx (B) kidneys. The ureteric bud is outlined in red. Scale bar, 50 μm. Increased TUNEL+ (green) cells occurred in the nephrogenic zone of Six2-TGCtg/+; DicerFlx/Flx (D and F) compared with Six2-TGCtg/+; DicerFlx/+ (C and E) kidneys. Co-labeling with Pax2 (red) marked the ureteric bud and metanephric mesenchyme. Scale bars: 100 (C and D) and 50 μm (E and F). (G) (Left) Representative DNA content histogram plots for E15.5 Six2-TGCtg/+; DicerFlx/+ and Six2-TGCtg/+; DicerFlx/Flx nephron progenitors, as determined by propidium iodide staining for DNA content and subsequent dual-color flow cytometric analysis gating on GFP+ nephron progenitors. The M1 gate denotes DNA content <2n. (Right) Frequency (%) of <2n DNA-containing components (i.e., apoptotic cells or cellular fragments) in Six2-TGCtg/+; DicerFlx/+ and Six2-TGCtg/+; DicerFlx/Flx nephron progenitors, determined by propidium iodide staining and dual-color flow cytometry (mean ± SE, for three independent experiments). There was a significant increase in the frequency of cells with <2n content in Six2-TGCtg/+; DicerFlx/Flx nephron progenitors (17 ± 3.1%) compared with Six2-TGCtg/+; DicerFlx/+ nephron progenitors (3.6 ± 1.5%; P < 0.02; t test; n = 10 embryos for each of three independent experiments).

Apoptosis in nephron progenitors was measured using two methods: the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and the determination of DNA content in GFP+ nephron progenitors by propidium iodide (PI) staining with dual color flow cytometry. TUNEL+ cells were largely limited to the developing renal stroma in control kidneys at E14.5 (Figure 4, C and E). In contrast, TUNEL+ cells were increased in the nephrogenic zone of Six2-TGCtg/+; DicerFlx/Flx kidneys (Figure 4, D and F). When Six2-TGCtg/+; DicerFlx/Flx sections were co-labeled with Pax2, which marks the ureteric bud and nephron progenitors, TUNEL+ cells were found among both the nephron progenitors and the stroma (Figure 4F). To assess this in a quantitative fashion, the DNA content of nephron progenitors was determined by dual color flow cytometry for GFP and PI staining (Figure 4G). The M1 gate denotes cells or cellular fragments with a DNA content of <2n in control and mutant nephron progenitors. There was a significant increase in the frequency of cells with <2n content in Six2-TGCtg/+; DicerFlx/Flx nephron progenitors (17 ± 3.1%) compared with Six2-TGCtg/+; DicerFlx/+ nephron progenitors (3.6 ± 1.5%; P < 0.02; t test, n = 10 embryos in each of three independent experiments). Together, these results are consistent with the hypothesis that increased apoptosis accounts for the loss of nephron progenitors in Six2-TGCtg/+; DicerFlx/Flx kidneys.

Identification of miRNA–mRNA Target Interactions in Nephron Progenitors

To identify miRNAs that are expressed during kidney development, miRNA expression in the embryonic kidney was profiled by LNA miRNA microarray (Exiqon), and these findings were compared with those of a recent large-scale mammalian small RNA cloning project.38 Eleven miRNAs that were identified by microarray and the small RNA cloning project were selected for further validation by Northern blot (Figure 5A)19 and LNA-ISH (Figure 5B). As expected, a number of these miRNAs were ubiquitously expressed in the developing kidney (mmu-let-7c, mmu-miR-130a, and mmu-miR-335), whereas others had a more specific expression pattern (mmu-miR-10a, mmu-miR-17-5p, mmu-miR-23b, mmu-miR-24, mmu-miR-26a, mmu-miR-30a, mmu-miR-30c, and mmu-miR-106b). Apart from the ubiquitously expressed miRNAs, there were several miRNAs present in nephron progenitors: mmu-miR-10a, mmu-miR-17-5p, mmu-miR-26a, and mmu-miR-106b.

Figure 5.

Figure 5.

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miRNA expression profiled in the embryonic kidney. (A) Northern blots for mmu-miR-30a, mmu-miR-106b, mmu-miR-130a, and mmu-miR-335 in a panel of wild-type adult and embryonic tissues confirmed the presence of these miRNAs in the embryonic kidney. 5S rRNA, loading control. (B) Expression of mmu-let-7c, mmu-miR-10a, mmu-miR-17-5p, mmu-miR-23b, mmu-miR-24, mmu-miR-26a, mmu-miR-30a, mmu-miR-30c, mmu-miR-106b, mmu-miR130a, and mmu-miR-335 in E14.5 wild-type CD-1 kidneys by LNA-ISH. Note an air bubble in the mmu-miR-10a figure. Scrambled oligonucleotide, negative control. Scale bar, 50 μm.

There is an emerging consensus that the key feature of miRNA target recognition is sequence complementarity in target mRNAs to the eight-nucleotide “seed” sequence of miRNAs.39 Conservation of specific miRNA target sites in mRNAs across species is also predictive of biologically relevant target mRNAs.40 Many bioinformatic tools have been developed to predict miRNA targets; however, individual tools often predict several hundred mRNA targets per miRNA, and its not yet apparent how many are true biologic interactions. One approach has been to use several tools in combination to increase the specificity of target predictions.41 The targets of the top 50 miRNAs expressed in E14.5 kidney by microarray were predicted using several well-described bioinformatics tools: MicroT, TargetScan, MiRanda, and MAMI.40,42–44 A total of 5920 unique miRNA–mRNA target interactions were predicted by more than one bioinformatics tool (Supplemental Table 1) and 75 unique interactions by all four tools (Supplemental Table 2).

These predictions were matched against the recent description of the nephron progenitor transcriptome at E11 and E15 by Brunskill et al.45 to generate a list of potential miRNA–mRNA interactions that can be experimentally validated. In the E11 kidney, a total of 135 miRNA–mRNA target interactions and 77 unique miRNA targets were predicted by two or more tools (Supplemental Table 3). Similarly in E15 kidneys, a total of 136 miRNA–target interactions and 77 unique miRNA targets were identified (Supplemental Table 4). These analyses identified several potential miRNA targets among the genes known to be required in the nephron progenitor population, such as Sall1, Osr1, and Eya1. However, none of these genes showed elevated expression in mutant kidneys (Figure 3), as would be predicted if these were biologically significant miRNA targets. The examination of other potential targets will be the subject of future studies.

Bim Expression Is Increased in Nephron Progenitors That Lack miRNAs

Of the potential miRNA targets identified, the pro-apoptotic protein Bim was a prime candidate (Figure 6A). Bim is a known miRNA target of the miR clusters: mmu-miR-106b∼25 and mmu-miR-17∼92.46,47 Bim exists in three common splice variants: BimS, BimL, and BimEL, with the Bims isoform being the most effective at promoting cell death. Western blot analysis of embryonic kidney lysates at different developmental stages showed elevated BimEL in mutant Six2-TGCtg/+; DicerFlx/Flx kidneys compared with controls at E15.5 and E16.5 (Figure 6B). By postnatal day 0, there were few remaining nephron progenitors in the mutant kidneys, and the overall Bim levels become similar in mutant and control kidneys. The expression of the pro-survival protein Bcl-2 remained constant in control and mutant kidneys across the different time points. Immunohistochemical staining substantiated an increase in Bim protein expression in nephron progenitors in Six2-TGCtg/+; DicerFlx/Flx kidneys compared with controls (Figure 6C). Interestingly, both Bcl-2 and Bim expression are largely confined to the nephron progenitor population, suggesting that the regulation of cell survival may be an important level at which progenitor populations are regulated during nephrogenesis.

Figure 6.

Figure 6.

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The expression of the candidate miRNA target, Bim, is increased in Six2-TGCtg/+; DicerFlx/Flx mutant kidneys. (A) Sequence alignment of the Bim 3′UTR and mmu-miR-10a, mmu-miR-17-5p, and mmu-miR-106b with two to three conserved target site matches for each miRNA (nucleotides in blue possess Watson-Crick pairing between the miRNA and Bim 3′UTR, whereas nucleotides in red are unpaired). (B) Western blot analysis showed an increase in the BimEL isoform in Six2-TGCtg/+; DicerFlx/Flx (M) kidneys compared with control kidneys (C) at E15.5 and E16.5. The BimL and BimS isoforms were relatively unchanged, as were Bcl2 and GAPDH. (C) Immunohistochemical staining for Bim and Bcl2 showed increased Bim levels specifically in nephron progenitors in mutant Six2-TGCtg/+; DicerFlx/Flx kidneys, whereas Bcl2 expression was constant. Scale bar, 20 μm.

DISCUSSION

Mammalian nephrogenesis proceeds through an iterative series of events, as nephron progenitors are induced at ureteric bud tips to form nephrons. We generated a mouse model that lacks miRNAs in Six2-expressing cells to show a requirement for miRNAs in the survival of nephron progenitors. The early depletion of nephron progenitors caused by apoptosis in Six2-TGCtg/+; DicerFlx/Flx kidneys is consistent with the findings recently reported by Nagalakshmi et al.48 This study extends those observations, with the initial description of miRNAs expressed during kidney development and the demonstration of increased expression of the pro-apoptotic Bim protein in the mutant kidneys. Furthermore, we identified several miRNAs (mmu-miR-10a, mmu-miR-17-5p, and mmu-miR-106b) in nephron progenitors that are predicted to target Bim. These results are consistent with a model in which miRNAs promote the survival of nephron progenitors by regulating the expression of Bim, as a means of regulating nephron endowment during kidney development.

Bim is a member of the large Bcl-2 family of pro-apoptotic and anti-apoptotic proteins, which share homology in conserved regions known as Bcl-2 homology domains.49,50 In broad terms, the determination of whether a cell undergoes apoptosis or survives is dependent on the pairing between Bcl-2 family members that promote cell death and those that promote survival. Bim binds to many of the Bcl-2 pro-survival proteins and is thought to release Bax or Bak proteins from their interaction with Bcl-2 to promote apoptosis.50

Bim null mice die at several months of age because of immune complex glomerulonephritis, which has been attributed to increased circulating plasma cells.51 Bcl-2–null mice show cystic kidney disease and apoptosis in nephron progenitors.52–54 Interestingly, the loss of a single Bim allele in Bcl-2–null mice is sufficient to rescue the phenotype of Bcl-2–null mice, suggesting that the gene dosage of Bim is critical during nephrogenesis.52 Given the specific expression of Bim and Bcl-2 in nephron progenitors (data herein52,55) and the observation that increased Bim expression occurs in association with apoptosis in the Six2-TGCtg/+; DicerFlx/Flx kidneys, it is intriguing to speculate that the balance between Bim and Bcl-2 activity in nephron progenitors regulates the survival of this cell population. In addition, this suggests a hypothesis implicating the survival of nephron progenitors in the cystic phenotype of Bcl-2–null mice.

Bim is a known miRNA target of the miR-17∼92, miR-106b∼25, and miR-106a∼363 clusters, which are likely derived from a unique gene that has evolved to result in three primary miRNA transcripts that express related miRNAs.56 The mmu-miR-106a∼363 cluster seems to be rarely expressed in adult tissues, and its functional role remains obscure. In contrast, both the miR-17∼92 and miR-106b∼25 clusters have been implicated in oncogenesis, and miR-17∼92 in particular is often amplified in lymphomas.57 Overexpression of the miR-17∼92 transcript in the B and T cell compartments of transgenic mice leads to suppression of Bim expression.47 Targeted deletion of miR-17∼92 results in elevated Bim levels and inhibition of B cell development.46

Aside from their importance in regulating Bim, members of both miRNA clusters have recently been described as differentially expressed in the blastocyst and germ layers of the early mouse embryo, and it has been suggested that these miRs regulate embryonic stem cell differentiation through the modulation of STAT3 in vitro.58 Transgenic overexpression of mmu-miR-17∼92 in embryonic lung epithelium promotes the persistence of a highly proliferative epithelial progenitors at the expense of differentiated proximal epithelial cells.59 These studies are suggestive of a role for these miRNA clusters in the regulation of stem cell populations, perhaps in part through their ability to modulate Bim activity.

Of the miRNAs identified in the expression profiling experiments, mmu-miR-10a possesses the most specific expression pattern in nephron progenitors and the branching ureteric bud. In a recent screen for miRNAs that modulate apoptosis in vitro, mmu-miR-10a was suggested to upregulate caspase-3 activity, an important downstream effector of apoptosis.60 In the zebrafish, dre-miR-10 has been shown to regulate Hox gene expression along the anterior–posterior axis.61 Mir-10b has also been implicated in tumor metastasis and invasiveness through the inhibition of HoxD10 and subsequent increase in RhoC expression.62 This last observation raises the question of whether mmu-miR-10a may be involved in regulating the mesenchymal to epithelial transition that nephron progenitors undergo to form developing nephrons.

In summary, our study provides evidence for a model in which miRNA-mediated regulation of the pro-apoptotic protein Bim affects the survival of nephron progenitors during kidney development. This mechanism represents a novel means by which congenital nephron endowment is determined and has significant implications for our understanding of the maintenance of this critical progenitor population during development.

CONCISE METHODS

Mouse Strains

Homozygous mice for the floxed Dicer allele11 were mated to mice carrying a Six2-TGC transgene.23 The Six2-TGC allele is a BAC transgenic allele using the Six2 promoter to drive a Tet-off-eGFPCre cassette; however, doxycycline fails to silence Cre activity in these mice.23 The resulting embryos were genotyped as described.19 Embryos with a wild-type Dicer allele or without the Six2-TGC allele were used as littermate controls. Lineage tracing was performed with the Rosa26-lacZ reporter allele.37 The Six2-TGCtg/+; Cre and the DicerFlx mice were maintained on a mixed background, whereas the Rosa26-lacZ reporter allele was on a C57/Bl6 background. The animal experiments were carried out in accordance with the policies of the Institutional Animal Care and Use Committee at Children's Hospital Boston.

Immunofluorescence

Kidneys from Six2-TGC embryos were frozen in Tissue-TEK 4583 OCT (Sakura Finetek, Torrance, CA) in a liquid nitrogen–cooled isopentane bath. To visualize GFP expression, the kidneys were fixed briefly in 4% paraformaldehyde before embedding in OCT. IF staining on cryosections was performed as described.19 The primary antibodies used were as follows: mouse monoclonal anti-pan-cytokeratin antibody 1:50 (Sigma Aldrich, St. Louis, MO), rabbit anti-Wt1 antibody 1:100 (Santa Cruz Biotechnologies, Santa Cruz, CA), and rabbit anti-Pax2 antibody 1:500 (Dr. Gregory Dressler, University of Michigan). The secondary antibodies used were as follows: goat anti-rabbit Texas Red or donkey anti-mouse Texas Red at 1:200 (Jackson Immunoresearch Laboratories, West Grove, PA). The slides were visualized with a Nikon Eclipse 80i microscope and photographed with a Qimaging Retiga 2000R Fast 1394 camera using NIS-Elements Basic Research 2.34 software (Micro Video Instruments, Avon, MA).

Histopathology and β-Galactosidase Staining

Kidneys from control littermates (Six2-TGCtg/+; DicerFlx/+ or DicerFlx/Flx) and mutant (Six2-TGCtg/+; DicerFlx/Flx) mice were paraffin-embedded and stained with hematoxylin and eosin. For β-galactosidase staining, the embryonic kidneys were fixed in 0.2% glutaraldehyde/2% formaldehyde/2 mM magnesium chloride, washed in PBS, cryopreserved in 30% sucrose/PBS, and frozen for cryosections. The cryosections were stained for β-galactosidase and counterstained with eosin.

In Situ Hybridization

Riboprobes were obtained for Six2 (G. Oliver, St. Jude Children's Research Hospital), Eya1 (R. Maas, Harvard Medical School), Osr1 (P. Danielian, Chester Beatty Laboratories), Wt1,63 Pax2 (G. Dressler, University of Michigan), Sall1 (Nishinakamura, Kumamoto University), FoxD1 (C. Mendelsohn, Columbia University), Pod1 (S. Quaggin, University of Toronto), raldh2 (A. McMahon, Harvard University), c-ret (F. Costantini, Columbia University), and wnt-4 (A. McMahon, Harvard University). Sense and antisense probes were synthesized and labeled with digoxigenin-UTP (Roche, Indianapolis, IN). The protocol for ISH was as described.64

Proliferation and Apoptosis

Timed pregnant females from Six2-TGCtg/+; DicerFlx/+ X DicerFlx/Flx matings were injected with bromodeoxyuridine (BrdU) at a dose of 100 μg/g body weight at E15.5 (Sigma-Aldrich). Embryonic kidneys were harvested 1 hour after injection and processed for paraffin-embedded sections. BrdU IHC was performed with an anti-BrdU-POD–conjugated antibody (Roche) with the IHC protocol described below.

TUNEL staining was performed with the Apoptag Plus Fluorescein In Situ Apoptosis Detection kit, as per the manufacturer's directions (Millipore, Billerica, MA) on control and mutant kidney cryosections at E14.5 and E16.5. Co-staining with a rabbit anti-Pax2 antibody (Dr. Gregory Dressler, University of Michigan) was performed sequentially after the TUNEL staining with the IF protocol above.

For flow cytometry analysis, E15.5 kidneys were dissected from control (Six2-TGCtg/+; DicerFlx/+) and mutant (Six2-TGCtg/+; DicerFlx/Flx) embryos. The kidneys were individually dissociated in Dispase 1 mg/ml (Life Technologies, Invitrogen, Carlsbad, CA) for 10 minutes at 37°C and fixed in 0.25% paraformaldehyde and then cold 65% (vol/vol) ethanol/PBS. Kidney cells isolated from the same genotype were pooled (from five to six embryos), washed in cold PBS, and stained with PI (50 mg/ml, 0.1% Triton X-100, 0.1 mM EDTA, and RNaseA 100 units/ml). The cell fraction containing <2n DNA was determined by flow cytometry (Becton Dickinson FACScan) as described previously.65 The frequency of cellular fragments, nonviable cells, and/or contaminating blood components containing <2n DNA was compared between pooled control and mutant kidneys for three independent experiments. The SEM was calculated, and the results were analyzed with the t test.

miRNA Expression

Total RNA was extracted from E14.5 kidneys using the Qiagen miRNeasy Mini kit (Qiagen, Valencia, CA). The expression of embryonic kidney miRNAs was profiled using Exiqon's miRCURY LNA Array microRNA profiling services (Exiqon, Vedbaek, Denmark).66,67 RNA samples were labeled with Hy3 and hybridized in triplicate to miRCURY LNA Arrays Version 8.1. The quantified signals were normalized with the global Lowess regression algorithm. Spike-in controls confirmed that labeling and hybridization to the arrays was successful. The miRNAs expressed in embryonic kidney were listed in descending order based on the average signal intensities for each miRNA from the three replicates (Supplemental Table 6). The fold increase in signal intensity over background was calculated, and a two-fold change in Hy3 signal intensity above background was used as the lower cut-off for the listed miRNAs. To confirm the similarity between the three replicates, scatterplots were performed using the log2-transformed average signal intensities of data from chip1 versus chip2, chip2 versus chip3, and chip3 versus chip1 (Supplemental Figure 4). The microarray results will be made publicly available through ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/).

The expression of individual miRNAs was confirmed by Northern blot and LNA-ISH as described previously except that the proteinase K step was shortened to 10 min.19 In brief, total RNA was isolated with Trizol (Invitrogen), run on a 15% Tris-borate-EDTA-urea polyacrylamide gel (BioRad, Hercules, CA), transferred to an Amersham Hybond N+ nylon membrane (GE Life Sciences, Piscataway, NJ), and hybridized overnight with P32-labeled oligonucleotides complementary to the mature miRNA sequence (Supplemental Table 7). The membrane was exposed to Kodak BioMax MS film (Sigma).

LNA probes complementary to the mature miRNA with approximately 30% LNA base insertions and a predicted Tm of 65 to 70°C were designed using LNA design tool (Integrated DNA Technologies, Coralville, IA) (Supplemental Table 7). A scrambled LNA oligo was used as a negative control (Exiqon).

Bioinformatic Analysis

Potential targets of the top 50 miRNAs from the miRNA microarrays were first individually mapped using four tools: TargetScan, miRBase/miRanda, microT, and MAMI.40,42–44 Custom written PERL scripts were used to generate predicted miRNA targets based on their identification by two, three, or all four target prediction algorithms.41 These targets were mapped against transcripts that had been reported in nephron progenitors at E11 and E15.45

Western Blot

Control and mutant embryonic kidneys were homogenized in high salt radioimmunoprecipitation assay buffer containing 500 mM NaCl, run on a reducing 12% SDS-PAGE, and blotted to an Immun-Blot polyvinylidene difluoride membrane (BioRad). The primary antibodies used were as follows: a rabbit anti-Bim antibody (Cell Signaling, Beverly, MA) at a concentration of 1:1000, a mouse monoclonal anti-Bcl2 antibody (Santa Cruz Biotechnologies), and a rabbit anti-GAPDH antibody (Abcam, Cambridge, MA). The signals were detected with a goat anti-rabbit-POD secondary antibody at 1:30,000 or goat anti-mouse-POD secondary antibody at 1:10,000 (GE Healthcare, Piscataway, NJ) and the Amersham ECL Advance Western Blotting detection kit (GE Healthcare) as per the manufacturer's directions.

IHC

Paraffin-embedded sections from control littermates and mutant mice at E14.5 and E16.5 were stained using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's directions. In brief, slides were deparaffinized in xylene and rehydrated, and antigen retrieval was performed by boiling in 10 mM sodium citrate buffer with 0.05% Tween-20. The slides were incubated with rabbit anti-Bim antibody 1:100 (Cell Signaling) or mouse monoclonal anti-Bcl2 antibody 1:100 (Santa Cruz Biotechnologies) at 4°C overnight. Antibody staining was visualized using Sigma Fast 3,3′-diaminobenzidine tablets as directed (Sigma Aldrich).

DISCLOSURES

None.

Acknowledgments

This work was supported by a National Institutes of Diabetes and Digestive and Kidney Diseases grant to J.A.K. J.H. and J.A.K. acknowledge the support of the Harvard Stem Cell Institute. J.H. and S.H. were supported by Kidney Research Scientist Core Education and National Training Program (KRESCENT) postdoctoral fellowships, and J.H. was a Fellow of the Pediatric Scientist Development Program (supported by the March of Dimes, Pediatric Chairs of Canada, and the SickKids Foundation). T.S. is the recipient of a Postdoctoral Fellowship Award from the American Heart Association Founders Affiliate. The authors thank Dr. Clifford Tabin (Harvard Medical School) for the DicerFlx/Flx mice, Dr. Andrew McMahon (Harvard University) for the Six2-TGC mice, and Dr. Gregory Dressler (University of Michigan) for the Pax2 antibody. We thank the Rodent Histopathology Core Facility of the Dana-Farber/Harvard Cancer (NIH-P30-CA-06516) for their technical support. This work was previously presented in part at the annual meeting of the American Society for Nephrology, November 2–5, 2007, San Francisco, CA, and was published in abstract form.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

Supplemental information for this article is available online at http://www.jasn.org/.

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