RESEARCH ARTICLE

Histone deacetylase 1 and 2 regulateWnt and p53 pathways in theureteric bud epitheliumShaowei Chen1, Xiao Yao1, Yuwen Li1, Zubaida Saifudeen1, Dimcho Bachvarov2 and Samir S. El-Dahr1,*

ABSTRACTHistone deacetylases (HDACs) regulate a broad range of biologicalprocesses through removal of acetyl groups from histones as well asnon-histone proteins. Our previous studies showed that Hdac1 andHdac2 are bound to promoters of key renal developmental regulatorsand that HDAC activity is required for embryonic kidney geneexpression. However, the existence of many HDAC isoforms inembryonic kidneys raises questions concerning the possiblespecificity or redundancy of their functions. We report here thattargeted deletion of both the Hdac1 and Hdac2 genes from theureteric bud (UB) cell lineage of mice causes bilateral renalhypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient tosustain normal renal development. In addition to defective cellproliferation and survival, genome-wide transcriptional profilingrevealed that the canonical Wnt signaling pathway is specificallyimpaired inUBHdac1,2−/− kidneys.Our results alsodemonstrate that lossof Hdac1 and Hdac2 in the UB epithelium leads to markedhyperacetylation of the tumor suppressor protein p53 on lysine 370,379 and 383; these post-translational modifications are known to boostp53 stabilityand transcriptional activity.Genetic deletionofp53partiallyrescues the development ofUBHdac1,2−/− kidneys. Together, these dataindicate that Hdac1 and Hdac2 are crucial for kidney development.They perform redundant, yet essential, cell lineage-autonomousfunctions via p53-dependent and -independent pathways.

KEY WORDS: Branching morphogenesis, Histone, Kidney, Uretericbud, Wnt, p53 (Trp53), Mouse

INTRODUCTIONHistonedeacetylases (HDACs) are a superfamilyof enzymes importantfor modulation of chromatin structure and function via removal ofacetyl moieties from lysine residues of histones as well as non-histoneproteins. In higher eukaryotes, 18 HDAC isoforms have beenidentified. Based on sequence homology to yeast HDAC genes, theyare divided into four classes: the class I RPD3-likeHDACs (1-3 and 8);the class II HDA1-like HDACs (4-7, 9 and 10); Sirtuins 1-7; and theclass IVHDAC11 (de Ruijter et al., 2003;Denu andGottesfeld, 2012).HDACs were originally assumed to be global transcriptional

co-repressors, but it subsequently became clear that HDACs regulategene expression in a highly selective manner and exhibit bothrepressive and activating effects. HDAC inhibition or deletion ofHDAC genes often results in alterations in a small subset of genes(<10%), and approximately as many are downregulated as areupregulated (Smith, 2008). Mechanistically, HDACs themselves

lack intrinsic DNA binding activity and are recruited to target genesthrough association with various transcriptional complexes (e.g. theSin3, NuRD, Co-REST and SMRT/N-CoR complexes) (Markset al., 2003). As such, the specificity of HDACs in gene regulationdepends on the partner proteins that they associate with underdifferent pathophysiological conditions. In addition to histones,HDACs can also deacetylate an increasing number of non-histoneproteins, including p53, STAT3, Yin Yang transcription factor(YY1), GATA1, E2F1 and Hsp90 (Glozak et al., 2005; Kruse andGu, 2009; Spange et al., 2009). Deacetylation of these proteins hasbeen shown to affect multiple aspects of their function, such asprotein stability, DNA binding affinity and transcriptional activity,adding an extra layer of complexity to the biological roles ofHDACs.

In line with recent studies showing that HDACs play both uniqueand redundant roles in the control of distinctive developmentalpathways during embryogenesis, our previous studies revealed thatthe expression of key developmental renal regulators (e.g. Osr1,Eya1, Pax2/8, Wt1 and Wnt9b) is dependent on intact HDACactivity (Chen et al., 2011; Haberland et al., 2009). Treatment ofcultured mouse embryonic kidneys with Scriptaid, a generalinhibitor of class I and II HDACs, or MS-275, a selective inhibitorof HDAC1-3, impairs ureteric bud (UB) branching morphogenesisand nephrogenesis, two central processes of metanephricdevelopment. Our results also demonstrated that Hdac1-3 areenriched in the undifferentiated metanephric mesenchyme (MM)and UB branches, but are reduced upon differentiation, implicatingtheir crucial role in mouse kidney development.

Among class I and class II HDACs, HDAC1 and HDAC2 areevolutionarily close to each other, sharing a high degree of sequencesimilarity (∼85% identity at the amino acid level) except for theirC-terminal domain. HDAC1 and HDAC2 can form homo- orheterodimers and are found together in almost all nuclear proteincomplexes, including three well-characterized co-repressorcomplexes: Sin3, NuRD and Co-REST (Brunmeir et al., 2009).Studies using a variety of genetic models of Hdac1 and Hdac2indicate that they exert both overlapping and non-redundantfunctions in different cell types at specific developmental stages(Haberland et al., 2010; Lagger et al., 2002; LeBoeuf et al., 2010;Ma et al., 2012; Montgomery et al., 2007; Ye et al., 2009). In thisstudy, by deleting different combinations of Hdac1 and Hdac2alleles in the UB cell lineage, we revealed the redundant yetessential cell-autonomous functions of Hdac1 and Hdac2 in theureteric epithelium during mouse kidney development. Moreover,our findings illustrate the developmental importance of HDAC-mediated control of p53 acetylation.

RESULTSConcurrent deletion of Hdac1 and Hdac2 in the UB celllineage causes renal hypodysplasiaIn order to delete the Hdac1 and Hdac2 genes specifically fromthe UB lineage, we crossed Hdac1flox/flox, Hdac2flox/flox andReceived 28 May 2014; Accepted 28 January 2015

1Department of Pediatrics, Section of Pediatric Nephrology, Tulane UniversityHealth Sciences Center, New Orleans, LA 70112, USA. 2Department of MolecularMedicine, Laval University, Quebec, QC, Canada G1R 2J6.

*Author for correspondence (seldahr@tulane.edu)

1180

© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

Hoxb7-CreEGFP transgenic mice (Montgomery et al., 2007; Zhaoet al., 2004). Previous studies have shown that Hoxb7-directed GFPexpression is observed in the Wolffian duct at embryonic day (E)10.0 and its derivatives, the UB and its branches, but not in the MMlineage (Zhao et al., 2004). To test the efficacy of Hoxb7-drivenCre-mediated excision, we examined the expression of Hdac1 andHdac2 proteins by immunohistochemistry in wild-type and mutantkidney tissues at E13.5. Consistent with our previous report, Hdac1and Hdac2 are expressed in both the UB and MM cells in wild-typemice (Fig. 1A,E). By contrast, in UBHdac1,2−/− mice (Hoxb7-Cretg/+;Hdac1flox/flox; Hdac2flox/flox), Hdac1 and Hdac2 are not detected in

the UB cells but are maintained in the surrounding mesenchymalcells (Fig. 1B,F). In accordance with the key functions of Hdac1 andHdac2 in histone deacetylation, the acetylation levels of histone H3(lysine 9 and 14, or lysine 9 specifically) and H4 (lysine 5, 8, 12 and16) are substantially increased in the ureteric cells of UBHdac1,2−/−

kidneys (Fig. 1I-N). Collectively, these results demonstrate efficientdeletion of Hdac1 and Hdac2 from the UB branches. It is worthnoting that knockout of Hdac1 and Hdac2 in oocytes leads to noapparent change in histone H3K9 acetylation (Ma et al., 2012).Thus, our results suggest that Hdac1 and Hdac2might have differenthistone residue specificity in different cell types at specificdevelopmental stages.

Our results revealed that mice with no more than three deletedalleles of Hdac1 and Hdac2 exhibit no significant abnormalities inkidney development (supplementary material Fig. S1); moreover,these mice survive to adulthood without any overt abnormalities ingrowth or development. By contrast, concurrent deletion of all fouralleles of Hdac1 and Hdac2 results in early postnatal lethality by2-4 weeks of age (supplementary material Fig. S2). Histologicalanalysis of kidney tissue from UBHdac1,2−/− mice at postnatal day(P) 0 showed absence of the nephrogenic zone, lack of cortico-medullary patterning, and the formation of multiple epithelial cysts(Fig. 2A-F). In line with the histological observations,immunofluorescence staining demonstrated that the UBHdac1,2−/−

neonates completely lack Six2-positive and Pax2-positive cells(Fig. 2G-J).

UBHdac1,2−/−kidneysexhibit stuntedUBgrowthandbranchingTo begin to define the embryological mechanisms leading to thisphenotype, we monitored UB morphogenesis in a real-time mannerin vivo and in vitro, taking advantage of the GFP fluorescence in theUB tissue driven by the Hoxb7 promoter. UBHdac1,2−/− miceexhibited attenuated UB branching as early as E13.5 (Fig. 3A), andstarted to show degeneration of the UB tissue after 1-2 days inculture (Fig. 3B). Previous studies have shown that Hdac1 andHdac2 regulate apoptosis and proliferation in a wide range of cells.To examinewhether increased apoptosis and decreased proliferationcontribute to the observed defects in UB branching morphogenesis,we examined the status of cell apoptosis and proliferation at E13.5,when the abnormal UB branching is first noted. Quantification ofactive caspase 3 (aCasp3)-positive cells revealed a significantincrease in the number of apoptotic UB cells in UBHdac1,2−/− versuswild-type kidneys at E13.5 (Fig. 4A-E). Consistently, staining ofphospho-histone γH2AX, a marker of DNA double-strand breaks,showed that UBHdac1,2−/− kidneys exhibited increased DNAdamage in UB cells (Fig. 4F-I). Analysis of phospho-histone H3(pH3), a marker of mitosis, revealed that the rate of cell proliferationwas decreased by 37.5% in UBHdac1,2−/− relative to wild-typekidneys (Fig. 4J-N). This is consistent with the known pro-proliferative functions of Hdac1 and Hdac2. Taken together, theseresults indicated that Hdac1 and Hdac2 are crucial for UB cellgrowth, survival and branching morphogenesis.

Genome-wide transcriptome analysis of UBHdac1,2−/− kidneysTo further elucidate the developmental pathways regulated by Hdac1and Hdac2, we carried out a genome-wide microarray analysis onRNA samples extracted from wild-type and UBHdac1,2−/− kidneys atE13.5. The raw and analyzed data have been deposited in the NCBIGene Expression Omnibus (GEO) under accession numberGSE35432. The results revealed that 496/41,000 probes (∼1.2%)are significantly altered in UBHdac1,2−/− kidneys (by ≥1.4-fold,P<0.05, n=4), of which 226 transcripts (0.55%) were upregulated

Fig. 1. UB-specific deletion of Hdac1 and Hdac2 causes hyperacetylationof histones H3 and H4 at E13.5. Wild-type (WT) and UBHdac1,2−/− kidneysectionswere immunostainedwith antibodies against Hdac1 (A,B),Hdac2 (E,F),acetylated histone H3 (I,J), histone H3 acetylated at K9 (K,L), acetylatedhistone H4 (M,N), cytokeratin (CK) (A,B,E,F,I-N) and with DAPI (C,D,G,H).UB, ureteric bud.

1181

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

(range 1.4- to 7.5-fold) and 270 (0.66%) downregulated (range 1.4-to 3.8-fold) (Fig. 5A; see GSE35432).To analyze whether certain pathways or biological processes are

especially sensitive to the loss of Hdac1 and Hdac2 in the UB cells,Ingenuity Pathway Analysis (IPA) was performed on thedifferentially expressed transcripts. This analysis indicated that themost significantly enriched genes participate in: (1) cellmorphology; (2) cellular growth and proliferation; (3) cellulardevelopment; (4) cell death and survival and (5) cellular movement(Fig. 5B). The most affected canonical pathways include: (1) basalcell carcinoma signaling; (2) Wnt/β-catenin signaling; (3) sonichedgehog signaling; (4) human embryonic stem cell pluripotencyand (5) tight junction signaling (Fig. 5C). The complete list of genes

for each category and pathway is shown in supplementary materialTables S1 and S2.

Further analysis using the Biological Networks Gene Ontology(BiNGO) tool revealed that many genes involved in: (1) tubedevelopment; (2) Wnt receptor signaling pathway; (3) ureteric buddevelopment; (4) cell-cell adhesion; (5) kidney development and (6)positive regulation of cell proliferation are downregulated inUBHdac1,2−/− kidneys (Table 1). Interestingly, we found decreasedexpression of a group of cytokeratins, including Krt7, Krt8, Krt18,Krt19 and Krt23 (Table 2), which is consistent with ourimmunostaining results using a pan-cytokeratin antibody (Fig. 1I-N).Cytokeratins, the largest intermediate filament protein group, arerecognized as peptide fingerprints for the classification of epithelialcells and are implicated in cytoplasmic organization and cellularcommunication. In embryonic kidneys, Krt8 and Krt18 arespecifically expressed in the ureteric epithelium, whereas Krt23 isspecifically expressed in the UB tips.

We validated the microarray results by quantitative real-time PCR(qPCR) and in situ hybridization (ISH) of known developmentalregulators in the E13.5 kidney. No change was observed in themRNA expression of Bmyc, cRet, Wnt11, Etv4, Emx2 and Six2between UBHdac1,2−/− and wild-type kidneys (note that Six2 wasdownregulated later at P0; Fig. 1). By contrast, Axin2, Shh, Tcf7,Wnt4 and Lef1were downregulated, and the stromal geneMeis1wasupregulated, confirming our microarray results (Fig. 5D and Fig. 6).It should be noted that qPCR and especially ISH demonstrated asignificant decrease of Wnt7b and Wnt9b, whereas the microarrayfailed to detect this decrease. This is likely to be a sensitivity issue:microarray of whole kidney RNA is not a sensitive approach todetect focal changes in gene expression in the ureteric epithelium.

β-catenin is downregulated in the UB cells of UBHdac1,2−/−

kidneysβ-catenin is a multifunctional protein that plays a crucial role in UBbranching morphogenesis as well as nephrogenesis. One role ofβ-catenin is to coordinate cell-cell adhesion through the formation

Fig. 2. Renal cystic dysplasia in newborn mice with combined Hdac1 andHdac2 deficiency in the ureteric cell lineage. (A,B) Gross anatomy of WTand UBHdac1,2−/− kidneys at P0. (C-F) PAS staining of kidney sections at P0shows loss of the nephrogenic zone and the formation of cysts in UBHdac1,2−/−

kidneys. (E,F) High-magnification images of C,D. The dashed green linedelineates the nephrogenic zone (NZ). (G-J) In UBHdac1,2−/− kidneys, thereis complete loss of Six2-positive and Pax2-positive cells. Note the cystsoriginating from the UB in UBHdac1,2−/− kidneys (H,J).

Fig. 3. Defective UB branching morphogenesis in UBHdac1,2−/− kidneys.(A) Heterozygous (HET; UBHdac1−/−,2+/− or UBHdac1+/−,2−/−) and UBHdac1,2−/−

kidneys from the same litters were dissected at E11.5, E12.5 and E13.5, andvisualized by Hoxb7-driven GFP fluorescence. Attenuated branching is seenin UBHdac1,2−/− fromE13.5. (B) The kidneys were isolated at E12.5 and culturedin vitro for up to 72 h. The UBHdac1,2−/− tissue shows evidence of degenerationafter 24-48 h in culture.

1182

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

of adherens junctions with E-cadherin and α-catenin. Anotherfunction of β-catenin is to regulate gene transcription, primarilythrough interactions with the Tcf/Lef family of transcription factors.This function is under the control of the canonical Wnt signalingpathway. Either deletion or overexpression of β-catenin in UB cellsleads to kidney hypoplasia (Bridgewater et al., 2008, 2011; Maroseet al., 2008). Hdac1 and Hdac2 have been reported to regulateβ-catenin in epidermal progenitor cells and oligodendrocytes. Inepidermal progenitor cells, Hdac1 and Hdac2 are required for theelevation of β-catenin during hair follicle fate acquisition.Epidermis that is deficient of Hdac1 and Hdac2 displays uniform,low-level expression of β-catenin (LeBoeuf et al., 2010).Conversely, deletion of Hdac1 and Hdac2 in oligodendrocytesresults in the stabilization and nuclear translocation of β-catenin,leading to activation of the canonical Wnt/β-catenin pathway (Yeet al., 2009). Our immunofluorescence results showed that β-cateninis dramatically decreased in the UB cells of UBHdac1,2−/− kidneys atE13.5 and thereafter (Fig. 7). These data are consistent with ourobservation made above that Wnt signaling is repressed inUBHdac1,2−/− kidneys.To exclude the possibility that we missed an initial activation of

Wnt signaling upon loss of Hdac1 and Hdac2 in our animal model,

we examined the acute response of cultured embryonic kidneys(E13.5) to HDAC inhibitor, using Axin2, a direct target of canonicalWnt signaling, as a read-out. As indicated by ISH, Axin2 isexpressed at a high level in the UB cells and at a low level in thesurrounding mesenchymal cells (Fig. 8A). As expected, Axin2 israpidly induced by the GSK3β inhibitor LiCl, which stabilizesβ-catenin and thus activates the canonical Wnt signaling pathway(Fig. 8A). We found that a 6-h treatment with HDAC inhibitorsubstantially decreases the mRNA level of Axin2 with or withoutLiCl (Fig. 8B,C). By contrast, expression of cRet is not altered(Fig. 8D). These results suggest that the repression of canonical Wntsignaling in the UB cells of UBHdac1,2−/− kidneys is likely to be adirect rather than secondary effect. As discussed below and shownin supplementary material Table S5, several genes in the Wntpathway are regulated by Hdac1 and Hdac2 and are also directlybound by p53 in the developing kidney, linking HDAC-Wnt to p53.

Deletion of Hdac1 and Hdac2 in the ureteric cells leads top53 hyperacetylationAt least two studies have shown that Hdac1 and Hdac2 areresponsible for deacetylation of p53 (Trp53) in epidermalprogenitor cells (LeBoeuf et al., 2010) and oocytes (Ma et al.,

Fig. 4. Conditional deletion of Hdac1 and Hdac2 increasescell apoptosis associated with DNA damage anddecreases cell proliferation in the UB lineage. (A-D)Apoptotic cells were identified using anti-active caspase 3(aCasp3) antibody. (E) Quantitative analysis of UB cellapoptosis as a percentage of the total number of UB cells.There is a ∼7-fold elevation in the level of UB cell apoptosis inUBHdac1,2−/− compared with wild-type kidneys: WT0.54±0.21% versus UBHdac1,2−/− 3.93±0.77% apoptoticureteric cells; n=5, *P=0.0029 (t-test), error bars indicates.e.m. Note that the baseline of apoptosis in wild-type kidneysis very low. (F-I) Representative images showing the inductionof DNA damage (phospho-histone γH2AX immunostaining) inUB cells of UBHdac1,2−/− kidneys. (J-M) Proliferating cells wereidentified using an anti-phospho-histone H3 (pH3) antibody.(N) Quantitative analysis showing that the UB cell proliferationrate, quantitated as the percentage of pH3-positive amongtotal UB cells, was decreased by ∼37.5% in UBHdac1,2−/−

kidneys: WT 6.78±1.00% versus UBHdac1,2−/− 4.24±0.31%proliferating ureteric cells; n=4, *P=0.0047 (t-test), error barsindicate s.e.m.

1183

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

2012). Acetylation is a key determinant of p53 function by increasingprotein stability, DNA binding affinity and transcriptional activity(Brooks and Gu, 2011; Meek and Anderson, 2009). Previous studiesin our laboratory demonstrated that p53 is a key renal developmentregulator that controls cell proliferation, differentiation and

apoptosis pathways (Hilliard et al., 2011, 2014; Saifudeen et al.,2002, 2009). Gain-of-function experiments showed that excessivep53 levels mediated by deletion of Mdm2 in the UB or MM led tobilateral renal dysplasia, which could be rescued by concurrentremoval of p53 (Hilliard et al., 2011, 2014). We therefore asked

Fig. 5. Aberrant gene expression in UBHdac1,2−/− kidneys. (A) Microarray analysis of gene expression in UBHdac1,2−/− kidneys at E13.5. A small subset ofgenes (1.2% of 41,000 probes) are significantly altered (>1.4-fold; P<0.05, n=4 experiments) in UBHdac1,2−/− kidneys. The heat maps display gene expression bylog2-transformed fold change (black=0); green indicates lower expression, red indicates higher expression. (B) Molecular functions analysis was performedwith IPA. Significantly (P<0.05) enriched biological processes are shown. (C) IPA depicts themost affected canonical pathways in UBHdac1,2−/− kidneys. Numbersabove bars indicate number of altered genes. (D) Validation of microarray results by real-time PCR. Data are presented as mean±s.e., n≥3, *P<0.05 (t-test).

1184

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

whether Hdac1 and Hdac2 modulate p53 acetylation as well as itsfunction in UB cells. Immunofluorescence revealed markedincreases in the levels of p53 acetylated at lysine 370, 379 and383 (corresponding to lysine 373, 382 and 386 in human,

respectively) in the ureteric epithelium of UBHdac1,2−/− kidneys atE13.5 (Fig. 9A-D). As anticipated, we also observed anaccumulation of total p53 protein in the ureteric epithelium(Fig. 9E,F).

Germline deletion of p53 partially rescues kidneydevelopment in UBHdac1,2−/− miceConsidering the observed upregulation of p53 in Hdac1/Hdac2-deficient UB cells, we hypothesized that p53 mediates the renaldefects observed in UBHdac1,2−/− mice, and employed a geneticrescue approach that involved crossing the Hoxb7-Cretg/+;Hdac1flox/flox; Hdac2flox/flox mice to p53−/− mice. Gross andhistological analyses of the kidney at P0 revealed that removal ofp53 on the UBHdac1,2−/− background partially rescued kidneydevelopment in a gene dosage-dependent manner (Fig. 10).Remarkably, Mendelian proportions of UBHdac1,2−/−; p53+/− andUBHdac1,2−/−; p53−/− mice were retrieved at P30, indicating thatelimination of one allele of p53 is sufficient to rescue the earlypostnatal lethality of UBHdac1,2−/− mice (Table 3). Thus, although aproportion of UBHdac1,2−/−; p53+/– and UBHdac1,2−/−; p53–/– miceexhibited no overt histological rescue, a measurable improvement inrenal survival must occur in these mice that allows their survival forat least 30 days. Remarkably, two of the 11 UBHdac1,2−/−; p53+/−

micewere still alive and fertile at 13 months of age and their kidneysexhibited relatively normal histology (supplementary materialFig. S3A-D). These results suggest that hyperacetylation of p53upon loss of Hdac1 and Hdac2 is one of the important mediators ofthe congenital renal dysgenesis observed in UBHdac1,2−/− mice.

To begin to identify the potential targets of p53 leading to therenal dysgenesis, we cross-referenced our UBHdac1,2−/− microarraydata with p53 ChIP-seq in E15.5 mouse kidneys (Li et al., 2013).Among the 496 altered genes in E13.5 UBHdac1,2−/− kidneys, 121are bound by p53 in their promoter regions (supplementary materialTable S3). The Hdac1/2-regulated and p53-bound genes includeLef1, Axin2, Tcf7 (Wnt signaling), Ptch1 (Shh signaling), severalpro-apoptosis and cell cycle regulatory genes, kinesin familymembers (important for ciliary function), calbindin and keratingenes. IPA revealed that the top five molecular and cellularfunctions regulated by these genes are: (1) cell death and survival;(2) molecular transport; (3) cellular growth and proliferation; (4)cellular development and (5) cell cycle (supplementary materialTable S4); and the top five affected canonical pathways are: (1) Vdr/Rxr activation; (2) neuroprotective role of Thop1 in Alzheimer’sdisease; (3) Wnt/β-catenin signaling; (4) protein kinase A signalingand (5) sonic hedgehog signaling (supplementary material Table S5and Fig. S4). Hypergeometric distribution analysis demonstratedthat cell death and survival genes are highly over-represented (51 of121 genes, P=0.00037).

Based on the above analyses, we further examined how deletionof p53 affected UB cell survival in UBHdac1,2−/− mice.Immunostaining results demonstrated that UBHdac1,2−/−; p53–/–

mice had a similar increase in γH2AX-positive UB cells, whereasthree of four UBHdac1,2−/−; p53–/– kidneys showed fewer aCasp3-positive UB cells, when compared with UBHdac1,2−/− kidneys atE14.5 (4.73±0.47%UBHdac1,2−/− versus 2.73±0.35%UBHdac1,2−/−;p53–/– apoptotic ureteric cells, P=0.0249) (Fig. 10C,D).

Direct effects of HDAC inhibition on cell growth and survivalTo test more directly the cell-autonomous role of p53hyperacetylation upon loss of HDAC activities, we treatedHCT116 p53 (TP53)+/+ and p53−/− isogenic human colon cancercells with the class I-specific HDAC inhibitor MS-275, which

Table 1. Selected functional categories of genes downregulated inUBHdac1,2–/– kidneys

GO ID P-value Description Genes in test set

35295 3.91E–09 Tubedevelopment

Gsta3, Bmp2,Foxa1, Gja1, Hp,Calb1, Shh,Epcam, Pgr,Wnt4, Arg2,Tgm2, Ptch1,Hhip, Adamts2

16055 1.82E–08 Wnt receptorsignalingpathway

Wnt2, Wnt10a,Fzd10, Wnt4,Tcf7, Lef1, Frzb,Axin2, Apcdd1,Shh

1657 2.65E–07 Ureteric buddevelopment

Epcam, Gsta3,Wnt4, Bmp2,Arg2, Calb1, Shh

16337 1.34E–06 Cell-cell adhesion Mia, Amigo2, Pvrl4,Cdh16, Cldn3,Pkhd1, Cldn1,Lef1, Thy1

1822 1.23E–02 Kidneydevelopment

Col4a4, Wnt4,Pkhd1, Shh

8284 1.05E–02 Positiveregulation ofcell proliferation

Wnt2, Cyp7b1,Fosl2, Btc, Tgm2,Avpr1a, Shh

Table 2. Selected mRNA transcripts decreased in UBHdac1,2–/– kidneys

Probe set IDGenesymbol Gene name

Foldchange P-value

A_52_P49014 Shh Sonic hedgehog –2.90 1.24E–05A_51_P287100 Cdh16 Cadherin 16 –2.82 2.18E–07A_51_P417643 Foxa1 Forkhead box A1 –1.84 9.52E–04A_51_P335801 Calb1 Calbindin 1 –1.73 6.72E–06A_51_P171616 Wnt10a Wingless-type

MMTVintegrationsite 10a

–1.66 3.68E–05

A_52_P377941 Pkhd1 Polycystic kidneyand hepaticdisease 1

–1.59 2.82E–03

A_52_P61864 Wnt2a Wingless-typeMMTVintegrationsite 2a

–1.44 2.56E–03

A_52_P244702 Tcf7 Transcriptionfactor 7

–1.43 6.24E–03

A_51_P501145 Lef1 Lymphoidenhancerbinding factor 1

–1.43 3.77E–03

A_52_P54176 Axin2 Axis inhibitionprotein 2

–1.42 1.79E–04

A_51_P130475 Wnt4 Wingless-typeMMTVintegration site 4

–1.41 2.70E–03

A_51_P312348 Krt7 Keratin 7 –2.19 5.26E–08A_51_P242399 Krt8 Keratin 8 –2.02 4.68E–07A_51_P324814 Krt18 Keratin 18 –2.20 3.33E–06A_51_P356642 Krt19 Keratin 19 –1.84 1.80E–03A_51_P287198 Krt23 Keratin 23 –1.55 1.79E–03

1185

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

preferentially inhibits HDAC1, 2 and 3. Light microscopy analysisrevealed that MS-275 induces notable growth arrest and cell death inboth p53+/+ and p53−/− cells, although p53−/− cells are clearly moreresistant to MS-275 than p53+/+ cells (supplementary materialFig. S5A).Next, we quantified the status of the cell cycle and of apoptosis

using flow cytometry. We found that the anti-proliferative effects ofMS-275 are not dependent on p53 function in HCT116 cells. MS-275 induced G1 and G2 phase arrest in both cell lines, with adecrease in S-phase cells to similar degrees (supplementary materialFig. S5B,D and Table S6), consistent with a previous report that p53is dispensable for cell cycle arrest in mouse embryonic fibroblasts

deficient for Hdac1 and Hdac2 (Wilting et al., 2010). By contrast,p53–/– cells underwent significantly less apoptosis than p53+/+ cells(3.52% versus 6.18%, respectively) after a 48-h treatment with10 µM MS-275 (supplementary material Fig. S5C,E), indicatingthat MS-275 induces apoptosis partially through p53 in HCT116cells. Western blot analyses confirmed that MS-275 effectivelyincreased the level of acetylated histone H3 and p53 in p53+/+ cellsand the level of acetylated histone H3 in p53–/– cells (supplementarymaterial Fig. S5F). Moreover, the total level of p53 is also increasedin p53+/+ cells (supplementary material Fig. S5F). Cleavage of poly(ADP-ribose) polymerase 1 (PARP1) by caspases is a hallmark ofapoptosis; it promotes apoptosis by preventing DNA repair undernormal conditions or stress. When we compared the HCT116 celllines, we identified a strong induction of cleaved PARP1 in p53+/+

cells but a much weaker induction in p53–/– cells (supplementarymaterial Fig. S5F). These findings are consistent with a recent reportfrom Sonnemann et al. (2014), showing that HDAC inhibition inHCT116 cells is partially dependent on p53.

DISCUSSIONThis study investigated the functions of Hdac1 and Hdac2 in theureteric epithelium during kidney development. Our resultsdemonstrate that: (1) Hdac1 and Hdac2 perform redundant yetessential functions in UB branching morphogenesis; (2) Hdac1 andHdac2 are required for the canonical Wnt signaling pathway inkidney development; and (3) Hdac1 and Hdac2 are required tosuppress hyperacetylation of p53 in the ureteric epithelium, whichpartially accounts for the UBHdac1,2−/− phenotype.

Hdac1 and Hdac2 perform redundant yet essential functionsin the UB lineageKidney development is dependent on reciprocal epithelial-mesenchymal interactions between the UB and the MM, whichdrive iterative branching of the UB and subsequent induction ofnephrons. Our previous studies showed that pharmacologicalinhibition of class I and II HDACs by Scriptaid, or of Hdac1, 2and 3 by MS-275, causes disruptions of the reciprocal signalingpathways, leading to growth arrest and apoptosis. As many HDACisoforms (e.g. Hdac1, 2 and 3) are expressed in the UB and MM, itwas important to determine the distinct functions of individualenzymes in specific cell lineages. In this study, we set out toelucidate the developmental roles of Hdac1 and Hdac2 in theureteric epithelium using a conditional knockout strategy. Althoughglobal deletion of eitherHdac1 orHdac2 leads to a lethal phenotype(Lagger et al., 2002; Montgomery et al., 2007), demonstrating theunique roles of these two enzymes in early embryogenesis,redundancy of Hdac1 and Hdac2 function has been found inmany somatic cell types (e.g. oligodendrocytes, Schwann cells,cardiomyocytes, basal cells and adipocytes) (Haberland et al., 2010;Jacob et al., 2011; LeBoeuf et al., 2010; Montgomery et al., 2007;Ye et al., 2009). In line with the previous studies, our results revealthat one allele of either Hdac1 or Hdac2 is sufficient to supportnormal kidney development, whereas loss of all four alleles leads torenal hypodysplasia and early postnatal lethality.

Hdac1 and Hdac2 are required for the canonical Wntsignaling pathway in kidney developmentGenome-wide profiling revealed that changes in gene expression arehighly specific in UBHdac1,2−/− mice at E13.5. Only 226 transcripts(0.55%) were upregulated and 270 transcripts (0.66%) weredownregulated (using a cut-off of 1.4-fold, P<0.05). This isconsistent with the increasing evidence showing that Hdac1 and

Fig. 6. In situ hybridization of renal developmental regulators in UB cellsat E13.5. (A-D) The expression of cRet, which encodes a tyrosine kinasereceptor for Gdnf, and its downstream targetWnt11 is unaltered in UBHdac1,2−/−

kidneys. (E-J)Axin2,Wnt9b andWnt7b are dramatically reduced inUBHdac1,2−/−

kidneys. (K,L) Expression of Emx2 is maintained in the UB epithelium inUBHdac1,2−/− kidneys.

1186

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

Hdac2 selectively control specific gene expression programs indifferent tissues. For example, deletion of Hdac1 and Hdac2 in theheart resulted in dysregulation of only ∼1.6% of the transcriptome(Montgomery et al., 2007). Given the apparent cell apoptosis andgrowth arrest observed, it is not surprising that genes regulating celldeath and survival and cellular growth and proliferation are amongthe most notably dysregulated functional categories. Moreinterestingly, we found that the canonical Wnt signaling pathwayis among the most affected pathways in UBHdac1,2−/− kidneys.The canonical Wnt signaling pathway controls both UB

branching morphogenesis and nephrogenesis. Specifically, Wnt7b

is expressed in the UB trunk and is essential for the establishment ofa cortico-medullary axis during later stages of kidney development.InWnt7bmutant mice, cortical epithelial development is normal butthe medullary zone fails to form (Yu et al., 2009). Wnt9b, anothermember of the Wnt family secreted by the UB trunk epithelium,plays an indispensable role in the activation of Wnt4, which is bothnecessary and sufficient for the induction of renal vesicles fromrenal progenitor cells. Wnt9b is also important for the proliferation/renewal of the renal progenitor cells. Genetic deletion of Wnt9b inmice results in failure of nephron induction and prematureexhaustion of the progenitor pool (Carroll et al., 2005; Karner

Fig. 7. β-catenin is downregulated in UBHdac1,2−/− kidneys. Immunofluorescence shows that β-catenin is initially expressed at normal levels in UBHdac1,2−/−

kidneys at E12.5 (A-F), but is decreased at E13.5 (G-L) and E14.5 (M-T). At E14.5, calbindin staining is too weak to label the UB cells in UBHdac1,2−/− kidneysand Hdac2 staining is used instead.

1187

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

et al., 2011). Our results show significant decreases in Wnt7b,Wnt9b and Wnt4 expression in UBHdac1,2−/− kidneys at E13.5,associated with the downregulation of a number of Wnt targetgenes, such as Axin2, Tcf7 and Lef1. Consistent with these findings,immunofluorescence results demonstrate that membrane-associatedβ-catenin is greatly decreased in the UB cells of UBHdac1,2−/−

kidneys. β-catenin exists in the cell in two pools: membraneassociated and cytosolic. On the membrane, as a component ofadherens junctions (AJs), β-catenin links cadherins to thecytoskeleton, whereas in the cytoplasm β-catenin is an essentialmediator of the Wnt signaling pathway (Perez-Moreno and Fuchs,2006). Although the interplay between the two pools remains to befully elucidated, time-lapse microscopy using photoactivatableGFP-tagged β-catenin has revealed that the membrane-associatedpool of β-catenin can be internalized together with E-cadherin,

accumulates at the perinuclear endocytic recycling compartment(ERC) upon AJ dissociation, and can be translocated into thenucleus upon Wnt pathway activation (Kam and Quaranta, 2009).Therefore, it is conceivable that decreased levels of membrane-associated β-catenin reduce the availability of cytosolic β-cateninand thus lead to the decrease in Wnt signaling in UBHdac1,2−/−

kidneys. Moreover, we observed that a 6-h treatment of culturedembryonic kidney with HDAC inhibitor leads to a prompt reductionof Axin2, a direct target of Wnt, supporting the idea that Hdac1 andHdac2 function in the activation of Wnt signaling in the embryonickidney.

It is also of note that Shh, a hedgehog homolog, is one of the mostsuppressed genes in UBHdac1,2−/− kidneys. During kidneydevelopment, Shh is initially expressed in the distal part of the UB,and then in the urothelium. Shh acts as a paracrine signal to promotemesenchymal cell proliferation, and regulates the pattern ofmesenchymal differentiation. Conditional deletion of Shh in the UBresults in renal hypoplasia, hydronephrosis and hydroureter innewborn pups (Yu et al., 2002). However, such a phenotype is notobserved in UBHdac1,2−/− mice. It is likely to be masked by the moresevere dysplastic phenotype of UBHdac1,2−/− kidneys, or the residuallevel of Shh is still sufficient to support mesenchymal cell proliferationand differentiation. Taken together, our results demonstrate that Hdac1and Hdac2 play a crucial role in maintaining essential ureteric genesfor renal growth and differentiation.

It should be noted that, although it might be difficult tocompletely dissociate the direct effects of HDAC inactivationfrom the secondary effects of cell loss due to apoptosis, there areseveral clues that support the notion that many of the geneexpression changes are specific. First, ISH and microarrays wereperformed at E13.5, prior to the observed degeneration of the UBtree. Several genes, such as Tcf7, Wnt7b, Wnt9b, Axin2 and Shh,were specifically altered. Second, if the effects of Hdac1/2inactivation on gene expression were generalized or non-specificdue to cell death, we would have expected a much more substantialchange in the number of genes altered, as opposed to the few percentof differentially expressed genes observed in the study. Third, ourresults cannot be fully explained by excessive cell death of UB tipcells since the expression of cRet, Wnt11, Bmyc and Etv4, fourspecific markers of UB tips, is unaltered in UBHdac1,2−/− mice.

Hdac1 and Hdac2 are required to suppress hyperacetylationof p53 in the ureteric epitheliumAlthough lysine acetylation was originally discovered in histones,these are not the only proteins that can be acetylated. p53 was thefirst non-histone protein found to be regulated by acetylation (Guand Roeder, 1997; Luo et al., 2000). The acetylation of multiplelysine residues in the C-terminus (K305, K370, K372, K373,

Fig. 8. Inhibition of HDAC activity in cultured metanephroi suppresses Axin2. Paired E13.5 metanephroi were cultured in the presence of 15 mM LiCl orvehicle control (PBS) (A), 2 µg/ml Scriptaid (HDACi) or Nullscript control (B,D), or in the presence of 15 mM LiCl with or without 2 µg/ml Scriptaid (HDACi) (C) for6 h. Expression of Axin2 (A-C) and cRet (D) was evaluated by whole-mount ISH. The HDAC inhibitor substantially decreases the Axin2 mRNA level, with orwithout LiCl, whereas cRet is unaffected.

Fig. 9. Deletion of Hdac1 and Hdac2 in UB cells causes p53hyperacetylation and accumulation. (A-D) Immunofluorescence detectionof p53 acetylation at lysine 370, 379 and 383 in wild-type and UBHdac1,2−/−

kidneys at E13.5. UBHdac1,2−/− kidneys display strong staining for lysine 370,379 (B) and 383 (D) p53 acetylation specifically in UB cells. (E,F) Detection ofp53 by DAB staining reveals elevated p53 expression in UB cells inUBHdac1,2−/− kidneys.

1188

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

K381, K382 and K386) and DNA-binding domain (K164) ofhuman p53 is significantly enhanced in response to stressand required for its stabilization, transcriptional activation andtranscription-independent function in apoptosis (Brooks and Gu,2011). p53 in which all eight lysine residues are substituted (8KR)

loses the ability to mediate cell cycle arrest and apoptosis (Tanget al., 2008). There is also evidence that HDAC1 is recruited to p53by an MDM2-containing protein complex, and HDAC1-mediateddeacetylation of p53 is required for degradation of p53 (Ito et al.,2002).

Fig. 10. Elimination of p53 partially rescues kidney development in UBHdac1,2−/− mice. (A) Representative images of kidneys from newborn mice of theindicated genotypes. Whole-mount (top), transverse sections (middle) and higher magnification (40x, bottom) are shown. (B) Quantification of the renalphenotype of mice of the indicated genotypes, as illustrated in A. The degree of morphological rescue was judged by microscopy and classified as:significant rescue, where kidneys are notably larger and form fewer cysts than UBHdac1,2−/−; p53+/+; moderate rescue, where kidneys (either unilateral orbilateral) are not notably larger but form fewer cysts; no overt rescue, where there is no obvious difference to UBHdac1,2−/−; p53+/+ kidneys. (C) Quantitativeanalysis of UB cell apoptosis (aCasp3 immunostaining) as a percentage of total UB cells indicates that there are fewer apoptotic cells upon p53 deletionfrom UBHdac1,2−/−. (D) By contrast, UBHdac1,2−/−; p53–/– mice show a similar elevation in γH2AX-positive UB cells to UBHdac1,2−/− mice. (C,D) Error bars denotemean±s.e.m.

1189

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

Consistent with two recent studies showing that double knockoutof Hdac1 and Hdac2 enhances p53 acetylation in mice epidermalprogenitor cells and oocytes, our results demonstrated that loss ofHdac1 and Hdac2 in the ureteric epithelium leads to markedhyperacetylation and accumulation of p53. However, to ourknowledge, the present study is the first to show rescue oforganogenesis, albeit partial, upon concomitant germline deletionof p53 in vivo. This finding is in line with our previous report thatexcessive p53 accumulation in the UB or renal progenitor cells(mediated by deletion of Mdm2) causes bilateral renal dysplasia,while concomitant elimination of p53 rescues the renal phenotypeand animal survival. With regards to the underlying cellularmechanisms, our immunostaining results demonstrate that p53 ispartially responsible for UB cell apoptosis in UBHdac1,2−/− kidneysat E14.5. In support of these results, bioinformatics analysisrevealed that 121/496 dysregulated genes in UBHdac1,2−/− kidneysare bound by p53 in their promoter regions and are therebypotentially regulated by p53. Moreover, over 40% of these genes arehighly enriched in the category of cell death and survival. As tothe apparent paradox of longer survival in the triple UBHdac1,2−/−;p53−/− null mutants yet no significant morphological rescue, wesuggest that ‘histological’ and ‘functional’ rescues might not beidentical in this setting. Loss of p53 might have improved functionalbut not structural variables, e.g. glomerular filtration or tubulartransport functions. This might account for the overall longersurvival of UBHdac1,2−/−; p53+/− or UBHdac1,2−/−; p53−/− mice,which did not show structural rescue.Together, these results suggest that Hdac1 and Hdac2 are required

to suppress the hyperacetylation and activation of p53 in the UBlineage, thus protecting cells from apoptosis in renal development.Further studies using p53 lysine-specific mutant mice are needed toaddress the question of whether the pro-apoptotic function of p53is dependent on its acetylation during kidney development. Insummary, our data reveal that Hdac1 and Hdac2 play an essentialrole in cell proliferation, survival and transcriptional regulation inthe ureteric epithelium during kidney development, and indicatethat this occurs, in part, through p53-mediated pathways.

MATERIALS AND METHODSMiceMice bearing conditional null alleles of Hdac1 and Hdac2 (Hdac1flox/flox

and Hdac2flox/flox) were obtained from the laboratory of Eric Olson(Montgomery et al., 2007) and were crossed to Hoxb7-CreEGFPtransgenic mice (Hoxb7-Cretg/+) (Zhao et al., 2004) to delete the Hdac1

and Hdac2 genes, singly or in combination, specifically in the UBepithelium. For the rescue experiment, p53–/– mice (Jackson Laboratory)were first crossed to the Hdac1flox/flox; Hdac2flox/flox mice, and then furthercrossed to Hoxb7-CreEGFP transgenic mice to remove p53 on theUBHdac1,2−/− background.

Histology and immunohistochemistryKidneys were fixed in 10% buffered formalin, embedded in paraffin, andsectioned at 4 μm. Histology analyses were performed by standard periodicacid-Schiff (PAS) staining and Hematoxylin and Eosin (H&E) staining.Immunofluorescence was performed as previously described (Chen et al.,2011). Primary antibodies and working concentrations are listed insupplementary material Table S7. The peroxidase-based Vectastain ABCElite Kit (Vector Laboratories) was used for DAB detection.

Organ cultureEmbryonic kidneys were aseptically micro-dissected from timed-pregnantmice and cultured on polycarbonate Transwell filters (0.4 μm pore size,Corning Costar) over medium [DMEM/F-12 containing 10% fetal bovineserum (FBS)] at 37°C and 5% CO2.

RNA extractionKidneys were harvested at E13.5 and stored in RNAlater RNA stabilizationreagent (Qiagen). Wild-type and UBHdac1,2−/− kidneys were each dividedinto four random pools (n=4). Total RNA was then isolated using theRNeasy Mini Kit (Qiagen).

Genome-wide microarray analysisMicroarray analysis was performed according to established protocols(Schanstra et al., 2007). Briefly, fluorescently labeled cRNAwas generatedfrom 0.5 μg total RNA in each reaction using the Agilent Fluorescent DirectLabel Kit and 1.0 mM Cyanine 3′- or 5′-labeled dCTP (PerkinElmer).Hybridization was performed using the Oligonucleotide MicroarrayHybridization and In Situ Hybridization Plus Kit (Agilent). The labeledcRNA was hybridized to Agilent 44K whole mouse genomeoligonucleotide microarray (containing ∼41,000 probes) as previouslydescribed (Schanstra et al., 2007). The arrays were scanned using a dual-laser DNA microarray scanner (Agilent). The data were then extracted fromimages using Feature Extraction software 6.1 (Agilent). Microarray data areavailable at GEO under accession number GSE35432.

Data analysisMultiExperiment Viewer v4.9 software was used to generate lists ofgenes differentially expressed between wild-type and UBHdac1,2−/−

kidneys, using P≤0.05 and a minimum 1.4-fold change in geneexpression. Genes were classified according to their function using IPAsoftware and BiNGO classification systems as previously described(Chen et al., 2011).

qPCRQuantitative real-time PCR (qPCR) was performed using the One-StepBrilliant Quantitative RT-PCRMaster Mix Kit (Applied Biosystems). Real-time PCR reaction mix contained 200 nm forward primer, 200 nm reverseprimer, and 60 ng total RNA. Relative levels of mRNAwere normalized toGapdh. The primers used for qPCR are listed in supplementary materialTable S8.

Whole-mount in situ hybridization (ISH)ISH was performed using digoxigenin-labeled antisense probes on kidneytissue fixed with 4% paraformaldehyde as previously described (Chen et al.,2011).

Cell culture and treatmentsHCT116 p53+/+ and p53–/– cells were obtained from Dr Hua Lu (TulaneUniversity, New Orleans, LA, USA, and Johns Hopkins University CellCenter,Baltimore,MD,USA).Cellswere grown in high-glucoseDMEMwith

Table 3. One-month (P30) survival rate of Hdac1,2 mutant offspringcarrying no, one or two p53 mutant alleles

GenotypeObserved/totalnumber of mice

Predicted/totalnumber of mice

UBHdac1,2−/−; p53+/+ 1/81* 5/81UBHdac1,2−/−; p53+/– 11/81 10/81UBHdac1,2−/−; p53–/– 5/81 5/81

The genotypes shown were generated by initial crossing of p53–/–; Hdac1flox/flox;Hdac2flox/flox andHoxb7-CreEGFPmice followed by cross-breeding of offspring.UBHdac1,2−/− refers toHoxb7-Cretg/+; Hdac1flox/flox; Hdac2flox/flox. The numbers ofliving mice of each genotype were counted at P30. Chi-square one-tailedanalysis was used to compare the observed values with those predicted byMendelian inheritance patterns. UBHdac1,2−/−; p53+/+ mice show postnatallethality (P=0.048 observed versus expected), whereas UBHdac1,2−/−; p53+/–

(P=0.408 observed versus expected) and UBHdac1,2−/−; p53–/– (P=0.500observed versus expected) mice were rescued from postnatal lethality.*Note that the one UBHdac1,2−/−; p53+/+ mouse observed at P30 was smaller andweak compared with its littermates, and later died at the age of 3 months.

1190

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

stable glutamine supplemented with 10% FBS and 10 mg/ml antibiotics(penicillin and streptomycin), under 5% CO2 and saturated moisture. Cellswere treated with vehicle control DMSOor 10 µMMS-275 for 24, 48 or 72 h.

Analysis of cell cycle and apoptosis using flow cytometryCells were treated with vehicle control DMSO or 10 µM MS-275 for 48 h.At the time of harvesting, cells were digested with 0.05% trypsin andresuspended in phosphate-buffered saline (PBS). For cell cycle analysis,2×105 cells were stained with 50 µg/ml propidium iodide (PI) using theCoulter DNA Prep Reagents Kit (Beckman Coulter). For evaluation of theearly stages of apoptosis, 2×105 cells were stained with Annexin V-FITCand PI using the Annexin V-FITC Apoptosis Detection Kit (Sigma). Cellswere then analyzed using a Beckman Coulter FC500 flow cytometer andCXP software (Beckman Coulter) for acquisition and analysis. The cellcycle distribution was further analyzed by ModFit LT 3.0 software (VeritySoftware House).

Western blotWhole-cell proteins were extracted using RIPA buffer (Invitrogen) andnuclear proteins were extracted using the Nuclear Extract Kit (ActiveMotif ). Primary antibodies and working concentrations are listed insupplementary material Table S7.

AcknowledgementsWe thank Dr Eric Olson for theHdac1flox/flox andHdac2flox/floxmice; Dr Carlton Batesfor the Hoxb7-CreEGFP transgenic mice; and Dr Hua Lu for providing HCT116 celllines. We acknowledge the support of the Tulane Renal and Hypertension Center ofExcellence, Center for StemCell Research & Regenerative Medicine and the Centerfor Gene Therapy.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsS.S.E. conceived the project, supervised the studies, analyzed the data, read, editedand approved the manuscript; S.C. and X.Y. performed the majority of theexperiments; D.B. performed the microarray experiments; S.C. and Y.L. performedmicroarray data analysis; Z.S. provided p53 knockout mice and analyzed anddiscussed the data; S.C. performed experiments, analyzed data and wrote themanuscript.

FundingThis work was supported by National Institutes of Health grants [R01DK079886,P50DK096373 and P30GM103337]. Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113506/-/DC1

ReferencesBridgewater, D., Cox, B., Cain, J., Lau, A., Athaide, V., Gill, P. S., Kuure, S.,Sainio, K. and Rosenblum, N. D. (2008). Canonical WNT/beta-catenin signalingis required for ureteric branching. Dev. Biol. 317, 83-94.

Bridgewater, D., Di Giovanni, V., Cain, J. E., Cox, B., Jakobson, M., Sainio, K.and Rosenblum, N. D. (2011). beta-catenin causes renal dysplasia viaupregulation of Tgf beta 2 and Dkk1. J. Am. Soc. Nephrol. 22, 718-731.

Brooks, C. L. andGu,W. (2011). The impact of acetylation and deacetylation on thep53 pathway. Protein Cell 2, 456-462.

Brunmeir, R., Lagger, S. and Seiser, C. (2009). Histone deacetylase HDAC1/HDAC2-controlled embryonic development and cell differentiation. Int. J. Dev.Biol. 53, 275-289.

Carroll, T. J., Park, J.-S., Hayashi, S., Majumdar, A. and McMahon, A. P. (2005).Wnt9b plays a central role in the regulation of mesenchymal to epithelialtransitions underlying organogenesis of the mammalian urogenital system. Dev.Cell 9, 283-292.

Chen, S., Bellew, C., Yao, X., Stefkova, J., Dipp, S., Saifudeen, Z., Bachvarov, D.and El-Dahr, S. S. (2011). Histone deacetylase (HDAC) activity is critical forembryonic kidney gene expression, growth, and differentiation. J. Biol. Chem.286, 32775-32789.

de Ruijter, A. J. M., van Gennip, A. H., Caron, H. N., Kemp, S. and vanKuilenburg, A. B. P. (2003). Histone deacetylases (HDACs): characterization ofthe classical HDAC family. Biochem. J. 370, 737-749.

Denu, J. M. and Gottesfeld, J. M. (2012). Minireview series on sirtuins: frombiochemistry to health and disease. J. Biol. Chem. 287, 42417-42418.

Glozak, M. A., Sengupta, N., Zhang, X. and Seto, E. (2005). Acetylation anddeacetylation of non-histone proteins. Gene 363, 15-23.

Gu, W. and Roeder, R. G. (1997). Activation of p53 sequence-specific DNA bindingby acetylation of the p53 C-terminal domain. Cell 90, 595-606.

Haberland, M., Montgomery, R. L. and Olson, E. N. (2009). The many roles ofhistone deacetylases in development and physiology: implications for diseaseand therapy. Nat. Rev. Genet. 10, 32-42.

Haberland, M., Carrer, M., Mokalled, M. H., Montgomery, R. L. and Olson, E. N.(2010). Redundant control of adipogenesis by histone deacetylases 1 and 2.J. Biol. Chem. 285, 14663-14670.

Hilliard, S., Aboudehen, K., Yao, X. and El-Dahr, S. S. (2011). Tight regulation ofp53 activity by Mdm2 is required for ureteric bud growth and branching. Dev. Biol.353, 354-366.

Hilliard, S. A., Yao, X. and El-Dahr, S. S. (2014). Mdm2 is required for maintenanceof the nephrogenic niche. Dev. Biol. 387, 1-14.

Ito, A., Kawaguchi, Y., Lai, C.-H., Kovacs, J. J., Higashimoto, Y., Appella, E. andYao, T.-P. (2002). MDM2-HDAC1-mediated deacetylation of p53 is required for itsdegradation. EMBO J. 21, 6236-6245.

Jacob, C., Christen, C. N., Pereira, J. A., Somandin, C., Baggiolini, A., Lotscher,P., Ozçelik, M., Tricaud, N., Meijer, D., Yamaguchi, T. et al. (2011). HDAC1 andHDAC2 control the transcriptional program of myelination and the survival ofSchwann cells. Nat. Neurosci. 14, 429-436.

Kam, Y. and Quaranta, V. (2009). Cadherin-bound beta-catenin feeds into the Wntpathway upon adherens junctions dissociation: evidence for an intersectionbetween beta-catenin pools. PLoS ONE 4, e4580.

Karner, C. M., Das, A., Ma, Z., Self, M., Chen, C., Lum, L., Oliver, G. andCarroll, T. J. (2011). Canonical Wnt9b signaling balances progenitor cellexpansion and differentiation during kidney development. Development 138,1247-1257.

Kruse, J.-P. and Gu, W. (2009). Modes of p53 regulation. Cell 137, 609-622.Lagger, G., O’Carroll, D., Rembold, M., Khier, H., Tischler, J., Weitzer, G.,

Schuettengruber, B., Hauser, C., Brunmeir, R., Jenuwein, T. et al. (2002).Essential function of histone deacetylase 1 in proliferation control and CDKinhibitor repression. EMBO J. 21, 2672-2681.

LeBoeuf, M., Terrell, A., Trivedi, S., Sinha, S., Epstein, J. A., Olson, E. N.,Morrisey, E. E. and Millar, S. E. (2010). Hdac1 and Hdac2 act redundantlyto control p63 and p53 functions in epidermal progenitor cells. Dev. Cell 19,807-818.

Li, Y., Liu, J., McLaughlin, N., Bachvarov, D., Saifudeen, Z. and El-Dahr, S. S.(2013). Genome-wide analysis of the p53 gene regulatory network in thedeveloping mouse kidney. Physiol. Genomics 45, 948-964.

Luo, J., Su, F., Chen, D., Shiloh, A. and Gu, W. (2000). Deacetylation of p53modulates its effects on cell growth and apoptosis. Nature 408, 377-381.

Ma, P., Pan, H., Montgomery, R. L., Olson, E. N. and Schultz, R. M. (2012).Compensatory functions of histone deacetylase 1 (HDAC1) and HDAC2 regulatetranscription and apoptosis during mouse oocyte development. Proc. Natl. Acad.Sci. USA 109, E481-E489.

Marks, P. A., Miller, T. and Richon, V. M. (2003). Histone deacetylases.Curr. Opin.Pharmacol. 3, 344-351.

Marose, T. D., Merkel, C. E., McMahon, A. P. and Carroll, T. J. (2008). beta-Catenin is necessary to keep cells of ureteric bud/Wolffian duct epithelium in aprecursor state. Dev. Biol. 314, 112-126.

Meek, D. W. and Anderson, C. W. (2009). Posttranslational modification ofp53: cooperative integrators of function. Cold Spring Harb. Perspect. Biol. 1,a000950.

Montgomery, R. L., Davis, C. A., Potthoff, M. J., Haberland, M., Fielitz, J., Qi, X.,Hill, J. A., Richardson, J. A. and Olson, E. N. (2007). Histone deacetylases 1and 2 redundantly regulate cardiac morphogenesis, growth, and contractility.Genes Dev. 21, 1790-1802.

Perez-Moreno, M. and Fuchs, E. (2006). Catenins: keeping cells from getting theirsignals crossed. Dev. Cell 11, 601-612.

Saifudeen, Z., Dipp, S. and El-Dahr, S. S. (2002). A role for p53 in terminalepithelial cell differentiation. J. Clin. Invest. 109, 1021-1030.

Saifudeen, Z., Dipp, S., Stefkova, J., Yao, X., Lookabaugh, S. and El-Dahr, S. S.(2009). p53 regulates metanephric development. J. Am. Soc. Nephrol. 20,2328-2337.

Schanstra, J. P., Bachvarova, M., Neau, E., Bascands, J. L. and Bachvarov, D.(2007). Gene expression profiling in the remnant kidney model of wild type andkinin B1 and B2 receptor knockout mice. Kidney Int. 72, 442-454.

Smith, C. L. (2008). A shifting paradigm: histone deacetylases and transcriptionalactivation. Bioessays 30, 15-24.

Sonnemann, J., Marx, C., Becker, S., Wittig, S., Palani, C. D., Kramer, O. H. andBeck, J. F. (2014). p53-dependent and p53-independent anticancer effects ofdifferent histone deacetylase inhibitors. Br. J. Cancer 110, 656-667.

Spange, S., Wagner, T., Heinzel, T. and Kramer, O. H. (2009). Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int. J. Biochem.Cell Biol. 41, 185-198.

1191

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT

Tang, Y., Zhao, W., Chen, Y., Zhao, Y. and Gu, W. (2008). Acetylation isindispensable for p53 activation. Cell 133, 612-626.

Wilting, R. H., Yanover, E., Heideman, M. R., Jacobs, H., Horner, J., van derTorre, J., DePinho, R. A. and Dannenberg, J.-H. (2010). Overlapping functionsof Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis. EMBO J. 29,2586-2597.

Ye, F., Chen, Y., Hoang, T., Montgomery, R. L., Zhao, X.-h., Bu, H., Hu, T., Taketo,M. M., van Es, J. H., Clevers, H. et al. (2009). HDAC1 and HDAC2 regulateoligodendrocyte differentiation by disrupting the beta-catenin-TCF interaction.Nat. Neurosci. 12, 829-838.

Yu, J., Carroll, T. J. and McMahon, A. P. (2002). Sonic hedgehog regulatesproliferation and differentiation of mesenchymal cells in the mouse metanephrickidney. Development 129, 5301-5312.

Yu, J., Carroll, T. J., Rajagopal, J., Kobayashi, A., Ren, Q. and McMahon, A. P.(2009). A Wnt7b-dependent pathway regulates the orientation of epithelial celldivision and establishes the cortico-medullary axis of the mammalian kidney.Development 136, 161-171.

Zhao, H., Kegg, H., Grady, S., Truong, H.-T., Robinson, M. L., Baum, M. andBates, C. M. (2004). Role of fibroblast growth factor receptors 1 and 2 in theureteric bud. Dev. Biol. 276, 403-415.

1192

RESEARCH ARTICLE Development (2015) 142, 1180-1192 doi:10.1242/dev.113506

DEVELO

PM

ENT