A Core Transcriptional Network Composed of Pax2/8, Gata3 and Lim1 Regulates Key Players of Pro/Mesonephros Morphogenesis

Developmental Biology 382 (2013) 555–566

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Developmental Biology

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Genomes and Developmental Control A core transcriptional network composed of Pax2/8, Gata3 and Lim1 regulates key players of pro/mesonephros morphogenesis

Sami Kamel Boualia a, Yaned Gaitan a, Mathieu Tremblay a, Richa Sharma a, Julie Cardin b, Artur Kania b, Maxime Bouchard a,n a Goodman Cancer Research Centre and Department of Biochemistry, McGill University, 1160 Pine Ave. W., Montreal, Quebec, Canada H3A 1A3 b Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada H2W 1R7, Department of Anatomy and Cell Biology, Division of Experimental Medicine, McGill University, Montréal, Quebec, Canada, H3A 2B2 and Faculté de médecine, Université de Montréal, Montréal, Quebec, Canada, H3C 3J7. article info abstract

Article history: Translating the developmental program encoded in the genome into cellular and morphogenetic Received 23 January 2013 functions requires the deployment of elaborate gene regulatory networks (GRNs). GRNs are especially Received in revised form crucial at the onset of organ development where a few regulatory signals establish the different 27 July 2013 programs required for tissue organization. In the renal system primordium (the pro/mesonephros), Accepted 30 July 2013 important regulators have been identified but their hierarchical and regulatory organization is still Available online 3 August 2013 elusive. Here, we have performed a detailed analysis of the GRN underlying mouse pro/mesonephros Keywords: development. We find that a core regulatory subcircuit composed of Pax2/8, Gata3 and Lim1 turns on a Kidney development deeper layer of transcriptional regulators while activating effector genes responsible for cell signaling Transcription and tissue organization. Among the genes directly affected by the core components are the key Pax2 developmental molecules Nephronectin (Npnt) and Plac8. Hence, the pro/mesonephros GRN links Gata3 together several essential genes regulating tissue morphogenesis. This renal GRN sheds new light on Lim1 CAKUT the disease group Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) in that gene mutations are expected to generate different phenotypic outcomes as a consequence of regulatory network deficiencies rather than threshold effects from single genes. & 2013 Elsevier Inc. All rights reserved.

Introduction specification (Koide et al., 2005) and in haematopoietic stem cell development (Pimanda and Gottgens, 2010). The comparative Throughout development, the progressive diversification and analysis of the GRNs characterized in these systems revealed an differentiation of cells requires a constant reorganization of their important level of conservation in the topology of the subcircuits underlying gene regulatory state. This progression is under the (network motifs) as well as a hierarchical organization of these control of gene regulatory networks (GRNs) comprised of transcrip- subcircuits (Alon, 2007; Davidson, 2010). The gradual deployment tion factors controlling regulatory and effector proteins, ultimately of the developmental program is indeed performed by sequential resulting in the activation of the terminal cellular differentiation activation of GRNs subcircuits, each responsible for the execution of program (Davidson and Erwin, 2006; Davidson, 2010). As such, a precise regulatory task. Subcircuits can be categorized as regula- GRNs effectively read and execute the developmental program tory and effectors to reflect the role they play in the hierarchy of encoded in the genome and convert it into concrete molecular GRN deployment (Davidson, 2010; Davidson and Erwin, 2006). machinery regulating cell and tissue organization. Among the regulatory subcircuits, kernels are critical evolutionary GRNs have been extensively studied in bacteria (Alon, 2007), conserved units found at the onset organ or structure formation. Drosophila (Levine and Davidson, 2005) and sea urchin (Davidson, The inactivation of any of their components will typically result in 2009), which are readily amenable to elaborate regulatory network major deficiencies in the structure they regulate (Davidson and studies. They have also been characterized in vertebrates, notably Erwin, 2006). Other regulatory units such as “plug-ins” (e.g. signal- in heart field formation (Cripps and Olson, 2002), in mesoderm ing pathways) and “on–off switches” linking subcircuits also perform an important role as flexible modular units. On the other hand, effector subcircuits perform cell biology functions such as cell shape Abbreviations: CND, common nephric duct; CAKUT, congenital anomalies of changes or proliferation, while differentiation gene batteries com- the kidney and urinary tract; GDNF, glial cell line-derived neurotrophic factor; prise the terminal differentiation molecules necessary for the cell to ChIP, chromatin immunoprecipitation n perform its ultimate role in the organism (Davidson, 2010; Davidson Corresponding author. Fax: +1 514 398 6769. E-mail address: [email protected] (M. Bouchard). and Erwin, 2006).

In the urogenital system, some crucial developmental regulators The analysis was done in triplicates (technical) with 3 biological have been identified but a clear understanding of the GRNs underlying samples of each genotype. The mouse beta-2 microglobulin (B2M) the development of this organ system is still lacking. Urogenital gene was used as internal control. The primers used are listed in system development is initiated with the formation of the nephric Table S3. duct by mesenchymal–epithelial transition and tubulogenesis of intermediate mesoderm progenitor cells (Bouchard, 2004; Dressler, Plasmids 2006). Nephric lineage induction is followed by the elongation of the nephric duct that induces adjacent mesonephric tubules to form the Evi1, Npnt, Wfdc2, Id4, Plac8 and Lim1 probes were generated by pro/mesonephros, and ultimately connects to the cloaca (bladder- RT-PCR using primers described in Table S3 and cloned in the urethra primordium) in the caudal part of the embryo trunk. Together, pGEM-T-easy or pLXSN vector. Murine Pax2 and Gata3 cDNA were the transcription factors Pax2 and Pax8 were identified as necessary subcloned in pMSCV-HA3-IRES-GFP vector (kindly provided by Dr. and sufficient for the specification of the nephric lineage and Jerry Pelletier, McGill University). Murine Lim1 was subcloned in subsequent pro/mesonephros morphogenesis (Bouchard et al., 2002). pLXSN-Flag vector. In addition to Pax2/8, the transcription factors Gata3 and Lim1 (Lhx1) are also crucial for pro/mesonephros development, as Gata3 regulates Luciferase reporter assay proliferation, elongation and guidance of the nephric duct (Chia et al., 2011; Grote et al., 2006), while Lim1 is necessary for nephric duct Enhancer sequences were cloned in pGL3-PolA Luciferase elongation and integrity (Kobayashi et al., 2005; Pedersen et al., 2005; reporter plasmid. Site directed mutagenesis was done by PCR to Shawlot and Behringer, 1995). Following nephric duct elongation, the generate mutated derivatives. Transcription factor expressing metanephric (or adult) kidney is induced as a diverticulum of the constructs (either Pax2, Gata3 or Lim1) and the Luciferase reporter nephric duct at the level of the hindlimbs. This crucial event is constructs were cotransfected in HEK293T cells with Lipofecta- regulated by the secreted molecule Gdnf in the mesenchyme, which mine 2000 (Invitrogen) according to the manufacturer's instruc- signals to the Ret receptor expressed in the nephric duct (Dressler, tions. Transient transactivation assays were performed on a 2006; Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996; LUMIstar Omega (BMG) using Dual-Glo Luciferase Assay System Schuchardt et al., 1994; Schuchardt et al., 1996). (Promega). In all samples the amount of total DNA was kept In the past, important transcriptional and signaling regulators constant with pGem4 and pCMV-Renilla vector was included for of nephric duct morphogenesis have been identified but relatively transfection efficiency. little is known about their regulatory organization and transcriptional output. However, evidence suggests that these regulators In situ hybridization may be part of a deep regulatory network conducting the different aspects of nephric duct morphogenesis. Here, we adopt a com- Embryos were dissected at E9.5 and fixed overnight in 4% prehensive approach to characterize the pro/mesonephros regu- paraformaldehyde at 4 1C. In situ hybridization was performed as latory network. We find that the pro/mesonephros GRN is described previously (Henrique et al., 1995) on cryosections from composed of a kernel sub-circuit that links to several key effectors E9.5 embryo with digoxigenin-dUTP RNA probes against Lim1 of nephric duct morphogenesis. (Bouchard et al., 2002), Gata3 (Grote et al., 2006), Emx2 (Yoshida et al., 1997), Pax2 (Bouchard et al., 2002), Pax8 (Bouchard et al., Materials and methods 2002), Pcdh19 (Gaitan and Bouchard, 2006), Evi1, Npnt, Wfdc2, Id4 and Plac8. Mice Cell culture and infection Pax2, Pax8, Gata3, Lim1, Emx2 and Pax2BACGFP mice were bred on a C3H/HeJ background for at least 6 generations. Genotyping of Murine inner medullary collecting duct cells (mIMCD3) (kindly these mice has been described previously (Bouchard et al., 2000, provided by Dr. Paul Goodyer, McGill University) were cultured in 2002, 2005; Grote et al., 2006; Hoyt et al., 1997; Miyamoto et al., 10% fetal bovine serum DMEM/F12 media (Wisent). Virus produc- 1997; Shawlot and Behringer, 1995; Yoshida et al., 1997). All in vivo tion was performed in HEK293T cells by cotransfection of pMSCV- studies were approved by the Animal Care Committee of McGill Pax2-HA3-IRES-GFP or pMSCV-Gata3-HA3-IRES-GFP, pVPack-GP University and strictly follow the guidelines from the Canadian and pVPack-VSV-G vectors (Stratagene). mIMCD3 cells were Council on Animal Care. infected for 48 h and sorted for GFP expression using a FACSAria (BD-Bioscience). mIMCD3-Gata3-HA cells were also stably trans- Microarray experiments fected with Lim1-Flag plasmid.

Microarray experiments for Pax2-regulated genes was reported Bioinformatic analysis previously (Grote et al., 2006) using either Pax2BACGFP;Pax2/ or Pax2+/+ cells from pro/mesonephros at E9.0–9.5 (13–20S). Briefly, Conserved genomic regions between mouse and human were embryo trunks were dissected and trypsinized. GFP+ cells (repre- identified by alignment of sequences of 50 kb upstream of the senting the whole pro-mesonephros epithelium) were sorted from coding regions (first ATG) using nBLAST (NCBI). Putative transcrip- surrounding GFP- trunk cells by flow cytometry. The mRNA tion factor binding sites were identified by probing conserved content of the cells was amplified by two rounds of linear RNA sequences for the presence of Pax2/5/8, Gata3 and Lim1 consensus amplification using T7-RNA polymerase recognition sequence DNA binding sequences using MacVector 8.0. The region covered inserted during reverse transcription. Microarray analysis was was increased in the absence of putative binding sites. The done on cDNA microarray slides containing 26,000 expressed consensus binding site sequence used are the following: Gata3, sequence tag (EST) clones from the BMAP and NIA libraries. (A/T)GATA(A/G) (Orkin, 1992), Pax2/8, (A/G)N(A/C/T)CANT(C/G)A Real-time quantitative PCR validation was done on sorted Pax2- (A/T)GCGT(A/G)(A/T)(A/C) allowing three mismatches (Boualia BACGFP;Pax2/ or Pax2+/+ cells at 18–21 somite stage, using et al., 2011) and Lim1, the core homeodomain consensus binding Green-to-go (Biobasic) on Realplex2 PCR machine (Eppendorf). sequence ATTA (Mochizuki et al., 2000). S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566 557

Chromatin immunoprecipitation triplicates. The qPCR results were accepted between 495% o105% efficiency (E¼1–10(1/slope)). All results were normalized Murine inner medullary collecting duct cells (mIMCD3) stably to an unrelated control region near the FoxO6 gene on chromo- expressing Pax2-HA, Gata3-HA and Lim1-Flag were cross-linked some 1. The final enrichment was calculated as the ratio of with 1% (w/v) formaldehyde for 10 min. The cells were collected normalized values for antibody over beads only. Primers were and sonicated to achieve DNA shearing to an average of 200 bp. designed around conserved DNA-binding consensus sites and are The chromatin was pre-cleared with protein G-Agarose beads listed in Table S2. (Roche, cat.11243233001) and precipitated overnight with an anti-HA (Abcam), an anti-Pax2 antibody (Covance PRB-276P, LN#143834801), an anti-Flag antibody (Sigma) or beads alone as a negative control. The antibody was retrieved with protein Results G-Agarose beads for 2 h. The HA immunoprectitations (Ab or beads only) were washed 8 10 min with 1 ml of Buffer I (low Pax2 transcriptional profile identifies molecular links to known salt: 0.1% SDS, 1% Triton-X, 20 mM Tris–HCl (pH8), 150 mM Nacl), kidney developmental regulators 8 10 min with 1 ml of Buffer II (high salt: 1% Triton-X, 20 mM Tris–HCl (pH8), 2 mM EDTA, 500 mM NaCl), 1 with 1 ml of We have previously reported that the transcription factors Pax2 Buffer III (250 mM LiCl, 1% Igepal, 1 mM EDTA, 1% Na-Desoxycho- and Pax8 are necessary and sufficient for renal lineage specifica- late, 10 mM Tris–HCl (pH8)), 1 with 1 ml of Buffer IV (1 Tris- tion in the embryo (Bouchard et al., 2002). To better understand EDTA), and de-crosslinked at 65 1C overnight in 150 ml of Buffer V the transcriptional program regulated by Pax2, we reanalyzed the

(1% SDS, 0.1 M NaHCO3). Pax2 immunoprecpitations (Ab and beads expression profile of Pax2-deficient pro/mesonephric cells (Grote only) were washed 6 with Buffer I, 6 with Buffer II, 1 with et al., 2006). In these experiments, pro/mesonephric cells were Buffer III, 1 with Buffer IV and de-crosslinked overnight in Buffer isolated using a BAC transgene expressing GFP under Pax2 control V. The Flag immunoprectitations (Ab and beads only) were washed (Pax2BACGFP) and samples were compared (1) between wild type with 7 with Buffer I, 8 with Buffer II, 1 with Buffer III, 1 and Pax2 mutant embryos at 13, 16 and 20 somite stages, with Buffer IV, and de-crosslinked overnight in Buffer V. The (2) between GFP-positive pro/mesonephric cells and GFP- samples were then treated with proteinase K (0.2 mg/ml) for 1 h negative trunk cells and (3) between 13 vs. 16 and 13 vs. 20 at 55 1C. Chromatin was isolated using the QIAquick PCR purifica- somite stage pro/mesonephric cells. We placed the emphasis on tion kit (Qiagen cat.28106). Quantitative PCR was performed on a genes differentially regulated by Pax2 at the 20 somite stage to minimum of 3 independent ChIP samples (biological replicates) reduce experimental stringency and identify additional differen- and compared to beads only experimental control. Each sample tially expressed genes (Figs. 1 and S1). This analysis identified a and control was analyzed in technical triplicates. Error bars refer large proportion of genes involved in transcription and signaling to biological triplicate data derived from the mean of all 3 technical (Fig. S1; Table S1), supporting a role for Pax2 in the early

Fig. 1. Genes differentially regulated by Pax2 and expressed in pro/mesonephros tissue. (A) Pax2-dependent genes were identified by cDNA microarray analysis and selected as being differentially regulated by Pax2 at 13, 16 and 20 somites, upregulated between 13 and 16 or 20 somites or specifically expressed in pro/mesonephros GFP+ tissue of Pax2BACGFP mice. Ratios are expressed as control/mutant (Pax2/), late/early or GFP+/GFP‐. The embryonic stages are defined as somite number(s). (B) Validation of microarray results for the genes identified in (A), using quantitative RT-PCR analysis on sorted GFP+ renal cells from 18 to 21 somite stage Pax2BACGFP embryos either wild type or mutant for Pax2. 558 S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566 deployment of the regulatory program of pro/mesonephros and C) (Bouchard et al., 2002; Grote et al., 2006; Kobayashi et al., 2005; morphogenesis. Miyamoto et al., 1997; Pedersen et al., 2005). Due to functional A selection for genes positively regulated by Pax2 revealed a redundancy between Pax2 and Pax8 in renal lineage specification number of transcription factor genes previously reported to play a (Bouchard et al., 2002), we opted to study the role of Pax genes by role in pro/mesonephros morphogenesis. As these genes represent removing an additional allele of Pax8 on a Pax2 null background, nodes in the pro/mesonephros developmental program, we effectively lowering Pax levels further, while still ensuring the forma- decided to focus on them for the purpose of primary network tion and morphogenesis of the pro/mesonephros. The analysis of characterization. Among the transcription factors requiring Pax2 Pax2/;Pax8+/ embryos confirmed the genetic dependency of function were Gata3 (as previously reported (Grote et al., 2006)) Gata3 on Pax gene function and further revealed a complete loss of but also Lim1 (Lhx1), Evi1, Id4 (Idb4) and Plac8 (Fig. 1). The latter Lim1 in Pax-deficient embryos (Fig. 2F). Similarly, upon removal of was recently identified as a Pax2-dependent transcriptional regulator Gata3, the expression of Lim1 was strongly reduced, while Pax2 required for renal development in zebrafish (Bedell et al., 2012). expression remained unchanged (Fig. 2HandI),confirming that In addition to transcription factors, we examined Pax2-dependent Pax2 acts genetically upstream of these genes. We next analyzed the genes for candidate effectors of pro/mesonephros morphogenesis. expression of Pax2 and Gata3 transcription factors in Lim1-deficient This analysis identified three genes previously associated with nephric embryos. Interestingly, Lim1 was found to be required for Gata3,but duct morphogenesis among the bestcandidatesdownstreamofPax2, not Pax2 expression, (Fig. 2K and L), placing it at a position equivalent namely Nephronectin (Npnt), Pcdh19 and Wfdc2 (Fig. 1). Npnt is to Gata3 in the transcriptional hierarchy. To validate the presence of specifically expressed in the nephric duct and acts as a ligand of the pro/mesonephric duct tissue in the region being investigated, adjacent mesenchymal alpha8/beta1 integrin complex (Brandenberger et al., sections to all in situ hybridization results described here stained 2001). Importantly, gene inactivation of either Nephronectin or alpha8 positive for Pax8 expression (Fig. 2D,G,J and M, data not shown). The integrin gene (Itga8) results in metanephric kidney agenesis (Linton fact that Pax8 was never found affected in these mutant backgrounds et al., 2007). Pcdh19 and Wfdc2 are potential nephric duct regulators also confirms Pax8 next to Pax2 at the onset of the transcriptional based on their strong expression previously reported in this tissue cascade of pro/mesonephros development. (Gaitan and Bouchard, 2006; Hellstrom et al., 2003). Together, these Together, these results identify a transcriptional cascade results identify important regulators of pro/mesonephros develop- whereby Pax2/8 act upstream of both Gata3 and Lim1, which in ment requiring Pax2 function for normal expression. turn require each other's function. From these data, we can define the Pax2/8-Gata3-Lim1 group as the core or “kernel” of the renal Hierarchical relationship between Pax2/8, Gata3 and Lim1 primordium gene regulatory network.

As the transcriptional profiling of Pax2-deficient pro/mesonephros The renal primordium kernel regulates a deeper GRN identified the key developmental regulators Gata3 and Lim1, we sought out to clarify the genetic relationship between these factors To study the depth of the renal primordium GRN, we next in the developing pro/mesonephros. We first assessed the expression assessed the regulation of additional transcription factors with of each transcript in wild type embryos by in situ hybridization. As reported roles in renal development. For this, we focused on previously reported, the expression of all three transcription factors transcriptional regulators identified among Pax2-regulated genes; was robust and specific to the pro/mesonephric epithelium (Fig. 2A Evi1, whose mutant show mesonephric hypoplasia (Hoyt et al., 1997),

Fig. 2. Genetic interaction between kidney development regulators. In situ hybridization for Pax2 (A,H and K), Gata3 (B,E and L), Lim1 (C,F and I), Pax8 (D,G,J and M) on E9.5 embryo sections of the indicated genotypes. The pro/mesonephros expression of both Gata3 and Lim1 requires Pax2/8 gene function. In turn, Gata3 and Lim1 require each other for sustained expression. Pax2 and Pax8 are unaffected by the loss of Gata3 or Lim1. S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566 559

Fig. 3. Altered expression pattern of transcriptional regulators of renal development. In situ hybridization for Evi1 (A,E,I and M), Id4 (B,F,J and N), Plac8 (C,G,K and O) and Emx2 (D,H,L and P) on E9.5 embryo sections of the indicated genotypes. Evi1 expression requires Lim1 gene function, while Pax2/8 plays a complementary role in its regulation. Conversely, Id4 and Plac8 require Pax2/8 and Gata3, whereas Lim1 plays a secondary role. Emx2 expression is lost in Lim1 mutant embryos, reduced in Pax2/8 deficient embryos and unaffected by Gata3 loss. the new renal developmental regulator Plac8 (Bedell et al., 2012)and on potential regulators of the cellular and tissue events necessary Id4, previously shown to be expressed in Xenopus pronephric for proper pro/mesonephric development. We focused on Npnt, development (Liu and Harland, 2003). To expand this analysis, we Pcdh19, and Wfdc2 three downstream cellular effectors previously included the transcription factor Emx2, another important regulator linked to kidney development that were identified differentially of kidney development that was recently shown to cooperate with regulated in Pax2 transcriptional profiling analysis. Pax2 in pro/mesonephros morphogenesis (Boualia et al., 2011). In situ We first confirmed the mesonephric expression of all three hybridization analysis of Evi1 expression in wild type embryos effector genes, which was found specific to the pro/mesonephros confirmed a specific localization of this gene in pro/mesonephric (Fig. 4A and C) (Gaitan and Bouchard, 2006). Strikingly, Wfdc2 tissue (Fig. 3A). In contrast, the nephric duct expression of Evi1 was expression was completely lost in Pax2/ Pax8+/ embryos severely compromised in Pax2/;Pax8+/ and completely abro- (Fig. 4E), while Npnt expression levels were strongly reduced gated in Lim1/ embryos (Fig. 3EandM)butnotsignificantly (Fig. 4F), identifying Pax gene requirement for full activation affected by Gata3 deficiency (Fig. 3I). In Pax2/;Pax8+/ embryos, and/or maintenance of these effectors genes. In contrast, Pcdh19 the rostral-most region maintained some Evi1 expression (not was not significantly affected in Pax2/8-deficient embryos shown), likely due to higher levels of Pax8 expression in this region. (Fig. 4D). In Gata3-/- embryos, Npnt and Pcdh19 levels of expression The wild type expression of Id4 and Plac8 was strong, and specificto were only slightly downregulated and Wfdc2 levels stayed unaf- the nephric duct (Fig. 3B and C) but greatly reduced in Lim1/ fected (Fig. 4G–I). As for Lim1/ embryos, the expression of embryos (Fig. 3N and O) and completely abrogated in the renal Pcdh19 and Npnt transcript expression was completely lost (Fig. 4J primordium of Pax2/ Pax8+/ and Gata3/ embryos (Fig. 3F,G,J and L), while Wfdc2 transcription was still detected (Fig. 4K), and K). The analysis of Emx2 expression levels confirmed its down- arguing for a critical role of Lim1 for proper Npnt and Pcdh19 regulation in Pax2/8-deficient embryos (Fig. 3H). Strikingly, Emx2 expression in the pro/mesonephros. expression was completely lost in Lim1-deficient nephric ducts Together, these results point to a genetic regulation of specific (Fig. 3P) but unaffected by Gata3-deficiency. Of interest, the expres- effector genes by single core regulators, as well as instances of sion of Pax2, Gata3 and Lim1 was found unaffected in Emx2-deficient coregulation by a combination of core transcriptional regulators. embryos at E9.5, confirming the downstream position of this Noteworthy, the regulation of specific effector genes occurs at transcription factor in the renal GRN (Fig. S2). different levels of the GRN kernel. Together, these results point to a relatively deep structure of the renal primordium GRN in which the kernel regulates at least Pax2 directly binds the Lim1 and Plac8 regulatory regions one additional layer of transcriptional regulators of renal primordium development. To assess whether the genetic regulatory interactions observed above are direct or not, we next used a Chromatin Immunopreci- Effector gene expression is regulated at different levels of the renal pitation (ChIP) approach. The number of cells of the renal primordium GRN primordium being limiting, we turned to the mouse inner collecting duct cell line (mIMCD3). As it is derived from the collecting Having established the genetic hierarchy within the transcrip- duct, this cell line is among the closest possible to nephric duct tional regulators, we wanted to further characterize their output cellular fate. Accordingly, it was shown to express endogenous 560 S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566

Fig. 4. Altered expression pattern of cellular effectors of kidney development. In situ hybridization for Pcdh19 (A,D,G and J), Wfdc2 (B,E,H and K) and Npnt (C,F,I and L) on E9.5 embryo sections of the indicated genotypes. Pcdh19 expression is mostly affected by Lim1 deﬁciency. Wfdc2 requires Pax2/8 but not Gata3 or Lim1. Npnt requires Lim1 function, while Pax2/8 and Gata3 play a complementary role.

Pax2 (Cai et al., 2005). Our analysis confirmed this expression but To validate further these positive sites we tested the enrichment of revealed very low levels of Gata3 and Lim1 expression (Fig. S3A, C, flanking regions less than 300 bp away (P2Lim1-3UP (upstream); D and E; mIMCD3-EV). P2Lim1-3D (downstream); P2Lim1-4UP; P2Lim1-4D) which failed To identify Pax2, Gata3 and Lim1 target genes in the same to be enriched by Pax2, re-enforcing the specificity of P2Lim1-3 system, we derived a stable mIMCD3 cell line expressing both HA- and P2Lim1-4 binding sites (Fig. 5B). The bioinformatics analysis of tagged Gata3 and Flag-tagged Lim1, as well as a Pax2-HA mIMCD3 100 kb upstream and 50 kb downstream of the Plac8 locus cell line (Fig. S3; mIMCD3-G3HA-L1Flag; mIMCD3-P2HA). As there identified 31 conserved regions, two of which contained putative was no established target binding sites of Pax2 in renal primor- Pax2 binding sites (Fig. 5D; P2Plac8-1 and P2Plac8-2). Chromatin dium genes, we first created an artificial positive control by immunoprecipitation identified only P2Plac8-2 as robustly bound generating a stable derivative of mIMCD3 Pax2-HA cells harboring by Pax2 with an enrichment ratio of 5.2 fold compared to the a concatemer of three strong Pax2/5/8 binding sites derived from control (Fig. 5E). Together, these data point to a direct binding of the CD19 locus (Kozmik et al., 1992). This stable cell line generated Pax2 to Lim1 and Plac8 regulatory regions. very robust and reproducible enrichment of more than 10 fold To assess whether the regions bound by Pax2 are in an active compared to beads only and was used as positive control in transcriptional state we used the H3K4me1 histone mark. Mono- subsequent experiments (Fig. S4A). ChIP against the endogenous methylation of histone H3 on lysine 4 has indeed been used to or HA-Pax2 gave similar results in this cell line. identify active enhancer sites in whole genome analyses (Heintz- To establish the direct regulatory role of Pax2, we focused on man 2009; Visel 2009). As expected, we found that H3K4me1 the genes Gata3, Lim1, Evi1, Id4, Npnt, Wfdc2 and Plac8. For each of mark was specifically enriched in regions bound by Pax2 (P2Lim1- these genes, putative binding sites conserved between mouse and 3, P2Lim1-4, P2Plac8-2) (Fig. 5C and F), demonstrating that these human were identified in genomic regions located within 50 kb of sites are bona fide enhancers. As expected for this chromatin mark, the translation start site. Each of these sites was tested either by the modification extended to the immediate neighboring regions endogenous Pax2 or Pax2-HA- mediated ChIP. Of those, we could of the bound sites (“UP” and “D” primer pairs) but not to more identify a very strong binding of Pax2 to Lim1 and Plac8 regulatory distant regions (Fig. 5B and F). regions whereas other genes failed to identify direct DNA-binding We next determined whether the identified Pax2 binding sites interactions (Fig. 5B D and S4A). The Lim1 locus contained 57 located in enhancer sequences were responsive to Pax2. For this, we conserved regions, four of which contained conserved putative cloned the P2Lim1-3, P2Lim1-4 and P2Plac8-2 regions in pGL3-PolA Pax2 binding sites (Fig. 5A; P2Lim1-1 to 4). Chromatin immuno- luciferase reporter vector and generated derivatives with point precipitation identified the conserved sites P2Lim1-3 and P2Lim1- mutations in Pax2 consensus binding sites. Co-transfections of the 4 as robustly bound by Pax2 with an enrichment ratio of 5.5 constructs with a Pax2-expression vector in HEK293T demonstrated fold and 13.6 fold compared to control, respectively (Fig. 5B), a strong activation by Pax2 that was significantly reduced in Pax2 whereas sites P2Lim1-1 and P2Lim1-2 failed to show enrichment. mutant derivatives (Fig. 5GandS8A). S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566 561

Fig. 5. Direct interaction between Pax2 and downstream regulated genes. (A and D) schematic representation of sequence conservation (thick bars) and Pax2 putative binding sites (asterisks) in the Lim1 (A) and Plac8 (D) loci. (B and E) Chromatin immunoprecipitation against Pax2 in mIMCD3‐Gata3‐HA‐Lim1‐Flag cells. Regions from the Lim1 (B) and Plac8 (E) loci containing putative Pax2 binding sites, or either 300 bp upstream (UP) or 300 bp downstream (D) sequences were ampliﬁed by real-time PCR. Data are expressed as fold enrichment relative to beads only and represent the average of experimental triplicates. (C and F) Chromatin immunoprecipitation against H3K4me1 marks (active enhancer) in the Lim1 (C) and Plac8 (F) loci. (G) Functional analysis of Pax2 binding sites in the Lim1 and Plac8 enhancer regions. The HEK293T cell line was cotransfected with Pax2 expressing vector and wild type or mutant enhancer constructs. Results are expressed as luciferase activity relative to PolA promoter activity and represent the average7SD of triplicate of 2 independent experiments. Luciferase reporter activities were normalized to that of an internal control (pCMV-Renilla).

Together, these results demonstrate a direct binding and control (G3Ret-2; Fig. 6AandB)(Grote et al., 2006, 2008). Reanalysis regulation of Pax2 to active enhancer sites of Lim1 and Plac8. of the Ret locus with the criteria described above identified a total of 4 putative Gata3 binding sites located in conserved regions. ChIP Gata3 binds the Ret and Lim1 regulatory regions analysis on these sites confirmed the binding of Gata3 to G3Ret-2 (2.9 fold enrichment) and further identified a strong binding of Gata3 We next turned to the identification of direct Gata3 binding sites to G3Ret-1 (4.0 fold enrichment) (Fig. 6B). These results validate the byChIP.Forthis,wetookadvantageofaGata3bindingsite ChIP approach for Gata3 binding sites and further support the direct previously associated with Ret gene expression to use as positive regulation of Ret by Gata3. 562 S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566

Fig. 6. Gata3 binds Ret and Lim1 regulatory regions. (A and D) schematic representation of sequence conservation (thick bars) and Gata3 putative binding sites (asterisks) in the Ret (A) and Lim1 (D) loci. (B and E) Chromatin immunoprecipitation against Gata3-HA in mIMCD3‐Gata3‐HA‐Lim1‐Flag cells on regions containing or ﬂanking (300 bp UP, D) conserved putative binding sites for Gata3 in the Ret (B) and Lim1 (E) loci. (C and F) Chromatin immunoprecipitation against H3K4me1 enhancer marks in the Ret (C) and Lim1 (F) loci. (G) Luciferase reporter analysis of Gata3 binding sites in the Ret and Lim1 enhancer regions by cotransfection with Gata3 expressing vector as in Fig. 5G.

We then performed ChIP on conserved Gata3 binding sites on demonstrated a direct role for Gata3 in the regulation of these genes differentially regulated by Pax2. This analysis identified a binding sites (Fig. 6G and S8B). Together, these results identify direct binding of Gata3 to Lim1. Bioinformatic analysis of the Lim1 Lim1 and Ret as direct regulatory targets of Gata3. locus identified 4 conserved Gata3 consensus binding sites (Fig. 6D). Of those, one was robustly enriched to 5.0 fold when Npnt Is a direct regulatory target of Lim1 compared to control conditions (Fig. 6E). This conserved site lies in a long 502 bp highly conserved stretch at 7.6 kb upstream of Lim1 The genetic regulation experiments determined that Lim1 was translation start site. The analysis of putative Gata3 binding sites required for the expression of Gata3, Id4, Evi1, Npnt and Pcdh19. for H3K4Me1 methylation mark confirmed the active state of Detailed ChIP analysis of the conserved genomic regions of these the G3-Ret1, G3Ret2 and G3Lim1-1 enhancer regions (Fig. 6C,F). genes positively identified a direct regulation of Npnt by Lim1. In addition, the functional analysis of these sites in HEK293T cells The bioinformatic analysis of 50 kb upstream of the Npnt locus S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566 563

enhancer conformation (Fig. 7C). The functional analysis of these sites further confirmed that Lim1 directly regulates their transcriptional activity (Fig. 7D and S8C). Together, these ChIP analyses successfully identified a cis- regulatory interaction of important renal GRN genes by the three kernel regulators. The identification of specific cis-regulatory modules demonstrates the direct role played by kernel regulators on downstream regulatory and effector genes and allows a first representation of the regulatory genome leading to renal GRN deployment.

Discussion

The early morphogenesis of organ systems is orchestrated by gene regulatory networks (GRN), which drive cellular organization and differentiation. In the urogenital system, we have previously identified the Pax2/8 and Gata3 transcription factor genes as crucial regulators of specification and morphogenesis at the earliest stages of organ development. We took advantage of these developmental nodes to elucidate the hierarchical organization of a first gene regulatory network underlying pro/mesonephros development. Our results reveal that Pax2/8 and Gata3 form a core or “kernel” GRN together with the transcription factor Lim1 (Fig. 8). Whereas the regulation of Gata3 by Pax2/8 was previously reported (Grote et al., 2006), this study identified several new gene interactions, one of which being the direct regulation of Lim1 by Pax2/8 genes. Following activation by Pax2/8, the regulatory molecules Gata3 and Lim1 maintain each other's expression (Fig. 8). Once established, this core renal GRN activates the expression of a deeper layer of gene regulatory molecules as well as a complex set of effector genes. For the purpose of characteriz- ing a first skeleton of the renal primordium GRN, we concentrated on downstream genes with a known or presumed role in pro/ mesonephros development. We could thus link the regulatory and effector genes Evi1, Id4, Npnt, Ret, Wfdc2, Pcdh19 and Plac8 to the Pax2/8-Gata3-Lim1 core renal GRN (Fig. 8).

Regulatory network of pro/mesonephros development

Gene regulatory “kernels” are deﬁned by a strong interrelationship between evolutionary conserved components of a network and a critical role of these components for structure formation (Davidson, 2010; Davidson and Erwin, 2006). They are impervious

Fig. 7. Lim1 is a direct regulator of Npnt expression. (A) Schematic representation to change as a result of high evolutionary pressure applied on the of sequence conservation (thick bars) and Lim1 putative binding sites (asterisks) in successful initiation of vital structure/organ development. A num- the Npnt locus. (B) Chromatin immunoprecipitation against Lim1-Flag in mIMCD3‐ ber of evidence support the notion that Pax2/8, Gata3 and Lim1 Gata3‐HA‐Lim1‐Flag cells on regions containing or flanking (300 bp UP, D) correspond to this definition and can therefore be considered as conserved putative binding sites for Lim1 in the Npnt locus. (C) Chromatin the “renal primordium GRN kernel”. Our data shows that both immunoprecipitation against H3K4me1 enhancer mark. (D) Functional analysis of Lim1 binding sites in the Npnt enhancer sequences by cotransfection with Lim1 expressing vector as in Fig. 5G. identified few conserved regions and was extended to 100 kb upstream and 50 kb downstream of the Npnt coding region. In the 15 conserved regions identified, 7 Lim1 putative binding sites were identified (using the generic homeodomain consensus sequence ATTA) (Mochizuki et al., 2000), 3 of which showed significant enrichment (L1Npnt-1 (2.8 fold), L1Npnt-2 (2.3 fold) and L1Npnt-3 (3.5 fold)) (Fig. 7A and B). The L1Npnt-1 site is located 14 kb upstream of the transcriptional start site, while the L1Npnt-2 and L1Npnt-3 sites are located in the 3′ untranslated Fig. 8. Model of renal gene regulatory network organization. A core regulatory subcircuit composed of Pax2/8, Gata3 and Lim1 turns on a deeper layer of region of Nephronectin (Fig. 7A). ChIP analysis with an anti- transcriptional regulators composed of developmental molecules, while activating fi H3K4Me1 antibody successfully ampli ed all three Lim1 target effector genes which are responsible for cell signaling and tissue organization (see sites in the Npnt locus, indicating that these sites are in an active text for details). 564 S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566

Gata3 and Lim1 require Pax2/8 function while the latter two are “evolutionary dynamic” gene pool. Evolutionary dynamic genes interdependent for normal expression in the renal primordium. are thought to provide an additional level of complexity in higher This close interrelationship is likely to lock the renal fate program organisms. Accordingly, ponzr1 morpholino knockdown in zebra- following lineage specification by Pax2/8 (Bouchard et al., 2002). fish leads to a pronephros phenotype characteristic of lower Importantly, each of them plays a different but essential role in vertebrates (Bedell et al., 2012). The fact that Plac8/ponzr1 expres- pro/mesonephros development. While Pax2/8 are required for sion and regulation by Pax2 has been conserved between zebrafish renal lineage specification and survival (Bouchard et al., 2002), and mammals is consistent with the establishment and main- Gata3 is necessary for duct guidance, proliferation control and to tenance of a more sophisticated regulatory subcircuit of renal prevent a premature differentiation of nephric duct cells (Chia development in recent evolution. et al., 2011; Grote et al., 2008; Grote et al., 2006). As for Lim1, it is required for nephric duct elongation and integrity (Kobayashi Regulation of signaling outputs et al., 2004; Pedersen et al., 2005; Shawlot and Behringer, 1995). As a consequence, embryos mutant for either of the genes fail to We have previously reported that Gata3 is required for Ret complete pro/mesonephros development and to initiate adult expression in the nephric duct and that this subcircuit is activated (metanephros) kidney development. In addition to this, all four by Pax2 and maintained by Wnt-β-catenin (Grote et al., 2006, transcription factors are evolutionary conserved in terms of 2008). More recently, we have shown that the regulation of Ret by expression, function and hierarchy in the renal primordium. Gata3 and retinoic acid signaling is necessary for the guidance and Evidence from chick, Xenopus or zebrafish systems identified fusion of the nephric duct to the cloaca (Chia et al., 2011). In the Pax2 and Pax8 as the earliest factors specifically expressed in the current network analysis, we reinforce the relationship between renal system (Heller and Brandli, 1999; Krauss et al., 1991; Mauch Gata3 and Ret by the identification of additional direct binding et al., 2000; Pfeffer et al., 1998; Puschel et al., 1992), while Gata3 sites in the Ret regulatory region. In addition to regulating Ret, the and Lim1 are expressed in the early pronephros (Carroll et al., pro/mesonephros GRN kernel indirectly regulates its ligand GDNF 1999; Sheng and Stern, 1999; Wingert and Davidson, 2011). expressed in the metanephric mesenchyme. Our data indeed Functional studies further suggest that these genes have conserved identify Nephronectin (Npnt) as a direct target of Lim1 in the their regulatory role throughout vertebrate evolution (Bouchard nephric duct. Npnt is the ligand of the alpha8-beta1 integrin et al., 2002; Carroll and Vize, 1999; Majumdar et al., 2000). complex (Brandenberger et al., 2001; Morimura et al., 2001). Interestingly, Pax, Gata and Lim1 factors have been identified Together, the Npnt-Alpha8 integrin signaling complex is necessary in other demonstrated or presumed GRN kernels. In the mouse, for Gdnf expression in the mesenchyme. Accordingly, mouse Pax2-deficient embryos completely lack kidneys, cochlea and mutants for either Npnt or Itga8 (alpha8 integrin gene) fail to cerebellum (Torres et al., 1995, 1996). In the mid-hindbrain induce metanephric kidney development (Linton et al., 2007; boundary organizer, which initiates posterior midbrain and cere- Muller et al., 1997). Hence the pro/mesonephros GRN regulates bellum development, Pax2 was identified as the primary regula- ureter budding in the epithelial (Ret) as well as the mesenchymal tory molecule working upstream of other regulators of the (Gdnf) tissue compartments. organizer (Ye et al., 2001). Along the same line, Gata3 acts as a One interesting observation resulting from our analysis is that lineage commitment factor for the Th2 hematopoietic lineage effector genes are activated at all levels of the network and not (Hosoya et al., 2010; Ting et al., 1996) and Lim1 is a crucial player only as an output to a previously established GRN. For example, of head organizer function (Shawlot and Behringer, 1995). Wfdc2 is exclusively regulated by Pax genes, while Npnt is mostly Together, these observations suggest that the transcription factors affected by loss of Lim1 function. Hence, the core components involved in the GRN kernel may have functional properties perform two parallel functions: (1) they unfold the lineage-specific generally applicable to other kernel subcircuits. transcriptional program to diversify and refine the transcriptional response and (2) they activate the lineage-specific effector and Effectors of the pro/mesonephros GRN differentiation genes. We believe that the coordination of both functions insures a balance between morphogenetic events and Evidence we have collected here, together with related data progressive cell differentiation. from the literature suggests that the renal GRN is relatively deep. A key finding of our study is the identification of direct In immediate response to pro/mesonephros GRN kernel is a wave interactions between kernel gene components and key regulators of transcriptional regulators and regulatory effectors. Among them of kidney development. This observation underscores the fact that are the transcriptional regulators Evi1, Id4 and Plac8, as well as the gene inactivation phenotypes cannot be taken in isolation but Pax2 regulated gene Emx2 (Boualia et al., 2011). Evi1 deficiency rather as a series of additive misregulatory events. This may have has been associated with mesonephros hypoplasia (Hoyt et al., direct consequences in the understanding of developmental dis- 1997), which may reflect a role in proliferation control. Id4 is a eases affecting the kidney and urinary tract. The terminology member of the inhibitor of differentiation gene family specifically Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) expressed in renal tissue (Liu and Harland, 2003). Interestingly, Id is used to describe a series of developmental defects affecting proteins have been shown to bind and inactivate Pax2/5/8 DNA- kidney and urinary tract development. It is characterized by a high binding activity (Roberts et al., 2001). This property raises the degree of intra and interfamilial variability (Weber, 2012). This interesting possibility that the activation of Id4 by Pax2 constitutes variability has been proposed to be the result of a threshold effect a negative feedback loop that serves to modulate the Pax2 for key regulatory molecules such that a given developmental transcriptional response. stage proceeds or fails depending on whether a given gene A similar subcircuit structure has been described for Plac8. This function is sufficient or not. In the context of the pro/mesonephros newly described transcriptional regulator protein is necessary for GRN, we propose that CAKUT variability would be better modeled glomerular development in zebrafish (Bedell et al., 2012). Inter- as network dynamics deficiencies in which the activation, main- estingly, Plac8 (ponzr1 in zebrafish) also requires Pax2 for normal tenance and feedback relations of a number of molecules deter- expression. In addition, the downregulation of ponzr1 leads to an mine whether or not a morphogenetic process is completed upregulation of Pax2, pointing to a negative feedback loop reg- successfully. Hence, one expects CAKUT to be caused by a number ulating Pax2 expression levels. Noteworthy, Plac8/ponzr1 appeared of different interrelated GRN members and lead to a spectrum of relatively recently during evolution and is therefore part of the possible phenotypes. S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566 565

Acknowledgments Hellstrom, I., Raycraft, J., Hayden-Ledbetter, M., Ledbetter, J.A., Schummer, M., McIntosh, M., Drescher, C., Urban, N., Hellstrom, K.E., 2003. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Res. 63, 3695–3700. We would like to thank Drs. M. Busslinger, (IMP, Vienna), R. Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., Ish-Horowicz, D., 1995. Behringer (U. Texas) and Amed Mansouri (MPI for Biophysical Expression of a Delta homologue in prospective neurons in the chick. Nature Chemistry, Göttingen, Germany) for providing mouse strains 375, 787–790. Hosoya,T.,Maillard,I.,Engel,J.D.,2010.Fromthecradletothegrave:activitiesofGATA-3 and Drs. J. Pelletier (McGill University) and P. Goodyer (McGill throughout T-cell development and differentiation. Immunol. Rev. 238, 110–125. University) for reagents. This work was supported by a grant from Hoyt, P.R., Bartholomew, C., Davis, A.J., Yutzey, K., Gamer, L.W., Potter, S.S., Ihle, J.N., the Canadian Institutes for Health Research (CIHR; MOP-84470). Mucenski, M.L., 1997. The Evi1 proto-oncogene is required at midgestation for neural, heart, and paraxial mesenchyme development. Mech. Dev. 65, 55–70. M.B. holds a Canada Research Chair in Developmental Genetics of Kobayashi, A., Kwan, K.M., Carroll, T.J., McMahon, A.P., Mendelsohn, C.L., Behringer, the Urogenital System. M.T. was supported by a Dr. Gerald B. Price R.R., 2005. Distinct and sequential tissue-specific activities of the LIM-class Fellowship (CRS), MICRTP and Fonds de Recherche Québec Santé homeobox gene Lim1 for tubular morphogenesis during kidney development. Development 132, 2809–2823. (FRQS) postdoctoral fellowships. Kobayashi, A., Shawlot, W., Kania, A., Behringer, R.R., 2004. Requirement of Lim1 for female reproductive tract development. Development 131, 539–549. Koide, T., Hayata, T., Cho, K.W., 2005. Xenopus as a model system to study transcriptional regulatory networks. Proc. Natl. Acad. Sci. USA 102, 4943–4948. Appendix A. Supporting information Kozmik, Z., Wang, S., Dorfler, P., Adams, B., Busslinger, M., 1992. The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. Mol. Cell – Supplementary data associated with this article can be found in Biol. 12, 2662 2672. Krauss, S., Johansen, T., Korzh, V., Fjose, A., 1991. Expression of the zebrafish paired the online version at http://dx.doi.org/10.1016/j.ydbio.2013.07.028. box gene pax[zf-b] during early neurogenesis. Development 113, 1193–1206. Levine, M., Davidson, E.H., 2005. Gene regulatory networks for development. Proc. Natl. Acad. Sci. USA 102, 4936–4942. Linton, J.M., Martin, G.R., Reichardt, L.F., 2007. The ECM protein nephronectin promotes kidney development via integrin alpha8beta1-mediated stimulation of Gdnf expression. Development 134, 2501–2509. References Liu, K.J., Harland, R.M., 2003. Cloning and characterization of Xenopus Id4 reveals differing roles for Id genes. Dev. Biol. 264, 339–351. fi Alon, U., 2007. Network motifs: theory and experimental approaches. Nat. Rev. Majumdar, A., Lun, K., Brand, M., Drummond, I.A., 2000. Zebra sh no isthmus Genet. 8, 450–461. reveals a role for pax2.1 in tubule differentiation and patterning events in the – Bedell, V.M., Person, A.D., Larson, J.D., McLoon, A., Balciunas, D., Clark, K.J., Neff, K.I., pronephric primordia. Development 127, 2089 2098. Nelson, K.E., Bill, B.R., Schimmenti, L.A., Beiraghi, S., Ekker, S.C., 2012. Mauch, T.J., Yang, G., Wright, M., Smith, D., Schoenwolf, G.C., 2000. Signals from The lineage-specific gene ponzr1 is essential for zebrafish pronephric and trunk paraxial mesoderm induce pronephros formation in chick intermediate – pharyngeal arch development. Development 139, 793–804. mesoderm. Dev. Biol. 220, 62 75. Boualia, S.K., Gaitan, Y., Murawski, I., Nadon, R., Gupta, I.R., Bouchard, M., 2011. Miyamoto, N., Yoshida, M., Kuratani, S., Matsuo, I., Aizawa, S., 1997. Defects of – Vesicoureteral reflux and other urinary tract malformations in mice compound urogenital development in mice lacking Emx2. Development 124, 1653 1664. heterozygous for Pax2 and Emx2. PLoS One 6, e21529. Mochizuki, T., Karavanov, A.A., Curtiss, P.E., Ault, K.T., Sugimoto, N., Watabe, T., Bouchard, M., 2004. Transcriptional control of kidney development. Differentiation Shiokawa, K., Jamrich, M., Cho, K.W., Dawid, I.B., Taira, M., 2000. Xlim-1 and LIM 72, 295–306. domain binding protein 1 cooperate with various transcription factors in the – Bouchard, M., Grote, D., Craven, S.E., Sun, Q., Steinlein, P., Busslinger, M., 2005. regulation of the goosecoid promoter. Dev. Biol. 224, 470 485. Identification of Pax2-regulated genes by expression profiling of the mid- Moore, M.W., Klein, R.D., Farinas, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L. hindbrain organizer region. Development 132, 2633–2643. F., Ryan, A.M., Carver_Moore, K., Rosenthal, A., 1996. Renal and neuronal – Bouchard, M., Souabni, A., Mandler, M., Neubuser, A., Busslinger, M., 2002. Nephric abnormalities in mice lacking GDNF. Nature 382, 76 79. lineage specification by Pax2 and Pax8. Genes Dev. 16, 2958–2970. Morimura, N., Tezuka, Y., Watanabe, N., Yasuda, M., Miyatani, S., Hozumi, N., Bouchard, M., St. Amand, J., Cote, S., 2000. Combinatorial activity of pair-rule Tezuka Ki, K., 2001. Molecular cloning of POEM: a novel adhesion molecule – proteins on the Drosophila gooseberry early enhancer. Dev. Biol. 222, 135–146. that interacts with alpha8beta1 integrin. J. Biol. Chem. 276, 42172 42181. Brandenberger, R., Schmidt, A., Linton, J., Wang, D., Backus, C., Denda, S., Muller, U., Muller, U., Wang, D., Denda, S., Meneses, J.J., Pedersen, R.A., Reichardt, L.F., 1997. Reichardt, L.F., 2001. Identification and characterization of a novel extracellular Integrin alpha8beta1 is critically important for epithelial-mesenchymal inter- – matrix protein nephronectin that is associated with integrin alpha8beta1 in the actions during kidney morphogenesis. Cell 88, 603 613. embryonic kidney. J. Cell Biol. 154, 447–458. Orkin, S.H., 1992. GATA-binding transcription factors in hematopoietic cells. Blood – Cai, Q., Dmitrieva, N.I., Ferraris, J.D., Brooks, H.L., van Balkom, B.W., Burg, M., 2005. 80, 575 581. Pax2 expression occurs in renal medullary epithelial cells in vivo and in cell Pedersen, A., Skjong, C., Shawlot, W., 2005. Lim 1 is required for nephric duct – culture, is osmoregulated, and promotes osmotic tolerance. Proc. Natl. Acad. Sci. extension and ureteric bud morphogenesis. Dev. Biol. 288, 571 581. USA 102, 503–508. Pfeffer, P.L., Gerster, T., Lun, K., Brand, M., Busslinger, M., 1998. Characterization of fi Carroll, T.J., Vize, P.D., 1999. Synergism between Pax-8 and lim-1 in embryonic three novel members of the zebra sh Pax2/5/8 family: dependency of Pax5 and – kidney development. Dev. Biol. 214, 46–59. Pax8 expression on the Pax2.1 (noi) function. Development 125, 3063 3074. Carroll, T.J., Wallingford, J.B., Vize, P.D., 1999. Dynamic patterns of gene expression Pichel, J.G., Shen, L., Sheng, H.Z., Granholm, A.C., Drago, J., Grinberg, A., Lee, E.J., in the developing pronephros of Xenopus laevis. Dev. Genet. 24, 199–207. Huang, S.P., Saarma, M., Hoffer, B.J., Sariola, H., Westphal, H., 1996. Defects in Chia, I., Grote, D., Marcotte, M., Batourina, E., Mendelsohn, C., Bouchard, M., 2011. enteric innervation and kidney development in mice lacking GDNF. Nature 382, – Nephric duct insertion is a crucial step in urinary tract maturation that is 73 76. regulated by a Gata3-Raldh2-Ret molecular network in mice. Development 138, Pimanda, J.E., Gottgens, B., 2010. Gene regulatory networks governing haemato- – 2089–2097. poietic stem cell development and identity. Int. J. Dev. Biol. 54, 1201 1211. fi Cripps, R.M., Olson, E.N., 2002. Control of cardiac development by an evolutionarily Puschel, A.W., Wester eld, M., Dressler, G.R., 1992. Comparative analysis of Pax-2 fi conserved transcriptional network. Dev. Biol. 246, 14–28. protein distributions during neurulation in mice and zebra sh. Mech. Dev. 38, – Davidson, E.H., 2009. Network design principles from the sea urchin embryo. Curr. 197 208. Opin. Genet. Dev. 19, 535–540. Roberts, E.C., Deed, R.W., Inoue, T., Norton, J.D., Sharrocks, A.D., 2001. Id helix-loop- Davidson, E.H., 2010. Emerging properties of animal gene regulatory networks. helix proteins antagonize pax transcription factor activity by inhibiting DNA – Nature 468, 911–920. binding. Mol. Cell. Biol. 21, 524 533. Davidson, E.H., Erwin, D.H., 2006. Gene regulatory networks and the evolution of Sanchez, M.P., Silos-Santiago, I., Frisen, J., He, B., Lira, S.A., Barbacid, M., 1996. Renal animal body plans. Science 311, 796–800. agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382, – Dressler, G.R., 2006. The cellular basis of kidney development. Ann. Rev. Cell. Dev. 70 73. Biol. 22, 509–529. Schuchardt, A., D_Agati, V., Larsson_Blomberg, L., Costantini, F., Pachnis, V., 1994. Gaitan, Y., Bouchard, M., 2006. Expression of the delta-protocadherin gene Pcdh19 Defects in the kidney and enteric nervous system of mice lacking the tyrosine – in the developing mouse embryo. Gene Expr. Patterns 6, 893–899. kinase receptor Ret. Nature 367, 380 383. Grote, D., Boualia, S.K., Souabni, A., Merkel, C., Chi, X., Costantini, F., Carroll, T., Schuchardt, A., D'Agati, V., Pachnis, V., Costantini, F., 1996. Renal agenesis and Bouchard, M., 2008. Gata3 acts downstream of beta-catenin signaling to hypodysplasia in ret-k- mutant mice result from defects in ureteric bud – prevent ectopic metanephric kidney induction. PLoS Genet. 4, e1000316. development. Development 122, 1919 1929. Grote, D., Souabni, A., Busslinger, M., Bouchard, M., 2006. Pax2/8-regulated Gata3 Shawlot, W., Behringer, R.R., 1995. Requirement for Lim1 in head-organizer – expression is necessary for morphogenesis and guidance of the nephric duct in function. Nature 374, 425 430. the developing kidney. Development 133, 53–61. Sheng, G., Stern, C.D., 1999. Gata2 and Gata3: novel markers for early embryonic polarity – Heller, N., Brandli, A.W., 1999. Xenopus Pax-2/5/8 orthologues: novel insights into and for non-neural ectoderm in the chick embryo. Mech. Dev. 87, 213 216. Pax gene evolution and identification of Pax-8 as the earliest marker for otic Ting, C.N., Olson, M.C., Barton, K.P., Leiden, J.M., 1996. Transcription factor GATA-3 is – and pronephric cell lineages. Dev. Genet. 24, 208–219. required for development of the T-cell lineage. Nature 384, 474 478. 566 S. Kamel Boualia et al. / Developmental Biology 382 (2013) 555–566

Torres, M., Gomez_Pardo, E., Gruss, P., 1996. Pax2 contributes to inner ear Ye, W., Bouchard, M., Stone, D., Liu, X., Vella, F., Lee, J., Nakamura, H., Ang, S.L., patterning and optic nerve trajectory. Development 122, 3381–3391. Busslinger, M., Rosenthal, A., 2001. Distinct regulators control the expression of Torres, M., Gomez-Pardo, E., Dressler, G.R., Gruss, P., 1995. Pax-2 controls multiple the mid-hindbrain organizer signal FGF8. Nat. Neurosci. 4, 1175–1181. steps of urogenital development. Development 121, 4057–4065. Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S., Aizawa, S., Weber, S., 2012. Novel genetic aspects of congenital anomalies of kidney and 1997. Emx1 and Emx2 functions in development of dorsal telencephalon. – urinary tract. Curr. Opin. Pediatr. 24, 212 218. Development 124, 101–111. Wingert, R.A., Davidson, A.J., 2011. Zebraﬁsh nephrogenesis involves dynamic spatiotemporal expression changes in renal progenitors and essential signals from retinoic acid and irx3b. Dev. Dyn. 240, 2011–2027.