Identification of a Novel Glial Cell Line-derived Neurotrophic Factor-inducible Gene Required for Renal Branching Morphogenesis
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m309629200
ISSN1083-351X
AutoresNaoyuki Fukuda, Masatoshi Ichihara, Takatoshi Morinaga, Kumi Kawai, Hironori Hayashi, Yoshiki Murakumo, Seiichi Matsuo, Masahide Takahashi,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoIn the developing kidney, activation of the rearrangement during transfection by glial cell line-derived neurotrophic factor (GDNF) is required for normal branching of the ureteric bud epithelium. By differential display analysis we identified a novel GDNF-inducible gene (named GZF1) with a BTB/POZ (broad complex, tramtrack, and bric-a-brac)/(poxvirus and zinc finger) domain and 10 tandemly repeated zinc finger motifs. The up-regulation of the GZF1 gene showed two peaks at 1 h and 24–48 h after GDNF stimulation by Northern blotting. The late induction was also found at protein levels by Western blotting with anti-GZF1 antibody. As observed for other proteins with the BTB/POZ domain, the GZF1 protein had strong transcriptional repressive activity. Intriguingly, its expression was detected at high levels in branching ureteric buds and collecting ducts of mouse metanephric kidney in which RET was also expressed. Antisense phosphorothioated oligodeoxynucleotides of the GZF1 gene markedly impaired the ureteric bud branching in the metanephric organ culture, suggesting that the induction of GZF1 expression via the GDNF/RET signaling system is required for renal branching morphogenesis. In the developing kidney, activation of the rearrangement during transfection by glial cell line-derived neurotrophic factor (GDNF) is required for normal branching of the ureteric bud epithelium. By differential display analysis we identified a novel GDNF-inducible gene (named GZF1) with a BTB/POZ (broad complex, tramtrack, and bric-a-brac)/(poxvirus and zinc finger) domain and 10 tandemly repeated zinc finger motifs. The up-regulation of the GZF1 gene showed two peaks at 1 h and 24–48 h after GDNF stimulation by Northern blotting. The late induction was also found at protein levels by Western blotting with anti-GZF1 antibody. As observed for other proteins with the BTB/POZ domain, the GZF1 protein had strong transcriptional repressive activity. Intriguingly, its expression was detected at high levels in branching ureteric buds and collecting ducts of mouse metanephric kidney in which RET was also expressed. Antisense phosphorothioated oligodeoxynucleotides of the GZF1 gene markedly impaired the ureteric bud branching in the metanephric organ culture, suggesting that the induction of GZF1 expression via the GDNF/RET signaling system is required for renal branching morphogenesis. The mammalian metanephric kidney develops from the metanephrogenic mesenchyme, which appears in the region of the posterior intermediate mesoderm (1.Saxen L. Organogenesis of the Kidney. Cambridge University Press, Cambridge, UK1987Crossref Google Scholar). It is well known that kidney development is induced by reciprocal signaling between the ureteric bud epithelium and the metanephric mesenchyme. On day 11 of mouse embryogenesis, the ureteric bud emerges near the caudal end of the Wolffian ducts and invades the metanephrogenic mesenchyme. Once the bud meets the mesenchyme, it begins to grow and branch repeatedly, thus generating the renal collecting duct system. At the same time, induction of the mesenchyme by signals from the bud initiates the condensation of the mesenchyme that differentiates to form the nephrons. In recent years, many important molecules that cooperatively function in the branching morphogenesis of the epithelial ureteric bud have been identified by in vitro and in vivo studies. These include transcription factors, secreted peptides, cell surface receptors, and extracellular matrices, which are expressed sequentially at specific sites of the developing kidney (2.Dressler G. Trends Cell Biol. 2002; 12: 390-395Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3.Piscione T.D. Rosenblum N.D. Differentiation. 2002; 70: 227-246Crossref PubMed Scopus (76) Google Scholar, 4.Vainio S. Lin Y. Nat. Rev. Genet. 2002; 3: 533-543Crossref PubMed Scopus (182) Google Scholar). Among them, glial cell line-derived neurotrophic factor (GDNF) 1The abbreviations used are: GDNFglial cell line-derived neurotrophic factorRETrearrangement during transfectionGFRα1GDNF family receptor α 1GZF1GDNF-inducible zinc finger gene 1GAL4BDGAL4 DNA-binding domainODNoligodeoxynucleotideTKthymidine kinaseEGFPenhanced green fluorescent proteinBTB/POZbroad complex, tramtrack, and bric-a-brac/poxvirus and zinc finger. and RET (rearrangement during transfection) have been recognized as critical regulators of ureteric bud branching. glial cell line-derived neurotrophic factor rearrangement during transfection GDNF family receptor α 1 GDNF-inducible zinc finger gene 1 GAL4 DNA-binding domain oligodeoxynucleotide thymidine kinase enhanced green fluorescent protein broad complex, tramtrack, and bric-a-brac/poxvirus and zinc finger. GDNF activates RET via its binding to glycosylphosphatidylinositol-linked cell surface protein, GFRα1 (GDNF family receptor α1) (5.Takahashi M. Cytokine Growth Factor Rev. 2001; 12: 361-373Crossref PubMed Scopus (364) Google Scholar, 6.Manie S. Santoro M. Fusco A. Billaud M. Trends Genet. 2001; 17: 580-589Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 7.Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1450) Google Scholar). The gene ablation studies revealed that GDNF, GFRα1, and RET are required for the development of the kidney and the enteric nervous system. Gdnf–/–, Gfrα1–/–, or Ret–/– mice showed kidney aplasia or severe hypodysplasia and lacked enteric neurons in the whole intestinal tract, resulting in their death soon after birth (8.Schuchardt A. D'Agati V. Larsson-Blomberg L. Costantini F. Pachnis V. Nature. 1994; 367: 380-383Crossref PubMed Scopus (1419) Google Scholar, 9.Sanchez M.P. Silos-Santiago I. Frisen J. He B. Lira S.A. Barbacid M. Nature. 1996; 382: 70-73Crossref PubMed Scopus (1044) Google Scholar, 10.Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Nature. 1996; 382: 73-76Crossref PubMed Scopus (1005) Google Scholar, 11.Moore M.W. Klein R.D. Farinas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. Rosenthal A. Nature. 1996; 382: 76-79Crossref PubMed Scopus (1084) Google Scholar, 12.Cacalano G. Farinas I. Wang L.C. Hagler K. Forgie A. Moore M. Armanini M. Phillips H. Ryan A.M. Reichardt L.F. Hynes M. Davies A. Rosenthal A. Neuron. 1998; 21: 53-62Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 13.Enomoto H. Araki T. Jackman A. Heuckeroth R.O. Snider W.D. Johnson Jr., E.M. Milbrandt J. Neuron. 1998; 21: 317-324Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). GDNF is secreted from the metanephric mesenchyme, and RET and GFRα1 are expressed on the cell surface of the branching ureteric bud epithelium (11.Moore M.W. Klein R.D. Farinas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. Rosenthal A. Nature. 1996; 382: 76-79Crossref PubMed Scopus (1084) Google Scholar, 14.Pachnis V. Mankoo B. Costantini F. Development. 1993; 119: 1005-1017Crossref PubMed Google Scholar, 15.Tsuzuki T. Takahashi M. Asai N. Iwashita T. Matsuyama M. Asai J. Oncogene. 1995; 10: 191-198PubMed Google Scholar, 16.Sainio K. Suvanto P. Davies J. Wartiovaara J. Wartiovaara K. Saarma M. Arumae U. Meng X. Lindahl M. Pachnis V. Sariola H. Development. 1997; 124: 4077-4087Crossref PubMed Google Scholar). Thus, it turned out that the interaction between the ureteric bud epithelium and the metanephric mesenchyme that leads to activation of the GDNF/RET signaling system is essential for normal development of the kidney. Based on these findings, much attention has been paid to the genes that regulate the expression of GDNF and RET. It was suggested that transcription factors Pax2 and Eya-1 are responsible for Gdnf gene expression and that Emx2 is required to maintain the expression of both Gdnf and Ret in mouse metanephric kidney (2.Dressler G. Trends Cell Biol. 2002; 12: 390-395Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3.Piscione T.D. Rosenblum N.D. Differentiation. 2002; 70: 227-246Crossref PubMed Scopus (76) Google Scholar, 4.Vainio S. Lin Y. Nat. Rev. Genet. 2002; 3: 533-543Crossref PubMed Scopus (182) Google Scholar). In addition, Batourina et al. (17.Batourina E. Gim S. Bello N. Shy M. Clagett-Dame M. Srinivas S. Costantini F. Mendelsohn C. Nat. Genet. 2001; 27: 74-78Crossref PubMed Scopus (225) Google Scholar) reported that vitamin A signaling from stromal mesenchyme is necessary for Ret expression. However, it remains unknown which gene expression downstream of the GDNF/RET signaling is critical for renal development. In this report, we describe a novel GDNF-inducible gene with a BTB/POZ domain and zinc finger motifs that are highly expressed in the ureteric bud of the metanephric kidney. Analysis by the metanephric organ culture suggested that this gene plays a crucial role in the ureteric bud branching. Cell Lines—TGW and NB39 human neuroblastoma cells, Neuro2a mouse neuroblastoma cells, and HEK293T human embryonic kidney cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Differential Display Analysis—Total RNAs were isolated from TGW cells treated with GDNF (100 ng/ml) for 2 h using an RNA purification kit (Qiagen). After treating total RNAs with RNase-free DNase I to eliminate contaminated chromosomal DNA, differential display polymerase chain reaction was performed using TaKaRa rhodamine fluorescence differential display system (TaKaRa). The fluorescence products were resolved by electrophoresis on denaturing urea-4% polyacrylamide gels. Differentially expressed bands were excised, sequenced, and compared with available public data bases. Nonredundant cDNA clones were used as probes for Northern blotting. Cloning of the Human GZF1 Gene—A human cDNA library was constructed using poly(A)+ RNA from TGW cells stimulated with GDNF and screened with a human GZF1 fragment isolated by differential display to obtain its full-length cDNA. Briefly, the TGW cDNAs were inserted into the ZAP Express predigested vector (Stratagene), and the resulting phage constructs were packaged using Gigapack III Gold packaging extract (Stratagene). The phages were infected into Escherichia coli XL1-Blue MRF′ strain, and the infected bacteria were plated for plaque hybridization. The library was screened with the [α-32P]dCTP-labeled human GZF1 fragment using the Gene Trapper cDNA positive selection system (Invitrogen). Northern Blotting—Total RNAs from GDNF-untreated or -treated TGW cells were separated on 1% agarose-formamide gels with formaldehyde and transferred onto Hybond-XL nylon membranes (Amersham Biosciences). The membranes containing RNAs from human and mouse multiple tissues were purchased from Clontech. A human or mouse GZF1 cDNA fragment utilized as a probe was labeled with [α-32P]dCTP using the High Prime DNA-labeling system (Roche Diagnostics), and Northern hybridization was performed at 68 °C for 2 h in QuikHyb solution (Stratagene). Antibodies—Rabbit anti-GZF1 polyclonal antibody was developed against the 19 carboxyl-terminal amino acids of GZF1 and affinity-purified with the immunized peptide. Anti-α-tubulin and anti-pan cytokeratin monoclonal antibodies were purchased from Sigma. Anti-Oct-1 monoclonal antibody was purchased from Santa Cruz Biotechnology. Rabbit anti-RET polyclonal antibody was described previously (18.Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Crossref PubMed Google Scholar). Western Blotting—TGW and NB39 cells were stimulated with GDNF (100 ng/ml) for 1, 24, 48, and 72 h after 24 h of starvation and lysed in SDS sample buffer (20 mm Tris-HCl, pH6.8, 2 mm EDTA, 2% SDS, 10% sucrose, 20 μg/ml bromphenol blue, and 80 mm dithiothreitol). The resulting lysates were analyzed by Western blotting as described previously (19.Watanabe T. Ichihara M. Hashimoto M. Shimono K. Shimoyama Y. Nagasaka T. Murakumo Y. Murakami H. Sugiura H. Iwata H. Ishiguro N. Takahashi M. Am. J. Pathol. 2002; 161: 249-256Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). GAL4-fused Reporter Gene-targeted Repression Assay—The generation of pGL3 luciferase reporter plasmids has been described previously (20.Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). HEK293T cells were cultured in 24-well tissue culture plates and cotransfected with 40 ng of luciferase reporter plasmid, 40 ng of pRL-TK plasmid (Promega), and 320 ng of pSRα-GAL4-GZF1 fusion construct or pSRα-GAL4 by the LipofectAMINE PLUS method (Invitrogen). The cells were harvested 48 h after transfection, and luciferase assays were performed as described previously (20.Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Cotransfection with the pRL-TK plasmid was used to normalize all luciferase values. Mutations in the BTB/POZ domain were introduced using the QuikChange™ site-directed mutagenesis kit (Stratagene). Immunohistochemistry—Embryos (13.5–14.5 days post coitus) of ICR mice were fixed in 4% paraformaldehyde and embedded in paraffin. Four-μm sections were deparaffinized in xylene and rehydrated through graded alcohols. In the case of RET staining, these sections were subjected to microwave pretreatment for 12 min in 10 mm citrate buffer (pH 6.0) and cooled at room temperature. Nonspecific binding sites were blocked with 10% goat serum for 30 min at room temperature. The sections were incubated with primary antibodies overnight at 4 °C, and endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 15 min. The slides were incubated with secondary antibody conjugated with peroxidase-labeled polymer (EnVision+, Dako), and the reaction products were visualized with diaminobenzidine and H2O2. Counterstaining was performed with hematoxylin. Transfection of the Antisense Oligodeoxynucleotide—Six antisense oligodeoxynucleotides (ODNs) were designed based on the predicted secondary structure of mouse GZF1 mRNA. We targeted the region near the initiation codon or the region forming loops. Six antisense phosphorothioated ODNs were synthesized and purified by high pressure liquid chromatography. One day before transfection, Neuro2a cells were plated at a density of 1.5 × 105 cells/well of a 24-well plate and transfected with each antisense ODN at a final concentration of 200 nm. Transfections were performed using the OligofectAMINE reagent (Invitrogen). The cells were harvested 48 h after transfection and analyzed by Western blotting with anti-GZF1 antibody. Metanephric Organ Culture—Metanephric organ culture was performed as described by Kanwar et al. (21.Kanwar Y.S. Kumar A. Ota K. Lin S. Wada J. Chugh S. Wallner E.I. Am. J. Physiol. 2002; 282: F953-F965Google Scholar). In brief, metanephroi were isolated from E11.5 ICR mouse embryos and cultured in Transwell-Clear (Costar) for 4 days in a humidified incubator with 95% air and 5% CO2 at 37 °C. The culture medium consisted of equal volumes of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 (Sigma) supplemented with transferrin (50 μg/ml), penicillin (100 μg/ml), and streptomycin (100 μg/ml). The medium was devoid of serum or any other growth factor. Sense, antisense, or scramble ODNs of mouse GZF1 were added to the medium daily at a concentration of 0.5–2.0 μm. The metanephric explants were photographed directly by stereomicroscope and then stained with anti-pan cytokeratin antibody to highlight ureteric bud derivatives. The rudiments in metanephric organ culture were fixed in 100% methanol, washed three times in phosphate-buffered saline, and permeabilized for 15 min in phosphate-buffered saline containing 0.2% Triton X-100. The rudiments were blocked with 3% bovine serum albumin in phosphate-buffered saline. The metanephroi were incubated with anti-pan cytokeratin antibody diluted 1:50 at 4 °C overnight and then with Cy3-conjugated donkey anti-mouse IgG diluted 1:100 at 4 °C overnight. After immunostaining, slides were mounted in PermaFluor (Shandon) and observed by a fluorescent microscope with a charge-coupled device camera (BX50, DP70, Olympus). To identify GDNF-inducible genes, we performed differential display analysis using RNA from a human neuroblastoma cell line, TGW, expressing RET and GFR α1. We detected 124 cDNA bands in which the intensity increased after GDNF stimulation. After isolating and sequencing these cDNA bands, 73 independent cDNA clones were identified and used for Northern blotting to confirm their increased expression after GDNF stimulation. As a consequence, we found the increased expression of 14 genes by GDNF, although the time course of the induction was different depending on the genes (data not shown). These included 10 known genes such as c-FOS, CREM, and cell division cycle-like kinase genes and four unpublished sequences. In this study, we focused on a new gene with a BTB/POZ domain and C2H2-type zinc finger motifs that was named GZF1 (GDNF-inducible zinc finger gene 1). We isolated its full-length cDNA from the library constructed from RNA of GDNF-treated TGW human neuroblastoma cells. The cDNA sequence revealed that it encodes a protein of 711 amino acids containing the BTB/POZ domain at the amino-terminal region and 10 C2H2-type zinc fingers (Fig. 1A). In addition, the GZF1 sequence contained a nuclear localization signal between the BTB/POZ domain and the zinc finger motifs. We also isolated its mouse ortholog cDNA from a mouse testis cDNA library with an amino acid sequence (706 amino acids) showing 84% identity with the human GZF1 sequence (Fig. 1A). The human and mouse GZF1 genes were located on chromosomes 20 and 2, respectively. When the GZF1 expression was examined in various human tissues, a single 4.8-kb mRNA was detected in brain, heart, skeletal muscle, kidney, and liver tissues (Fig. 1B). In addition, it was expressed at relatively high levels in brain and kidney tissue among human fetal tissues examined (Fig. 1B). The GZF1 gene expression was also detected in several mouse tissues including kidney and in different stages of mouse embryos (7–17-day embryos). (Fig. 1C). The induction of the GZF1 gene in TGW cells showed two peaks at 1 h and at 24–48 h after GDNF stimulation by Northern blotting (Fig. 2A). This pattern of the GZF1 gene induction by GDNF was confirmed by reverse transcriptase-PCR analysis (data not shown). To further investigate the GZF1 protein expression, we developed a rabbit polyclonal antibody against the 19 carboxyl-terminal amino acids of human GZF1. The antibody specifically detected 95- and 115-kDa proteins in TGW cells and a 115-kDa protein in Neuro2a mouse neuroblastoma cells (Fig. 2B). Because we were not able to detect either N-linked or O-linked glycosylation in these proteins (data not shown), the difference between expected and apparent molecular masses of GZF1 (80 kDa as expected molecular mass and 95 and 115 kDa as apparent molecular masses) may be caused by their peculiar protein conformations or other modifications. When the GZF1 expression was examined in GDNF-stimulated TGW and NB39 human neuroblastoma cells in which both RET and GFRα1 are expressed, its expression significantly increased 24–72 h after stimulation (Fig. 2C). The early induction of the GZF1 protein was not observed, probably because of transient up-regulation of GZF1 mRNA for a short period at an early phase (∼1–2 h after GDNF stimulation). To confirm that the bands detected by the antibody represent the GZF1 protein, we constructed the EGFP-GZF1 fusion gene and transfected it into HEK293T cells. Although the fusion gene encodes a protein of 976 amino acids in which the expected molecular mass is 107 kDa, Western blotting with anti-GZF1 and anti-GFP antibodies revealed that the fusion protein was detected mainly as a 140-kDa band (Fig. 2D). The difference (33 kDa) between expected and apparent molecular masses of the EGFP-GZF1 fusion protein was almost the same as that between expected and apparent molecular masses of the endogenous GZF1 protein (80 and 115 kDa), indicating that the bands recognized by anti-GZF1 antibody represent the GZF1 protein. The 95-kDa protein detected in human cells may be translated from another methionine (e.g. codon 192) that is present only in the human GZF1 gene or may be influenced by differences in modification or processing, although further investigation is necessary. Cell fractionation experiments revealed that the GZF1 protein was detected in both nuclear and cytosolic fractions (Fig. 2E), although the nuclear expression was predominant. Because it is known that the BTB/POZ domain is involved in transcriptional repression (22.Melnick A. Carlile G. Ahmad K.F. Kiang C.L. Corcoran C. Bardwell V. Prive G.G. Licht J.D. Mol. Cell. Biol. 2002; 22: 1804-1818Crossref PubMed Scopus (173) Google Scholar), we next investigated the transcriptional activity of GZF1 by the luciferase reporter gene assay. The human GZF1 cDNA was fused to the GAL4 DNA-binding domain (Fig. 3A, GAL4BD) in the expression plasmid containing the SRα promoter, and the resulting construct was transfected into HEK293T cells together with the luciferase reporter plasmid containing five tandem repeats of the GAL4-binding sequence, a binding site for the serum response element (SRE) and the thymidine kinase minimal (TKm) promoter (Fig. 3A) (20.Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). As a consequence, it turned out that expression of GZF1 reduced the luciferase activity by ∼80% as compared with that by expression of the GAL4BD only (Fig. 3C). It was reported that two conserved charged residues within the BTB/POZ domains of PLZF and Bcl-6 proteins are important for their transcriptionally repressive activity (22.Melnick A. Carlile G. Ahmad K.F. Kiang C.L. Corcoran C. Bardwell V. Prive G.G. Licht J.D. Mol. Cell. Biol. 2002; 22: 1804-1818Crossref PubMed Scopus (173) Google Scholar). Thus, we replaced the corresponding amino acids (aspartic acid 32 and lysine 50 in Fig. 3B) in GZF1 with asparagine and aspartic acid, respectively (designated D32N and K50D). As shown in Fig. 3C, the introduction of each mutation resulted in a 1.6–1.7-fold increase of the luciferase activity. Moreover, when both mutations were introduced into GZF1, the activity further increased (about 2.2-fold), suggesting the importance of these two charged amino acids for transcriptional repressive activity of GZF1. To elucidate the importance of GZF1 on kidney development, we stained the mouse embryonic kidney with anti-GZF1 antibody. Interestingly, the ureteric bud epithelia of 13.5–14.5-day embryonic metanephroi were strongly stained (Fig. 4, B and D). Using serial sections, we detected the expression of GZF1 and RET in the same ureteric buds of 13.5–14.5-day metanephroi (Fig. 4, A–D). Both nuclear and cytoplasmic staining of GZF1 were observed in the ureteric buds (Fig. 4, B and D). Like RET, the GZF1 expression significantly decreased in the kidney after birth (data not shown). When immunostaining was performed with protein A-purified rabbit IgG instead of the anti-GZF1 antibody as a negative control, no staining was observed in these tissues (data not shown). Finally, we investigated whether antisense phosphorothioated ODNs of the GZF1 gene impair the ureteric bud branching in the metanephric organ culture. We designed six antisense ODNs based on the mouse sequence (Fig. 5A). When mouse neuroblastoma cells (Neuro2a cells) were cultured in the presence of each antisense ODN for 2 days, it turned out that two antisense ODNs (designated AS-4 and AS-5) significantly decreased the expression of the mouse GZF1 protein in Neuro2a cells (Fig. 5B). In addition, sense and scramble ODNs corresponding to the AS-4 sequence (SS-4 and SC-4) did not affect the expression of GZF1 (Fig. 5B). Thus, metanephroi isolated from 11.5-day ICR mouse embryos were incubated in the presence of AS-4 or AS-5 for 4 days, and the ureteric bud branching was observed by staining with anti-pan cytokeratin antibody. Both AS-4 (Fig. 5C) and AS-5 (data not shown) markedly impaired the branching, whereas it normally occurred in the presence of SS-4 or SC-4 (Fig. 5C). On the other hand, four other antisense ODNs (AS-1, -2, -3, and -6) that did not decrease the GZF1 protein expression in Neuro2a cells showed no effect on the ureteric bud branching (data not shown). These findings demonstrated that the GZF1 gene is a novel GDNF-inducible gene that is required for renal branching morphogenesis. In the past 10 years, a variety of factors including transcription factors and secreted peptides have been reported to be involved in normal development of metanephric kidney (2.Dressler G. Trends Cell Biol. 2002; 12: 390-395Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3.Piscione T.D. Rosenblum N.D. Differentiation. 2002; 70: 227-246Crossref PubMed Scopus (76) Google Scholar, 4.Vainio S. Lin Y. Nat. Rev. Genet. 2002; 3: 533-543Crossref PubMed Scopus (182) Google Scholar). Genetic evidence has established a crucial role for the GDNF/RET signaling in the ureteric bud development and branching. GDNF that is secreted from the metanephric mesenchyme is essential for correct outgrowth and branching of the ureteric bud in which RET and GFRα1 are expressed (8.Schuchardt A. D'Agati V. Larsson-Blomberg L. Costantini F. Pachnis V. Nature. 1994; 367: 380-383Crossref PubMed Scopus (1419) Google Scholar, 9.Sanchez M.P. Silos-Santiago I. Frisen J. He B. Lira S.A. Barbacid M. Nature. 1996; 382: 70-73Crossref PubMed Scopus (1044) Google Scholar, 10.Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Nature. 1996; 382: 73-76Crossref PubMed Scopus (1005) Google Scholar, 11.Moore M.W. Klein R.D. Farinas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. Rosenthal A. Nature. 1996; 382: 76-79Crossref PubMed Scopus (1084) Google Scholar, 12.Cacalano G. Farinas I. Wang L.C. Hagler K. Forgie A. Moore M. Armanini M. Phillips H. Ryan A.M. Reichardt L.F. Hynes M. Davies A. Rosenthal A. Neuron. 1998; 21: 53-62Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 13.Enomoto H. Araki T. Jackman A. Heuckeroth R.O. Snider W.D. Johnson Jr., E.M. Milbrandt J. Neuron. 1998; 21: 317-324Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). It was demonstrated that several transcription factors including Pax2, Pax8, Eya-1, and Emx-2 are expressed in nephric duct and metanephric mesenchyme in spatial and temporal fashions and regulate the GDNF and/or RET expression in the developing kidney (23.Brophy P.D. Ostrom L. Lang K.M. Dressler G.R. Development. 2001; 128: 4747-4756PubMed Google Scholar, 24.Bouchard M. Souabni A. Mandler M. Neubuser A. Busslinger M. Genes Dev. 2002; 16: 2958-2970Crossref PubMed Scopus (412) Google Scholar, 25.Miyamoto N. Yoshida M. Kuratani S. Matsuo I. Aizawa S. Development. 1997; 124: 1653-1664Crossref PubMed Google Scholar, 26.Xu P.X. Adams J. Peters H. Brown M.C. Heaney S. Maas R. Nat. Genet. 1999; 23: 113-117Crossref PubMed Scopus (564) Google Scholar). In addition, vitamin A signaling from the stromal mesenchyme (a third renal cell type) that activates the retinoic acid receptors, Rarα and Rarβ2, was required for RET expression in the ureteric bud (17.Batourina E. Gim S. Bello N. Shy M. Clagett-Dame M. Srinivas S. Costantini F. Mendelsohn C. Nat. Genet. 2001; 27: 74-78Crossref PubMed Scopus (225) Google Scholar), indicating that there are kidney-specific regulatory mechanisms for the activation of the GDNF/RET signaling system. Moreover, the forkhead/winged helix transcription factor, Foxd1 (27.Hatini V. Huh S.O. Herzlinger D. Soares V.C. Lai E. Genes Dev. 1996; 10: 1467-1478Crossref PubMed Scopus (424) Google Scholar), and the basic helix-loop-helix transcription factor, Pod1 (28.Quaggin S.E. Schwartz L. Cui S. Igarashi P. Deimling J. Post M. Rossant J. Development. 1999; 126: 5771-5783PubMed Google Scholar), appeared to control the spatially restricted Ret expression during renal development. Foxd1–/– and Pod–/– mice showed a transition in Ret expression from the normal restricted pattern at tips of the branching ureteric buds to ectopic expression throughout the collecting duct system, resulting in decreased ureteric bud branching and renal hypoplasia. However, what gene expression (as regulated by the GDNF/RET signaling) is required for normal ureteric bud branching has not been elucidated so far. In this study, we identified for the first time a GDNF-inducible gene, GZF1, involved in ureteric bud branching. The facts that RET and GZF1 are highly expressed in the same ureteric buds and that antisense ODNs of GZF1 markedly impaired their branching in the metanephric organ culture strongly supported the view that GZF1 is an important branching factor that functions downstream of the GDNF/RET signaling pathway. The GZF1 gene contained the BTB/POZ domain as well as zinc finger motifs. It was reported that the BTB/POZ domains were found in 0.6 and 0.8% of total genes in the human and mouse genome, respectively (29.Waterston R.H. Lindblad-Toh K. Birney E. Rogers J. Abril J.F. Agarwal P. Agarwala R. Ainscough R. Alexandersson M. An P. Antonarakis S.E. Attwood J. Baertsch R. Bailey J. Barlow K. Nature. 2002; 420: 520-562Crossref PubMed Scopus (5420) Google Scholar). BTB/POZ domain-containing proteins comprise a large and diverse family of factors involved in multiple cellular processes including development and cell growth (30.Dent A.L. Shaffer A.L. Yu X. Allman D. Staudt L.M. Science. 1997; 276: 589-592Crossref PubMed Scopus (772) Google Scholar, 31.Horowitz H. Berg C.A. Development. 1996; 122: 1859-1871Crossref PubMed Google Scholar, 32.Shaknovich R. Yeyati P.L. Ivins S. Melnick A. Lempert C. Waxman S. Zelent A. Licht J.D. Mol. Cell. 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Thus, further investigation of GZF1 functions and identification of the genes in which expression is regulated by GZF1 would promote our understanding of mechanisms of renal branching morphogenesis. We thank Y. Sawa, N. Asano, T. Katahara, M. Kondo, K. Imaizumi, K. Uchiyama, and M. Kozuka for excellent technical assistance.
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