Implication of Wt1 in the Pathogenesis of Nephrogenic Failure in a Mouse Model of Retinoic Acid-Induced Caudal Regression Syndrome
2005; Elsevier BV; Volume: 166; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)62349-8
ISSN1525-2191
AutoresHerman Tse, Maran Bo Wah Leung, Adrian S. Woolf, Aswin Menke, Nicholas D. Hastie, J. A. Gosling, Chi-Pui Pang, Alisa S.W. Shum,
Tópico(s)Prenatal Screening and Diagnostics
ResumoRenal malformations are common human birth defects that sometimes occur in the context of the caudal regression syndrome. Here, we found that exposure of pregnant mice to all-trans retinoic acid, at a time when the metanephros has yet to form, causes a failure of kidney development along with caudal regression. Maternal treatment with Am580 (retinoic acid receptor α agonist) also induced similar patterns of kidney maldevelopment in the fetus. In metanephroi from retinoic acid-treated pregnancies, renal mesenchyme condensed around the ureteric bud but then failed to differentiate into nephrons, instead undergoing involution by fulminant apoptosis to produce a renal agenesis phenotype. Results of whole organ cultures in serum-free medium, and also tissue recombination experiments, showed that the nephrogenic defect was intrinsic to the kidney and that it resided in the metanephric mesenchyme and not the ureteric bud. Renal mesenchyme from control embryos expressed Wilms' tumor 1 (Wt1), but this transcription factor, which is indispensable for kidney development, failed to express in metanephroi of retinoic acid-exposed embryos. Wt1 expression and organogenesis were both restored, however, when metanephroi from retinoic acid-treated pregnancies were grown in serum-containing media. Our data illuminate the pathobiology of a severe, teratogen-induced kidney malformation. Renal malformations are common human birth defects that sometimes occur in the context of the caudal regression syndrome. Here, we found that exposure of pregnant mice to all-trans retinoic acid, at a time when the metanephros has yet to form, causes a failure of kidney development along with caudal regression. Maternal treatment with Am580 (retinoic acid receptor α agonist) also induced similar patterns of kidney maldevelopment in the fetus. In metanephroi from retinoic acid-treated pregnancies, renal mesenchyme condensed around the ureteric bud but then failed to differentiate into nephrons, instead undergoing involution by fulminant apoptosis to produce a renal agenesis phenotype. Results of whole organ cultures in serum-free medium, and also tissue recombination experiments, showed that the nephrogenic defect was intrinsic to the kidney and that it resided in the metanephric mesenchyme and not the ureteric bud. Renal mesenchyme from control embryos expressed Wilms' tumor 1 (Wt1), but this transcription factor, which is indispensable for kidney development, failed to express in metanephroi of retinoic acid-exposed embryos. Wt1 expression and organogenesis were both restored, however, when metanephroi from retinoic acid-treated pregnancies were grown in serum-containing media. Our data illuminate the pathobiology of a severe, teratogen-induced kidney malformation. Renal malformations are among one of the most common congenital malformations in humans and are the major cause of chronic renal failure in children.1Woolf AS Welham SJM Hermann MM Winyard PJD Maldevelopment of the human kidney and lower urinary tract: an overview.in: Vize PD Woolf AS Bard JBL The Kidney: From Normal Development to Congenital Disease. Elsevier Science/Academic Press, Amsterdam2003: 377-393Crossref Scopus (43) Google Scholar Renal agenesis represents the most severe phenotype, with bilateral agenesis occurring in ∼1 in 10,000 births, and unilateral agenesis occurs in ∼1 in 1000 individuals. Strictly speaking, agenesis implies that the metanephric kidney precursor has never formed; it is now recognized, however, that an agenesis-like phenotype can be the end-result of prenatal involution of malformed embryonic kidneys,2Belk RA Thomas DFM Mueller RF Godbole P Markham AF Weston MJ A familial study and the natural history of prenatally detected unilateral multicystic dysplastic kidney.J Urol. 2002; 167: 666-669Abstract Full Text Full Text PDF PubMed Google Scholar, 3Hiraoka M Tsukahara H Ohshima Y Kasuga K Ishihara Y Mayumi M Renal aplasia is the predominant cause of congenital solitary kidneys.Kidney Int. 2002; 61: 1840-1844Crossref PubMed Scopus (79) Google Scholar a process probably driven by excessive apoptosis.4Winyard PJD Nauta J Lirenman DS Hardman P Sams VR Risdon AR Woolf AS Deregulation of cell survival in cystic and dysplastic renal development.Kidney Int. 1996; 49: 135-146Crossref PubMed Scopus (151) Google Scholar Other types of malformation include renal hypoplasia (too few glomeruli and nephron tubules) and dysplasia (incomplete and metaplastic differentiation, often with cysts); in addition, the polycystic kidney diseases can be considered a failure of terminal epithelial differentiation and some types manifest antenatally. There are three general causes of renal malformations:1Woolf AS Welham SJM Hermann MM Winyard PJD Maldevelopment of the human kidney and lower urinary tract: an overview.in: Vize PD Woolf AS Bard JBL The Kidney: From Normal Development to Congenital Disease. Elsevier Science/Academic Press, Amsterdam2003: 377-393Crossref Scopus (43) Google Scholar 1) mutation in genes expressed during metanephric development such as the Wilms' tumor 1 (Wt1)5Kreidberg JA Sariola H Loring JM Maeda M Pelletier J Housman D Jaenisch R WT1 is required for early kidney development.Cell. 1993; 74: 679-691Abstract Full Text PDF PubMed Scopus (1717) Google Scholar and Paired box 2 (PAX2)6Porteous S Torban E Cho NP Cunliffe H Chua L McNoe L Ward T Souza C Gus P Giugliani R Sato T Yun K Favor J Sicotte M Goodyer P Eccles M Primary renal hypoplasia in humans and mice with PAX2 mutations: evidence of increased apoptosis in fetal kidneys of Pax2(1Neu)+/− mutant mice.Hum Mol Genet. 2000; 9: 1-11Crossref PubMed Scopus (170) Google Scholar transcription factors; 2) impairment of fetal urinary flow caused by physical obstruction in lower urinary outflow tract;7Woolf AS Thiruchelvam N Congenital obstructive uropathy—its origin and contribution to end-stage renal failure in children.Adv Ren Replace Ther. 2001; 8: 157-163Abstract Full Text PDF PubMed Scopus (60) Google Scholar, 8Nyirady P Thiruchelvam N Fry CH Godley ML Winyard PJD Peebles DM Woolf AS Cuckow PM Effects of in utero bladder outflow obstruction on fetal sheep detrusor contractility, compliance and innervation.J Urol. 2002; 168: 1615-1620Abstract Full Text Full Text PDF PubMed Google Scholar and 3) changes in the embryonic milieu associated with exposure to formal teratogens9Nathanson S Moreau E Merlet-Benichou C Gilbert T In utero and in vitro exposure to β-lactams impairs kidney development in the rat.J Am Soc Nephrol. 2000; 11: 874-884PubMed Google Scholar or more subtle factors, such as alterations in maternal diet.10Welham SJM Wade A Woolf AS Protein restriction in pregnancy is associated with increased apoptosis of mesenchymal cells at the start of rat metanephrogenesis.Kidney Int. 2002; 61: 1231-1242Crossref PubMed Scopus (156) Google Scholar In many cases, the renal malformation is not an isolated anomaly, but occurs as part of a multiorgan congenital syndrome. For instance, renal agenesis and other renal malformations can occur in the context of the caudal regression syndrome,11Rubenstein MA Bucy JG Caudal regression syndrome: the urologic implications.J Urol. 1975; 114: 934-937PubMed Google Scholar, 12Andrish J Kalamchi A MacEwen GD Sacral agenesis: a clinical evaluation of its management, heredity, and associated anomalies.Clin Orthop Relat Res. 1979; 139: 52-57PubMed Google Scholar, 13Sparnon AL Ahmed S Urological anomalies in the caudal regression syndrome.Aust N Z J Surg. 1984; 54: 365-367Crossref PubMed Scopus (7) Google Scholar which is defined by a characteristic pattern of malformations including agenesis of the lower vertebral column, imperforate anus, and caudal spinal cord malformations that often appear as a terminal myelomeningocele.12Andrish J Kalamchi A MacEwen GD Sacral agenesis: a clinical evaluation of its management, heredity, and associated anomalies.Clin Orthop Relat Res. 1979; 139: 52-57PubMed Google Scholar, 14Carson JA Barnes PD Tunell WP Smith EI Jolley SG Imperforate anus: the neurological implication of sacral abnormalities.J Pediatr Surg. 1984; 19: 838-842Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 15Bohring A Lewin SO Reynolds JF Voigtländer T Rittinger O Carey JC Köpernik M Smith R Zackai EH Leonard NJ Gritter HL Bamforth JS Okun N McLeod DR Super M Powell P Mundlos S Hennekam RCM van Langen IM Viskochil DH Wiedemann H-R Opitz JM Polytopic anomalies with agenesis of the lower vertebral column.Am J Med Genet. 1999; 87: 99-114Crossref PubMed Scopus (52) Google Scholar, 16Pang D Sacral agenesis and caudal spinal cord malformations.Neurosurgery. 1993; 32: 755-779Crossref PubMed Scopus (220) Google Scholar However, the reason for, and the mechanism of, this particular association are unclear. There is emerging evidence that retinoids play an essential role in renal organogenesis. Vitamin A (retinol) is transferred to the embryo from the maternal circulation and then converted into its bioactive metabolite all-trans retinoic acid (RA). The RA signal is then transduced via nuclear RA receptors (RARs) that form heterodimers with retinoid x receptors (RXRs) to activate or suppress transcription of target genes. Rats made severely vitamin A-deficient delivered pups with renal malformations,17Wilson JG Warkany J Malformations in the genito-urinary tract induced by maternal vitamin A deficiency in the rat.Am J Anat. 1948; 83: 357-407Crossref PubMed Scopus (139) Google Scholar which could be rescued by restoration with vitamin A during gestation.18Wilson JG Roth CB Warkany J An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation.Am J Anat. 1953; 92: 189-217Crossref PubMed Scopus (651) Google Scholar Renal agenesis occurs in RARαγ−/− mouse double mutants, whereas renal hypoplasia occurs in RARαβ2−/− and RXRα/RARα−/− embryos,19Mendelsohn C Lohnes D Décimo D Lufkin T LeMeur M Chambon P Mark M Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants.Development. 1994; 120: 2749-2771Crossref PubMed Google Scholar, 20Kastner P Mark M Ghyselinck N Krezel W Dupé V Grondona JM Chambon P Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development.Development. 1997; 124: 313-326Crossref PubMed Google Scholar showing that retinoid signal is involved in various developmental processes during kidney development. RARs have also been implicated in embryonic modeling of the lower urinary tract, including ureter and bladder trigone.21Batourina E Choi C Paragas N Bello N Hensle T Costantini FD Schuchardt A Bacallao RJ Mendelsohn CI Distal ureter morphogenesis depends on epithelial cell remodelling mediated by vitamin A and Ret.Nat Genet. 2002; 32: 109-115Crossref PubMed Scopus (141) Google Scholar Batourina and colleagues22Batourina E Gim S Bello N Shy M Clagett-Dame M Srinivas S Costantini F Mendelsohn C Vitamin A controls epithelial/mesenchymal interactions through Ret expression.Nat Genet. 2001; 27: 74-78Crossref PubMed Scopus (230) Google Scholar reported that RARs expressed in the stromal mesenchyme were involved in a reciprocal signaling loop with RET, a receptor for glial cell line-derived neurotrophic factor (GDNF). Ret is expressed in ureteric bud (UB) branch tips and is up-regulated by exposure of murine metanephric explants to vitamin A or RA; the same maneuvers enhance UB branching and nephron formation in a dose-dependent manner.22Batourina E Gim S Bello N Shy M Clagett-Dame M Srinivas S Costantini F Mendelsohn C Vitamin A controls epithelial/mesenchymal interactions through Ret expression.Nat Genet. 2001; 27: 74-78Crossref PubMed Scopus (230) Google Scholar, 23Vilar J Gilbert T Moreau E Merlet-Bénichou A Metanephros organogenesis is highly stimulated by vitamin A derivatives in organ culture.Kidney Int. 1996; 49: 1478-1487Crossref PubMed Scopus (99) Google Scholar, 24Moreau E Vilar J Lelièvre-Pégorier M Merlet-Bénichou C Gilbert T Regulation of c-ret expression by retinoic acid in rat metanephros: implications in nephron mass control.Am J Physiol. 1998; 275: F938-F945PubMed Google Scholar It has also been found in the rat that the number of nephrons per kidney correlated positively with circulating vitamin A concentrations and that mild vitamin A deficiency leads to an inborn nephron deficit.25Lelièvre-Pégorier M Vilar J Ferrier M-L Moreau E Freund N Gilbert T Merlet-Bénichou C Mild vitamin A deficiency leads to inborn nephron deficit in the rat.Kidney Int. 1998; 54: 1455-1462Crossref PubMed Scopus (227) Google Scholar Such experimental observations, which suggest that the vitamin A axis can modify nephron number, would have major clinical implications if applicable to humans because it is established that mild hypoplasia (ie, having approximately half the normal number of nephrons per kidney) is associated with arterial hypertension.26Keller G Zimmer G Mall G Ritz E Amann K Nephron number in patients with primary hypertension.N Engl J Med. 2003; 348: 101-108Crossref PubMed Scopus (930) Google Scholar Although vitamin A or RA deficiency will lead to a spectrum of malformations affecting a number of organ systems, such as the kidney, heart, eye, and nervous system,17Wilson JG Warkany J Malformations in the genito-urinary tract induced by maternal vitamin A deficiency in the rat.Am J Anat. 1948; 83: 357-407Crossref PubMed Scopus (139) Google Scholar, 27Warkany J Roth C Congenital malformations induced in rats by maternal vitamin A deficiency. 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Effect of varying the preparatory diet upon the yield of abnormal young.J Nutrition. 1948; 35: 1-12Google Scholar, 28Dickman EC Thaller C Smith SM Temporally-regulated retinoic acid depletion produces specific neural crest, ocular and nervous system defects.Development. 1997; 124: 3111-3121PubMed Google Scholar, 29White JC Highland M Clagett-Dame M Abnormal development of the sinuatrial venous valve and posterior hindbrain may contribute to late fetal resorption of vitamin A-deficient rat embryos.Teratology. 2000; 62: 374-384Crossref PubMed Scopus (26) Google Scholar there is evidence for both humans and animal models that high vitamin A or RA intake is teratogenic for the same organ systems, including the kidney.30Robens JF Teratogenic effects of hypervitaminosis A in the hamster and the guinea pig.Toxicol Appl Pharmacol. 1970; 16: 88-99Crossref PubMed Scopus (37) Google Scholar, 31Shenefelt RE Morphogenesis of malformations in hamsters caused by retinoic acid: relation to dose and stage at treatment.Teratology. 1972; 5: 103-118Crossref PubMed Scopus (446) Google Scholar, 32Bernhardt IB Dorsey DJ Hypervitaminosis A and congenital renal anomalies in a human infant.Obstet Gynecol. 1974; 43: 750-755PubMed Google Scholar, 33Rothman KJ Moore LL Singer MR Nguyen US Mannino S Milunsky A Teratogenicity of high vitamin A intake.N Engl J Med. 1995; 333: 1369-1373Crossref PubMed Scopus (589) Google Scholar Moreover, disturbing retinoid signaling either by homologous disruption of retinoid receptors19Mendelsohn C Lohnes D Décimo D Lufkin T LeMeur M Chambon P Mark M Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants.Development. 1994; 120: 2749-2771Crossref PubMed Google Scholar, 20Kastner P Mark M Ghyselinck N Krezel W Dupé V Grondona JM Chambon P Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development.Development. 1997; 124: 313-326Crossref PubMed Google Scholar or by maternal treatment with RAR selective agonists34Elmazar MMA Reichert U Shroot B Nau H Pattern of retinoid-induced teratogenic effects: possible relationship with relative selectivity for nuclear retinoid receptors RARα, RARβ, and RARγ.Teratology. 1996; 53: 158-167Crossref PubMed Scopus (44) Google Scholar can induce renal malformations in the fetus. Taken together, these findings indicate that the level of retinoid signal is required to be tightly regulated for normal kidney development; when RA concentrations deviate from normal, in either direction, abnormal development occurs. Recently, our laboratory has demonstrated that pregnant mice treated with RA at mid-gestation gave birth to fetuses with a spectrum of malformations, including lower vertebral agenesis, imperforate anus, and terminal myelomeningocele,35Shum ASW Poon LLM Tang WWT Koide T Chan BWH Leung YC Shiroishi T Copp AJ Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo.Mech Dev. 1999; 84: 17-30Crossref PubMed Scopus (82) Google Scholar which closely resembled caudal regression syndrome in humans. In these experiments, it was also noted (A.S.W.S., personal observation) that renal malformations, including renal agenesis, were generated. In the current study, we have used the RA-induced caudal regression mouse model to study the cellular and molecular bases of severe renal malformations. Guidelines for the use and care of laboratory animals, as set by The Chinese University of Hong Kong, were followed. Except for experiments to determine the dose response of Wt1 mutants to RA, all experiments were conducted using ICR (Institute of Cancer Research) mice housed at The Chinese University of Hong Kong under a light-dark cycle with the dark period from 1:00 a.m. to 11:00 a.m. Timed mating was performed by pairing mice for 2 hours immediately before commencement of the light cycle. Fertilization was assumed to occur at 10:00 a.m., which was regarded as embryonic day (E) 0. Wt1(+/−) mutant mice in C57/BL6 background were maintained at the MRC Human Genetics Unit. Fetuses were obtained from timed mating between Wt1(+/−) male and wild-type C57/BL6 female mice. In the Wt1 mutant allele, 10 bp in the Wt1 gene are replaced by the neomycin cassette.5Kreidberg JA Sariola H Loring JM Maeda M Pelletier J Housman D Jaenisch R WT1 is required for early kidney development.Cell. 1993; 74: 679-691Abstract Full Text PDF PubMed Scopus (1717) Google Scholar The genotype of the fetus was determined by amplification of DNA prepared from the liver using polymerase chain reaction (PCR), with primers that detected part of the neomycin cassette and part of Wt1.36Menke AL Ijpenberg A Fleming S Ross A Medine CN Patek CE Spraggon L Hughes J Clarke AR Hastie ND The wt1-heterozygous mouse; a model to study the development of glomerular sclerosis.J Pathol. 2003; 200: 667-674Crossref PubMed Scopus (34) Google Scholar To determine the stage specificity of the effect of RA on kidney development, a single dose of 100 mg/kg body weight of RA (Sigma, St. Louis, MO) suspended in peanut oil was injected intraperitoneally into pregnant ICR female mice at 4-hour intervals from 8 days 20 hours to 9 days 16 hours, whereas control mice received an equivalent volume of suspension vehicle. To determine a dose response, pregnant ICR female mice received a dose of 10, 50, 100, or 125 mg/kg body weight of RA or Am580 (RARα agonists)34Elmazar MMA Reichert U Shroot B Nau H Pattern of retinoid-induced teratogenic effects: possible relationship with relative selectivity for nuclear retinoid receptors RARα, RARβ, and RARγ.Teratology. 1996; 53: 158-167Crossref PubMed Scopus (44) Google Scholar, 37Delescluse C Cavey MT Martin B Bernard BA Reichert U Maignan J Darmon M Shroot B Selective high affinity retinoic acid receptor α or β-γ ligands.Mol Pharmacol. 1991; 40: 556-562PubMed Google Scholar suspended in peanut oil, whereas pregnant C57/BL6 female mice received 50 mg/kg body weight of RA at 9 days 0 hours. To perform an initial assessment of effects on renal development, E18 fetuses were delivered by cesarean section and examined for anomalies. Kidney phenotypes were designated, using gross external morphology and paraffin sections (data not shown) as either: normal, agenesis (ie, no kidney tissue was found at E18), or nonagenesis renal malformation; the latter comprised a variety of phenotypes including hypoplasia (small kidneys containing normal-looking structures), dysplasia (kidneys containing poorly differentiated structures), or polycystic-like kidneys (differentiation appeared normal apart from cystic tubules). Injection of 125 mg/kg RA at 9 days 0 hours resulted in bilateral absence of kidneys in all fetuses, and hence this dosage and time point were used in all subsequent experiments to determine the cellular and molecular mechanisms of RA-induced renal agenesis. Embryos at different developmental stages were fixed in Bouin's solution and prepared as 7-μm-thick paraffin sections stained with hematoxylin and eosin (H&E) for histological examination of early kidney development. To identify the nature of cell death in the metanephros of the embryo at E11.00, embryos were fixed in 4% paraformaldehyde and prepared as 10-μm-thick paraffin sections cut transversely through the hindlimb bud region. Apoptotic cells with DNA breaks were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, using fluorescein-conjugated avidin to detect the biotinylated dUTP-antibody complex according to Gavrieli and colleagues,38Gavrieli Y Sherman Y Ben-Sasson SA TdT mediated dUTP-biotin nick end labeling of tissue sections (TUNEL).J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9198) Google Scholar with modifications as described in Shum and colleagues.35Shum ASW Poon LLM Tang WWT Koide T Chan BWH Leung YC Shiroishi T Copp AJ Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo.Mech Dev. 1999; 84: 17-30Crossref PubMed Scopus (82) Google Scholar DNA was counterstained using propidium iodide. At least five embryos were examined in each group. The expression of Wt1,39Armstrong JF Pritchard-Jones K Bickmore WA Hastie HD Bard JBL The expression of the Wilms' tumor gene, WT1, in the developing mammalian embryo.Mech Dev. 1992; 40: 85-97Crossref Scopus (513) Google Scholar Pax2,40Dressler GR Deutsch U Chowdhury K Nornes HO Gruss P Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system.Development. 1990; 109: 787-795PubMed Google Scholar Gdnf,41Matsushita N Fujita Y Tanaka M Nagatsu T Kiuchi K Cloning and structural organization of the gene encoding the mouse glial cell-line-derived neurotrophic factor, GDNF.Gene. 1997; 203: 149-157Crossref PubMed Scopus (39) Google Scholar and c-Ret42Pachnis V Mankoo B Costantini F Expression of the c-ret proto-oncogene during mouse embryogenesis.Development. 1993; 119: 1005-1017Crossref PubMed Google Scholar in embryos at E11.00 was studied by whole mount in situ hybridization using digoxigenin-labeled probes according to Wilkinson.43Wilkinson DG Whole mount in situ hybridisation of vertebrate embryos.in: Wilkinson DG In Situ Hybridisation. IRL Press, Oxford1992: 75-83Google Scholar At least 12 embryos were examined in each group. Hybridized embryos were embedded in gelatin and cut into 50-μm-thick vibratome sections for detailed examination of spatial distribution patterns of these genes. To confirm whether Wt1 and Gdnf mRNA transcripts could be detected in metanephroi at E11.00 and E12.00, isolated metanephroi of embryos from the same litter were pooled together; total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. The cDNA was then amplified by real-time quantitative PCR (40 cycles comprising of denaturation at 95°C for 15 seconds and annealing at 54°C for 1 minute) using SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and the following primers: Wt1 (forward: 5′-CAG ATG TAA TTC TAC AGG CGA TTG-3′; reverse: 5′-TCC CTA CAT ACT TGC AGA TTC AAA-3′), Gdnf (forward: 5′-CCT GAA GAT TAT CCT GAC CA-3′; reverse: 5′-CTT TCC CTC TGG AAT TCT CT-3′), and β-actin (forward: 5′-TGT TAC CCA CTG GGA CGA CA-3′; reverse: 5′-GGG GTG TTG AAG GTC TCA AA-3′) as internal control. The fluorescent-labeled PCR products were detected by ABI Prism 7000 sequence detection system (Applied Biosystems). The standard curve was prepared by using the threshold cycle (CT values) from dilutions of known concentrations of DNA obtained from respective cDNA plasmids. Three separate trials of PCR were performed for each sample. Each sample consisted of one litter of embryos. Three to four litters of embryos in each group were assessed. Embryos were dissected out in L-15 medium (Life Technologies Inc., Carlsbad, CA) at E11.00, when the UB had just penetrated into the metanephric mesenchyme (MM) but not yet branched. The metanephros was isolated with the aid of a 25-gauge needle fitted into a 1-ml syringe. After enzymatic digestion with 0.05% trypsin (Sigma) and 0.01% pancreatin (Sigma) in Dulbecco's modified Eagle's medium/F12 medium (Life Technologies Inc.) at room temperature for 10 to 15 minutes, the MM and UB were mechanically separated with a pair of electrically sharpened tungsten needles. Different modes of tissue recombination, consisting of RA-treated and control tissues, were performed, with three pieces of MM from RA-exposed or control embryos placed in close proximity to one UB. The recombinants were cultured on the Millicell-CM 0.4-μm culture plate insert (Millipore, Bedford, MA) in a hormonally defined serum-free medium composed of Dulbecco's modified Eagle's medium/F12 medium supplemented with HEPES (15 mmol/L; Life Technologies Inc.), transferrin (5 μg/ml; Sigma), triiodothyronine (3.2 pg/ml; Sigma), prostaglandin E1 (25 ng/ml; Sigma), hydrocortisone (5 μg/ml; Sigma) and insulin (5 μg/ml; Sigma) at the air-medium interphase at 37°C in a 5% CO2 incubator. This medium could support normal metanephros differentiation.44Avner ED Ellis D Temple T Jaffe R Metanephric development in serum-free organ culture.In Vitro. 1982; 18: 675-682Crossref PubMed Scopus (59) Google Scholar The recombinants were cultured for 7 days and the culture medium was refreshed every 48 hours. The first day of culture was designated day 0. In another set of experiments, three pieces of isolated control or RA-treated MM were placed in close proximity to a piece of spinal cord obtained from the cervical region of normal embryo at E11.00. The recombinants were cultured in the same conditions as described above. At least six recombinants were examined in each group. To study the development of the metanephros in the absence of any influence from surrounding tissues, metanephroi were dissected out from control and RA-exposed embryos between E11.25 and E15.25 at 1-day intervals and cultured in the hormonally defined medium alone or supplemented with 10% or 20% fetal bovine serum (Life Technologies Inc.), under the same conditions as the recombinants, except the culture was terminated after 2 or 5 days. The explants were analyzed in either one of the following ways. To determine whether there was any mesenchymal differentiation, after 5 days in culture, the explant was fixed in Bouin's fixative overnight and prepared as 7-μm-thick paraffin sections stained with H&E, and analyzed by light microscopy. To examine branching of the UB, after 5 days in culture, the explant was fixed in a mixture of methanol:acetone (1:1) for 20 minutes at −20°C. The ureteric epithelium was labeled by a mouse monoclonal antibody against calbindin-D (1:250, C-8666; Sigma), followed by detection using fluorescein-conjugated anti-mouse IgG secondary antibody (1:200; Jackson ImmunoResearch, West Grove, PA), and then examined by confocal microscopy. To measure the expression levels of Wt1 and β-actin (internal control) mRNA at 2 day after culture, real-time quantitative RT-PCR was performed as described above. Three separate trials of PCR were performed for each sample. Each sample consisted of 7 to 22 explants obtained in the same batch of culture. There were three to six samples in each group. The frequency and severity of renal malformations in response to RA or Am580 treatment at different dosages and time points were analyzed by Jonckheere-Terpstra test using StatXact 4 for Windows software (Cytel Software Corp., Cambridge, MA). The susceptibility of Wt1 mutant and wild-type embryos to RA-induced renal malformations was analyzed by Fisher's exact test (two-sided) using StatXact4 for Windows software. The expression levels of Wt1 and Gdnf, relative to β-actin, between different treatment groups were analyzed by Student's t-test using SPSS software (SPSS, Chicago, IL). The significance level was set at a P value of <0.05. We have previously found that the severity of caudal regression induced by RA, measured in terms of the body axial level being truncated, was highly developmental stage- and dose-dependent.35Shum ASW Poon LLM Tang WWT Koide T Chan BWH Leung YC Shiroishi T Copp AJ Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo.Mech Dev. 1999; 84: 17-30Crossref PubMed Scopus (82) Google Scholar To determine the stage when kidney development might be most sensitive to the teratogenic effect of RA, a time-response study was conducted. We found that treatment of pregnant mice with 100 mg/kg of RA between 8 days 20 hours and 9 days 12 hours, other than inducing caudal regression, also resulted in a spectrum of renal malformations in fetuses examined at E18 (Table 1). The frequency of malformations was dependent on the time of RA treatment, such that teratogen administration at earlier time points resulted in a significantly higher (P < 0.0001) percentage of embryos with renal malformations; moreover, l
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