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Toshifumi Fukuda,1,,
Kumi Kawai,2 Akari Nakamura,1 Kei Kurokawa,2 Yoshiki Murakumo,1 Masatoshi Ichihara,1 and Masahide Takahashi1,2*
Department of Pathology,1 Division of Molecular Pathology, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan2
Received 26 November 2003/ Returned for modification 24 January 2004/ Accepted 26 June 2004
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ABSTRACT |
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INTRODUCTION |
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s for complex formation with Ret. GDNF, neurturin, artemin, and persephin preferentially bind to GFR1, GFR2, GFR3, and GFR4, respectively, and show specific biological roles in vivo (1). Gene knockout studies revealed that Ret, Gdnf, and Gfr1 are indispensable for the development of the kidney and the enteric nervous system (ENS). Ret–/– (55), Gdnf–/– (44, 52, 54), and Gfr1–/– (10) mice died within 12 to 24 h after birth, lacked enteric neurons posterior to the stomach, and showed kidney aplasia or severe hypoplasia.
To date, intracellular signaling via activated Ret has been studied extensively, with a variety of cell lines and primary culture cells. Specific tyrosine residues in the Ret intracellular domain are phosphorylated by GDNF stimulation and have been identified as docking sites for many adaptor or effector proteins. For example, Tyr905, Tyr1015, and Tyr1096 represent docking sites for GRB7/GRB10, phospholipase-C
, and GRB2, respectively (2, 9, 49). Intriguingly, Tyr1062 is a multidocking site for several signaling molecules, including SHC, FRS2, DOK family proteins, IRS1/2, Enigma, and protein kinase C (3-5, 15, 22, 35, 38, 42, 43, 45). Consistent with these findings, it turned out that representative intracellular signaling pathways, such as the RAS/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/AKT, Jun-associated N-terminal kinase (JNK), and ERK5 pathways are activated via phosphorylated Tyr1062 (7, 14, 23, 24, 57). Activation of these pathways leads to Ret-mediated biological responses such as cell transformation, survival and migration of neuronal cells, and tubule formation of kidney-derived cells (5, 17, 46, 61).
RET loss-of-function mutations have been identified in human Hirschsprung's disease, a congenital malformation associated with the absence of intrinsic ganglion cells in the distal gastrointestinal tract (16, 53). The mutations cause Ret dysfunction by various mechanisms (8, 11, 12, 20, 29, 31, 32, 50, 51). We and other investigators reported that a few mutations identified in Hirschsprung's disease could affect signaling via Tyr1062 in Ret (20, 28). In this study, to elucidate the in vivo role of signaling via Tyr1062, knock-in mice in which Tyr1062 was replaced with phenylalanine were generated. Our results reveal that signaling via Tyr1062 is necessary for normal development of the ENS and the kidney. In addition, because the abnormalities of homozygous mutant mice were milder than those of Ret-deficient mice, it is likely that signaling via other tyrosine residues also plays a role in organogenesis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genotype screening. The genomic DNA of the offspring was extracted from their tails. Mice with the mutant Ret allele were screened by genomic PCR followed by SspI digestion. PCR was performed with the forward primer in exon 19 (19f; 5'-ACTACTTGGACCTGGCTGCATCCA-3') and the reverse primer in intron 19 (19r; 5'-AGAAAAGGGTTCGGAGGAGGCTTTGGTGTC-3') (Fig. 1A).
Transfection. A plasmid containing the entire coding sequence of the human RET gene (long isoform) was produced as described previously (5). Each mutation (isoleucine for leucine at codon 1061 and phenylalanine for tyrosine at codon 1062) was introduced by the QuikChangeII site-directed mutagenesis kit (Stratagene) according to the manufacturer's instruction. Plasmid DNAs (4 µg) were transfected into SK-N-MC human primitive neuroectodermal tumor cells (5 x 105) with the Lipofectamine 2000 reagent (Invitrogen). The cells were maintained in a humidified incubator with 95% air and 5% CO2 at 37°C. After 24 h of incubation, the medium was replaced with serum-free Dulbecco's modified Eagle's medium (DMEM) and kept for 3 h. Then the transfected cells were treated with GDNF (100 ng/ml) for 15 min, lysed in sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris-HCl [pH 6.8], 5 mM EDTA, 2% SDS, 10% glycerol, 20 µg of bromophenol blue per ml) containing 80 mM dithiothreitol and subjected to Western blot analysis as described below.
Tissue preparation for protein and RNA extraction. After body weight measurement, mice were sacrificed under general anesthesia. A complete autopsy was performed, and organs were resected, cut into 5-mm3 specimens, and quickly frozen for protein and RNA extraction.
Gene expression analysis. Total cellular RNA was isolated from frozen tissues with the RNeasy minikit (Qiagen). RNA was reverse transcribed with avian myeloblastosis virus reverse transcriptase XL (TaKaRa) for 30 min at 55°C. The resulting cDNA was subjected to 30 cycles of PCR consisting of 30 s at 94°C, 30 s at 56°C, and 30 s at 72°C. The PCR primer sets were as follows: mouse Ret short isoform (Ret9) (forward primer, 5'-CCAGCTATGTGTCCACAGCCGTGC-3', and reverse primer, 5'-CATGATGGGGGTAGGGTGCAAAGG-3'); mouse Ret long isoform (Ret51) (forward primer, 5'-CCGATGGCACTAGCACTGGGTTCC-3', and reverse primer, 5'-ATTTTGCCGCTGAGGGTGAAACCA-3'); and mouse ß-actin (forward primer, 5'-AGCTGCCTGACGGCCAGGTC-3', and reverse primer, 5'-GCTCAGGAGGAGCAATGATC-3').
Antibodies. Anti-Ret rabbit polyclonal antibody was developed against the carboxyl-terminal 19 amino acids as described previously (47). Polyclonal anti-Akt, anti-phospho-Akt, anti-Erk, and anti-phospho-Erk antibodies were purchased from Cell Signaling Technology. Monoclonal antiphosphotyrosine antibody was purchased from Upstate Biotechnology. Monoclonal anti-ß-actin antibody was purchased from Sigma.
Protein analysis. Frozen mouse tissues were homogenized in SDS sample buffer containing 80 mM dithiothreitol. The lysates, containing 30 µg of protein, were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore). Membranes were blocked for 1 h at room temperature in 3% ovalbumin in TPBS (phosphate-buffered saline containing 0.05% Tween 20) with gentle shaking and incubated with the primary antibody overnight at 4°C. After washing the membranes with TPBS three times, they were incubated with the secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit immunoglobulin G- horseradish peroxidase; Dako) for 1 h at room temperature. The reaction was examined by an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences) according to the manufacturer's instructions.
Primary culture of dorsal root ganglia. Dorsal root ganglia were obtained from mice at postnatal day 13.5 under a stereoscopic microscope and washed twice with ice-cold physiological saline and twice with Ham's F-12 (Gibco). They were digested with 0.15% collagenase and 0.05% trypsin-EDTA, and mechanically triturated. Isolated cells were suspended in F-12, and 1.5 volumes of Dulbecco's modified Eagle's medium supplemented with 8% calf serum were added. The cells were plated on 35-mm-diameter tissue culture dishes coated with filtrated 0.01% poly-L-lysine (Sigma) and maintained in a humidified incubator with 95% air and 5% CO2 at 37°C. The medium was replaced with serum-free Dulbecco's modified Eagle's medium/F-12 (at a ratio of 1.5 to 1) 48 h after the initial plating and kept for 24 h. Then cultured cells were treated with GDNF (100 ng/ml) for 15 min, lysed in SDS sample buffer, and subjected to Western blot analysis.
Histology and immunohistochemistry. Organs were resected as described above. The stomach was incised along the greater curvature. The intestine was linearized and sectioned in 20 to 30 pieces of 1-cm-long fragments. The kidney was cut along the longitudinal axis to yield the greatest section.
Embryos were taken from female mice at gestational day 13.5 (E13.5) and E17.5 and sacrificed by cervical joint dislocation. Genomic DNA was extracted from the amnion, and the genotype was screened as described above.
All tissues and embryos were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 µm for light microscopic analysis. Longitudinal sections of the stomach, transverse sections of the colon and small intestine, and sagittal sections of the kidney and embryos were prepared. Hematoxylin and eosin staining was performed by a conventional method. For immunohistochemistry, slides were deparaffinized in xylene and rehydrated in a graded series of ethanol (100, 90, and 70%). For antigen retrieval, they were incubated in 50 mM Tris-HCl (pH 7.6) containing 5% trypsin at 37°C for 10 min. Nonspecific binding sites were blocked with 10% normal goat serum for 10 min. The sections were incubated with the primary antibody (rabbit anti-peripherin antibody, Chemicon) overnight at 4°C, and endogenous peroxidase was inhibited with 0.3% hydrogen peroxide in methanol for 15 min. The slides were incubated with the secondary antibody conjugated to peroxidase-labeled polymer (EnVision+, rabbit; Dako) for 30 min at room temperature. The reaction products were visualized with diaminobenzidine. Counterstaining was performed with hematoxylin.
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RESULTS |
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The targeting construct contained a neomycin resistance cassette for positive selection (Fig. 1A). Genomic DNA from ES clones obtained by G418 selection was analyzed by Southern blotting with the 3' and Neo probes (Fig. 1A). From these results, two ES clones (330 and 400) were expected to harbor a targeted mutant Ret allele (Fig. 1B). Both clones were used successfully to generate chimeric mice, and germ line transmission of the mutant allele was achieved. Genomic DNA fragments containing tyrosine 1062 codons were amplified from tail DNAs of wild-type, heterozygous, and homozygous mutant mice and digested with SspI. As expected, approximately 270-bp and 140-bp fragments were obtained from the wild-type and mutant alleles, respectively (Fig. 1C). The mutations introduced at codons 1061 and 1062 in the mutant allele were confirmed by sequencing the amplified fragments.
When heterozygous mutant mice were mated, wild-type, heterozygous, and homozygous mice were born at the expected frequencies (data not shown). Homozygous mice exhibited obvious abdominal distension around day 10 and died by 27 days of age (Fig. 2A). There was no significant difference in body weight among the three genotypes at birth, although homozygous mice displayed severe growth retardation at day 13.5 (Fig. 2B). No difference in gross appearance, body weight, or survival rate was observed between wild-type and heterozygous mutant mice at least up to 18 months of age (data not shown).
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Defects of the ENS in Ret Y1062F knock-in mice. The abdomen of homozygous Y1062F mice was distended macroscopically. Autopsy revealed severe dilation of the small intestine in all homozygous mutant mice (Fig. 4A). About 25% of the homozygous mice showed total colon stenosis, whereas the remaining mice displayed dilation of the oral part of the colon and stenosis of its anal part (Fig. 4B).
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Renal hypoplasia in Ret Y1062F knock-in mice. Intriguingly, kidneys developed in all homozygous Y1062F knock-in mice, although their sizes were significantly smaller than those of wild-type and heterozygous mice (Fig. 7A and B). The adrenal gland, ureter, and urinary bladder developed normally in homozygous mutant mice, and urine production was observed. Because homozygous mice exhibited growth retardation after birth, the kidney weight-to-body weight ratio was analyzed to evaluate the degree of kidney hypoplasia. The ratio in homozygous mice was about half of that in the wild-type and heterozygous mutant mice from birth onward (Fig. 7B). No significant difference in the heart weight-to-body weight ratio was observed among the three genotypes (Fig. 7C).
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DISCUSSION |
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pathways (60). These pathways are activated via phosphorylated tyrosine residues in the Ret intracellular domain. For example, Tyr1015 is a binding site for PLC that is responsible for protein kinase C activation (9). Phosphorylated Tyr1062 binds several adaptor and effector proteins including SHC, FRS2, IRS1/2, and the DOK protein family, resulting in activation of RAS/ERK, phosphatidylinositol 3-kinase/AKT, and JNK (7, 22-24, 35, 42, 43, 45, 57). In addition, Tyr1096, which is only present in the carboxyl-terminal sequence of the long Ret isoform (Ret51), is a binding site for GRB2 and appears to activate partially, but not fully, the RAS/ERK and phosphatidylinositol 3-kinase/AKT pathways (7). The Y1062F knock-in mice that we generated in this study confirmed the importance of the signal via Tyr1062 in the development of the ENS and the kidney. However, significant differences in phenotype were observed between Y1062F knock-in and Ret-deficient mice. First, although about 40% of the knock-in mice exhibited a lack of enteric neurons posterior to the stomach, as observed in Ret-deficient mice, the rest of them had enteric neurons in the small intestine and colon to various extents, accompanying the reduction in the size and number of ganglion cells. Many reports suggested that GDNF/Ret signaling is required for the proliferation, migration, and survival of the ENS progenitors (18, 21, 25, 27, 30, 46, 62, 64, 65). The fact that the ENS progenitors were few in the intestines of E13.5 and E17.5 homozygous Y1062F embryos supported this view. In addition, these findings are consistent with a report showing that the phosphatidylinositol 3-kinase and ERK pathways play crucial roles in the migratory response of enteric neural crest cells (ENCC) to GDNF (46). However, the signal via Tyr1062 was not necessarily a sole residue for complete migration and proliferation of ENCC, because the development of enteric neurons was detected in the intestine of some homozygous Y1062F mice. Thus, signaling via distinct tyrosine residues such as Tyr1015 or Tyr1096 may also play a role in the migration and proliferation of ENCC.
Maina et al. (39) reported knock-in mice in which the multifunctional docking sites of Met receptor tyrosine kinase were replaced with specific binding motifs for phosphatidylinositol 3-kinase, Src or Grb2. These mutant mice retained normal signaling via Gab1, but differentially recruited specific effectors. While the mutants with optimal Grb2 binding motifs developed normally, the mutants with phosphatidylinositol 3-kinase or Src binding motifs resulted in severe loss of function but displayed different phenotypes and rescue of distinct tissues. The rescue of placenta and myoblast proliferation versus axon growth by Src and phosphatidylinositol 3-kinase binding sites, respectively, indicated that Met-mediated developmental events require the activation of specific pathways. The introduction of these types of mutations in the surrounding sequence of Tyr1062 in Ret may elucidate specific roles for each signaling in organogenesis.
In addition to GDNF, neurturin that is another member of the GDNF protein family that activates Ret though binding to GFR2. Although GDNF and neurturin have similar effects on ENS progenitors in vitro (27), Gdnf–/– (44, 52, 54) and Neurturin–/– (26) mice have strikingly different phenotypes. Recently, it was reported that neurturin is essential for maintaining the size of enteric neurons, whereas GDNF availability determines neuron number by controlling ENS progenitor proliferation (21). Our findings that both enteric neuron number and size were markedly reduced in homozygous Y1062F mice suggested that the action of both GDNF and neurturin was impaired in them.
The difference in the extent of aganglionosis observed in individual mutant mice suggests the involvement of factors other than Ret in the development of enteric neurons. The spatial and temporal regulation of Gdnf expression along the developing bowel has recently been reported (46, 66). Gdnf mRNA upregulation was detected in the stomach of E9.0 to 9.5 and the cecum of E11.5 to 13.5 embryos and appeared to function as a chemoattractant of ENS progenitors in vivo, leading to the ordered colonization of the esophagus, stomach, and small intestine (46). Thus, the difference in the level of Gdnf upregulation in the developing bowel of individual Y1062F mice may affect the proliferation of Ret-expressing ENS progenitors in the stomach as well as their migration towards the intestine during embryogenesis.
In addition, it is possible that other signals produced by the microenvironment of the developing gut play some role in the migration of Ret-expressing ENS progenitors in Y1062F mice. In this regard, endothelin-3 (ET-3) is a candidate molecule because ET-3 itself is involved in the pathogenesis of aganglionosis in human Hirschsprung's disease and a high level of ET-3 expression was observed in the cecum and the colon during embryogenesis (33, 37, 41, 58, 63). In fact, it was recently demonstrated that ET-3 regulates GDNF-induced ENCC proliferation and migration by inhibiting protein kinase A activity (6). ET-3 promoted GDNF-induced ENCC proliferation but inhibited their migration. In addition, we recently reported that serine 696 phosphorylation in the juxtamembrane region of Ret by protein kinase A promoted lamellipodium formation in neuroectodermal cells, which is a critical event for neuritegenesis (19). Thus, it is likely that modulation of Ret function by ET-3 is crucial for normal development of the ENS.
Unlike Ret-deficient mice, kidneys developed in all of the homozygous Y1062F mice, although the kidney weight-to-body weight ratio of the homozygous mice was about half that of the wild-type and heterozygous mutant mice. Although the early stages of metanephric development in homozygous Y1062F mice, such as evagination of the ureteric bud and initial branching, occurred normally, defects in ureteric bud branching at later stages of nephrogenesis were observed. It was reported that half of the Ret-deficient mice had the defects in ureteric bud formation from the Wolffian duct (56). Approximately 15% formed a ureteric bud, but it failed to grow into the metanephric mesenchyme. In the rest of the Ret-deficient mice, the ureteric bud succeeded in reaching the metanephric mesenchyme, but the growth and branching of the ureteric bud were severely retarded and often abnormal (56). These findings suggested that signaling other than that via Tyr1062 is critical for ureteric bud formation and evagination during early nephrogenesis stages.
Ret is translated into two major isoforms, Ret9 and Ret51, by alternative splicing (36). The Ret isoforms differ only in their carboxyl-terminal amino acid sequences. The carboxyl-terminal 51 amino acids in Ret51 are replaced with nine unrelated amino acids in Ret9 (59). Recently, de Graaff et al. (13) generated mutant mice that expressed single Ret isoforms and showed that Ret9 and Ret51 have different signaling properties in vivo. Monoisomeric Ret9 mice, which lack Ret51, appeared normal, whereas monoisomeric Ret51 animals, which lack Ret9, exhibited kidney hypodysplasia and lacked enteric ganglia from the colon. As in homozygous Y1062F mice, ureteric bud branching was impaired in monoisomeric Ret51 mice, although histologic abnormalities in the kidney, such as cystic changes of the tubules, appeared to be found more frequently in the latter. On the other hand, most of the homozygous Y1062F mice had more severe defects in the ENS than monoisomeric Ret51 mice.
Because the sequence divergence of the two isoforms occurs one amino acid after Tyr1062, one hypothesis to explain the differential signaling properties of Ret9 and Ret51 is that the sequence differences that are carboxyl terminal to Tyr1062 may modulate the efficiency of the complex formation of signaling molecules. The fact that monoisomeric Ret51 mice and homozygous Y1062F mice had similar kidney phenotypes supported the view that the signal via Tyr1062 in Ret51 is not sufficient for ureteric bud branching at late embryogenesis. It is interesting that the specific sequence that is carboxyl terminal to Tyr1062 observed in each isoform can affect the binding ability of the phosphotyrosine-binding and SH2 domains of SHC (48).
In this study, we elucidated the in vivo role of signaling via Tyr1062 by targeted mutagenesis. This signaling appeared to play a crucial role in the migration and/or proliferation of ENS progenitors because the neuron number and distribution in the intestine of Y1062F homozygous mice were markedly reduced. In addition, signaling via Tyr1062 was required for ureteric bud branching at later stages of nephrogenesis but not for ureteric bud formation, evagination, and first branching at its early stages. To explain the difference in phenotype between Ret-deficient mice and Y1062F mutant mice, it would be interesting to generate mutant mice in which other tyrosine residues, such as Tyr1015 and Tyr1096, are replaced.
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ACKNOWLEDGMENTS |
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This work was supported in part by Grants-in-Aid for COE Research, Scientific Research (A), and Scientific Research on Priority Areas Cancer from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from the Uehara Memorial Foundation.
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FOOTNOTES |
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M.J. and T. F. contributed equally to this study.
Present address: Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Aoba, Sendai, Japan.
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REFERENCES |
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140 bp. Heterozygous mice (He) gave both bands. (D) Analysis of intracellular signaling via Ret with the L1061I or L1061I/Y1062F mutation. Lysates from GDNF-treated (100 ng/ml, 15 min) and untreated SK-N-MC cells that were transiently transfected with wild-type RET (designated MC [RET]), L1061I mutant RET, or L1061I/Y1062F mutant RET were immunoblotted with anti-Ret, anti-Akt, anti-phospho-Akt, anti-Erk, and anti-phospho-Erk antibodies.
