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Carcinogenesis |
Departments of Experimental Pathology [T. K., H. M., O. H.] and Cell Biology [O. M., J. K., T. N.], Cancer Institute, Toshima-ku, Tokyo 170-8455; Department of Molecular Genetics, Tohoku University School of Medicine, Sendai 980-8575 [T. N.]; and CREST, Japan Science and Technology Corporation, Kawaguchi 332-0012 [T. N.], Japan
ABSTRACT
Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis (TSC), a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, we have generated a Tsc2 mutant mouse. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, 
80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants died around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibited an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis.
INTRODUCTION
Human TSC,3 an autosomal dominant multiorgan disorder caused by a mutation in either the TSC1 (human chromosome 9q34) or TSC2 (human chromosome 16p13.3) gene, is characterized by phacomatosis with manifestations that include mental retardation and seizures (1, 2, 3) . It has been reported that hamartomas that develop in TSC patients have LOH at one of two TSC loci (4) , suggesting both to be tumor suppressor genes fitting Knudson’s two-hit hypothesis (5) .
In the Eker rat, a germ-line retrotransposon insertion (intron 30) in a homologue of the human TSC gene 2 (Tsc2) causes RCs with a complete penetrance in all heterozygotes (6, 7, 8, 9) . At the histological level, multiple bilateral RCs develop through multiple stages from early preneoplastic lesions (e.g., phenotypically altered tubules) to adenomas in virtually all heterozygotes by the age of 1 year (6) . LOH and intragenic somatic mutations of wild-type Tsc2 are characteristics of Eker rat RCs, indicating that a second hit of the Tsc2 gene is a cause of RC development (10, 11, 12) . We have generated transgenic Eker rats carrying wild-type Tsc2 as a transgene and demonstrated that phenotypes of the Eker rat are suppressed by extra copies of the wild-type Tsc2 gene (13) . This demonstration finally confirmed that tumors in the Eker rat are caused by the germ-line mutation of Tsc2. Phenotypes of the Eker rat differ from those of human TSC, including the incidence of RCs (in man, angiomyolipomas are more common renal lesions), although subependymal and subcortical hamartomas resembling those in TSC were reported quite recently in the Eker rat (1 , 14) . Thus, a mutation of the same gene causes diverse phenotypes between species.
TSC2/Tsc2 encodes tuberin, which is highly conserved in vertebrates (15 , 16) . It contains Rap1-GAP homology region near its COOH terminus (3) . Recently, Rap1-GAP activity (17) and Rab5-GAP activity along with rabaptin 5 binding (18) of tuberin have been demonstrated. Transcriptional activator activities of tuberin have also been reported (19 , 20) . Because TSC patients with alterations of either of the two predisposing genes (TSC1 and TSC2) exhibit identical symptoms, the products of these two genes (hamartin and tuberin) are thought to be involved in a common biological pathway (2) . Indeed, direct interactions between tuberin and hamartin have been reported previously (21) . However, the precise functions of tuberin and the molecular mechanisms of tumor development associated with Tsc2 mutations have not been fully elucidated.
In this study, we have generated a Tsc2 knockout mouse to characterize the function of tuberin in vivo and to elicit insights into tumorigenesis caused by Tsc2 gene inactivation. A comparison of the phenotypes of Tsc2 mutant mice with those of human TSC and the Eker rat will provide some insights into species-specific differences in tumorigenesis caused by tuberin deficiency. Moreover, in comparison with the rat system, one of the advantages of the mouse system is the availability of various genetically modified lines for genetic cross experiments (22) . The pathway in which tuberin is involved might be elucidated through such genetic analysis.
MATERIALS AND METHODS
Construction of a Targeting Vector.
Overlapping phage clones covering the mouse Tsc2 gene were isolated from a 129/Sv mouse genomic library using rat Tsc2 cDNA as a probe (15)
. A Tsc2-targeting vector, pKO-LT, was constructed to delete a 3-kb fragment of the mouse Tsc2 gene that ranged from a HindIII site in exon 2 to a PstI site in exon 5 (Fig. 1A)
. An open reading frame of ß-gal (lacZ) with a NLS was fused to the Tsc2 gene in frame at the 3' end of a 7-kb 5' homologous region. For positive selection, a PGK-neo cassette flanked by a pair of loxP sequences was introduced between NLS-lacZ and a 1.5-kb 3' homologous region (23)
. For negative selection, a DTA-expressing cassette without a polyadenylic acid addition signal was fused to the 3' end of the 3' homologous region (24)
. Transcriptional orientations of PGK-neo and DTA cassettes were the opposite and the same, respectively, with respect to that of the Tsc2 gene.
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, probe A). One homologous recombinant (clone 44) was identified and used for blastocyst injection, which was performed as described previously (25)
. Male chimeras were bred to C57BL/6J females to obtain F1 mice. F2 offspring and embryos were obtained by F1 heterozygous intercrosses.
Genotyping.
For genotyping by Southern blot analysis, 10 µg of DNA samples prepared from mouse tail tips were digested with BamHI and separated by 1% agarose gel electrophoresis. Filter preparation, hybridization, and washing were carried out as described previously (6)
. A 0.7-kb PstI fragment containing exons 6 and 7 and a 1.3-kb NcoI-BamHI fragment containing exon 1a of the Tsc2 gene were used as the 3'outside and 5' inside probes, respectively (Fig. 1A)
. To genotype F2 embryos by PCR, DNA was prepared from yolk sacs or embryonic tails by proteinase K digestion. Two primer sets, LZF2 and LZR2 for mutated allele-specific amplification (224 bp) and MTSC3 and MTSC9 for wild-type allele-specific amplification (160 bp), were used. PCR (35 cycles) was carried out as described previously, except for the use of Taq DNA polymerase (Toyobo, Tokyo, Japan; Ref. 15
), and the products were analyzed by a 3% Nusieve agarose gel (FMC BioProducts, Rockland, ME) electrophoresis. Sequences and locations of the primers were as follows: LZF2, 5'-GGTAAACTGGCTCGGATTAG-3' in the lacZ coding region; LZR2, 5'-CCATCAGTTGCTGTTGACTG-3' in the lacZ coding region; MTSC3, 5'-AATGCCCTAAGTGCAACCTG-3' in intron 2; and MTSC9, 5'-CTACATGGTAAGTCCCTGTC-3' in intron 2.
Analyses of RNA and Protein.
For RT-PCR, first-strand cDNA was synthesized from 5 µg of total RNA from whole embryos at E 9.5 (noon of the day on which a vaginal plug was detected was defined as E 0.5). PCR (35 cycles) was carried out as described for genotyping (15)
. Primers used for the amplification of wild-type Tsc2 cDNA were MTSC10 (sense; 5'-TGTGCAGAAGGCAAACAGAC-3' in exon 1) and MTSC11 (antisense; 5'-CATGTTGTAGATGGCGCTGT-3' in exon 8). Primers used for the amplification of mutant cDNA were MTSC10 (sense) and LZRT (antisense, 5'-GACGTTGTAAAACGACGGGA-3' in lacZ). Amplified products were analyzed by 2% Nusieve agarose gel electrophoresis. For the detection of tuberin, whole E9.5 embryos of comparable body size were directly homogenized in SDS-sample buffer [2.3% SDS, 0.0625 M Tris-HCl (pH 6.8), 5% ß-mercaptoethanol, and 10% glycerol]. Comparable amounts of protein from each were separated on 5% SDS-PAGE, transferred onto nylon membrane, and probed with anti-human tuberin NH2-terminal and COOH-terminal peptide antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) known to cross-react with mouse tuberin.
Carcinogen Treatment.
Female C57BL/6J mice were bred with Tsc2 heterozygous mutant (Tsc2+/-) F1 males. At E 14 or E 15, ENU solution (10 mg/ml in saline) was i.p. injected into bred females at a final dose of 50 mg/kg body weight (26)
. Offspring were sacrificed at the ages of 11 weeks or 6 months and examined for tumor formation.
Histological Analyses.
Whole mount X-gal staining of embryos was carried out using a standard protocol (27)
after fixation in 4% paraformaldehyde/PBS (4°C; 20 min). Immunohistochemical analysis of embryos was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) after fixation in Bouin’s solution (4°C; overnight) and standard sectioning in paraffin. For orientation, sections were stained with H&E. The primary antibodies used were a mouse monoclonal anti-rat PCNA antibody (Novocastra Laboratories, Newcastle, United Kingdom), a mouse monoclonal anti-rat class III ß-tubulin antibody (Babco, Richmond, CA), a rabbit polyclonal anti-nestin antibody (28)
, and mouse monoclonal anti-MAP2 antibodies (clone AP-20 and HM-2; Sigma, St. Louis, MO). Tissues from adult mice were examined by H&E staining as described previously (10)
.
Analysis of LOH.
For detection of LOH by PCR, tissues were fixed in 10% buffered formalin and sectioned (10 µm thick). Tumor samples and comparable amounts of normal kidney cortex areas were dissected separately from sections with needles under a stereomicroscope. Samples were dissolved in 25 µl of proteinase K solution [1 mg/ml proteinase K, 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.5% Tween 20], digested overnight at 55°C, and heated at 95°C for 20 min. Aliquots (5 µl) of each mixture were subjected to PCR (35 cycles) in a 25-µl reaction volume containing two primer sets, MTSC3 and MTSC9 for the wild-type allele and LZF2 and LZR2 for the mutated allele. Amplified products were separated by 3% Nusieve agarose gel electrophoresis, transferred onto Nylon membranes, and hybridized with 32P-labeled internal oligonucleotides [MTSC14 (5'-CCACCAAGGTAAGTCTCTCA-3') for the wild-type allele and NZF4 (5'-TGAATTATGGCCCACACCAG-3') for the mutated allele]. For the detection of LOH by Southern blot analysis, membrane filters of tissue DNAs were prepared after BamHI digestion and hybridized as described for genotyping. For both PCR and Southern blot analysis, radioactivity was measured with a BAS2000 Bio-Image Analyzer (Fuji Film, Tokyo, Japan).
RESULTS
Generation of Tsc2 Mutant Mice.
To generate a line of Tsc2 mutant mice, we constructed a targeting vector, pKO-LT, in which a fragment from codon 74 in exon 2 to codon 164 in exon 5 of the mouse Tsc2 gene was deleted (Fig. 1A)
. A NLS-LacZ coding fragment was introduced in frame at exon 2 to analyze the expression profile of the Tsc2 gene (Fig. 1A)
. By gene targeting, one ES clone (clone 44) was identified as a homologous recombinant that gave rise to germ-line transmission of the mutated Tsc2 allele after blastocyst injection and subsequent mating of chimeras with C57BL/6J females (Fig. 1, A and B)
. PCR genotyping analysis of embryos at E 9.5 from F1 heterozygous intercrosses revealed the existence of homozygous mutant embryos (Tsc2-/-; Fig. 1C
). These embryos were subjected to Western blot analysis using anti-tuberin NH2 terminus or COOH terminus antibodies. The 180-kDa band of normal tuberin was absent in Tsc2-/- embryos in both cases, confirming the inactivation of the Tsc2 gene (Fig. 1D)
.
The expression of the mutated Tsc2 allele was examined using RT-PCR and whole mount X-gal staining of Tsc2+/-embryos (Fig. 1, E and F)
. When the primer set encompassing exons 1–8 of the Tsc2 gene was used, the predicted 741-bp product derived from wild-type Tsc2 mRNA was amplified in wild-type (Tsc2+/+) as well as in Tsc2+/- embryos (Fig. 1E)
. A 196-bp product was amplified in a Tsc2+/- embryo-specific manner using a lacZ coding region-specific primer, suggesting that splicing occurred between exon 1 and the modified exon 2 of the mutated Tsc2 allele (Fig. 1E)
. Sequence analysis of this 196-bp product confirmed in frame fusion of the Tsc2 transcript with the NLS-lacZ at the 3' end of exon 2 (data not shown). These results indicated that the mutated Tsc2 allele expressed Tsc2-lacZ chimeric mRNA, possibly resulting in the expression of a fusion protein consisting of the NH2-terminal 73 amino acid residues of tuberin and NLS-ß-gal. Tsc2+/- embryo-specific expression of ß-gal was detected by whole mount staining of E 11.5 embryos with X-gal (Fig. 1F)
. Although a detailed histological examination at the single cell level has not been fully accomplished, organs including the heart and central nervous system were stained as reported in an earlier work by in situ hybridization analysis of Tsc2 gene expression in mouse embryos (29)
.
Development of RCs and Hepatic Hemangiomas in Tsc2 Heterozygous Mutant Mice.
Although Tsc2+/- mice were born without any apparent phenotype, they developed spontaneous bilateral and multiple RCs with age (Fig. 2A)
. Macroscopically visible RCs developed in most Tsc2+/- mice (20 of 21 cases) by the age of 6 months, irrespective of the sex. The earliest occurrence of a macroscopically visible RC was seen in a female mouse at the age of 14 weeks (data not shown). By 10 months of age, virtually all Tsc2+/- mice developed RCs. None of the Tsc2+/+ mice developed RCs, even after the age of 1 year (n = 43). Macroscopically, the RCs were mostly cystic, with a diameter of 1–2 mm (Fig. 2A)
. Histological examination revealed RCs to be located exclusively in the cortex region, and the majority showed a papillar growth within the cysts (Fig. 2B)
. Some tubular-type RCs were also observed (Fig. 2C)
. As seen in the Eker rat (30)
, the development of RCs in Tsc2+/- mice was accelerated by transplacental ENU-treatment of embryos at E 14.0 or E 15.0 (n = 7; Fig. 2D
). All ENU-treated Tsc2+/- offspring developed macroscopically visible RCs by the age of 11 weeks. Thus, the results indicate that a Tsc2 germ-line mutation in the mouse can cause hereditary RCs, as seen in the rat.
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; data not shown). Although substantial amounts of the PCR product from the wild-type Tsc2 allele were amplified from RCs, these were thought to be derived from contaminating normal tissues, which could not be completely eliminated during sample preparation. On Southern blot analysis of larger RCs, 4 of 11 cases showed loss of the wild-type Tsc2 allele (Fig. 2F
; data not shown). Thus, we infer that the wild-type Tsc2 allele may be lost during the development of RCs in Tsc2+/- mice.
Examination of aged Tsc2+/- mice (age > 1.5 years) revealed hepatic hemangiomas in 18 of 23 cases (80%; Fig. 2G
). These hemangiomas developed in both sexes and in all hepatic lobes. Histologically, they resembled cavernous hemangiomas consisting of a thin layer of endothelium and large cavities filled with blood. Thus far, our analysis has not detected loss of the wild-type Tsc2 allele in these hemangiomas (data not shown). The development of macroscopically visible hemangiomas in Tsc2+/- mice was not obvious at 10 months of age. In contrast to the Tsc2+/- mice, none of the Tsc2+/+ mice over 1.5 years of age (n = 29) developed any hepatic hemangiomas. These results indicate that a Tsc2 germ-line mutation causes hepatic hemangiomas in the mouse.
Embryonic Lethality of Tsc2 Homozygous Mutants.
Although intercrossing of F1Tsc2+/- mice was carried out extensively (111 offspring from 17 intercrosses), we could not obtain any Tsc2-/-offspring (43 Tsc2+/+ mice and 68 Tsc2+/- mice), indicating the embryonic lethality of Tsc2 homozygous mutants. Therefore, we analyzed the genotype of embryos from F1 heterozygous intercrosses to determine the lethal stage (Table 1)
. At E 10.0–10.5, 16% of all surviving embryos (7 of 44) were homozygous mutants. On the other hand, 5 of 6 Tsc2-/- embryos were dead at E 11.0–11.5. Therefore, we concluded that most Tsc2-/- embryos died at around E 10.5. However, dead or resorbed Tsc2-/- embryos were already present at E 9.0–9.5, and some Tsc2-/- embryos survived to E 11.5–12.5, suggesting that there might be considerable variation in the lethal stage.
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. The most prominent feature found in a significant proportion (50%) but not all of nonresorbed Tsc2-/- embryos at E 9.0–11.5 was the nonclosure of the neural tube in the head region (Figs. 3
and 4
).
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. Fig. 4A
shows the most severe consequence of nonclosure found in an E 11.5 Tsc2-/- embryo by transverse sectioning. In the head region, the subventricular layer of the neural tube was completely exposed to body surface in this embryo. However, even in this case, the neural tubes in homozygous Tsc2 mutants formed the neuroepithelial architecture. To assess the early neuronal differentiation status in Tsc2-/- embryos, we performed immunohistochemical analysis for several neuronal markers (class III ß-tubulin, nestin, and MAP2; Refs. 31, 32, 33
) and PCNA. In the nonclosed neural tube of Tsc2-/- embryos (E 9.5), nestin-positive cells showing a process-like staining pattern were prominent throughout the mantle layer as seen in Tsc2+/+ or Tsc2+/- embryos (Fig. 4C)
. In E 11.5 and E 12.5 embryos, class III ß-tubulin- and MAP2-positive cells also accumulated in the marginal layer, regardless of the Tsc2 genotype (Fig. 4B
; data not shown). In contrast to these neuronal markers, PCNA-positive cells were localized mainly in the subventricular layer in Tsc2+/+, Tsc2+/-, and Tsc2-/- embryos (Fig. 4C)
. These results indicated that cell division in the ventricular layer, cell migration to the marginal layer, and cell differentiation to a neural lineage still occur in the neural tube of Tsc2-/- embryos.
At the histological level, hearts of E 11.5 (two cases) and E 12.5 (one case) Tsc2-/- embryos exhibited abnormal myocardia (Fig. 5
; data not shown). Although these two E 11.5 embryos had a smaller body size, the size of their hearts was comparable to the size of those of control embryos (Fig. 5B
, top panels; data not shown). Myocardia of these E 11.5 and E 12.5 Tsc2-/- embryos were thickened and associated with increased cell density (Fig. 5A
, bottom panels, Fig. 5B
, top panels). Consequently, ventricular cavities were narrowed (Fig. 5A
, top panels). Endocardia of those hearts became less recognizable than normal controls because of their tight association with thickened myocardia (Fig. 5A
, bottom panels, Fig. 5B
, top panels). Such myocardial abnormalities might have resulted from altered cell proliferation activity of cardiomyocytes in the outer mantle layer or trabeculae. However, no particular differences were found on PCNA staining between Tsc2-/- and Tsc2+/+ or Tsc2+/-hearts (Fig. 5B)
. Differences between the developing hearts of E 9.0–10.5 Tsc2-/- embryos and control Tsc2+/+ or Tsc2+/- embryos were not obvious on histological examination (data not shown). Thus, the myocardial anomaly became apparent at later stages (E 11.5–12.5) in Tsc2-/- embryos.
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In the present study, we generated and initially characterized a Tsc2 mutant mouse strain. The most notable phenotype of the Tsc2+/- mice was the development of RCs histologically resembling those in the Eker rat (6) . Consistent with the tumor suppressor characteristics of the TSC2/Tsc2 gene, our LOH analysis detected a second hit in the wild-type Tsc2 allele in those RCs, as seen in hamartomas from TSC patients and RCs from the Eker rat (4 , 5 , 10 , 11) . As in the Eker rat, the development of hamartomas characteristic of human TSC was not observed in Tsc2+/- mice. The similar RC development in both mouse and rat Tsc2 mutants suggests that some species-specific mechanism may participate in the occurrence of phenotypic differences caused by tuberin deficiency.
However, there were also several differences in the incidences of extrarenal lesions between our Tsc2+/- mice and Eker rats (34) . For example, a high incidence of hepatic hemangiomas was seen, although the underlying mechanism remains to be determined. Such hepatic hemangiomas have not been detected in the Eker rat. In contrast, in Tsc2+/- mice, we have not observed the pituitary adenomas or subependymal nodules that often develop in the Eker rat (14 , 34) . Although we have identified a few cases of Tsc2+/- mice exhibiting tumors in the spleen, uterus, and foot pad, 4 the incidences of these tumors were not as high as those of tumors in the spleen and uterus in the Eker rat (34) . Thus, differences in the cell-type specificity of tumorigenesis caused by tuberin deficiency might exist not only between man and rodents but also between rat and mouse.
Because all of the Tsc2-/- embryos died in utero, an essential role for tuberin function in mouse development was revealed (6) . Although we have not detected the common lethal defect, the phenotypes of the Tsc2-/- embryos did provide some insights into the physiological functions of tuberin. The most characteristic histological feature seen in Tsc2-/- embryos in later stages was anomalies of the myocardium. The increased cell density associated with cardiac anomalies might have been caused by loss of the growth-suppressive function of tuberin. Because myocardia in Tsc2-/- embryos at earlier stages (E 9.0–10.5) exhibited a normal architecture with substantial trabeculation and the hearts of Tsc2-/- embryos beat, early differentiation of cardiomyocytes into functional heart muscle occurs normally. However, late-stage differentiation or maturation processes in cardiac development may be disturbed by tuberin deficiency. Because human neonates with TSC often develop cardiac rhabdomyomas that originate primarily in the ventricle (1) , we infer that the cardiac anomaly in Tsc2-/- embryos is directly linked to the tuberin deficiency and that tuberin function is important for the growth and differentiation of cardiomyocytes. Recently, Pajak et al. (35) reported that the DNA synthetic activity of Tsc2-/- rat cardiomyocytes in the embryonic primary culture is sustained, suggesting the involvement of tuberin in cardiac development.
Nonclosure of the neural tube in the brain region was another prominent feature found in Tsc2-/- embryos. Considering its occurrence in E 10–11.5 Tsc2-/- embryos and the developmental progress of other tissues in each embryo, this neural tube abnormality may not be simply a consequence of whole embryonic growth retardation. Further investigation of the specific role of Tsc2 in neural tube development and the presence of modifier gene(s) is needed, because nonclosure was seen in only 50% of Tsc2-/- embryos. In contrast to the heart anomaly in Tsc2-/- embryos, which might result in the failure of heart activity, the neural tube nonclosure itself might not give rise to embryonic lethality. Indeed, some homozygous mutants were found to have a closed neural tube. In addition, mouse embryos can be born without a forebrain-midbrain region, as seen in Lim1-deficient mice (36)
. However, a contribution of tuberin function to the development of the nervous system might be expected because TSC patients frequently develop lesions such as cortical tubers and subependymal nodules in the brain (1)
. Histological studies including immunostaining for various neuronal and glial markers suggested that the failure of cell differentiation and/or migration defect may be associated with the development of those lesions (37
, 38)
. However, the unclosed neural tubes in Tsc2-/- embryos demonstrated substantially organized neuroepithelial structures. Moreover, our immunohistochemical analysis for several neuronal differentiation markers and PCNA suggests that, at least in the mouse, the potential to differentiate into early neuronal lineage was retained normally by neuronal stem cells in Tsc2-/- embryos. Because of the embryonic lethality, the effects of tuberin deficiency in mice in later embryonic stages or in adulthood could not be analyzed. To circumvent embryonic lethality, conditional gene targeting of Tsc2 is necessary for a more complete functional analysis of tuberin (22
, 39)
.
In summary, we have established a mouse Tsc2 mutant line and demonstrated spontaneous renal carcinogenesis and hepatic hemangiomatosis in heterozygotes as well as embryonic lethality in homozygotes. Extensive studies of this mouse system, using the genetic advantage of this species, should facilitate the elucidation of the physiological function of tuberin in cell growth and differentiation and the molecular mechanisms of tumorigenesis associated with tuberin deficiency.
ACKNOWLEDGMENTS
We thank I. Komuro and M. Miyagawa for histological analysis; Y. Tomooka for anti-nestin antibody; H. Yamanaka, Y. Sugitani, T. Takahara, Y. Hirayama, and E. Kobayashi for excellent technical assistance, and other members of the Hino and Noda laboratories for stimulating discussions and invaluable advice. We also thank Drs. H. Sugano, T. Kitagawa, and A. G. Knudson for encouragement throughout this work.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grants-in Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare of Japan and the Organization for Pharmaceutical Safety and Research. 
2 To whom requests for reprints should be addressed, at Department of Experimental Pathology, Cancer Institute, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan.
3 The abbreviations used are: TSC, tuberous sclerosis; RC, renal carcinoma; E, embryonic day; LOH, loss of heterozygosity; GAP, GTPase-activating protein; NLS, nuclear localization signal; DTA, diphtheria toxin A fragment; RT-PCR, reverse transcription-PCR; ENU, N-ethyl-N-nitrosourea; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopy-ranoside; PCNA, proliferating cell nuclear antigen; MAP2, microtubule-associated protein 2; ß-gal, ß-galactosidase; ES, embryonic stem.
Received 11/ 2/98. Accepted 1/18/99.
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 B. Gan, X. Peng, T. Nagy, A. Alcaraz, H. Gu, and J.-L. Guan Role of FIP200 in cardiac and liver development and its regulation of TNF{alpha} and TSC-mTOR signaling pathways J. Cell Biol., October 9, 2006; 175(1): 121 - 133. [Abstract] [Full Text] [PDF]
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C. Wilson, C. Bonnet, C. Guy, S. Idziaszczyk, J. Colley, V. Humphreys, J. Maynard, J. R. Sampson, and J. P. Cheadle Tsc1 Haploinsufficiency without Mammalian Target of Rapamycin Activation Is Sufficient for Renal Cyst Formation in Tsc1+/- Mice Cancer Res., August 15, 2006; 66(16): 7934 - 7938. [Abstract] [Full Text] [PDF]
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C. Thivierge, A. Kurbegovic, M. Couillard, R. Guillaume, O. Cote, and M. Trudel Overexpression of PKD1 Causes Polycystic Kidney Disease Mol. Cell. Biol., February 15, 2006; 26(4): 1538 - 1548. [Abstract] [Full Text] [PDF]
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 L. Ma, J. Teruya-Feldstein, N. Behrendt, Z. Chen, T. Noda, O. Hino, C. Cordon-Cardo, and P. P. Pandolfi Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression Genes & Dev., August 1, 2005; 19(15): 1779 - 1786. [Abstract] [Full Text] [PDF]
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B. D. Manning, M. N. Logsdon, A. I. Lipovsky, D. Abbott, D. J. Kwiatkowski, and L. C. Cantley Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2 Genes & Dev., August 1, 2005; 19(15): 1773 - 1778. [Abstract] [Full Text] [PDF]
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C. Wilson, S. Idziaszczyk, L. Parry, C. Guy, D. F.R. Griffiths, E. Lazda, R. A.L. Bayne, A. J.H. Smith, J. R. Sampson, and J. P. Cheadle A mouse model of tuberous sclerosis 1 showing background specific early post-natal mortality and metastatic renal cell carcinoma Hum. Mol. Genet., July 1, 2005; 14(13): 1839 - 1850. [Abstract] [Full Text] [PDF]
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B. Govindarajan, D. J. Brat, M. Csete, W. D. Martin, E. Murad, K. Litani, C. Cohen, F. Cerimele, M. Nunnelley, B. Lefkove, et al. Transgenic Expression of Dominant Negative Tuberin through a Strong Constitutive Promoter Results in a Tissue-specific Tuberous Sclerosis Phenotype in the Skin and Brain J. Biol. Chem., February 18, 2005; 280(7): 5870 - 5874. [Abstract] [Full Text] [PDF]
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L. Meikle, J. R. McMullen, M. C. Sherwood, A. S. Lader, V. Walker, J. A. Chan, and D. J. Kwiatkowski A mouse model of cardiac rhabdomyoma generated by loss of Tsc1 in ventricular myocytes Hum. Mol. Genet., February 1, 2005; 14(3): 429 - 435. [Abstract] [Full Text] [PDF]
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 K. J. Sales, S. Battersby, A. R. W. Williams, R. A. Anderson, and H. N. Jabbour Prostaglandin E2 Mediates Phosphorylation and Down-Regulation of the Tuberous Sclerosis-2 Tumor Suppressor (Tuberin) in Human Endometrial Adenocarcinoma Cells via the Akt Signaling Pathway J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6112 - 6118. [Abstract] [Full Text] [PDF]
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M. Cully, A. Elia, S.-H. Ong, V. Stambolic, T. Pawson, M.-S. Tsao, and T. W. Mak grb2 heterozygosity rescues embryonic lethality but not tumorigenesis in pten+/- mice PNAS, October 26, 2004; 101(43): 15358 - 15363. [Abstract] [Full Text] [PDF]
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 W. G. Kaelin Jr. The Von Hippel-Lindau Tumor Suppressor Gene and Kidney Cancer Clin. Cancer Res., September 15, 2004; 10(18): 6290S - 6295S. [Abstract] [Full Text] [PDF]
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 K.-S. Au, A. T. Williams, M. J. Gambello, and H. Northrup Molecular Genetic Basis of Tuberous Sclerosis Complex: From Bench to Bedside J Child Neurol, September 1, 2004; 19(9): 699 - 709. [Abstract] [PDF]
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D. K. Scheidenhelm and D. H. Gutmann Mouse Models of Tuberous Sclerosis Complex J Child Neurol, September 1, 2004; 19(9): 726 - 733. [Abstract] [PDF]
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 E. Kleymenova, J. I. Everitt, L. Pluta, M. Portis, J. R. Gnarra, and C. L. Walker Susceptibility to vascular neoplasms but no increased susceptibility to renal carcinogenesis in Vhl knockout mice Carcinogenesis, March 1, 2004; 25(3): 309 - 315. [Abstract] [Full Text] [PDF]
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V. Murthy, S. Han, R. L. Beauchamp, N. Smith, L. A. Haddad, N. Ito, and V. Ramesh Pam and Its Ortholog Highwire Interact with and May Negatively Regulate the TSC1{middle dot}TSC2 Complex J. Biol. Chem., January 9, 2004; 279(2): 1351 - 1358. [Abstract] [Full Text] [PDF]
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 M. Tachataki, R. M.L. Winston, and D. M. Taylor Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo Mol. Hum. Reprod., October 1, 2003; 9(10): 593 - 601. [Abstract] [Full Text] [PDF]
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W. Ma, L. Tessarollo, S.-B. Hong, M. Baba, E. Southon, T. C. Back, S. Spence, C. G. Lobe, N. Sharma, G. W. Maher, et al. Hepatic Vascular Tumors, Angiectasis in Multiple Organs, and Impaired Spermatogenesis in Mice with Conditional Inactivation of the VHL Gene Cancer Res., September 1, 2003; 63(17): 5320 - 5328. [Abstract] [Full Text] [PDF]
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B. Govindarajan, M. C. Mizesko, M. S. Miller, H. Onda, M. Nunnelly, K. Casper, D. Brat, C. Cohen, and J. L Arbiser Tuberous Sclerosis-associated Neoplasms Express Activated p42/44 Mitogen-activated Protein (MAP) Kinase, and Inhibition of MAP Kinase Signaling Results in Decreased in Vivo Tumor Growth Clin. Cancer Res., August 1, 2003; 9(9): 3469 - 3475. [Abstract] [Full Text] [PDF]
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M. Y. Liu, L. Poellinger, and C. L. Walker Up-Regulation of Hypoxia-inducible Factor 2{alpha} in Renal Cell Carcinoma Associated with Loss of Tsc-2 Tumor Suppressor Gene Cancer Res., May 15, 2003; 63(10): 2675 - 2680. [Abstract] [Full Text] [PDF]
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D. M. Hockenbery Nailing Down a Link between Tuberin and Renal Cysts Am. J. Pathol., February 1, 2003; 162(2): 369 - 371. [Full Text] [PDF]
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L. A. Haddad, N. Smith, M. Bowser, Y. Niida, V. Murthy, C. Gonzalez-Agosti, and V. Ramesh The TSC1 Tumor Suppressor Hamartin Interacts with Neurofilament-L and Possibly Functions as a Novel Integrator of the Neuronal Cytoskeleton J. Biol. Chem., November 8, 2002; 277(46): 44180 - 44186. [Abstract] [Full Text] [PDF]
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A. Jaeschke, J. Hartkamp, M. Saitoh, W. Roworth, T. Nobukuni, A. Hodges, J. Sampson, G. Thomas, and R. Lamb Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent J. Cell Biol., October 28, 2002; 159(2): 217 - 224. [Abstract] [Full Text] [PDF]
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 K. S. Mcdorman and D. C. Wolf Use of the Spontaneous Tsc2 Knockout (Eker) Rat Model of Hereditary Renal Cell Carcinoma for the Study of Renal Carcinogens Toxicol Pathol, October 1, 2002; 30(6): 675 - 680. [Abstract] [PDF]
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 L Khare, A Astrinidis, W Senapedis, P D Adams, and E P. Henske Expression of wild type and mutant TSC2, but not TSC1, causes an increase in the G1 fraction of the cell cycle in HEK293 cells J. Med. Genet., September 1, 2002; 39(9): 676 - 680. [Full Text] [PDF]
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E. A. Goncharova, D. A. Goncharov, A. Eszterhas, D. S. Hunter, M. K. Glassberg, R. S. Yeung, C. L. Walker, D. Noonan, D. J. Kwiatkowski, M. M. Chou, et al. Tuberin Regulates p70 S6 Kinase Activation and Ribosomal Protein S6 Phosphorylation. A ROLE FOR THE TSC2 TUMOR SUPPRESSOR GENE IN PULMONARY LYMPHANGIOLEIOMYOMATOSIS (LAM) J. Biol. Chem., August 16, 2002; 277(34): 30958 - 30967. [Abstract] [Full Text] [PDF]
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 H.-S. Yoon, T. J. Monks, J. I. Everitt, C. L. Walker, and S. S. Lau Cell proliferation is insufficient, but loss of tuberin is necessary, for chemically induced nephrocarcinogenicity Am J Physiol Renal Physiol, August 1, 2002; 283(2): F262 - F270. [Abstract] [Full Text] [PDF]
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 K. B.S. Pasumarthi and L. J. Field Cardiomyocyte Cell Cycle Regulation Circ. Res., May 31, 2002; 90(10): 1044 - 1054. [Abstract] [Full Text] [PDF]
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D. J. Kwiatkowski, H. Zhang, J. L. Bandura, K. M. Heiberger, M. Glogauer, N. el-Hashemite, and H. Onda A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells Hum. Mol. Genet., March 1, 2002; 11(5): 525 - 534. [Abstract] [Full Text] [PDF]
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 J. YU, A. ASTRINIDIS, and E. P. HENSKE Chromosome 16 Loss of Heterozygosity in Tuberous Sclerosis and Sporadic Lymphangiomyomatosis Am. J. Respir. Crit. Care Med., October 15, 2001; 164(8): 1537 - 1540. [Abstract] [Full Text] [PDF]
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J. L. Arbiser, R. Yeung, S. W. Weiss, Z. K. Arbiser, M. B. Amin, C. Cohen, D. Frank, S. Mahajan, G. S. Herron, J. Yang, et al. The Generation and Characterization of a Cell Line Derived from a Sporadic Renal Angiomyolipoma : Use of Telomerase to Obtain Stable Populations of Cells from Benign Neoplasms Am. J. Pathol., August 1, 2001; 159(2): 483 - 491. [Abstract] [Full Text]
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T. Kobayashi, O. Minowa, Y. Sugitani, S. Takai, H. Mitani, E. Kobayashi, T. Noda, and O. Hino A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice PNAS, June 28, 2001; (2001) 151033798. [Abstract] [Full Text] [PDF]
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X. Gao and D. Pan TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth Genes & Dev., June 1, 2001; 15(11): 1383 - 1392. [Abstract] [Full Text] [PDF]
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K. B. S. Pasumarthi, H. Nakajima, H. O. Nakajima, S. Jing, and L. J. Field Enhanced Cardiomyocyte DNA Synthesis During Myocardial Hypertrophy in Mice Expressing a Modified TSC2 Transgene Circ. Res., May 26, 2000; 86(10): 1069 - 1077. [Abstract] [Full Text] [PDF]
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V. Murthy, L. A. Haddad, N. Smith, D. Pinney, R. Tyszkowski, D. Brown, and V. Ramesh Similarities and differences in the subcellular localization of hamartin and tuberin in the kidney Am J Physiol Renal Physiol, May 1, 2000; 278(5): F737 - F746. [Abstract] [Full Text] [PDF]
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D. M. Juriloff and M. J. Harris Mouse models for neural tube closure defects Hum. Mol. Genet., March 1, 2000; 9(6): 993 - 1000. [Abstract] [Full Text] [PDF]
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T. Kobayashi, O. Minowa, Y. Sugitani, S. Takai, H. Mitani, E. Kobayashi, T. Noda, and O. Hino A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice PNAS, July 17, 2001; 98(15): 8762 - 8767. [Abstract] [Full Text] [PDF]
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with numbers. The coding region for ß-gal with a NLS (NLS-lacZ) and expression cassettes for neomycin-resistant (neo) and DTA genes are shown by 
. 
and 
, loxP sequences flanking the neo cassette and the position of a 0.7-kb fragment used as a screening probe (probe A), respectively. The lengths of BamHI (B) fragments from wild-type (
, the wild-type allele; 
, the mutated allele) below each allele. B, Southern blot analysis of tail DNAs from F1 littermates after digestion by BamHI. Right, positions of fragments from wild-type (wt; 
