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

Abstract

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1(+/-)) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5-11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1(+/-) mice was apparently slower than that in Tsc2(+/-) mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.

Figures

Figure 1

Inactivation of mouse Tsc1 gene. (A) Structure of targeting vector and wild-type and mutant alleles. Exons are denoted with filled boxes with numbers, and restriction enzyme sites (E, EcoRI; V, EcoRV; X, XbaI) are shown. Expression cassettes for the neomycin resistance gene (neo) and diphtheria toxin A-chain gene (DTA), and the coding sequence for EGFP preceded with an IRES (IRES-EGFP) are shown as open boxes. Length (kb) of restriction fragments and positions of probes (5′ and 3′ probes) used for genotyping are denoted by arrows and hatched boxes, respectively. (B) Southern blot analysis of ES cells. XbaI- or EcoRI-digested DNAs from homologous recombinant (lane 2) and control (lane 1) ES cells were probed with probes shown in A. Sizes (kb) of bands are shown. (C) PCR genotyping of F2 embryos. (Left) Schematic representation of primers and product size (bp). (Right) A representative result of PCR genotyping of E10.0 embryos obtained by double heterozygous breeding. Wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) embryos were present in this litter. (D) Northern blot analysis of E11.0 embryos. (Upper) The expression profile of Tsc1 mRNA (≈8.0 kb) in wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) embryos. Probe used was a Tsc1 cDNA fragment covering the 3′ region downstream from exon 8. (Lower) The result with a GAPDH cDNA probe using the same blot.

Figure 2

Analysis of homozygous Tsc1 mutant embryos. (A) Macroscopic appearance of heterozygous (+/−) and homozygous Tsc1 mutant (−/−) embryos at E12.0 with heartbeat. Arrows point to the unclosed neural tube exhibiting exencephaly. (B) Histology of head region of heterozygous (+/−) and homozygous (−/−) mutant embryos at E12.0 with heartbeat shown by HE staining. Field of homozygous mutant is enlarged with twice the magnitude of that of the heterozygous mutant. Arrows point to the unclosed region of the neural fold exposed to the outside. (C) Immunostaining of neural tube with anti-class III β-tubulin antibody (TuJ1). Sections of the head region from heterozygous (+/−) and homozygous (−/−) mutant embryos at E12.0 with heartbeat were stained with TuJ1. After immunostaining, a counterstain with HE was performed. Positive signals were seen as brown staining. VZ, ventricular zone; MZ, marginal zone. (D) Histological analysis of heart from wild-type (+/+) and homozygous Tsc1 mutant (−/−) embryos at E12.0 with heartbeat shown by HE staining. (Lower) Enlarged view of the region enclosed with a rectangle in the upper panel. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (Scale bars: B and C = 200 μm; D = 100 μm.)

Figure 3

Renal tumors in Tsc1+/− mice. (A) A phenotypically altered renal tubule (arrow) shown by HE staining. (B) Macroscopic renal cysts (arrows) developed in a 15-month-old mouse. (C and D) Histology of a renal cystadenoma shown by HE staining. D is an enlarged view of region enclosed with a rectangle in C. (E) Histology of a renal adenoma exhibiting tubular structure shown by HE staining. (F) Loss of wild-type Tsc1 allele in a renal tumor of Tsc1+/− mice. Representative result of LOH analysis is shown. Lanes T, tumor; N, normal counterpart; −, no input DNA sample. Products from mutant allele (M, 220 bp) and from wild-type allele (W, 192 bp) are indicated. (G) Macroscopic renal cysts (arrows) developed in an 11-week-old mouse transplacentally treated with ENU. (H) Histology of a renal cystadenoma developed by ENU treatment shown by HE staining. (Scale bars = 50 μm.)

Figure 4

Extra-renal tumors in Tsc1+/− mice. (A) Macroscopic view of a hepatic hemangioma (arrows) developed in a 17-month-old mouse. (B) Histology of a hepatic hemangioma shown by HE staining. (C) Macroscopic view of a tail of 15-month-old mouse showing a knob (arrow). (D and E) Histology of a hemangioma developed in the knob in C shown by HE staining. E is an enlarged view of the region enclosed with a rectangle in D. (F) Macroscopic view of a uterus leiomyoma/leiomyosarcoma developed in a 16-month-old mouse (arrows). (G and H) Histology of the leiomyoma/leiomyosarcoma in F shown by HE staining. In G, * and T indicate the endometrium and tumor region, respectively. Arrowheads in H point to mitotic cells. (I) Loss of wild-type Tsc1 allele in extra-renal tumor of Tsc1+/− mice. Representative results of LOH analysis are shown. Lanes T, tumor; N, normal counterpart. M, size marker; −, no input DNA sample. Products from mutant allele (Mut., 220 bp) and from wild-type allele (Wild, 192 bp) are indicated. (Scale bars: B, D, E, and G = 100 μm; H = 10 μm.)