Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis

Abstract

Peutz-Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine/threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3. However, several studies have produced results that are not consistent with a link between LKB1 gene mutation and PJS. We constructed a knockout gene mutation of Lkb1 to determine whether it is the causative gene of PJS and to examine the biological role of the Lkb1 gene. Lkb1(-/-) mice died in utero between 8.5 and 9.5 days postcoitum. At 9.0 days postcoitum, Lkb1(-/-) embryos were generally smaller than their age-matched littermates, showed developmental retardation, and did not undergo embryonic turning. Multiple gastric adenomatous polyps were observed in 10- to 14-month-old Lkb1(+/-) mice. Our results indicate that functional Lkb1 is required for normal embryogenesis and that it is related to tumor development. The Lkb1(+/-) mouse is suitable for studying molecular mechanism underlying the development of inherited gastric tumors in PJS.

Figure 1

Mouse Lkb1 gene targeting. (a) Structures of mouse Lkb1 gene (exons 1–10), targeting vector, and the predicted structures of targeted and null alleles, which can be generated from targeted allele by Cre-loxP-mediated recombination. In the targeting vector, the neomycin gene (neo^r) flanked by two loxP sequences was inserted in the HindIII site at intron 1, and one more loxP sequence was inserted in another HindIII site just after exon 8. Localization of each PCR primer and probes for Southern blots used to detect each allele are indicated. The positions of restriction sites of BamHI (B) and KpnI (K) are also indicated. (b) PCR analysis (PCR, Left) and Southern blot analysis (STN, Right) of ES cell clones. In Southern analysis, DNAs from each ES cell clone (whose clone number is indicated above the gel) and normal mouse tissue (Normal) were digested with KpnI and hybridized with probe A. To detect targeted alleles by PCR, LOXP3 S2 and MPJ37 primers were used. The bands derived from wild-type allele (W) and targeted allele (T) are indicated by arrows. (c) Southern blot analysis of Lkb1 3-loxP heterozygous mice, which were generated from two independent ES cell clones (clones 256 and 358). Southern blot analysis was performed as in b. (d) PCR analysis of Lkb1^+/− offspring generated by pCre-pac plasmid injection into pronuclei of fertilized C57BL/6J eggs that were inseminated with Lkb1 3-loxP heterozygous mutant spermatozoa. MPJ69 and MPJ37 primers were used to detect the null allele (N), which lacks exons 2–8, flanked by loxP sequences.

Figure 2

Morphology of Lkb1^−/− embryo and expression of Lkb1 in wild-type embryo. (a–d) Morphology of Lkb1^−/− embryos. Wild-type (a) and Lkb1^−/− (c and d) at 9.0 dpc and wild-type (b) at 8.5 dpc. The size of Lkb1^−/− embryo was similar to that of normal embryos of 8.0–8.5 dpc. Note the Lkb1^−/− embryos did not undergo embryonic turning and had very small somite (s). (e–g) Immunostaining of Lkb1 in wild-type embryo at 8.5 dpc (e) and 9.0 dpc (f and g) using anti-Lkb1 antibody. The heart (g) and nucleated embryonic blood cells (f) of wild-type embryo at 9.0 dpc immunostained for Lkb1. S: somite. Only one nucleated embryonic blood cell (e) of wild-type embryo at 8.5 dpc was immunostained for Lkb1. (Magnifications: a–d, ×40; e and f, ×400; g, ×100.)

Figure 3

Macroscopic and histological appearance of Lkb1^+/− mice. (a–c) Macroscopic appearance of polyps in Lkb1^+/− mice. Gross appearance of the polyps (P) at junction of pyloric antrum (PA) and duodenum (D). Pylorus (Py) (a) 10 months, (b) 14 months. (c) The size of the polyp is almost equivalent to that of the glandular stomach at 14 months of age. Note that the duodenum and small intestine contained no food. S: glandular stomach; P: polyps. (d–i) Histological examination of polyps at 14 months. (d and f–i) Hematoxylin and eosin staining. (e) Azan stain. (d) Low-power section of the polyp protruding into the gastric lumen. (e) The core of the polyp is formed by a tree-like branching of the muscularis mucosa with connective tissue (arrow). (f) The polyps consist of two types of cells: surface mucous epithelium-like cells (SC) and pyloric gland-like cells (PC) and form glandular tubules like gastric pits (LGP). (g) Surface mucous epithelium-like cells of columnar or high columnar type (SC). Pyloric gland-like cells have large nuclei and pale cytoplasm (PC). (h) High nuclear density, nuclear atypia, and mitosis (arrowhead) are observed in a proportion of proliferating cells. (i) Pyloric gland-like cells also contain eosinophilic material in the cytoplasm. (Magnifications: a–c, ×2; d, ×5; e, ×20; f, ×100; g–i, ×200).

Figure 4

Northern blot and reverse transcriptase–PCR analysis of Lkb1 in polyps. (a) Lanes 1–6: 20 μg total RNA was isolated from 18-month-old Lkb1^+/− and wild-type mice. Lanes 1 and 2: glandular stomach of wild-type (wi). Lanes 3 and 4: normal parts of the glandular stomachs of Lkb1^+/− (He). Lanes 5 and 6: gastric polyps of Lkb1^+/−. Lanes 3 and 5, and 4 and 6: RNAs from the same mouse. The mouse Lkb1 cDNA was used for probe. Blots were reprobed for β-actin, which was used as the loading control. (b) Lanes 1–6: amplification by primer pair 1, LKS1 located in exon 1 and MPJ18 located in exon 6. Lanes 7–12: amplification by primer pair 2, MPJ4 located in exon 5 and LKAS1 located in exon 9. Lanes 13–18: amplification by primer pair 3 LKS1 located in exon 1 and LKAS1 located in exon 9. Lanes 1, 2, 7, 8, 13, and 14: glandular stomach of wild-type (wi); lanes 3, 4, 9, 10, 15, and 16: normal part of the glandular stomach of Lkb1^+/− (He); lanes 5, 6, 11, 12, 17, and 18: gastric polyps of Lkb1^+/−. Note that for lanes 15–18 the mutant mRNAs (about 500 bp) from targeted allele, deleted exons 2–8, were expressed in heterozygous mice.