{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T21:54:52Z","timestamp":1792706092122,"version":"4.5.0"},"reference-count":24,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5533","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2001,8,17]]},"abstract":"<jats:p>\n            The\n            <jats:italic>LKB1<\/jats:italic>\n            tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine\/threonine kinase of unknown function. Here we show that mice with a targeted disruption of\n            <jats:italic>Lkb1<\/jats:italic>\n            die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor (\n            <jats:italic>VEGF<\/jats:italic>\n            ) expression, including a marked increase in the amount of\n            <jats:italic>VEGF<\/jats:italic>\n            messenger RNA. Moreover, VEGF production in cultured\n            <jats:italic>\n              Lkb1\n              <jats:sup>\u2212\/\u2212<\/jats:sup>\n            <\/jats:italic>\n            fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place\n            <jats:italic>Lkb1<\/jats:italic>\n            in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.\n          <\/jats:p>","DOI":"10.1126\/science.1062074","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:39:52Z","timestamp":1027762792000},"page":"1323-1326","source":"Crossref","is-referenced-by-count":269,"title":["Vascular Abnormalities and Deregulation of\n            <i>VEGF<\/i>\n            in\n            <i>Lkb1<\/i>\n            -Deficient Mice"],"prefix":"10.1126","volume":"293","author":[{"given":"Antti","family":"Ylikorkala","sequence":"first","affiliation":[{"name":"Molecular and Cancer Biology Program, Haartman Institute and Biomedicum Helsinki, Post Office Box 63, University of Helsinki, Helsinki 00014, Finland."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Derrick J.","family":"Rossi","sequence":"additional","affiliation":[{"name":"Molecular and Cancer Biology Program, Haartman Institute and Biomedicum Helsinki, Post Office Box 63, University of Helsinki, Helsinki 00014, Finland."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nina","family":"Korsisaari","sequence":"additional","affiliation":[{"name":"Molecular and Cancer Biology Program, Haartman Institute and Biomedicum Helsinki, Post Office Box 63, University of Helsinki, Helsinki 00014, Finland."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keijo","family":"Luukko","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, University of Bergen N-5009 Bergen, Norway."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kari","family":"Alitalo","sequence":"additional","affiliation":[{"name":"Molecular and Cancer Biology Program, Haartman Institute and Biomedicum Helsinki, Post Office Box 63, University of Helsinki, Helsinki 00014, Finland."},{"name":"Helsinki University Central Hospital Laboratory Diagnostics, Post Office Box 401, Helsinki 00029 HYKS, Finland."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"Henkemeyer","sequence":"additional","affiliation":[{"name":"Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75235\u20139133, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomi P.","family":"Ma\u0308kela\u0308","sequence":"additional","affiliation":[{"name":"Molecular and Cancer Biology Program, Haartman Institute and Biomedicum Helsinki, Post Office Box 63, University of Helsinki, Helsinki 00014, Finland."},{"name":"Helsinki University Central Hospital Laboratory Diagnostics, Post Office Box 401, Helsinki 00029 HYKS, Finland."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/34432","volume":"391","author":"Hemminki A.","year":"1998","unstructured":"Hemminki A., et al., Nature 391, 184 (1998).","journal-title":"Nature"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0925-4773(99)00050-7"},{"key":"e_1_3_1_4_2","unstructured":"Two independent targeting strategies were used. The first resulted in a deletion of genomic sequences encompassing exons 2 to 7 (out of a total of 10); the second based on Cre\/LoxP methodology resulted in the inversion of these sequences. In both cases we used a 6.3-kb Nsi I\u2013Hind III (5\u2032) fragment and 2.0-kb Bam HI\u2013Bam HI (3\u2032) fragment of genomic sequence. Positive (PGK-Neomycin) and negative (PGK-HSV-tk) selection markers were used in both strategies. The target vectors were electroporated into embryonic stem (ES) cells and correctly targeted clones were identified by Southern blotting with 5\u2032 and 3\u2032 external probes. ES cell clones were injected into C57BL\/6 blastocysts. Several chimeric offspring were found to transmit targeted alleles in the germ line. Germ line inactivation of Lkb1 with LoxP\/Cre was achieved by crossing targeted animals to PGK-Cre mice (22). Stable inversion of the targeted sequences was then achieved by breeding the PGK-Cre transgene out of subsequent generations. Lkb1 \u2212\/\u2212 animals obtained with both strategies were found to have identical phenotypes and both were used in this study."},{"key":"e_1_3_1_5_2","unstructured":"Supplementary Web material is available on Science Online at www.sciencemag.org\/cgi\/content\/full\/293\/5533\/1323\/DC1."},{"key":"e_1_3_1_6_2","unstructured":"For whole-mount in situ hybridization embryos were fixed overnight in 4% paraformaldehyde bleached for 5 hours in 7% H 2 O 2 \u201393% methanol treated with 5 \u03bcg\/ml proteinase K and postfixed in 4% paraformaldehyde\u20130.2% glutaraldehyde. Overnight hybridization at 63\u00b0C with digoxygenin\u2013uridine triphosphate\u2013labeled antisense probe in 50% formamide 0.75% NaCl 10 mM Pipes (pH 6.8) 1 mM EDTA 100 \u03bcg\/ml tRNA 0.05% heparin 0.1% bovine serum albumin 1% SDS was followed by ribonuclease A\u2013ribonuclease T1 treatment extensive washes and blocking in 10% goat serum. Embryos were incubated overnight (4\u00b0C) with alkaline phosphatase (AP)\u2013conjugated anti-digoxigenin (Roche Molecular Biochemicals) and AP activity was then detected with BM purple."},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/343657a0"},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1101\/gad.1.1.29","volume":"1","author":"Joyner A. L.","year":"1987","unstructured":"Joyner A. L., Martin G. R., Genes Dev. 1, 29 (1987).","journal-title":"Genes Dev."},{"key":"e_1_3_1_9_2","unstructured":"A. Ylikorkala et al. data not shown."},{"key":"e_1_3_1_10_2","unstructured":"For whole-mount immunostainings E8.5 or E9.5 embryos were fixed in 4% paraformaldehyde bleached for 5 hours in 5% H 2 O 2 \u201395% methanol and blocked overnight (4\u00b0C) in phosphate-buffered saline containing 3% milk and 0.1% Triton X-100. After an overnight (4\u00b0C) incubation with primary antibodies (PECAM-1 Pharmingen; smooth-muscle actin Sigma) the embryos were treated with peroxidase-conjugated secondary antibodies and developed in 3 3\u2032-diaminobenzidine tetrahydrochloride (Sigma)."},{"key":"e_1_3_1_11_2","unstructured":"TUNEL labeling and in situ hybridization were performed on paraffin-embedded and paraformaldehyde-fixed 7-\u03bcm tissue sections. TUNEL analysis was performed as instructed by the manufacturer (In Situ Cell Death Detection Kit Roche) and sections were counterstained with Hoechst 33342 (Sigma). In situ hybridization was performed as described (2) with Vegf flk-1 and flt-1 probes (23)."},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1242\/dev.118.2.489"},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1002\/aja.1002030109"},{"key":"e_1_3_1_14_2","doi-asserted-by":"publisher","DOI":"10.1038\/380435a0"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1038\/380439a0"},{"key":"e_1_3_1_16_2","unstructured":"E9.5 embryos were minced into small fragments and cultured in individual wells on 48-well plates in Dulbecco's modified Eagle's medium containing 15% fetal calf serum glutamine penicillin and streptomycin for 5 days. Cells were trypsinized and replated and nonadherent cells were removed 1 hour later. For the VEGF analysis 25 000 MEFs from individual embryos in passage 3 were plated on 24-well plates and cultured in normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions for 24 hours. Conditioned medium was removed and VEGF concentration was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems). Remaining cells were lysed immediately in SDS sample buffer subjected to SDS-PAGE analysis and immunoblotted with antibodies to glucose transporter 1 (Chemicon)."},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","first-page":"4604","DOI":"10.1128\/MCB.16.9.4604","volume":"16","author":"Forsythe J. A.","year":"1996","unstructured":"Forsythe J. A., et al., Mol. Cell. Biol. 16, 4604 (1996).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_18_2","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1182\/blood.V89.2.503","volume":"89","author":"Gleadle J. M.","year":"1997","unstructured":"Gleadle J. M., Ratcliffe P. J., Blood 89, 503 (1997).","journal-title":"Blood"},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","first-page":"9102","DOI":"10.1073\/pnas.94.17.9102","volume":"94","author":"Gnarra J. R.","year":"1997","unstructured":"Gnarra J. R., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 9102 (1997).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_20_2","first-page":"2299","volume":"56","author":"Siemeister G.","year":"1996","unstructured":"Siemeister G., et al., Cancer Res. 56, 2299 (1996).","journal-title":"Cancer Res."},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"27509","DOI":"10.1074\/jbc.272.44.27509","volume":"272","author":"Pal S.","year":"1997","unstructured":"Pal S., Claffey K. P., Dvorak H. F., Mukhopadhyay D., J. Biol. Chem. 272, 27509 (1997).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","first-page":"1447","DOI":"10.1053\/gast.2000.20228","volume":"119","author":"Giardiello F. M.","year":"2000","unstructured":"Giardiello F. M., et al., Gastroenterology 119, 1447 (2000).","journal-title":"Gastroenterology"},{"key":"e_1_3_1_23_2","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1023\/A:1008868325009","volume":"7","author":"Lallemand Y.","year":"1998","unstructured":"Lallemand Y., Luria V., Haffner-Krausz R., Lonai P., Transgenic Res. 7, 105 (1998).","journal-title":"Transgenic Res."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","first-page":"2077","DOI":"10.1084\/jem.178.6.2077","volume":"178","author":"Kaipainen A.","year":"1993","unstructured":"Kaipainen A., et al., J. Exp. Med. 178, 2077 (1993).","journal-title":"J. Exp. Med."},{"key":"e_1_3_1_25_2","unstructured":"We thank J. Partanen for discussions and reagents P. Lonai for the PGK-Cre mice strain and B. Tj\u00e4der and S. R\u00e4s\u00e4nen for technical assistance. Supported by the Finnish Medical Foundation Vuorisalo Fund Orion-Farmos Research Foundation Emil Aaltonen Foundation Finnish Cancer Foundation Ida Montin Foundation and Academy of Finland. A.Y. and D.J.R. are in Helsinki Biomedical Graduate School and N.K. is in the Helsinki Graduate School in Biotechnology and Molecular Biology."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.1062074","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T21:28:16Z","timestamp":1704835696000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.1062074"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2001,8,17]]},"references-count":24,"journal-issue":{"issue":"5533","published-print":{"date-parts":[[2001,8,17]]}},"alternative-id":["10.1126\/science.1062074"],"URL":"https:\/\/doi.org\/10.1126\/science.1062074","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2001,8,17]]}}}