Vascular Abnormalities and Deregulation of VEGF in Lkb1 -Deficient Mice

Author:

Ylikorkala Antti1,Rossi Derrick J.1,Korsisaari Nina1,Luukko Keijo2,Alitalo Kari13,Henkemeyer Mark4,Mäkelä Tomi P.13

Affiliation:

1. Molecular and Cancer Biology Program, Haartman Institute and Biomedicum Helsinki, Post Office Box 63, University of Helsinki, Helsinki 00014, Finland.

2. Department of Anatomy and Cell Biology, University of Bergen N-5009 Bergen, Norway.

3. Helsinki University Central Hospital Laboratory Diagnostics, Post Office Box 401, Helsinki 00029 HYKS, Finland.

4. Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75235–9133, USA.

Abstract

The LKB1 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 Lkb1 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 ( VEGF ) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1 −/− fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference24 articles.

1. Hemminki A., et al., Nature 391, 184 (1998).

2. Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development

3. 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–Hind III (5′) fragment and 2.0-kb Bam HI–Bam HI (3′) 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′ and 3′ 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 −/− animals obtained with both strategies were found to have identical phenotypes and both were used in this study.

4. Supplementary Web material is available on Science Online at www.sciencemag.org/cgi/content/full/293/5533/1323/DC1.

5. For whole-mount in situ hybridization embryos were fixed overnight in 4% paraformaldehyde bleached for 5 hours in 7% H 2 O 2 –93% methanol treated with 5 μg/ml proteinase K and postfixed in 4% paraformaldehyde–0.2% glutaraldehyde. Overnight hybridization at 63°C with digoxygenin–uridine triphosphate–labeled antisense probe in 50% formamide 0.75% NaCl 10 mM Pipes (pH 6.8) 1 mM EDTA 100 μg/ml tRNA 0.05% heparin 0.1% bovine serum albumin 1% SDS was followed by ribonuclease A–ribonuclease T1 treatment extensive washes and blocking in 10% goat serum. Embryos were incubated overnight (4°C) with alkaline phosphatase (AP)–conjugated anti-digoxigenin (Roche Molecular Biochemicals) and AP activity was then detected with BM purple.

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