Norrin/Frizzled4 signalling in the preneoplastic niche blocks medulloblastoma initiation.

The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.

High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes.

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival.

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.