Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections

Increased Skin Tumor Incidence and Keratinocyte Hyper-Proliferation in a Mouse Model of Down Syndrome.

Down syndrome (DS) is a genetic disorder caused by the presence of an extra copy of human chromosome 21 (Hsa21). People with DS display multiple clinical traits as a result of the dosage imbalance of several hundred genes. While many outcomes of trisomy are deleterious, epidemiological studies have shown a significant risk reduction for most solid tumors in DS. Reduced tumor incidence has also been demonstrated in functional studies using trisomic DS mouse models. Therefore, it was interesting to find that Ts1Rhr trisomic mice developed more papillomas than did their euploid littermates in a DMBA-TPA chemical carcinogenesis paradigm. Papillomas in Ts1Rhr mice also proliferated faster. The increased proliferation was likely caused by a stronger response of trisomy to TPA induction. Treatment with TPA caused hyperkeratosis to a greater degree in Ts1Rhr mice than in euploid, reminiscent of hyperkeratosis seen in people with DS. Cultured trisomic keratinocytes also showed increased TPA-induced proliferation compared to euploid controls. These outcomes suggest that altered gene expression in trisomy could elevate a proliferation signalling pathway. Gene expression analysis of cultured keratinocytes revealed upregulation of several trisomic and disomic genes may contribute to this hyperproliferation. The contributions of these genes to hyper-proliferation were further validated in a siRNA knockdown experiment. The unexpected findings reported here add a new aspect to our understanding of tumorigenesis with clinical implications for DS and demonstrates the complexity of the tumor repression phenotype in this frequent condition

Survival, tumor initiation, and number in DMBA-TPA treated mice.

<p>Tumor number and size were measured twice a week from the initial DMBA treatment for 20 weeks. (A) WT (n = 20) survived significantly longer than Ts1Rhr (n = 21) mice. **p = 0.007 by Log-rank test. (B) The tumor initiation plotted versus time (weeks) for WT and Ts1Rhr mice. (C) Tumor number per mouse plotted versus time was not different in Ts and WT mice (error bar is SE). From week 9 on, Ts1Rhr developed significantly more tumors per individual compared to WT (ANOVA, *p<0.04).</p

Ts1Rhr mice demonstrate increased proliferation and TPA-induced hyperkeratosis <i>in vivo</i> and <i>in vitro</i>.

<p>(A) The average tumor size (mm) ± S.E. was plotted versus time in WT and Ts groups. * T-test, p<0.05. (B) Tumors showed increased relative growth rate in Ts compared to WT (*p<0.028). (C) H&E stain of WT and Ts skin without TPA treatment (WT-NT and Ts-NT). H&E stain of TPA-treated skin (WT-TPA and Ts-TPA). (D) There is no difference in thickness of the epidermis between WT (n = 12) and Ts (n = 14) without TPA treatment (NT group). The epidermis of Ts (n = 14) is significantly thicker than that of WT (n = 16) (*P = 0.023, T-test). (E) EdU staining of keratinocytes +/- TPA treatment. (F) Quantitation of cells in (2E) showed significantly more EdU positive cells in Ts cultures following TPA treatment (*p = 0.043, T-test). NT, no TPA treatment; TPA, treatment with TPA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146570#sec002" target="_blank">Methods</a>).</p

Microarray analysis and RT-PCR validation of gene expression in keratinocytes.

<p>(A) TPA treated and untreated keratinocytes isolated from trisomic (Ts) and euploid (WT) mice are compared in this figure (n = 4 in each group). Differentially expressed genes are depicted in the Venn diagram. (B) Gene Set Enrichment Analysis (GSEA) was used to identify gene sets that exhibited significant overlaps with those gene differentially expressed between WT-NT and Ts-NT. Enrichment plot (left panel) and Heat map (right panel) for the Chr21q22 region gene set from GSEA analysis of WT-NT vs. Ts-NT keratinocytes. The relative ratios of transcripts are shown for trisomic genes. (C) <i>PigP</i>, (D) <i>Ttc3</i> and (E) <i>Ets2</i> in WT and Ts keratinocyte cultures before and after TPA treatment (*p<0.01). (F) Gene enrichment analysis found genes in the signature of head and neck squamous cell carcinoma significantly upregulated in the Ts-TPA group vs. WT-TPA (left panel). A heat map shows core genes upregulated in the Ts-TPA group (right panel).</p

Evaluation of the impact of alternative fuel use on the emissions and performance of a service-exposed T56 engine

Alternative fuel sources are becoming an operational reality; these fuels have the potential to reduce emissions, improve combustion characteristics and to increase fuel supply security. A test with a T56 turboprop engine was performed to demonstrate that a CHEFA/JP8 (Camelina Hydroprocessed Ester and Fatty Acids and standard JP8) fuel blend would meet operational requirements. The primary test objective was to assess whether a fuel change had an immediate impact on the engine condition, performance, emissions or vibration characteristics. This paper presents test results comparing engine performance with JP8 and a 50/50 blend of JP8 and CHEFA. Comparison runs were conducted before and after a 20 hour ground durability test with the CHEFA fuel blend. A nearly time-expired, nacelle-dressed T56 on an outdoor test stand was tested. The engine was equipped with minimallyintrusive non-standard pressure, temperature and emissions monitoring equipment, and a field vibration assessment suite in addition to the standard flight instrumentation. This paper discusses the test plan, data acquisition methods, results and data repeatability. The performance and emissions results are compared to the changes predicted theoretically from the fuel properties. Observations from the borescope inspections before, during and after the 20 hour durability test are also presented. The lessons learned in this test could be applied to future fuel or process-change tests, and the results provide a performance baseline for engine health assessment. Copyright \ua9 2012 by Her Majesty the Queen in Right of CanadaPeer reviewed: YesNRC publication: Ye