Human FTO gene variants are associated with body mass index and type 2 diabetes. Because the obesity-associated SNPs are intronic, it is unclear whether changes in FTO expression or splicing are the cause of obesity or if regulatory elements within intron 1 influence upstream or downstream genes. We tested the idea that FTO itself is involved in obesity. We show that a dominant point mutation in the mouse Fto gene results in reduced fat mass, increased energy expenditure, and unchanged physical ...[more]
Project description:We have identified an ENU-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype overlapping including cardiac, tracheo-esophageal, anorectal, axial skeletal antero-posterior patterning defects, limb malformations, presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6, SPC6) that fails to complement a null allele, which also has a similar phenotype. The mutation ablates a predicted disulfide bond in the P domain, and results in reduced secretion, abnormal cellular localisation, and loss of proprotein convertase activity. We analysed whole genome gene expression in a mouse line carrying ENU-generated point mutation in Pcsk5 gene on a mixed background : 25% C57BL6/J on C3H/HeH (mutation originally introduced into C57BL6/J). We show that GDF11, a regulator of Hox expression, anteroposterior patterning and nephrogenesis, is a PCSK5 target, and that Pcsk5 mutation results in abnormal expression of several paralogous caudal Hox genes (Hoxa, Hoxc, Hoxd), and Mnx1 (Hlxb9) that are necessary for caudal embryo development. Our data establishes novel and pleiotropic functions for Pcsk5 in mammalian development and the coordinated expression of caudal Hox genes; and identifies GDF11 as a novel cleavage target for PCSK5. We propose that Pcsk5, at least in part via GDF11, coordinately regulates the expression of caudal Hox paralogs, to control antero-posterior patterning, nephrogenesis, and skeletal and anorectal development.
Project description:The mammalian suprachiasmatic nucleus (SCN) drives daily rhythmic behavior and physiology, yet a detailed understanding of its coordinated transcriptional programmes is lacking. To reveal the true nature of circadian variation in the mammalian SCN transcriptome we combined laser-capture microdissection (LCM) and RNA-Seq over a 24-hour light / dark cycle. We show that 7-times more genes exhibited a classic sinusoidal expression signature than previously observed in the SCN. Another group of 766 genes unexpectedly peaked twice, near both the start and end of the dark phase; this twin-peaking group is significantly enriched for synaptic transmission genes that are crucial for light-induced phase-shifting of the circadian clock. 342 intergenic non-coding RNAs, together with novel exons of annotated protein-coding genes, including Cry1, also show specific circadian expression variation. Overall, our data provide an important chronobiological resource (www.wgpembroke.com/shiny/SCNseq/) and allow us to propose that transcriptional timing in the SCN is gating clock resetting mechanisms. Pooled dissected tissue of the suprachiasmatic nucleus from five adult male mice provided one of three replicates for each of six time points over a 12:12 light/dark (LD) cycle (ZT2, 6, 10, 14, 18 and 22). Each biological replicate was sequenced over 3 separate lanes using Illumina HiSeq.
Project description:Mutants of zebrafish affected in neural crest development (cls/sox10, hps/erbb3, low/tfap2a, nac/mitfa, pfe/csfr1, shd/ltk, spa/kit tdo/trpm7) were compared to their wild type siblings or to wild type controls during development at 28hpf, 48hpf and 4 dpf.
Project description:Collect tissues from control C57kBL/6 and the following genetically engineered mice-apoE knockout,apoA1 knockout, apoA1 transgene, apoB knockout, LDLR knockout.Both male and female mice fed chow or atherogenic diet will be used. Animals from each group were pooled into 2 pools, one containing 4 mice and the other containing 5 mice
Project description:The aim of the experiment was to identify genes in the mouse suprachiasmatic nucleus whose expression was altered by exposure to a light pulse of 400 lux at CT16 (subjective night). C57BL/6 male mice were entrained to a 12:12 LD cycle and given a 30 min 400 lux light pulse at CT16. Punches of SCN tissue were collected at 30 (LP30), 60 (LP60)and 120 (LP120) mins after the start of the light pulse and flash frozen on dry ice. Sham SCN punches (Sham) were collected from mice not exposed to light at CT16. Total RNA from individual tissue samples was extracted using RNeasy micro kit from Qiagen using the lipid tissue protocol according to manufacturer's recommendations. Individual samples were analyzed for quality using an Agilent Bioanalzyer and only samples with a RIN of 8 and above were used. Samples were quantified by Nanodrop and 300ug of total RNA was used to prepare probes for hybridisation to Affymetrix Mouse Exon arrays. Probe preparation and array hybridisation was carried out according to Affymetrix protocols. Each array represents an individual SCN tissue punch.<br><br>
Project description:Wnt Phospho protein profiling comparing control and Fto deficient cell after Wnt3a stimulation Control vs Fto deficient MEFs treated with Wnt 3a condioned medium for 40 minutes. 6 replicates per array