Project description:All vertebrates share a segmented body axis. Segments form from the rostral end of the presomitic mesoderm (PSM) with a periodicity that is regulated by the segmentation clock. The segmentation clock is a molecular oscillator that exhibits dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanism by which NICD stability is regulated in this context is unclear. We identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle-dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations and an increase in somite size.
Project description:Notch intracellular domain (NICD) is the active form of the Notch receptor. In this mouse model, NICD is inserted in the Rosa26 locus downstream of a loxP-STOP-LoxP (lsl) sequence and therefore NICD expression is dependant on Cre recombinase expression (Mono transgenic control Rosa26-lsl-NICD) . These mice are crossed with the AFP-Cre strain that expresses Cre in hepatoblasts due to its regulation by the AFP promoter and albumin enhancer (Double transgenic mutant AFP-Cre/Rosa26-lsl-NICD). Newborn mice at day 0 and day 2 are sacrificed and liver RNA samples from control monotransgenic Rosa26-lsl-NICD and from bitransgenic AFP-Cre/Rosa26-lsl-NICD (AFP-NICD) are obtained. Whole genome expression profiling of these samples is submitted.
Project description:The study deals with the elucidation of potential interaction partners of the intracellular domain of the transmembrane receptor Notch1, termed NICD. The NICD is released from the cytoplasmic tail of the Notch receptor by gamma-secretase treatment and translocated as a transcription factor to the nucleus. Here, virally transduced recombinant NICD constructs (wild-type and deltaEP hyperactive mutant) were employed for AP-MS with the aim of identifying novel NICD interactors in a murine T cell leukemia cell line.
Project description:Selective isolation of total RNA and then whole genome expression analysis of sprouting neurons in peri-infarct cortex of the adult rat after stroke, compared to adjacent neurons that have not sprouted a new connection, in young adult (2 months) and aged (2 years) animals. Two different fluorescent conjugates of the tracer cholera toxin B (CTb), CTb-Alexa 488 and CTb-Alexa 647 (Molecular Probes), were sequentially injected into peri-infarct cortex at the sites of post-stroke axonal sprouting using a picospritzer pressure injection system. After survival periods of 7 days and 21 days (separate cohorts of animals), CTb-647 was injected exactly within CTb-488 injection site. Seven days after the second tracer injection, laser capture microdissection (LCM) was used to capture ~300 neurons for each cell type. Total RNA was isolated from captured cells and subjected to two rounds of T7 amplification.
Project description:Selective isolation of total RNA and then whole genome expression analysis of sprouting neurons in peri-infarct cortex of the adult rat after stroke, compared to adjacent neurons that have not sprouted a new connection, in young adult (2 months) and aged (2 years) animals. Two different fluorescent conjugates of the tracer cholera toxin B (CTb), CTb-Alexa 488 and CTb-Alexa 647 (Molecular Probes), were sequentially injected into peri-infarct cortex at the sites of post-stroke axonal sprouting using a picospritzer pressure injection system. After survival periods of 7 days and 21 days (separate cohorts of animals), CTb-647 was injected exactly within CTb-488 injection site. Seven days after the second tracer injection, laser capture microdissection (LCM) was used to capture ~300 neurons for each cell type. Total RNA was isolated from captured cells and subjected to two rounds of T7 amplification.
Project description:Notch intracellular domain (NICD) is the active form of the Notch receptor. In this mouse model, NICD is inserted in the Rosa26 locus downstream of a loxP-STOP-LoxP (lsl) sequence and therefore NICD expression is dependant on Cre recombinase expression. These mice are crossed with the AFP-Cre strain that expresses Cre in hepatoblasts due to its regulation by the AFP promoter and albumin enhancer. Mice from 6 to 12 months are sacrificed and liver RNA samples from control monotransgenic Rosa26-lsl-NICD and confirmed HCC lesions from bitransgenic AFP-Cre/Rosa26-lsl-NICD (AFP-NICD) are obtained. Exon expression profiling of these samples are submitted.