Project description:Transcriptional targets of Xenopus EBF3 in the presence of Noggin : Untreated animal caps vs. Dexamethasone (DEX) treated animal caps
Project description:Transcriptional targets of Xenopus EBF3 in the absence of Noggin : Untreated animal caps vs. Dexamethasone (DEX) treated animal caps
Project description:To determine the positions of promoters and enhancers in developing Xenopus laevis epithelial progenitors, we performed ChIPseq on the histone modifications H3K4me3 and H3K27ac. We also performed ChIPseq on the transcription factors foxj1 (in the presence or absence of rfx2), myb (in the presence or absence of multicilin), and rad21. Some embryos were harvested as wild-types; in other experiments, we injected embryos with mRNAs encoding FLAG-foxj1 (with and without rfx2 morpholino) or GFP-myb (with and without an inducible form of multicilin (mcidas-HGR)). We then isolated epithelial progenitors surgically and, when injected with multicilin, induced at mid-stage 11. We then harvested chromatin at 9 hours after induction (roughly stage 18) and performed ChIPseq using antibodies against endogenous targets (H3K4me3, H3K27ac, rad21) or protein tags (FLAG, GFP). We then sequenced these libraries, aligned the reads to the X. laevis genome (version 9.1) with bwa mem and called peaks with HOMER, using input as background.
Project description:ADAMs are transmembrane metalloproteases that control cell behavior by cleaving both cell adhesion and signaling molecules. The cytoplasmic domain of ADAMs can regulate the proteolytic activity by controlling the subcellular localization and/or the activation of the protease domain. Here we show that the cytoplasmic domain of ADAM13 is cleaved and translocates into the nucleus. Preventing this translocation renders the protein incapable of promoting cranial neural crest (CNC) cell migration in vivo, without affecting its proteolytic activity. In addition, the cytoplasmic domain of ADAM13 regulates the expression of multiple genes in the CNC. This study shows that the cytoplasmic domain of ADAM metalloproteases can perform essential functions in the nucleus of cells and may contribute substantially to the overall function of the protein. Total RNA from cranial neural crest explants dissected from Xenopus laevis embryos at stage 15 to 17. Embryos were injected with either control morpholino (CMO), morpholino to ADAM13 (MO13) or MO13 and a fusion between GFP- and the cytoplasmic domain of ADAM13 (GFP-C13). Triplicate microarray correspond to independent experiments labeled DA4, DA5 and DA2. For each sample 1 is CMO, sample 2 is MO13 and sample 3 is MO13+C13. Embryos were injected with 10 ng of morpholino and 0.5 ng of GFP-C13 mRNA. Supplementary files: [1] matrix2_sig-genes.txt CNC array: Genes significantly affected by the loss of ADAM13 p<0.01. Normalized probe set generated by GC-RMA. [2] matrix3_sig-genes.txt CNC array: Genes significantly affected by the loss of ADAM13 and the addition of GFP-C13 p<0.01. Normalized probe set generated by GC-RMA.
Project description:Anti GFP AP-MS of GFP-fusions for Ccdc78 and Ccdc33 from excised cultured Xenopus animal caps rich in multi-ciliated cells. Control samples were anti-GFP pull-downs from similar tissue expressing only GFP. Samples were prepared by in-gel digest, and there are 3 injections for each sample (labeled a,b,c). Filenames with dates from 8/2024 are Ccdc33 pull-downs and matched controls. Samples with dates 6/24 and 7/24 are Ccdc78 pulldowns and matched controls.
Project description:The Notch signaling pathway functions in a number of processes during embryologic development, especially the maintenance or aquisition of cell fate. We purturb the Notch signalling pathway in embryonic Xenopus laevis in order to 1) better characterize the downstream targets of Notch signalling, and 2) determine the extent to which early embryos can recover from transient purturbations to critical signalling pathways, if at all. Xenopus laevis embryos were unilaterally injected at the two cell stage with either GFP, GFP and ICD (Notch intracellular domain, an up-regulator of the Notch pathway), or GFP and DBM (domain-binding mutant, a downregulator of the Notch pathway). At stages 18, 28, and 38, for each injection, pooled total RNA from 10 embryos was extracted. Extraction was performed for three biological replicates for each time/injection condition. cDNA from total RNA was hybridized on Affymetrix Xenopus laevis Genome 2.0 arrays.