Project description:Shh signal mediated by Gli family of transcription factors regulates digit growth and patterning in early limb development. Shh expression in the posterior margin of the limb bud defines the zone of polarizing activity. However, much less is know about downstream targets that mediate Shh signal functions. In this dataset, we include the expression data obtained from dissected anterior and posterior halves of mouse limb bud respectively. These data are used to obtain 889 transcripts that were upregulated 1.3 fold or more in the posterior limb bud, and 1189 transcripts that were enriched in the anterior limb bud at 1.3 fold or more. Two samples were analyzed. We generate pairwise comparisons between anterior and posterior limb tissues. Genes with a fold-change ≥1.3 were selected.
Project description:We wanted to identify differentially expressed genes in wild-type forelimbs and forelimbs briefly exposed to Shh signaling. E10.25 forelimbs were cultured in control media and media containing cyclopmaine (inhibits the Hedgehog pathway) Generated two wild-type (control) and two cyclopmaine biological replicates for RNA-seq UT-Genome and Analysis Facility
Project description:Limb buds were dissected from E10.75 mouse embryos and stored in RNAlater reagent (Qiagen), for genotyping. For each replicate, RNA was isolated from pools of six limb buds either of wild type or homozygous mutants, using RNeasy micro-kit (Qiagen). cRNA was synthesized according to the manufacturers instructions (Ambion) and hybridized to the GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix), which interrogates ca 39,000 transcripts. Three independent RNA extractions, cDNA synthesis and array hybridizations were performed. The expression arrays data were normalized and scaled to signal intensity of 100 using GCOS 1.2 software (Affymetrix). Expression levels were analyzed using GeneSpring software (Silicon Genetics, Redwood City, CA) and Matlab 2009 (Math Works, Inc., MA). To identify differentially expressed transcripts, pair-wise comparison analysis was performed with GCOS 1.2 software (Affymetrix). A 77% cutoff in consistency of change (at least seven out of nine comparisons were either increased or decreased) was applied. Only genes that satisfied the pair-wise comparison test and displayed ?1.5 fold change in expression were considered for further analysis.
Project description:Limb buds were dissected from E10.75 mouse embryos and stored in RNAlater reagent (Qiagen), for genotyping. For each replicate, RNA was isolated from pools of 6 limb buds either of wild type or homozygous mutants using RNeasy micro-kit (Qiagen). rRNA was depleted using RiboMinusTM Human/Mouse Transcriptome Isolation Kit (Invitrogen). After cRNA amplification, single or double-stranded cDNA was generated using The GeneChip Whole Transcript Amplified Double-Stranded Target Assay kit (Affymetrix) according to manufacturers instructions. cDNA was fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to oligonucleotide tiling arrays. The control genomic DNA samples were fragmented with DNAse I. RNA-chip data were computed at the exon level, by averaging the normalized intensities of all probes falling within the exon. As a complement, array data were quantile normalized within cDNA/genomic DNA replicate groups and scaled to medial feature intensity of 10 using TAS software (Affymetrix). For each genomic position, a dataset was generated consisting of all probes mapping within a sliding window of 80 bp. The averaged ratios were plotted along the genomic DNA sequence using Integrated Genome Browser (IGB) software (Affymetrix).
Project description:Limb buds were dissected from E10.75 mouse embryos, fixed in 1% formaldehyde for 15 min at room temperature, washed 3 times with cold PBS and stored at -800C. Pools of 16 limb buds were used for each ChIP-chip experiment. ChIP was performed according to (Lee et al., 2006) using 2 ?g of anti-CTCF antibodies (A300-543A, Bethyl Laboratories) and EZview Red Protein G/A Affinity Gel (Sigma). Immunoprecipitated and whole cell extract DNA (input) were treated with RNaseA, proteinase K and purified by 2 rounds of extraction with phenol:chloroform: isoamyl alcohol. ChIP and input DNA were amplified using Ligation-Mediated PCR (Lee et al., 2006). PCR was limited to 15 cycles. 1.5 ?g of ChIP and input DNA were fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to the Chromosome 2 and X tiling arrays (Affymetrix). Tiling arrays data were quantile normalized within cDNA/genomic DNA or ChIP/ input replicate groups using R packages, STARR and Ringo (Zacher et al., 2010; Toedling et al., 2007). The ratio of probe intensity between the experiment and the control groups were computed considering the median values over replicates. The ratios were smoothened by computing the running medians with a half window size set to 150 bp and a minimum of 5 probes per window. To identify enriched regions a minimum of 3 consecutive probes with a smoothed ratio exceeding a threshold have been considered. The threshold has been fixed by taking the 99th percentile of the estimated null distribution of the ratios. Only ChIP enriched regions with score log2?1.5 and width ?150 bp were considered for further analysis. As a complement, array data were quantile normalized within ChIP/input replicate groups and scaled to medial feature intensity of 500 using TAS software (Affymetrix). For each genomic position, a dataset was generated consisting of all probes mapping within a sliding window of 250 bp. The averaged ratios were plotted along the genomic DNA sequence using Integrated Genome Browser (IGB) software (Affymetrix).
Project description:Analysis of mouse limb bud (E10.5) lacking the Bhlha9 gene. Bhlha9 knockout mouse shows syndactyly and poliosis in the limb. This microarray results provides insight into the molecular mechanisms underlying Bhlha9 function in the limb development DNA microarray analysis was performed using Affymetrix mouse genome 430 2.0 array. RNA samples were obtained from the whole limb bud of the E10.5 wild-type and Bhlha9 knockout embryos described above. Total RNA (200 ng) was reverse-transcribed and biotinylated using the GeneChip 3′ IVT Express Kit (Affymetrix). The microarray data were summarized using the MAS 5.0 method.
Project description:Comparing gene expression of cells from the E10.5 limb bud ZPA and the rest of the E10.5 limb bud from Shhgfpcre heterozygotes separated by FACS. Experiment Overall Design: 8 samples, 4 ZPA and 4 rest of the limb
Project description:POR is the obligate electron donor for all microsomal P450 enzymes. POR was conditionally knocked out during limb development at E9.5. The experiment was set up to answer two questions. First, to investigate which genes are differentially expressed in POR deficient limb buds and are influenced by a metabolically inactive cytochrome P450 system. Second, to assess which P450 enzymes are expressed at all at E12.5 during limb development and therefore which metabolic pathways involving this group of enzymes are present at this stage. Differentially expressed genes were analysed by comparing 5 wild type control samples with 5 conditional knock out samples (whole forelimb buds each). Present calls of control samples were screened for annotated P450s to establish a list of expressed P450 enzymes at E12.5.
Project description:The basic helix-loop-helix transcription factor Twist1 has a well-documented role in mesenchymal populations of the developing embryo, such as endocardial cushion (ECC) mesenchymal cells and limb buds, and during cancer development and progression. Whether Twist1 regulates the same transcriptional targets in different cell types has yet to be investigated. Through chromatin immunoprecipitation followed by sequencing (Chip-seq) analysis, the cell type-specific genome-wide occupancy of Twist1 was investigated in ECCs, limb buds and mouse peripheral nerve sheath tumor (PNST) cells. Twist1 binds mainly in a cell type-specific manner, with very few common genomic regions occupied by Twist1 in different cell types. Genes associated with binding peaks in each cell type are related to known Twist1 cellular functions in ECCs, limb buds, and cancer cells. We found that cell type-specific binding of Twist1 may be influenced by histone modifications or co-factors. Binding regions were located in several Wnt pathway associated genes, supporting a link between Twist1 and Wnt signalling in ECCs, limb buds, and PNST cells. These data suggest that similar functions are regulated by Twist1 in ECCs, limb buds, and PNST cells in a cell type-specific manner, and provide insights into possible mechanisms utilized for cell type-specificity of Twist1 binding. We compare Twist1 genome occupancy in mouse embryonic day (E) 12.5 endocardial cushion mesenchymal cells, E10.5 forelimb buds, and a mouse peripheral nerve sheath tumor cell line.