Project description:We have analysed and compared mRNA expression between wt embryos and embryos deficient for Arid3b in E9.5 heads, with the aim of identifying differentially expressed genes that could give us a clue to the functions of Arid3b during development. Heads were dissected from E9.5 embryos (including head and first branchial arch). Four wild-type and four mutant embryos were collected and pooled for each of the microarray triplicates.
Project description:We have analysed and compared mRNA expression between wt embryos and embryos deficient for Arid3b in E9.5 hearts, with the aim of identifying differentially expressed genes that could give us a clue to the functions of Arid3b during development. Hearts were dissected from E9.5 embryos (including the heart tube and the pharyngeal mesoderm and endoderm located beneath). Four wild-type and four mutant embryos were collected and pooled for each of the microarray triplicates.
Project description:We have analysed and compared mRNA expression between wt embryos and embryos deficient for Arid3b in E9.5 trunks, with the aim of identifying differentially expressed genes that could give us a clue to the functions of Arid3b during development. Trunks were dissected from E9.5 embryos (including the body from the otic vesicle to somite 15 and forelimbs). Four wild-type and four mutant embryos were collected and pooled for each of the microarray triplicates.
Project description:We have analysed and compared mRNA expression between wt embryos and embryos deficient for Arid3b at E9.0 days of development, with the aim of identifying differentially expressed genes that could give us a clue to the functions of Arid3b during development. Four wild-type and four mutant samples, consisting of 3-4 embryos each, were used.
Project description:A heterobimetallic Mn/Fe cofactor is found in the R2 subunit of class Ic ribonucleotide reductases (R2c) and R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (μ-oxo/μ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (μ-hydroxo) and a fatty acid ligand. Here we identify a second coordination sphere residue which directs the divergent reactivity of the protein scaffold. The residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group, but not in R2c. Mutation of the glycine to leucine converts the resting state R2lox cofactor into an R2c-like cofactor, a µ-oxo/µ-hydroxo bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in wild-type R2lox, demonstrating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine mutant no longer functions as a two-electron oxidant. Specifically the mass spectrometry data here deposited tracks the abundance of the cross-linked peptide AVIRAATVYNMIVE-AVTLD, which was used as surrogate marker for the abundance of the internal V72-Y162 ether cross-link, which is directly impacted by the metalation state of the protein as well as the residue present in position 68 (G or L). Taken together, our data demonstrate that the residue preceding the N-terminal metal ligand directs the cofactor’s reactivity towards one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.
Project description:Heterozygous loss-of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, peripheral nerves and several organ systems like eyes, ears, nose and heart. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition is unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7Whi/+ and Chd7Whi/Whi) mouse embryos at day 9.5, the time point of neural crest cell migration. We identified 98 genes showing greater than two fold differences in expression (log2 fold-change) and a P-value to false discovery rate (FDR) < 0.05 between wild-type and Chd7Whi/Whi embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance like semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 and one of its target genes, namely semaphorin3a in Xenopus laevis embryos, we could show abnormalities in the migration of neural crest cells in vivo. Additionally, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7 negative CHARGE patients suggesting a role for SEMA3A in the pathogenesis of CHARGE syndrome. To identify genes that are affected by the absence of functional Chd7 at the time point of neural crest cell migration, the expression profiles of E9.5 wild-type, Chd7Whi/+ and Chd7Whi/Whi female mouse embryos were compared by whole-genome microarray analysis. Mouse embryos of the same sex were used to avoid sex-dependent gene expression effects. We performed microarray analysis by using the Agilent-026655 Whole Mouse Genome Microarray 4x44K v2 (Agilent) on four biological replicates from each group.
Project description:RNA was isolated from three pairs of wild-type and Brpf1-deficient E8.75 mouse embryos for microarray analysis. The cDNA samples were then labeled and mixed for hybridization with one array. The data from three arrays were obtained and used for bioinformatic analysis.
Project description:This study was aimed at identifying Tbx1 dosage-dependent genes in vivo, so we performed a transcriptome analysis of Tbx1 mutants with nine different genotypes corresponding to different Tbx1 mRNA dosages. RNA isolated from whole E9.5 embryos with 9 different genotypes was hibridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays. For each genotype, we used two embryos (biological replicates), each hybridized to one array, thus we analyzed a total of 18 arrays. Tbx1 gene dosage
Project description:Mammalian heart development is built on highly conserved molecular mechanisms with polygenetic perturbations resulting in a spectrum of congenital heart diseases (CHD). However, the transcriptional landscape of cardiogenic ontogeny that regulates proper cardiogenesis remains largely based on candidate-gene approaches. Herein, we designed a time-course transcriptome analysis to investigate the genome-wide expression profile of innate murine cardiogenesis ranging from embryonic stem cells to adult cardiac structures. This comprehensive analysis generated temporal and spatial expression profiles, prioritized stage-specific gene functions, and mapped the dynamic transcriptome of cardiogenesis to curated pathways. Reconciling the bioinformatics of the congenital heart disease interactome, we deconstructed disease-centric regulatory networks encoded within this cardiogenic atlas to reveal stage-specific developmental disturbances clustered on epithelial-to-mesenchymal transition (EMT), BMP regulation, NF-AT signaling, TGFb-dependent induction, and Notch signaling. Therefore, this cardiogenic transcriptional landscape defines the time-dependent expression of cardiac ontogeny and prioritizes regulatory networks at the interface between health and disease. To interrogate the temporal and spatial expression profiles across the entire genome during mammalian heart development, we designed a time-course microarray experiment using the mouse model at defined stages of cardiogenesis, starting with embryonic stem cells (ESC, R1 stem cell line), early embryonic developmental stages: E7.5 whole embryos, E8.5 heart tubes, left and right ventricle tissues at E9.5, E12.5, E14.5, E18.5 to 3 days after birth (D3) and adult heart (Figure 1A). At each time point, microarray experiments were performed on triplicate biological samples. Starting at E9.5, tissue samples from left ventricles (LV) and right ventricles (RV) were microdissected for RNA purification and microarray analysis to determine spatially differential gene expression between LV and RV during heart development.
Project description:The study sought to determine whether deletion of individual seed families within the miR-17~92 cluster affected the expression of the remaining microRNAs of the cluster. There were 8 samples analyzed. The reference is the wild-type embryo.