Transcription profiling of zebrafish embryos following cardiac troponin T2 knockdown
Ontology highlight
ABSTRACT: We examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since the fish obtain sufficient oxygen via diffusion. Morpholino antisense knockdown of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. We concluded that absence of hemodynamic force induces endothelial CXCR4a up-regulation and promotes recovery of blood flow. We used microarrays to define the genetic signatures of the development of collateral vessels in zebrafish. Experiment Overall Design: One cell zebrafish embryos were injected with morpholino antisense against control or cardiac troponin t2, which prevents cardiac development. Whole embryo RNA was extracted at 36, 48, and 60h post fertilization and transcriptionally profiled.
Project description:We examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since the fish obtain sufficient oxygen via diffusion. Morpholino antisense knockdown of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. We concluded that absence of hemodynamic force induces endothelial CXCR4a up-regulation and promotes recovery of blood flow. We used microarrays to define the genetic signatures of the development of collateral vessels in zebrafish. Keywords: Time course comparison of wild type zebrafish embryos and morpholino antisense against troponin t2 treated embryos.
Project description:Human cardiac Troponin I epitope peptides were incubated with monoclonal anti-cardiac Troponin I antibody. Immune complexes were subjected to nano electrospray mass spectrometry in order to determine apparent binding energies and dissociation constants of immune complex dissociation reactions in the gas phase.
Project description:Transcriptional profiling of embryonic zebrafish injected with a control morpholino or a morpholino that causes exclusion of TNNT2 exon 13. Zebrafish embryos were injected with a antisense morpholino oligo that induced missplicing of exon 13 of TNNT2 (TNNT2sp) or a control morpholino. At 96hpf these embryos were euthanized and RNA was collected.
Project description:A study of cardiac troponin I evolution in mammals with high heart rates, including shrews, moles, and bats. With genomic, mRNA and protein level analyses we showed repeated loss of the N-terminal extension. Here, liquid chromatography with tandem mass spectrometry was used to verify cardiac troponin I (TNNI3) protein identity in ∼22 kDa bands from Pyrenean desman (Galemys pyrenaicus) and northern short-tailed shrew (Blarina brevicauda) hearts.
Project description:[1] Gene expression profiling in Gata4, Mef2a knockdown in HL-1 cells. HL-1 cells infected with adenovirus expressing either control siRNA or Gata4, and Gata4 & Mef2a siRNA. [2] Genome-wide maps of cardiac transcription factors binding region in HL-1 cells. We performed bio-ChIP-seq using streptavidin beads immunoprecipitation of biotinylated 5 cardiac transcription factors (fbio) and P300 antibody ChIP-seq. We used a dox-inducible dual adenovirus system to express biotinylated Core Cardiac TFs in HL1. One adenovirus expressed the dox-activated reverse tet activator protein (rtTA) and the E. coli biotinylating enzyme BirA from the cardiac specific rat troponin T promoter. The second virus expressed a Core Cardiac TF fused to a C-terminal flag-bio epitope tag, where bio is the 15 amino acid substrate for BirA. This in vivo biotinylation approach permits pulldown of different factors to be performed under identical, stringent conditions, and circumvents limitations posed by available antibodies. We titrated adenovirus and dox doses to express GATA4flbio and MEF2Aflbio at near endogenous levels. The same conditions were then used to express SRFflbio, TBX5flbio, and NKX2-5flbio. Reference: 12. de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100: 7480-7485. [1] Gene expression profiling: Identify differentially expressed genes after siRNA Gata4 or Gata4_Mef2a double knockdown in HL-1 cells [2] Genome occupancy profiling: Identify cardiac active enhancers by identified 5 cardiac transcription factors and P300 peaks.
Project description:Changes in the gene expression in the heart of knock-in mouse model of dilated cardiomyopathy caused by delK210 mutation in cardiac troponin T.
Project description:A zebrafish model of arterial tortuosity syndrome (ATS) was generated by knocking down the slc2a10/glut10 gene using antisense morpholino oligonucleotides (MO). Control morpholino treated embryos were used as controls. The samples were collected for gene expression profiling at 48 hours post fertilization. Experimental details and analyzed data are available in Willaert et al. Human Molecular Genetics 2011; doi: 10.1093/hmg/ddr555 Two-condition experiment, slc2a10 MO (7.5ng) treatment vs control MO (5ng) treatment. Biological replicates: 3 slc2a10 MO replicates, compared to a pooled sample of 3 control MO replicates with dye swap.
Project description:We have characterised the zebrafish ortholog, setb, and investigated its role in embryogenesis. Phylogenetic analysis showed that zebrafish Setb has an amino acid sequence identity of approximately 96% with the mammalian orthologs. Whole mount immunofluorescence analysis revealed that Setb is expressed mainly in the eye, the lateral line neuromasts and the olfactory pit. Knockdown of setb using antisense morpholino oligonucleotides resulted in increased apoptosis, reduced cell proliferation and severe morphological defects. The morphant phenotypes were partially rescued when setb MO1 was co-injected with human set mRNA. In vivo labelling of hair cells in the lateral line of setb morphants with the vital fluorescent dye FM1-43 showed a significant decreased number of functional neuromasts. Gene expression analysis of setb morphants, employing DNA microarrays revealed a role of Setb in neurogenesis and the mechanosensory lateral line system. 48hpf control morpholino- and setb MO1-injected embryos in triplicates
Project description:This study examined the effects of genetic knockdown of autophagy genes on vertebrate cardiac development We performed microarray studies comparing the hearts of control zebrafish embryos to the hearts of embryos with decreased expression of the autophagy genes atg5, becn1 or atg7. The results provide insight into the role of autophagy in developmental morphogenesis. Hearts were purified from 3 day-old zebrafish embryos injected with control or autophagy gene-specific morpholino oligonucleotides. RNA was prepared from all samples and hybridized to zebrafish-specific Affymetricx arrays.