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  • Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm. 21383318

    In mature human sperm, genes of importance for embryo development (i.e., transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent, key factors that may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, whereas H3K36me3 is located in the 3' coding region of heavily transcribed genes in somatic cells, H3K36me3 is present in the promoters of "silent" developmental regulators in sperm, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tDNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation. 19056886

    During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior-posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3(-/-) embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease. 21719443

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Oxidative stress has been associated with the etiology of both sporadic and monogenic forms of PD. The transcription factor Nrf2, a conserved global regulator of cellular antioxidant responses, has been implicated in neuroprotection against PD pathology. However, direct evidence that upregulation of the Nrf2 pathway is sufficient to confer neuroprotection in genetic models of PD is lacking. Expression of the PD-linked gene encoding α-synuclein in dopaminergic neurons of Drosophila results in decreased locomotor activity and selective neuron loss in a progressive age-dependent manner, providing a genetically accessible model of PD. Here we show that upregulation of the Nrf2 pathway by overexpressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of α-synuclein-expressing flies. Similar benefits are observed upon RNA-interference-mediated downregulation of the prime Nrf2 inhibitor, Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the α-synuclein-induced dopaminergic neuron loss is suppressed by Maf-S overexpression or keap1 heterozygosity. Our data validate the sustained upregulation of the Nrf2 pathway as a neuroprotective strategy against PD. This model provides a genetically accessible in vivo system in which to evaluate the potential of additional Nrf2 pathway components and regulators as therapeutic targets.
    Document Type:
    Reference
    Product Catalog Number:
    AB152
    Product Catalog Name:
    Anti-Tyrosine Hydroxylase Antibody
  • Genetic analysis of yeast Yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor. 15611160

    Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes. The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding. Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes. In this study we have investigated the physiological requirements for Yip1p action. Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability. We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3580
    Product Catalog Name:
    Anti-Green Fluorescent Protein Antibody
  • Genetic and maternal effects on valproic acid teratogenesis in C57BL/6J and DBA/2J mice. 20457659

    Valproic acid (VPA) is used worldwide to treat epilepsy, migraine headaches, and bipolar disorder. However, VPA is teratogenic and in utero exposure can lead to congenital malformations. Using inbred C57BL/6J (B6) and DBA/2J (D2) mice, we asked whether genetic variation could play a role in susceptibility to VPA teratogenesis. Whereas B6 fetuses were more susceptible than D2 fetuses to digit and vertebral malformations, D2 fetuses were more susceptible to rib malformations. In a reciprocal cross between B6 and D2, genetically identical F1 mice carried in a B6 mother had a greater percentage of vertebral malformations following prenatal VPA exposure than F1 mice carried in a D2 mother. This reciprocal F1 difference is known as a maternal effect and shows that maternal genotype/uterine environment is an important mediator of VPA teratogenecity. VPA is a histone deacetylase inhibitor, and it is possible that the differential teratogenesis in B6 and D2 is because of strain differences in histone acetylation. We observed strain differences in acetylation of histones H3 and H4 in both embryo and placenta following in utero VPA exposure, but additional studies are needed to determine the significance of these changes in mediating teratogenesis. Our results provide additional support that genetic factors, both maternal and fetal, play a role in VPA teratogenesis. Lines of mice derived from B6 and D2 will be a useful model for elucidating the genetic architecture underlying susceptibility to VPA teratogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis. 25122120

    Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5
  • Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy. 19359597

    The phenotypic hallmark of arrhythmogenic right ventricular cardiomyopathy, a genetic disease of desmosomal proteins, is fibroadipocytic replacement of the right ventricle. Cellular origin of excess adipocytes, the responsible mechanism(s) and the basis for predominant involvement of the right ventricle are unknown. We generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters alpha-myosin heavy chain (alphaMyHC), Nkx2.5, or Mef2C. We conditionally expressed the reporter enhanced yellow fluorescent protein while concomitantly deleting the desmosomal protein desmoplakin in cardiac myocyte lineages using the Cre-LoxP technique. Lineage tracer mice showed excess fibroadiposis and increased numbers of adipocytes in the hearts. Few adipocytes in the hearts of alphaMyHC-regulated lineage tracer mice, but the majority of adipocytes in the hearts of Nkx2.5- and Mef2C-regulated lineage tracer mice, expressed enhanced yellow fluorescent protein. In addition, rare cells coexpressed adipogenic transcription factors and the second heart field markers Isl1 and Mef2C in the lineage tracer mouse hearts and in human myocardium from patients with arrhythmogenic right ventricular cardiomyopathy. To delineate the responsible mechanism, we generated transgenic mice expressing desmosomal protein plakoglobin in myocyte lineages. Transgene plakoglobin translocated to nucleus, detected by immunoblotting and immunofluorescence staining and coimmunoprecipitated with Tcf7l2, a canonical Wnt signaling transcription factor. Expression levels of canonical Wnt/Tcf7l2 targets bone morphogenetic protein 7 and Wnt5b, which promote adipogenesis, were increased and expression level of connective tissue growth factor, an inhibitor of adipogenesis, was decreased. We conclude adipocytes in arrhythmogenic right ventricular cardiomyopathy originate from the second heart field cardiac progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin.
    Document Type:
    Reference
    Product Catalog Number:
    05-511
    Product Catalog Name:
    Anti-TCF-4 Antibody, clone 6H5-3
  • Genetic knock-down of HDAC7 does not ameliorate disease pathogenesis in the R6/2 mouse model of Huntington's disease. 19484127

    Huntington's disease (HD) is an inherited, progressive neurological disorder caused by a CAG/polyglutamine repeat expansion, for which there is no effective disease modifying therapy. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression. Administration of histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) have consistently shown therapeutic potential in models of HD, at least partly through increasing the association of acetylated histones with down-regulated genes and by correcting mRNA abnormalities. The HDAC enzyme through which SAHA mediates its beneficial effects in the R6/2 mouse model of HD is not known. Therefore, we have embarked on a series of genetic studies to uncover the HDAC target that is relevant to therapeutic development for HD. HDAC7 is of interest in this context because SAHA has been shown to decrease HDAC7 expression in cell culture systems in addition to inhibiting enzyme activity. After confirming that expression levels of Hdac7 are decreased in the brains of wild type and R6/2 mice after SAHA administration, we performed a genetic cross to determine whether genetic reduction of Hdac7 would alleviate phenotypes in the R6/2 mice. We found no improvement in a number of physiological or behavioral phenotypes. Similarly, the dysregulated expression levels of a number of genes of interest were not improved suggesting that reduction in Hdac7 does not alleviate the R6/2 HD-related transcriptional dysregulation. Therefore, we conclude that the beneficial effects of HDAC inhibitors are not predominantly mediated through the inhibition of HDAC7.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60
  • Genetic modification of donor hepatocytes improves therapeutic efficacy for hemophilia B in mice. 20412633

    Hepatocyte transplantation (Tx) holds promise for curing genetic liver diseases. However, a limited number of donor hepatocytes can be transplanted into the host liver. Recipient preconditioning and donor cell engineering are under investigation to improve cell engraftment. In theory, genetically engineered cells secreting therapeutic proteins with superior function could compensate for poor engraftment efficiency. We have generated a bioengineered human coagulation factor IX (FIX) with augmented specific activity (named FIX-Triple). The aim of this study was to evaluate therapeutic efficacy of cell therapy using hemophilia B (HB) as a disease model by transplanting FIX-Triple-secreting hepatocytes. The donor hepatocytes were isolated from FIX-Triple knock-in (KI) or FIX-WT (wild-type) KI mice and transplanted intrasplenically into FIX knock-out (KO) mice. FIX-Triple KI recipients exhibited fourfold higher plasma FIX clotting activity than FIX-WT KI recipients. By repeated Txs, the clotting activity of FIX-Triple KI recipients even increased to more than 10% of normal mouse plasma. The engraftment and FIX production efficiencies of transplanted cells were equivalent between the FIX-WT KI and FIX-Triple KI donors. A hemostatic function assay showed that FIX-Triple KI recipients with repeated Txs had more enhanced clot kinetics and a greater maximum rate of thrombus generation than those with a single Tx. Moreover, FIX inhibitors in these recipients rarely developed. In conclusion, hepatocyte Tx with genetically engineered donor cells is an effective therapeutic strategy for HB.
    Document Type:
    Reference
    Product Catalog Number:
    AP160
    Product Catalog Name:
    Rabbit Anti-Mouse IgG Antibody
  • Genetic modulation of soluble Aβ rescues cognitive and synaptic impairment in a mouse model of Alzheimer's disease. 24899710

    An unresolved debate in Alzheimer's disease (AD) is whether amyloid plaques are pathogenic, causing overt physical disruption of neural circuits, or protective, sequestering soluble forms of amyloid-β (Aβ) that initiate synaptic damage and cognitive decline. Few animal models of AD have been capable of isolating the relative contribution made by soluble and insoluble forms of Aβ to the behavioral symptoms and biochemical consequences of the disease. Here we use a controllable transgenic mouse model expressing a mutant form of amyloid precursor protein (APP) to distinguish the impact of soluble Aβ from that of deposited amyloid on cognitive function and synaptic structure. Rapid inhibition of transgenic APP modulated the production of Aβ without affecting pre-existing amyloid deposits and restored cognitive performance to the level of healthy controls in Morris water maze, radial arm water maze, and fear conditioning. Selective reduction of Aβ with a γ-secretase inhibitor provided similar improvement, suggesting that transgene suppression restored cognition, at least in part by lowering Aβ. Cognitive improvement coincided with reduced levels of synaptotoxic Aβ oligomers, greater synaptic density surrounding amyloid plaques, and increased expression of presynaptic and postsynaptic markers. Together these findings indicate that transient Aβ species underlie much of the cognitive and synaptic deficits observed in this model and demonstrate that significant functional and structural recovery can be attained without removing deposited amyloid.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple