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Haematologica Jan 2015Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal...
Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages.
Topics: Animals; Cell Differentiation; Cell Lineage; Cells, Cultured; Embryo, Nonmammalian; Embryonic Development; Erythrocytes; Gene Expression Regulation, Developmental; Hematopoiesis; Humans; Immunoenzyme Techniques; Macrophages; Mesoderm; Morpholinos; Myeloid Cells; RNA, Messenger; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish; Zebrafish Proteins
PubMed: 25281506
DOI: 10.3324/haematol.2014.104091 -
Nucleic Acid Therapeutics Apr 2021Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the gene, resulting in the loss of dystrophin from...
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMD) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.
Topics: Animals; Cell Line; Cyclin-Dependent Kinase 4; Dogs; Dystrophin; Exons; Genetic Therapy; Humans; Mice; Morpholinos; Muscular Dystrophy, Duchenne; Myoblasts; Oligonucleotides, Antisense; Peptides; RNA Splice Sites; Telomerase
PubMed: 33567244
DOI: 10.1089/nat.2020.0907 -
Developmental Biology Oct 2019The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the...
The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the availability of a relatively large collection of transcriptomic datasets to identify common signatures that characterize morpholino-injected animals (morphants). In addition to the previously reported activation of tp53 expression, we observed increased expression of the interferon-stimulated genes (ISGs), isg15 and isg20, the cell death pathway gene casp8, and other cellular stress response genes including phlda3, mdm2 and gadd45aa. Studies involving segmentation stage embryos were more likely to show upregulation of these genes. We also found that the expression of these genes could be upregulated by increasing doses of an egfl7 morpholino, or even high doses of the standard control morpholino. Thus, these data show that morpholinos can induce the expression of ISGs in zebrafish embryos and further our understanding of morpholino effects.
Topics: Animals; Down-Regulation; Embryo, Nonmammalian; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Interferons; Morpholinos; Mutation; Phenotype; Stress, Physiological; Tumor Suppressor Protein p53; Up-Regulation; Zebrafish; Zebrafish Proteins
PubMed: 31201802
DOI: 10.1016/j.ydbio.2019.06.008 -
PLoS Genetics Oct 2017
Topics: Animals; Female; Genetic Techniques; Male; Morpholinos; Zebrafish
PubMed: 29049395
DOI: 10.1371/journal.pgen.1007000 -
Cell Cycle (Georgetown, Tex.) Nov 2016CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as...
CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae. This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation.
Topics: Animals; Bromodeoxyuridine; Cell Death; Cell Proliferation; Cyclin-Dependent Kinase 9; Embryo, Nonmammalian; Flavonoids; Immunohistochemistry; Kaplan-Meier Estimate; Larva; Morpholinos; Phenotype; Piperidines; Protein Kinase Inhibitors; Survival Analysis; Zebrafish
PubMed: 27715402
DOI: 10.1080/15384101.2016.1231283 -
Restorative treatments of dystrophin expression in Duchenne muscular dystrophy: A systematic review.Annals of Clinical and Translational... Sep 2020To evaluate the effect of pharmacological treatments that increase the synthesis of dystrophin in Duchenne muscular dystrophy (DMD). Systematic searches were carried out... (Meta-Analysis)
Meta-Analysis
To evaluate the effect of pharmacological treatments that increase the synthesis of dystrophin in Duchenne muscular dystrophy (DMD). Systematic searches were carried out in MEDLINE, EMBASE, and Web of Science, and in gray literature from inception to December 2019. Clinical trials addressing the effect of restorative treatments of dystrophin expression in children and adolescents with DMD on functional outcomes {(6-minute walking distance [6MWD], other timed functional tests [TFTs], The North Star Ambulatory Assessment)}, dystrophin expression, cardiorespiratory function, and biochemical tests were included. The DerSimonian-Laird method was used to calculate the pooled estimates for functional outcomes. Eleven studies were included in the systematic review and five in the meta-analysis. Eteplirsen showed a significant effect on 6MWD, Δ6MWD = 67.3 m (95% CI: 27.32, 107.28), and Δ6MWD = 151.0 m (95% CI: 36.15, 265.85) at 48 weeks and 3 years, respectively. In the systematic review, analyzing individually the clinical trials using Ataluren and Drisapersen showed a nonsignificant effect on 6MWD. However, the meta-analysis showed a significant effect on 6MWD for Ataluren and Drisapersen, Δ6MWD = 18.3 m (95% CI: 1.0, 35.5) and Δ6MWD = 21.5 m (95% CI: 4.7, 38.3), respectively. There were no significant differences according to baseline age for Drisapersen. Similarly, the meta-analysis showed effect in TFT with Ataluren. All drugs induced a partial synthesis of dystrophin, and exon skipping was obtained with Eteplirsen and Drisapersen. Eteplirsen also improved forced vital capacity (Δ%pFVC = 1.8%) and maximal inspiratory pressure (Δ%pMIP = 4.4%). Eteplirsen and Ataluren could modestly reduce disease progression. However, more trials are needed to confirm its efficacy, as well as quality of life and cost-utility studies.
Topics: Dystrophin; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides; Outcome Assessment, Health Care; Oxadiazoles
PubMed: 33325654
DOI: 10.1002/acn3.51149 -
Journal of Managed Care & Specialty... Apr 2020No funding contributed to the writing of this commentary. Brandsema reports consulting for Alexion, Audentes, AveXis, Biogen, Cytokinetics, PTC Therapeutics, Sarepta,... (Review)
Review
No funding contributed to the writing of this commentary. Brandsema reports consulting for Alexion, Audentes, AveXis, Biogen, Cytokinetics, PTC Therapeutics, Sarepta, and WaVe and has received research funding as a site investigator from Alexion, AveXis, Biogen, CSL Behring, Cytokinetics, Fibrogen, Pfizer, PTC Therapeutics, Sarepta, Summit, and WaVe.
Topics: Cost-Benefit Analysis; Humans; Insurance Coverage; Insurance, Pharmaceutical Services; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides; Pregnenediones; United States; United States Food and Drug Administration
PubMed: 32223600
DOI: 10.18553/jmcp.2020.26.4.366 -
ELOVL5 Participates in Embryonic Lipid Determination of Cellular Membranes and Cytoplasmic Droplets.International Journal of Molecular... Jan 2021Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the...
Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (-Mo), Mo antisense oligonucleotides for the thalassemic β-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects ( > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and -Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased ( < 0.05) in -Mo-derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the -Mo-derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.
Topics: Animals; Blastocyst; Cattle; Cell Membrane; Cytoplasm; Embryonic Development; Fatty Acid Elongases; Female; Gene Knockdown Techniques; Humans; Lipid Metabolism; Morpholinos; Pregnancy; beta-Globins
PubMed: 33525659
DOI: 10.3390/ijms22031311 -
PloS One 2017Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it...
Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it has been implicated in the regulation of pancreatic and endothelial cell differentiation, the phenotypic consequences of Atoh8 loss are uncertain. Conclusions from knockout studies in the mouse differ widely depending on the targeting strategy used, while atoh8 knockdown by interfering morpholino oligonucleotides (morpholinos) in zebrafish has led to a range of developmental defects. This study characterised zebrafish embryos homozygous for atoh8sa1465, a loss-of-function allele of atoh8, in order to provide genetic evidence for the developmental role of Atoh8 in this species. Embryos homozygous for atoh8sa1465 present normal body morphology, swimbladder inflation, and heart looping, and survive to adulthood. These embryos do not develop pericardial oedema by 72 hpf and are not sensitised to the loss of Fog1 protein, suggesting that this previously described abnormality is not a specific phenotype. Vascular patterning and primitive haematopoiesis are unaffected in atoh8sa1465/sa1465 mutant embryos. Together, the data suggest that Atoh8 is dispensible for zebrafish development under standard laboratory conditions.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Gene Expression Regulation, Developmental; Heart; Hematopoiesis; Homozygote; Morpholinos; Mutation; Neovascularization, Physiologic; Phenotype; Zebrafish; Zebrafish Proteins
PubMed: 28182631
DOI: 10.1371/journal.pone.0171143 -
Cell Reports Jun 2019Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors...
Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA.
Topics: Animals; Binding Sites; Chromatin; Endoderm; Enhancer Elements, Genetic; Female; Forkhead Transcription Factors; Genome; Histones; Male; Morpholinos; Otx Transcription Factors; RNA Polymerase II; T-Box Domain Proteins; Transcriptome; Xenopus; Xenopus Proteins; Zygote
PubMed: 31167141
DOI: 10.1016/j.celrep.2019.05.013