Little, S. C. & Mullins, M. C. Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning. Development 131, 5825-5835

William Penn University, Filadelfia, Pennsylvania, United States
Development (Impact Factor: 6.46). 01/2005; 131(23):5825-35. DOI: 10.1242/dev.01464
Source: PubMed


In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.

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    • "Artifacts and erroneous interpretations have occurred with MOs (e.g., Ross et al. 2001; Little and Mullins, 2004), as they have occurred in genetics and will occur with the emerging genome modification technologies. Gene editing nucleases promise to revolutionize developmental biology but, at least for the time being, cannot replace MOs in many applications. "
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    ABSTRACT: For over 15 years, antisense morpholino oligonucleotides (MOs) have allowed developmental biologists to make key discoveries regarding developmental mechanisms in numerous model organisms. Recently, serious concerns have been raised as to the specificity of MO effects, and it has been recommended to discontinue their usage, despite the long experience of the scientific community with the MO tool in thousands of studies. Reviewing the many advantages afforded by MOs, we conclude that adequately controlled MOs should continue to be accepted as generic loss-of-function approach, as otherwise progress in developmental biology will greatly suffer.
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    • ").The most important issues to consider in RNA rescue experiments are: (1) achievement of appropriate levels of injected MO and mRNA by co-injecting different concentrations of both components (Little and Mullins, 2004), (2) making sure that injected synthetic mRNA does not include the MO target sequence (Eisen and Smith, 2008). For TB MOs against the 5 UTR sequence, the open reading frame can be cloned by PCR into a standard transcription vector (Hyatt and Ekker, 1999). "
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    ABSTRACT: Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and zebrafish. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos (MOs) into early stage embryos. Validation of the MO needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of zebrafish models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification.
    Frontiers in Genetics 04/2013; 4(74):1-17. DOI:10.3389/fgene.2013.00074
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    • "brain and eyes), and an abnormal accumulation of cells caudal to the urogenital pore visible at the 26-somite stage [22 hours post fertilization, (hpf)] [51]. In some cases, including the chordin-null mutant, multiple ventral fin folds are observed [52]. We tested whether the four small molecule flavonoids ventralized zebrafish embryos as would be predicted for BMP pathway agonists by treating wild-type embryos from 2 to 50 hpf. "
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    ABSTRACT: Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.
    PLoS ONE 03/2013; 8(3):e59045. DOI:10.1371/journal.pone.0059045 · 3.23 Impact Factor
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