Browsing by Subject "Xenopus"
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Item Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting Agent(Public Library of Science, 2012-08-21) Cha, Hye Ji; Byrom, Michelle; Mead, Paul E.; Ellington, Andrew D.; Wallingford, John B.; Marcotte, Edward M.Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.Item Group II Intron-Based Gene Targeting Reactions in Eukaryotes(Public Library of Science, 2008-09-01) Mastroianni, Marta; Watanabe, Kazuo; White, Travis B.; Zhuang, Fanglei; Vernon, Jamie; Matsuura, Manabu; Wallingford, John; Lambowitz, Alan M.Background: Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes. Methodology/Principal Findings: By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg2+ concentrations. By supplying additional Mg2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization. Conclusions/Significance: Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.Item Partial Least Squares Regression for Modeling of Complex Morphogenetic Cell Behaviors During Neural Tube Closure(2020-05) Kim, Juliana; Wallingford, John B.During neural tube closure (NTC), a sheet-like structure of cells folds into a tube that forms the brain and spinal cord. Neural tube defects can arise when cells fail to constrict their apical surfaces, a process dependent on Shroom3. Yet, we lack understanding how Shroom3-constriction is achieved. Mechanisms of cell constriction in the developing embryo vary by organism and stage of development. Each observed constriction mechanism has been associated with a different model, the best-established being “ratchet-like” medioapical constriction, purse-string junctional constriction, and pulsed medioapical constriction. While these models help us distinguish mechanisms by attributes we can visually observe, we need methods to describe more complex mechanisms, with precision. Here, we present a framework to characterize cell constrictions quantitatively, according to cell parameters that are readily measurable and associated with morphogenesis, including protein localization, cell polarity, cell shape descriptors, and cell rearrangement. We use partial least squares regression (PLS) to understand which parameters are associated with cellular apical area changes and apply our framework to in-vivo time-lapse imaging of Xenopus tropicalis during NTC. Using PLS, we generate a model of cell constrictions in wild-type and Shroom3 mutant cells during NTC. Our comparisons highlight possible modes of apical constriction across developmental contexts.Item Transcriptional control of epithelial morphogenesis(2013-05) Chung, Mei-I; Wallingford, John B.How tissues and organs develop into their final shape during embryogenesis is a fascinating and long-standing question in developmental biology. Tissue morphogenesis is driven by a variety of events at the cellular level and individual cell shape change is one of the central morphogenetic engines. Thus, it is crucial to understand what signals specify the correct cell behavior in specific groups of cells during development. For my doctoral studies, I have focused on two cell shape change events, apical constriction and cilia assembly. First, we present data demonstrating that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut, where Shroom3 transcription requires the Pitx1 transcription factor. We identified a Pitx-responsive regulatory element in the genomic DNA upstream of Shroom3, and showed that Pitx proteins directly activated Shroom3 transcription in Xenopus. Moreover, we showed that ectopic expression of Pitx proteins was sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrated new breadth to the requirements for Shroom3 in morphogenesis, and also provided a cell-biological mechanism for Pitx transcription factors action during morphogenesis. Next, we focused on understanding the transcriptional regulation of ciliogenesis. We first showed that Rfx2 transcription factor broadly controlled ciliogenesis, and by RNA- and ChIP-sequencing, we showed that Rfx2 directly regulated a wide range of genes encoding diverse ciliogenic machinery. Finally, in addition to ciliogenesis regulation, a large number of non-ciliary genes in our Rfx2 dataset led us to identify a novel role of Rfx2 in controlling insertion of multi-ciliated cells into the overlying mucociliary epithelium. Moreover, we showed here that Slit2, a target of Rfx2, was involved in multi-ciliated cell movements, possibly through mediating cortical E-cadherin level. This work allowed us to begin building a genetic network controlling multi-ciliated cells in mucociliary epithelium. Together, we showed a transcriptional regulation of apical constriction driving gut morphogenesis and a comprehensive transcriptional network that governs multi-ciliated cell development.