Biology subjects

Takahashi, Y.

Publications and source records attributed to Takahashi, Y..

5 recordsLinked to original sources

bioRxiv2018

Blood flow-mediated gene transfer and siRNA-knockdown in the developing vasculature in a spatio-temporally controlled manner in chicken embryos

We describe a method by which early developing vasculature can be gene-manipulated independently of the heart in a spatio-temporally controlled manner. Lipofectamine 2000 or 3000, an easy-to-use lipid reagent, has been found to yield a high efficiency of transfection when co-injected with GFP DNA within a critical range of lipid concentration. By exploiting developmentally changing patterns of vasculature and blood flow, we have succeed in controlling the site of transfection: injection with a lipid-DNA cocktail into the heart before or after the blood circulation starts results in a limited and widely spread patterns of transfection, respectively. Furthermore, a cocktail injection into the right dorsal aorta leads to transgenesis of the right half of embryonic vasculature. In addition, this method combined with the siRNA technique has allowed, for the first time, to knockdown the endogenous expression of VE-cadherin (also called Cdh5), which has been implicated in assembly of nasant blood vessels: when Cah5 siRNA is injected into the right dorsal aorta, pronounced defects in the right half of vasculature are observed without heart defects. Whereas infusion-mediated gene transfection method has previously been reported using lipid reagents that were elaborately prepared on their own, Lipofectamine is an easy-use reagent with no requirement of special expertise. The methods reported here would overcome shortcomings of conventional vascular-transgenic animals, such as mice and zebrafish, in which pan-endothelial enhancer-driven transgenesis often leads to the heart malformation, which, in turn, indirectly affects peripheral vasculature due to flow defects. Since a variety of subtypes in vasculature have increasingly been appreciated, the spatio-temporally controllable gene manipulation described in this study offers a powerful tool.\n\nResearch HighlightsO_LIBlood flow-mediated transfection enables site-specific transgenesis in vessels.\nC_LIO_LIThis transfection technique allows local knockdown of endogenous gene(s) by siRNA.\nC_LIO_LIKnockdown of endogenous VE-cadherin causes vascular defects without heart failure.\nC_LI

developmental biology↗

bioRxiv2018

Comparison of the 3-D patterns of the parasympathetic nervous system in the lung at late developmental stages between mouse and chicken

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.\n\nHighlightsO_LI3-D patterns of parasympathetic nerves are visualized in mouse and chicken lungs.\nC_LI\n\nComparison of these patterns reveals three prominent similarities between mouse and chicken: O_LIVAChT-positive postganglionic fibers and ganglia are widely distributed in the lung.\nC_LIO_LIGas exchange units are devoid of parasympathetic nerves.\nC_LIO_LIParasympathetic nerves are in close association with smooth muscle cells.\nC_LI

developmental biology↗

bioRxiv2018

Secondary neurulation-fated cells in the tail bud undergo self-renewal and tubulogenesis regulated by a Sox2 gradient

During amniote development, anterior and posterior components of the neural tube form by primary neurulation (PN) and secondary neurulation (SN), respectively. Unlike PN, SN proceeds by the mesenchymal-to-epithelial transition of SN precursors in the tail bud, a critical structure for the axial elongation. Our direct cell labeling delineates non-overlapping territories of SN- and mesodermal precursors in the chicken tail bud. SN-fated precursors are further divided into self-renewing and differentiating cells, a decision regulated by graded expression levels of Sox2. Whereas Sox2 is confined to SN precursors, Brachyury (T) is widely and uniformly distributed in the tail bud, indicating that Sox2+/Brachyury+ cells are neural-fated and not mesodermal. These results uncover multiple steps during the neural posterior elongation, including precocious segregation of SN precursors, their self-renewal, and regulation by graded Sox 2.

developmental biology↗

bioRxiv2017

N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1{triangleup} strains but not in slx5{triangleup} strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

molecular biology↗

bioRxiv2017

Multidisciplinary field research in Kabwe, Zambia, towards better understanding of lead contamination of the city - A short report from a field survey

Heavy metal contamination is a serious issue in many post-mining regions around the world. Kabwe town, Zambia, is known as one of the most polluted cities in the world, where high lead (Pb) levels have been reported in soils, plants, animals and human blood. Multidisciplinary approaches are critically needed to understand the current situation and to remediate the polluted area. In the current research, a large-scale preliminary field survey was performed to understand the current situation in Kabwe and to plan future mitigation approaches. Three aspects were mainly observed; 1) plant communities during the dry season in Kabwe city, 2) spectral images of the land surfaces in various locations in Kabwe and 3) Pb concentrations in soils and water. Overall, >15 different plant species were observed and many of them maintained their green colour even during the dry season. Some tree species, for example, Caesalpiniaceae and Fabaceae families may be utilised as phytostabilization approaches although their impacts on the soil Pb mobility should be further studied. For the spectral images, we used a handmade portable spectrometer, and our obtained spectral images showed typical curves observed from soils. These data may be used to understand the distribution of different soil types in this area, using aboveground images such as satellite images. For Pb concentrations in soils, extremely high total Pb levels (>1,000 ppm) was observed only within 2 km from the mining site. There was a weak but a positive correlation between the total and soluble Pb thus further study should also focus on the mobility of Pb from soils to plant ecosystems.

ecology↗