Biomedical subjects

Yukinori Endo

Publications and source records attributed to Yukinori Endo.

6 recordsLinked to original sources

PubMed2006

Inhibition of rho GTPases by RNA interference.

Selective down-modulation or silencing of individual members of the Rho-GTPase family is now practical using RNA interference. Transfection of mammalian cells with an individual siRNA duplex or siRNA pools can suppress expression of a specific isoform to understand its function. By adjusting the dose of siRNA, intermediate levels of suppression can be attained to test the biological role of different levels of a GTPase such as Rac. Nevertheless, there are significant potential pitfalls, including "off-target" effects of the siRNA on other genes. Besides demonstrating successful, noncytotoxic suppression of protein and activity levels of a specific GTPase, controls are essential to establish specificity. In this chapter, we provide methods for selective knockdown of expression by siRNA and confirmation of the effectiveness of Rho GTPase silencing, as well as descriptions and some examples of controls for specificity that include evaluations of dose-response, negative and positive controls, GTPase specificity, confirmation by using more than one siRNA for the same gene, rescue by a mutated siRNA-resistant cDNA encoding the target gene, and complementary supporting evidence. Selective silencing of specific Rho family GTPases should provide increasing insight into the regulatory and functional roles of each isoform in a wide variety of biological processes.

Cell Line↗

PubMed2005

A Rac switch regulates random versus directionally persistent cell migration.

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

Cell Culture Techniques↗

PubMed2005

Regulation of Slug transcription in embryonic ectoderm by beta-catenin-Lef/Tcf and BMP-Smad signaling.

Neural crest is formed at the boundary of epidermal and neural ectoderm. To understand the molecular mechanism of neural crest formation, we focused on the transcriptional regulation of the Slug gene. In the upstream sequence of the chicken Slug gene, we have identified potential binding sites for transcription factors, such as Lef/Tcf and Smad1. Transgenic mouse embryos carrying the chicken Slug promoter-reporter gene showed a crest-specific activation of the reporter, suggesting the isolated sequence included the cis-regulatory elements to receive Slug-inducing signals in the mouse neural crest. While these potential cis-regulatory elements could be recognized and activated by corresponding transcription factors, such as Lef1 and Smad1, Wnt-Lef-beta-catenin signal failed to induce endogenous Slug expression in quail neural plate tissue prepared from forebrain and midbrain levels. In contrast, Slug expression and subsequent epithelial-mesenchymal transition were effectively induced by BMP4. Consistently, while we could detect phosphorylation of Smad1 in the ectoderm including the neural plate and the neural fold region, the activation of a reporter gene for a detection of canonical Wnt signal activation was below the level of detection at the forebrain and midbrain levels. These observations indicated that in the anterior ectoderm BMP signal has a predominant role for Slug expression.

Animals↗

PubMed2004

Multiple roles of Sox2, an HMG-box transcription factor in avian neural crest development.

Expression of Sox2, which encodes an HMG-box-type transcription factor, is down-regulated in the neural plate when neural crest segregates from dorsal neural tube and remains low during crest cell migration. Sox2 expression is subsequently up-regulated in some crest-derived cells in the developing peripheral nervous system and is later restricted to glial sublineages. Misexpression of Sox2 and mutant forms of Sox2 both in neural plate explants and in embryonic ectoderm reveals that Sox2 inhibits neural crest formation as a transcriptional activator. Similar manipulation of Sox2 function in migratory and postmigratory neural crest-derived cells indicates that Sox2 regulates proliferation and differentiation in developing peripheral nervous system. Developmental Dynamics 229:74-86, 2004.

Animals↗

PubMed2003

Deltex/Dtx mediates NOTCH signaling in regulation of Bmp4 expression in cranial neural crest formation during avian development.

NOTCH signaling plays a key role in cell fate determination in both vertebrates and invertebrates. It is well known that Su(H)/RBP-J is a major mediator of NOTCH signaling. In a previous study, it was shown that NOTCH signaling was involved in cranial neural crest formation in avian embryos. However, Su(H)/RBP-J activity did not appear to be required in this process. In this study, the Deltex/Dtx gene was focussed on as a potential mediator of NOTCH signaling in neural crest formation. At the time of neural crest formation, quail Deltex2 was expressed throughout the ectoderm. Misexpression of a dominant-negative form of Deltex in the ectoderm caused reduced expression of Slug, a neural crest marker. Dominant-negative Deltex expression reduced the expression of Bmp4, a neural crest inducer, whereas co-transfection of Bmp4 with dominant-negative Deltex rescued Slug expression. In parallel, Hairy2 expression in the epidermis was regulated by a Su(H)-dependent pathway. These results indicate that NOTCH signaling has dual functions mediated by either Su(H) or Deltex in the avian embryonic ectoderm.

Animals↗

PubMed2002

Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development.

Neural crest is induced at the junction of epidermal ectoderm and neural plate by the mutual interaction of these tissues. In previous studies, BMP4 has been shown to pattern the ectodermal tissues, and BMP4 can induce neural crest cells from the neural plate. In this study, we show that epidermally expressed Delta1, which encodes a Notch ligand, is required for the activation and/or maintenance of Bmp4 expression in this tissue, and is thus indirectly required for neural crest induction by BMP4 at the epidermis-neural plate boundary. Notch activation in the epidermis additionally inhibits neural crest formation in this tissue, so that neural crest generation by BMP4 is restricted to the junction.

Animals↗