Xenopus 배아 발생 과정에서 TGF-beta 신호전달경로를 조절하는 분자들의 기능에 관한 연구

Abstract

TGF-beta signaling plays major roles in various developmental processes including germ-layer specification, patterning, left-right asymmetry, and organogenesis. TGF-beta signals are propagated into the nucleus by Smad complex activated by TGF-beta receptors, and many regulators have been implicated in the regulation of Smad activation by the receptors. In this study, I analyzed the developmental functions of regulators of TGF-beta signaling including XDab2 and Xdpcp in Xenopus development in order to gain a better understanding of the molecular mechanisms underlying the germ-layer specification and angiogenesis in Xenopus embryos.1. Role of XDab2 in Xenopus embryonic angiogenesisThe molecular mechanisms governing the formation of the embryonic vascular system remain poorly understood. Here, I show that Disabled-2 (Dab2), a cytosolic adaptor protein, has a pivotal role in the blood vessel formation in Xenopus early embryogenesis. Xenopus Disabled-2 (XDab2) is spatially localized to the blood vessels including the intersomitic veins (ISV) in early embryos. Both antisense morpholino oligonucleotide (MO)-mediated knockdown and overexpression of XDab2 inhibit the formation of ISV, which arise from angiogenesis. In addition, I found that activin-like signaling is essential for this angiogenic event. Functional assays in Xenopus animal caps reveal that activin-like signals induce VEGF expression and this induction can be inhibited by XDab2 depletion. However, XDab2 MO has no effects on the induction of other target genes by activin-like signals. Furthermore, I show that the disruption of the sprouting ISV in XDab2-depleted embryos can be rescued by coexpression of VEGF. Taking together, I suggest that XDab2 regulates the embryonic angiogenesis by mediating the VEGF induction by activin-like signaling in Xenopus early development. 2. Role of Xdpcp in Xenopus germ-layer specificationPhosphotyrosine binding (PTB) domains, which are found in a large number of proteins, have been implicated in signal transduction mediated by growth factor receptors. However, the in vivo roles of these PTB-containing proteins remain to be investigated. Here, I show that Xdpcp (Xenopus dok-PTB containing protein) has a pivotal role in regulating mesendoderm formation in Xenopus, and negatively regulates the activin/nodal signaling pathway. I isolated cDNA for xdpcp and examined its potential role in Xenopus embryogenesis. I found that Xdpcp is strongly expressed in the animal hemisphere at the cleavage and blastula stages. The overexpression of xdpcp RNA affects activin/nodal signaling, which causes defects in mesendoderm formation. In addition, loss of Xdpcp function by injection of morpholino oligonucleotides (MO) leads to the expansion of the mesodermal territory. Moreover, I found that the axis duplication by ventrally forced expression of activin is recovered by coexpression with Xdpcp. In addition, Xdpcp inhibits the phosphorylation and nuclear translocation of Smad2. Furthermore, I also found that Xdpcp interacts with Alk4, a type I activin receptor, and inhibits activin/nodal signaling by disturbing the interaction between Smad2 and Alk4. Taken together, these results indicate that Xdpcp regulates activin/nodal signaling that is essential for mesendoderm specification.