@misc{oai:ir.soken.ac.jp:00001392, author = {飯岡, 英和 and イイオカ, ヒデカズ and IIOKA, Hidekazu}, month = {2016-02-17, 2016-02-17}, note = {Gastrulation is one of the most important developmental events for many multicellular organisms. In the amphibian embryos, mesodermal cells involute to the inside of the embryo and migrate along the blastocoel roof (BCR) to establish the three germ layer structure and body axes. These processes involve several morphogenetic cell movements including convergent extension. In convergent extension, cells are polarized, elongated mediolaterally, and then intercalated each other.
　It is known that Wnt signaling pathways play an essential role in the regulation of convergent extension movements. Wnts are a family of secreted proteins that regulate many biological processes. Functional analyses in Xenopus suggest that the Wnt family can be divided into two functionally distinct groups. The first group of Wnts induces a secondary axis when ectopically expressed in Xenopus embryos. They activate the canonical Wnt/β-catenin pathway and induce transcription of target genes such as siamois and Xnr3. The second group of Wnts, which include Wnt5a and Wnt11, activates the noncanonical Wnt signaling pathway that controls morphogenetic cell movements. The noncanonical Wnt pathway branches into two cascades. One is the PCP (Planar Cell Polarity) pathway, and the other is the Wnt/Ca²⁺ pathway. Previous data showed that Wnt/PCP pathway has been implicated in the regulation of convergent extension. One of the PCP signaling components Dishevelled (Dsh) is essential for convergent extension. However, it is largely unknown how the PCP pathway regulates convergent extension.
　Because active cell movements occur during convergent extension, the regulation of cytoskeletal dynamics may be important for the regulation of this process. It has been shown that Dsh activates Rho family small GTPases, suggesting that the PCP pathway may be involved in the cytoskeletal regulation. In order to investigate the regulatory mechanisms of actin cytoskeleton during gastrulation, He cloned a gene encoding an actin-binding protein MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) from a Xenopus embryonic cDNA library. MARCKS was first identified as a PKC (Protein Kinase C) substrate in mammalian cells. It attaches with the plasma membrane through N-terminal myristoylation. He showed that loss of MARCKS function by MO (Morpholino oligonucleotide) in Xenopus embryo induced a gastrulation defect phenotype without affecting mesoderm induction. To elucidate why MARCKS MO caused gastrulation defect, cell biological analyses were conducted. During convergent extension, MARCKS MO inhibited polarization and intercalation of mesodermal cells. He performed further observation at the cellular level. As a result, cell adhesion on fibronectin, protrusive activity of mesodermal cells and cortical actin formation in the cells were also inhibited by MARCKS MO. Furthermore, He found that activation of the PCP pathway promoted formation of filopodia- and lamellipodia-like structures in ectoderm explant cells, and MARCKS MO inhibited this activity. These results indicate that MARCKS regulates cortical actin dynamics, and it is requisite for the morphological processes regulated by the PCP signaling pathway. In addition, MARCKS MO also severely impaired neural tube closure without affecting the neural induction. It is consistent with the phenotype of mice deficient in MARCKS. MARCKS function may be conserved in vertebrates. Taken together, MARCKS is an essential molecule not only for gastrulation movements but also neural tube closure through controlling the cortical actin formation. These results are shown and discussed in Chapter 2.
　It is known that Dsh is translocated to the plasma membrane in response to Wnt signaling in animal cap cells. In this thesis, He showed the bipolor localization of Dsh in mesodermal cells during convergent extension. These data indicate that the regulation of Dsh localization is important for the regulation of convergent extension. But its regulatory mechanism is unknown. Thus, He analyzed molecular mechanism to regulate Dsh localization and identified three proteins involved in the PCP pathway, PKCδ, G_α11 (G₁₁) and G_αi1 (G_i1). First He identified PKCδ as an essential factor to regulate Dsh localization and showed that it physically interacted with Dsh. Loss of PKCδ function induced a gastrulation defective phenotype without affecting mesoderm induction. Confocal microscopic analyses revealed that both PKCδ and Dsh were translocated from the cytoplasm to the plasma membrane by Fz7 signaling. In addition, loss of PKCδ function reduced the signal-dependent Dsh translocation. These results indicate that PKCδ regulates Dsh localization under the control of Wnt signaling. Next, He focused on heterotrimeric G protein α subunits. Injections of antisense MOs against G₁₁ or G_i1 caused a phenotype in the body axis elongation and/or gastrulation defect. In addition, these MOs inhibited elongation of DMZ explants. These results suggested these G proteins might be required for convergent extension. Thus, He investigated functions of these G proteins in the PCP pathway and found that G_i1 and G₁₁ are necessary for the membrane localization of Dsh. G₁₁ MO reduced both hyperphosphorylation of Dsh and the protrusive activity induced by the PCP pathway, whereas G_i1 MO did not. These data indicate that both G_i1 and G₁₁ are required for the Dsh translocation, but these molecules may play distinct roles. These results are shown and discussed in Chapter 3.
　This work has demonstrated that the PCP pathway regulates convergent extension movements through cytoskeletal regulation, and identified molecules essential for the intracellular signaling components in this pathway. These findings may contribute to understand the mechanisms of convergent extension movements and the other developmental processes in which the PCP pathway is involved., application/pdf, 総研大甲第875号}, title = {Cytoskeletal regulation by Wnt signaling in Xenopus gastrulation movements}, year = {} }