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ラパログ依存的な新規細胞間接着制御系による中胚葉遊走の解析

宇佐美, 知沙 大阪大学

2022.06.15

概要

細胞間接着は多細胞生物の構造を確立し維持する上で非常に重要である。その中でも主要な役割を担っているカドヘリンはカルシウムイオン依存性の細胞間接着分子であり、細胞外ドメインを介して近隣細胞のカドヘリンと結合し、細胞同士を接着させる。カドヘリンの発現量や活性は動的に制御されており、そのダイナミクスはエピボリー運動や神経堤細胞の遊走、収斂伸長運動といった様々な発生プロセスに関与することが知られている。

動的な細胞間接着制御が重要な発生プロセスの一つとして、原腸形成運動が挙げられる。原腸形成は初期胚における主要な発生イベントの1つであり、組織の大きな変形を伴い、前後軸・神経パターン・原腸といった基本的なボディプランが構築される。

アフリカツメガエルの原腸形成過程において、中胚葉はblastocoel roof (BCR)上を動物極方向へと遊走する。この時、中胚葉−BCR間の接着頻度が遊走を制御していると考えられているが、接着頻度と遊走の関係を直接的に解析する実験手法は確立されていない。そこで本研究では、小分子化合物ラパマイシンのアナログであるラパログを用いて、異なる組織間の接着を特異的に誘起する実験系を新たに開発した。

まず、ラパログ特異的に細胞間接着を誘起させるための細胞膜タンパク質であるriCAM1とriCAM2を作製した。細胞にこれらのタンパク質を発現させ、カルシウムイオンを含まないメディウム中で解離させた状態でラパログを添加すると、細胞間接着が誘起された。また、接着パターンを解析した結果、ラパログはriCAM1発現細胞とriCAM2発現細胞間の接着のみを誘起し、同種のriCAMを発現している細胞間の接着には影響を与えなかった。

続いて、BCRにriCAM1、中胚葉にriCAM2を独立に発現させ、ラパログを添加することでこれらの組織間の接着を特異的に増強させると、中胚葉の遊走が強く阻害された。また、riCAMの発現とラパログの添加は、中胚葉とBCR間の組織境界であるBrachet’s cleftの形成には影響を及ぼさなかった。さらに、riCAM1を発現しているBCR断片上に riCAM2を発現している中胚葉組織をマウントし、組織境界における細胞の挙動を観察した結果、ラパログが両組織間の接着頻度を増強させることを定量的に明らかにした。

これらの結果は、中胚葉−BCR間の接着頻度が中胚葉遊走を制御するというモデルを強く支持している。さらに、本実験系がin vivoにおいて組織間接着を特異的に制御できる有用な手法であることを示している。

Cell-cell adhesion is very important in establishing and maintaining the structure of multicellular organisms. Cadherins, which play a major role in this process, are Ca2+-dependent intercellular adhesion molecules that bind to cadherins of neighboring cells via their extracellular domains, resulting in cell-cell adhesion. Cadherin expression and activity are dynamically regulated, and its dynamics are known to be involved in various developmental processes such as epiboly, neural crest cell migration, and convergent extension.

One developmental process in which dynamic regulation of intercellular adhesion is important is the gastrulation movement. Gastrulation is one of the major developmental events in the early embryo and involves major tissue deformation and the establishment of basic body plans such as the anterior- posterior axis, neural patterns, and archenteron.

During the gastrulation of the Xenopus laevis, the mesoderm migrates toward the animal pole on the blastocoel roof (BCR). Although the frequency of adhesion between mesoderm and BCR is thought to regulate migration, no direct experimental method has been established to analyze the relationship between the frequency of adhesion and migration. In this study, I developed a new experimental system that specifically induces adhesion between different tissues using rapalog, an analog of rapamycin.

First, I produced two membrane proteins, riCAM1 and riCAM2, to induce cell-cell adhesion specifically with rapalog. When cells expressed these proteins and dissociated them in Ca2+-free medium and added rapalog, intercellular adhesion was induced. Analysis of adhesion patterns showed that rapalog induced adhesion only between riCAM1- and riCAM2-expressing cells and did not affect adhesion between cells expressing the same riCAM.

When riCAM1 and riCAM2 were independently expressed in the BCR and mesoderm, respectively, and adhesion between these tissues was specifically enhanced by the addition of rapalog, migration of the mesoderm was strongly inhibited. Expression of riCAM and addition of rapalog had no effect on the formation of Brachet's cleft, the tissue boundary between the mesoderm and BCR. Furthermore, I mounted mesodermal tissue expressing riCAM2 on BCR fragments expressing riCAM1 and observed cell behavior at the tissue boundary, quantitatively demonstrating that rapalog enhanced the frequency of adhesion between the two tissues.

These results strongly support the model that mesoderm-BCR adhesion frequency regulates mesoderm migration. Furthermore, they demonstrate that this experimental system is a useful method for specifically regulating intertissue adhesion in vivo.

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