Microsurgical Manipulations to Isolate Collectively Migrating Mesendoderm.
Microsurgical Manipulations to Isolate Collectively Migrating Mesendoderm.
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DOI：
10.1101/pdb.prot097378
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发表时间：
2022-11-01
影响因子：
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通讯作者：
Davidson, Lance A
中科院分区：
文献类型：
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作者：
Davidson, Lance A
中内胚层外套膜闭合完成了非洲爪蟾胚胎的原肠胚形成运动，并提供了一个无与伦比的机会来研究间充质组织内的集体细胞行为。这些组织的自由边缘片状集体运动与上皮组织的运动形成对比，因为中内胚层细胞不受紧密连接或粘附连接的约束，但在几个小时内以连贯和持久的模式迁移。中内胚层细胞是非洲爪蟾胚胎中最大的运动细胞，并完成500 µm的迁移路径。当中内胚层在刚性玻璃基质上培养时，这些细胞的长度可超过100 µm，并显示出高度持久的领先片状伪足，从尖端到基部可超过20 µm。这些大的集体迁移的细胞提供了一个独特的成像机会，可视化极化的粘合剂和细胞骨架结构与高数值孔径物镜。早期胚胎中的中内胚层细胞起源于整个边缘区周围，也可以通过它们的来源沿动物-植物轴沿着来区分。这里我们使用术语中内胚层，但注意这些细胞的替代术语可以包括：头部中胚层，内中胚层和前索中胚层。本方案总结了中内胚层组织外植体和“开窗”胚胎的显微外科制备。解剖中内胚层外套膜碎片所需的技能略大于分离动物帽外胚层所需的技能，可以在两周内掌握;分离中内胚层“环”或准备开窗胚胎所需的技能明显更大，可能需要几周的培训。
Mesendoderm mantle closure completes the gastrulation movements of the Xenopus laevis embryo and provides an unparalleled opportunity to study collective cell behaviors within a mesenchymal tissue. Free-edge sheet-like collective movements of these tissues contrast with movements of epithelial tissues in that mesendodermal cells are not constrained by tight junctions or adherens junctions, yet migrate in a coherent and persistent mode over several hours. Mesendoderm cells are the largest motile cells in the Xenopus embryo and complete a 500 µm migratory path. When mesendoderm is cultured on rigid glass substrates, these cells can exceed 100 µm in length and exhibit a highly persistent leading lamellipodia that can exceed 20 µm from tip to base. These large collectively migrating cells provide a unique imaging opportunity to visualize polarized adhesive and cytoskeletal structures with high numerical aperture objectives. Mesendodermal cells in the early embryo originate from around the entirety of the marginal zone and may also be distinguished by their source along the animal-vegetal axis. Here we use the term mesendoderm but note alternative terms for these cells can include: head mesoderm, endomesoderm, and prechordal mesoderm. This protocol summarizes microsurgical preparation of mesendoderm tissue explants and ‘windowed’ embryos. Skills needed to dissect fragments of the mesendoderm mantle are marginally greater than those needed to isolate animal cap ectoderm and can be mastered within two weeks; skills needed to isolate the mesendoderm “ring” or to prepare windowed embryos are significantly greater and may require several weeks training.