Microsurgical Methods to Isolate and Culture the Early Gastrula Dorsal Marginal Zone.
Microsurgical Methods to Isolate and Culture the Early Gastrula Dorsal Marginal Zone.
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DOI：
10.1101/pdb.prot097360
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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
该方案描述了边缘带外植体的制备，该外植体可用于揭示在背部组织中正常工作的细胞行为和组织运动。背部外植体的组成反映了在背部组织中存在的不同的祖细胞集，包括中内胚层、头部中胚层、前索状中胚层、有瓶状细胞的内胚层、预期脊索的轴向中胚层、体节的轴向中胚层、侧板中胚层、神经外胚层和外胚层。与类器官不同，背缘区(DMZ)外植体是“器官类型”的，因为除了从胚胎中分离组织所需的操作外，显微手术不会破坏自然组织。器官型DMZ外植体从早期原肠胚中分离出来，保留了天然边缘区域存在的边界和紧密的组织联系。根据不同的阶段，外植体和组织分离株中可以保持图案和细胞的特性。与构图相似，局部细胞的运动和行为可能被保留，然而，它们集体运动的大规模生物力学影响可能与自然边缘区域的运动不同。例如，DMZ外植体中的内卷通常被抑制，排除了在原肠形成过程中实现的中胚层和预期的神经外胚层的两层结合。DMZ外植体可以通过多种方式安装和成像，展示一个或多个有趣的细胞行为或集体运动，如中轴和轴旁中胚层的内侧细胞嵌入、瓶状细胞的顶端收缩和中内胚层的定向迁移。扁平的DMZ外植体还可用于研究新的组织定义边界的出现，如脊索-体细胞边界、外胚层-中胚层边界、中内胚层-中胚层边界。显微手术分离DMZ外植体所需的技能比解剖动物帽外胚层所需的技能更高，但可以在几周内掌握。
This protocol describes the preparation of marginal zone explants that can be used to to expose cell behaviors and tissue movements that normally operate in dorsal tissues. The composition of dorsal explants reflect the diverse set of progenitor cells that can be found in dorsal tissues including mesendoderm, head mesoderm, prechordal mesoderm, endoderm with bottle cells, axial mesoderm of the prospective notochord, paraxial mesoderm of the somites, lateral plate mesoderm, neural ectoderm, and ectoderm. Unlike an organoid, the dorsal marginal zone (DMZ) explant is ‘organotypic’ in that microsurgery does not disrupt native tissue organization beyond those manipulations needed to dissect the tissue from the embryo. An organotypic DMZ explant dissected from the early gastrula preserves boundaries and close tissue associations present in the native marginal zone. Depending on the stage, patterning and cell identities can be maintained in the explants and tissue isolates. Similar to patterning, local cell movements and behaviors may be preserved, however, the large-scale biomechanical impact of their collective movements may be altered from those in the native marginal zone. For instance, involution is typically inhibited in the DMZ explant precluding the two layer association of mesoderm and prospective neural ectoderm that is achieved during gastrulation. The DMZ explant may be mounted and imaged in a variety of ways, exposing one or more interesting cell behaviors or collective movements such as mediolateral cell intercalation in the axial and paraxial mesoderm, apical constriction of bottle cells, and directional migration of mesendoderm. The flattened DMZ explant can also be used to study emergence of new tissue-defining boundaries such as the notochord-somite boundary, the ectoderm-mesoderm boundary, and the mesendoderm-mesoderm boundary. Skills needed to microsurgically isolate the DMZ explant are greater than those needed to dissect animal cap ectoderm but can be mastered in a few weeks.