Microsurgical Methods to Make the Keller Sandwich Explant and the Dorsal Isolate.
Microsurgical Methods to Make the Keller Sandwich Explant and the Dorsal Isolate.
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DOI：
10.1101/pdb.prot097386
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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
本方案总结了背侧边缘区夹层外植体（又名“Keller夹层”）和背侧分离体的制备。Keller三明治由两个早期原肠期背缘区（DMZ）外植体组装而成。在内化之前分离的DMZ外植体维持平面图案过程，并阻断可能在内化前后组织之间交换的径向信号。DMZ外植体在原肠胚后期分离，但随后打开和变平，可能具有平面和径向图案。在Keller三明治中，非军事区外植体的上皮边缘愈合，两个非军事区外植体的深层之间形成基底接触。背侧分离体是从中期到晚期原肠期胚胎中分离出来的，经过复归和肠原肠形成。由原肠胚形成运动产生的背侧内胚层、中胚层和外胚层之间的细菌层接触在背侧分离体中保持。这两种外植体可用于研究与趋同延伸相关的组织、细胞和亚细胞过程，从模式到细胞行为，以及它们的集体生物力学。解剖凯勒三明治所需的技能比解剖动物帽外胚层所需的技能更大，可以在几周内掌握；解剖背分离物所需的技能与解剖动物帽所需的技能相似，可以在一周内学会。
This protocol summarizes preparation of the dorsal marginal zone sandwich explant (a.k.a. the “Keller Sandwich”) and the dorsal isolate. The Keller sandwich is assembled from two early gastrula stage dorsal marginal zone (DMZ) explants. DMZ explants isolated before involution maintain planar patterning processes and block radial signals that might be exchanged between pre- and post-involution tissues. DMZ explants isolated later in gastrulation, but subsequently opened and flattened may have both planar and radial patterning. The epithelial margins of DMZ explants in Keller sandwiches heal and basal contacts form between the deep layers of the two DMZ explants. The dorsal isolate is dissected from mid- to late-stage gastrula stage embryos after involution and archenteron formation. Germ-layer contacts between dorsal endoderm, mesoderm, and ectoderm generated by gastrulation movements are maintained in the dorsal isolate. These two explants can be used to study tissue-, cell-, and subcellular-processes relevant to convergent extension, from patterning to cell behaviors, and their collective biomechanics. Skills needed to dissect the Keller sandwich are greater than those needed to dissect animal cap ectoderm and can be mastered in a few weeks; skills needed to dissect the dorsal isolate are similar to those needed to dissect animal caps and can be learned in a week.