Department of Biology and Neuroscience Program, Hamline University;
Department of Genetics, Cell Biology and Development, University of Minnesota; Developmental Biology Center, University of Minnesota.
J Vis Exp. 2022 Jun 27(184). doi: 10.3791/63799.
During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.
在脊椎动物发育过程中，神经嵴细胞（NCC）广泛迁移并分化为各种细胞类型，这些细胞类型有助于构建颅面骨骼和周围神经系统等结构。虽然了解 3D 胚胎中的 NCC 迁移至关重要，但在 2D 培养中分离迁移细胞有利于可视化和功能特征分析，从而补充胚胎研究。本方案演示了一种从鸡颅神经褶中分离初级 NCC 培养物的方法。将神经褶外植体接种到纤维连接蛋白包被的基质上，可使迁移的 NCC 从中逸出。这会产生分散的、贴壁的 NCC 群体，可以通过染色和定量形态分析进行评估。这种简化的培养方法具有高度的适应性，可与其他技术结合使用。例如，通过延时成像可以评估 NCC 的迁出和迁移行为，或者通过包括抑制剂或对基因表达的实验操作（例如，DNA、莫洛尼或 CRISPR 电穿孔）来对其进行功能研究。由于其多功能性，该方法为研究颅 NCC 发育提供了一个强大的系统。
