Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
Methods Mol Biol. 2021;2258:43-56. doi: 10.1007/978-1-0716-1174-6_4.
Development of multicellular organisms depends on the proper establishment of signaling information in space and time. Secreted molecules called morphogens form concentration gradients in space and provide positional information to differentiating cells within the organism. Although the key molecular components of morphogen pathways have been identified, how the architectures and key parameters of morphogen pathways control the properties of signaling gradients, such as their size, speed, and robustness to perturbations, remains challenging to study in developing embryos. Reconstituting morphogen gradients in cell culture provides an alternative approach to address this question. Here we describe the methodology for reconstituting Sonic Hedgehog (SHH) signaling gradients in mouse fibroblast cells. The protocol includes the design of morphogen sending and receiving cell lines, the setup of radial and linear gradients, the quantitative time-lapse imaging, and the data analysis. Similar approaches could potentially be applied to other cell-cell communication pathways.
多细胞生物的发育依赖于信号信息在空间和时间上的正确建立。称为形态发生素的分泌分子在空间中形成浓度梯度，并为生物体内部的分化细胞提供位置信息。尽管已经确定了形态发生素途径的关键分子成分，但形态发生素途径的结构和关键参数如何控制信号梯度的特性，例如它们的大小、速度和对扰动的鲁棒性，在发育中的胚胎中仍然难以研究。在细胞培养中重建形态发生素梯度提供了一种解决这个问题的替代方法。在这里，我们描述了在小鼠成纤维细胞中重建 Sonic Hedgehog (SHH) 信号梯度的方法。该方案包括设计形态发生素发送和接收细胞系、建立径向和线性梯度、定量延时成像以及数据分析。类似的方法可能可以应用于其他细胞间通讯途径。
