Department of Cell and Molecular Biology, St. Jude Children's Research Hospital.
Department of Cell and Molecular Biology, St. Jude Children's Research Hospital; St. Jude Graduate School of Biomedical Sciences.
J Vis Exp. 2022 Jun 16(184). doi: 10.3791/64100.
Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.
发育组织模式形成和发育后组织稳态依赖于称为形态发生素的细胞信号的受控传递。形态发生素以浓度和时间依赖性方式发挥作用，指定特定的转录程序，指导和强化细胞命运。确保适当的形态发生素信号阈值的一种机制是通过专门的纤毛状突起（称为胞突）传递信号蛋白。胞突非常细（直径≤200nm），可以生长到数百微米的长度，这使得它们在固定图像分析中难以保存。本文描述了一种精细处理小鼠胚胎的改良方法，用于固定、免疫染色和厚切片，以便使用标准共焦显微镜观察胞突。该方案已成功用于可视化在小鼠神经管发育过程中连接不同细胞信号隔室的胞突。该技术还可以适应于检测不同组织类型的胞突，以促进以前所未有的分辨率对发育信号的探究。
