Department of Surgery, Keck School of Medicine of USC.
Department of Surgery, Keck School of Medicine of USC; Department of Pediatrics, Keck School of Medicine of USC; Heart Institute, Children's Hospital Los Angeles;
J Vis Exp. 2021 Dec 10(178). doi: 10.3791/63247.
Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches. This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.
非经典 Wnt 信号调节胚胎发生过程中祖细胞的细胞内肌动蛋白丝组织和极化迁移。这个过程需要信号发送细胞和信号接收细胞之间复杂和协调的旁分泌相互作用。鉴于这些相互作用可以发生在不同谱系的各种类型的细胞之间，体内评估细胞特异性缺陷可能具有挑战性。本研究描述了一种高度可重复的方法来评估体外旁分泌非经典 Wnt 信号。该方案的设计具有以下能力：（1）对任何两种感兴趣的细胞类型之间的非经典 Wnt 信号进行功能和分子评估；（2）在非经典 Wnt 信号通路中剖析信号发送分子与信号接收分子的作用；（3）使用标准的分子或药理学方法进行表型挽救实验。该方案用于评估神经嵴细胞 (NCC) 介导的成肌细胞中的非经典 Wnt 信号。NCC 的存在与成肌细胞中更多的鬼笔环肽阳性细胞质丝状伪足和片状伪足以及划痕愈合测定中成肌细胞迁移的改善有关。Wnt5a-ROR2 轴被确定为 NCC 和第二心脏场 (SHF) 心肌细胞祖细胞之间的关键非经典 Wnt 信号通路。总之，这是一种高度可行的方案，可以研究体外旁分泌非经典 Wnt 信号机制。
