Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening.
Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening.
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通过大规模并行克隆筛选揭示β-连环蛋白依赖性Wnt 信号传导的动力学和进化。
DOI：
10.1039/c4ib00050a
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Allbritton,NancyL
中科院分区：
文献类型：
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作者：
Shah,PavakK;Walker,MatthewP;Sims,ChristopherE;Major,MichaelB;Allbritton,NancyL
Wnt/β-catenin信号传导由于其在调节发育、组织再生和疾病中的作用而受到显著关注。转录报告基因已被广泛用于研究活细胞和整个生物体中的Wnt/β-catenin信号转导，并已被应用于了解胚胎发育，探索肿瘤发生和开发治疗方法。报道细胞系中的多克隆异质性在历史上被视为在开发新型细胞系和基于荧光素酶的测定中要克服的挑战，并且通常采用单克隆报道细胞系来减少这种变异性。在短（<6）代和长（> 25）代时间尺度上筛选用Wnt/β-连环蛋白信号传导的报道基因感染的A375细胞系。为了表征这些时间尺度上的表型差异，开发了基于微制造细胞阵列的筛选，使得能够并行表征1119个克隆集落。该筛选在25代后显示表型分化。<6 generations at a similar scale to that observed in monoclonal cell lines cultured for >不仅观察到报告动力学差异很大，而且观察到单克隆细胞系具有看似相反的信号传导表型。此外，这些观察结果揭示了A375细胞中Wnt信号传导的世代依赖性趋势，这提供了对正反馈和自我抑制途径机制的深入了解。
Wnt/β-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/β-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/β-catenin signaling were screened over short (<6) and long (>25) generational timescales. To characterize phenotypic divergence over these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1119 clonal colonies in parallel. This screen revealed phenotypic divergence after <6 generations at a similar scale to that observed in monoclonal cell lines cultured for >25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provides insight into the pathway's mechanisms of positive feedback and self-inhibition.