Resolving in vivo gene expression during collective cell migration using an integrated RNAscope, immunohistochemistry and tissue clearing method.
Resolving in vivo gene expression during collective cell migration using an integrated RNAscope, immunohistochemistry and tissue clearing method.
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DOI：
10.1016/j.mod.2017.06.004
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发表时间：
2017-12
影响因子：
2.6
通讯作者：
Kulesa PM
中科院分区：
文献类型：
--
作者：
Morrison JA;McKinney MC;Kulesa PM
在集体细胞迁移过程中，单个细胞表现出多种行为，这使我们对细胞群体的方向决策和凝聚力的理解变得复杂。体内基因和蛋白质表达分析将有助于揭示潜在的分子机制。然而，由于难以整合单细胞检测方法以及同时读取胚胎深处的信号，这方面的信息一直有限。在此，我们优化并整合了RNAscope多重荧光原位杂交、免疫组织化学和组织透明化技术，以观察完整鸡胚深处单个细胞内的转录本和蛋白质定位。利用标准共聚焦显微镜，我们在2日龄透明化鸡胚中，在蛋白质标记的HNK1阳性迁移性颅神经嵴细胞内同时观察多达3个基因的mRNA表达。分别使用斑点计数和多线轨迹图方法对相邻细胞之间或亚区域内测量的基因表达差异进行定量。这些方法的优化和整合为集体细胞迁移提供了一种改进的三维体内分子研究方法，并为拓展到更广泛的胚胎和成年模型系统奠定了基础。
During collective cell migration individual cells display diverse behaviors that complicate our understanding of group cell decisions of direction and cohesion. In vivo gene and protein expression analyses would shed light on the underlying molecular choreography. However, this information has been limited due to difficulties to integrate single cell detection methods and the simultaneous readout of signals deep within the embryo. Here, we optimize and integrate multiplex fluorescence in situ hybridization by RNAscope, immunohistochemistry, and tissue clearing to visualize transcript and protein localization within single cells deep within intact chick embryos. Using standard confocal microscopy, we visualize the mRNA expression of up to 3 genes simultaneously within protein labeled HNK1-positive migrating cranial neural crest cells within 2 day old cleared chick embryos. Gene expression differences measured between adjacent cells or within subregions are quantified using spot counting and polyline kymograph methods, respectively. This optimization and integration of methods provide an improved 3D in vivo molecular interrogation of collective cell migration and foundation to broaden into a wider range of embryo and adult model systems.
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DOI：
10.1073/pnas.1700307114
发表时间：
2017-02-28
影响因子：
11.1
作者：
Nerurkar, Nandan L.;Mahadevan, L.;Tabin, Clifford J.
通讯作者：
Tabin, Clifford J.
影响因子：
3.7
作者：
Grabinski TM;Kneynsberg A;Manfredsson FP;Kanaan NM
通讯作者：
Kanaan NM
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
1.2
作者：
Jolly, Sarah;Fudge, Alexander;Li, Huiliang
通讯作者：
Li, Huiliang
哺乳动物大脑的可扩展且兼容 Dil 的光学间隙
影响因子：
2.9
作者：
Hou B;Zhang D;Zhao S;Wei M;Yang Z;Wang S;Wang J;Zhang X;Liu B;Fan L;Li Y;Qiu Z;Zhang C;Jiang T
通讯作者：
Jiang T