Department of Biochemistry and Molecular Biology, University of TexasGraduate School of Biomedical Sciences, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.
Genetics. 2010 Nov;186(3):943-57. doi: 10.1534/genetics.110.121822. Epub 2010 Sep 2.
Robust mechanisms for tissue repair are critical for survival of multicellular organisms. Efficient cutaneous wound repair requires the migration of cells at the wound edge and farther back within the epidermal sheet, but the genes that control and coordinate these migrations remain obscure. This is in part because a systematic screening approach for in vivo identification and classification of postembryonic wound closure genes has yet to be developed. Here, we performed a proof-of-principle reporter-based in vivo RNAi screen in the Drosophila melanogaster larval epidermis to identify genes required for normal wound closure. Among the candidate genes tested were kinases and transcriptional mediators of the Jun N-terminal kinase (JNK) signaling pathway shown to be required for epithelial sheet migration during development. Also targeted were genes involved in actin cytoskeletal remodeling. Importantly, RNAi knockdown of both canonical and noncanonical members of the JNK pathway caused open wounds, as did several genes involved in actin cytoskeletal remodeling. Our analysis of JNK pathway components reveals redundancy among the upstream activating kinases and distinct roles for the downstream transcription factors DJun and DFos. Quantitative and qualitative morphological classification of the open wound phenotypes and evaluation of JNK activation suggest that multiple cellular processes are required in the migrating epidermal cells, including functions specific to cells at the wound edge and others specific to cells farther back within the epidermal sheet. Together, our results identify a new set of conserved wound closure genes, determine putative functional roles for these genes within the migrating epidermal sheet, and provide a template for a broader in vivo RNAi screen to discover the full complement of genes required for wound closure during larval epidermal wound healing.
组织修复的稳健机制对于多细胞生物的生存至关重要。有效的皮肤伤口修复需要细胞在伤口边缘和表皮片中更远的部位迁移，但控制和协调这些迁移的基因仍然不清楚。部分原因是，尚未开发出一种用于体内鉴定和分类胚胎后伤口闭合基因的系统筛选方法。在这里，我们在黑腹果蝇幼虫表皮中进行了基于报告基因的体内 RNAi 筛选实验，以鉴定正常伤口闭合所需的基因。在测试的候选基因中，有丝裂原活化蛋白激酶（MAPK）和 Jun N 末端激酶（JNK）信号通路的转录调节剂，这些基因在发育过程中上皮片迁移是必需的。还靶向了参与肌动蛋白细胞骨架重塑的基因。重要的是，JNK 通路的经典和非经典成员的 RNAi 敲低都会导致伤口张开，参与肌动蛋白细胞骨架重塑的几个基因也是如此。我们对 JNK 通路成分的分析揭示了上游激活激酶之间的冗余性，以及下游转录因子 DJun 和 DFos 的不同作用。对张开伤口的表型进行定量和定性形态分类，并评估 JNK 激活情况表明，迁移的表皮细胞中需要多种细胞过程，包括伤口边缘细胞特有的功能和表皮片中更远部位细胞特有的功能。总之，我们的研究结果确定了一组新的保守的伤口闭合基因，确定了这些基因在迁移的表皮片中的潜在功能作用，并为更广泛的体内 RNAi 筛选提供了模板，以发现幼虫表皮伤口愈合过程中所需的完整基因。
