A Targeted UAS-RNAi Screen in Drosophila Larvae Identifies Wound Closure Genes Regulating Distinct Cellular Processes
A Targeted UAS-RNAi Screen in Drosophila Larvae Identifies Wound Closure Genes Regulating Distinct Cellular Processes
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DOI：
10.1534/genetics.110.121822
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发表时间：
2010-11-01
期刊：
影响因子：
3.3
通讯作者：
Galko, Michael J.
中科院分区：
文献类型：
--
作者：
Lesch, Christine;Jo, Juyeon;Galko, Michael J.
强大的组织修复机制对于多细胞生物体的生存至关重要。有效的皮肤伤口修复需要在伤口边缘和更远的表皮层内的细胞迁移，但控制和协调这些迁移的基因仍然不清楚。这在一定程度上是因为系统的筛选方法，在体内鉴定和分类胚后伤口闭合基因尚未开发。在这里，我们在果蝇幼虫表皮中进行了一个基于原理验证的体内RNAi筛选，以确定正常伤口闭合所需的基因。在测试的候选基因中，有Jun N-末端激酶（JNK）信号通路的激酶和转录介质，这些激酶和转录介质被证明是发育过程中上皮片迁移所需的。还靶向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敲低引起开放性伤口，与肌动蛋白细胞骨架重塑相关的几个基因也是如此。我们对JNK通路组分的分析揭示了上游激活激酶之间的冗余以及下游转录因子DJun和DFos的不同作用。开放性伤口表型的定量和定性形态学分类和JNK活化的评价表明，在迁移的表皮细胞中需要多个细胞过程，包括对伤口边缘的细胞特异性的功能和对表皮层内更远的细胞特异性的其他功能。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，确定这些基因在迁移的表皮片推定的功能作用，并提供了一个模板，更广泛的体内RNAi筛选，以发现完整的补充，在幼虫表皮伤口愈合过程中伤口闭合所需的基因。
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.