A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole
A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole
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DOI：
10.1016/j.ydbio.2017.08.012
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发表时间：
2018-01-15
影响因子：
2.7
通讯作者：
Mochii, Makoto
中科院分区：
文献类型：
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作者：
Sato, Kentaro;Umesono, Yoshihiko;Mochii, Makoto
创伤的快速愈合和随后顶端上皮帽(AEC)的形成被认为是成功的两栖动物附属物再生所必需的。尽管AEC在肢体再生中起着重要的作用，但其在尾巴再生中的作用以及调控伤口愈合和AEC形成的机制尚不清楚。我们先前发现非洲爪哇ES1基因主要在损伤区域表达，包括尾巴再生后的AEC。在这项研究中，我们建立并鉴定了在ES1基因调控序列(ES1：EGFP)控制下携带增强型绿色荧光蛋白(EGFP)基因的非洲爪哇转基因品系。在胚胎的几个区域可以清楚地看到EGFP报告基因的表达，然后在幼虫中下降到检测不到的水平，重复了内源ES1的表达。蝌蚪尾巴切断后，EGFP在残肢表皮边缘重新激活，然后在覆盖截肢表面的伤口表皮(WE)表达增强。随着残肢开始再生，EGFP的表达开始局限于最远端的表皮区域，包括AEC。EGFP优先在WE和AEC的基底层或深层细胞中表达，而不在表层细胞中表达。我们进行了一项小规模的药理学筛选，以寻找影响尾部切除后残端表皮中EGFP表达的化学物质。用ERK、转化生长因子-β或活性氧簇(ROS)信号转导抑制剂处理可减弱EGFP的表达。这些处理也损害了截肢表面的伤口闭合，提示这三种信号活动是ES1在WE中表达和尾部截尾后伤口成功愈合所必需的。这些发现表明ES1：EGFP是分析创伤愈合和附件再生调控分子机制的有用工具。
Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp).The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC.We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-beta or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation.These findings showed that es1:egfp Xenopus laeuis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.