GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo
GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo
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DOI：
10.1016/j.modgep.2006.08.003
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发表时间：
2007-01-01
影响因子：
1.2
通讯作者：
Baeg, Gyeong-Hun
中科院分区：
文献类型：
--
作者：
Bach, Erika A.;Ekas, Laura A.;Baeg, Gyeong-Hun
JAK/STAT信号通路在果蝇的许多发育过程中起着重要作用。JAK/STAT途径参与这些过程的机制是最近研究的主题。然而，尚未开发出反映所有果蝇组织中JAK/STAT途径活性的报告基因。通过将Stm 92 E靶基因Socs 36 E的片段（其含有至少两个推定的Stat 92 E结合位点）置于GFP的上游，我们产生了可用于监测体内JAK/STAT途径活性的三种构建体。这些构建体的不同之处在于Stat 92 E结合位点的数量和GFP的稳定性。2XSTAT 92 E-GFP和10 XSTAT 92 E-GFP构建体分别含有2个和10个Stat 92 E结合位点，驱动增强的GFP的表达，而10 XSTAT 92 E-DGFP驱动不稳定的GFP的表达。我们表明，这些报告基因在胚胎中表达的重叠模式与Stat 92 E蛋白和JAK/STAT信号是必需的组织。此外，这些报告准确地反映了JAK/STAT途径的活性在幼虫阶段，因为它们的表达模式重叠的活化配体未配对的成虫盘。此外，STAT 92 E-GFP报告基因被异位JAK/STAT信号激活。STAT 92 E-GFP荧光响应于幼虫眼盘中的异位upd和成体脂肪体中JAK激酶跳房子的错误表达而增加。最后，这些报告基因被Stat 92 E特异性激活，因为在stat 92 E纯合突变组织中，STAT 92 E-GFP报告基因的表达在细胞内自主丧失。总之，我们已经产生了体内GFP报告基因，其准确地反映了多种组织中JAK/STAT途径活化。这些报告基因是进一步研究和理解JAK/STAT信号在果蝇中作用的重要工具。(c)2006 Elsevier B. V.保留所有权利。
JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the Subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stm92E target gene Socs36E, which contains at least two Putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These Constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. in sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila. (c) 2006 Elsevier B.V. All rights reserved.