El Haj A J, Tamone S L, Peake M, Sreenivasula Reddy P, Chang E S
School of Biological Sciences, University of Birmingham, UK.
Gene. 1997 Nov 12;201(1-2):127-35. doi: 10.1016/s0378-1119(97)00437-x.
The role of ecdysteroids in modulating exoskeletal growth during the moult cycle of Crustacea has been well described. However, little is known about the action of ecdysteroids at the level of gene transcription and regulation in Crustacea. This paper reports the cloning of an ecdysteroid responsive gene, HHR3, a potential Manduca sexta MHR3 homologue in the American lobster, Homarus americanus. Levels of HHR3 expression are up-regulated in response to in vivo injections of premoult concentrations (10(-6) M) of 20-hydroxyecdysone in the epidermal and muscle tissue of the lobster after 6 h. Maximal mRNA levels are observed after 21 h before returning to basal levels. In muscle tissue, elevated levels of HHR3 mRNA follow a time course similar to elevated actin mRNA expression in response to hormonal injection. In contrast, in eyestalk tissue, the HHR3 levels decline up to 21 h post-injection before rising to basal levels after 48 h. Eyestalk, epidermal and leg muscle tissue was extracted over the moult cycle to determine the levels of expression. In muscle, HHR3 is high during the premoult period that corresponds to the period of the moult cycle when the ecdysteroid titre is high. In the epidermis, HHR3 levels are also high during the premoult with elevated levels maintained into the postmoult period. In the eyestalk, mRNA levels of HHR3 show an opposite pattern of expression with low levels during premoult and postmoult and high levels found during the intermoult period. Our results provide novel evidence for an ecdysteroid responsive gene in a crustacean that has many similarities to MHR3 in Manduca and DHR3 in Drosophila melanogaster. This raises the question of whether a similar cascade of ecdysteroid responsive genes exist in other members of Arthropoda such as the Crustacea, as has been demonstrated in Drosophila. In addition, we provide further evidence for negative feedback regulation of ecdysteroids at the site of moult-inhibiting hormone (MIH) production in the lobster eyestalk.
蜕皮类固醇在调节甲壳纲动物蜕皮周期中外骨骼生长过程中的作用已得到充分描述。然而，关于蜕皮类固醇在甲壳纲动物基因转录和调控水平上的作用却知之甚少。本文报道了一个蜕皮类固醇反应基因HHR3的克隆，它可能是美洲龙虾美洲螯龙虾中烟草天蛾MHR3的同源物。在龙虾的表皮和肌肉组织中，体内注射蜕皮前浓度（10⁻⁶ M）的20-羟基蜕皮酮6小时后，HHR3的表达水平上调。21小时后观察到最大mRNA水平，之后又恢复到基础水平。在肌肉组织中，HHR3 mRNA水平升高的时间进程与注射激素后肌动蛋白mRNA表达升高的时间进程相似。相比之下，在眼柄组织中，注射后21小时内HHR3水平下降，48小时后上升至基础水平。在整个蜕皮周期中提取眼柄、表皮和腿部肌肉组织以确定表达水平。在肌肉中，HHR3在蜕皮前期含量较高，这与蜕皮周期中蜕皮类固醇滴度较高的时期相对应。在表皮中，HHR3水平在蜕皮前期也较高，且在蜕皮后期仍维持在较高水平。在眼柄中，HHR3的mRNA水平呈现相反的表达模式，在蜕皮前期和蜕皮后期含量较低，而在蜕皮间期含量较高。我们的研究结果为甲壳纲动物中一个与烟草天蛾中的MHR3以及黑腹果蝇中的DHR3有许多相似之处的蜕皮类固醇反应基因提供了新的证据。这就提出了一个问题，即像在果蝇中所证明的那样，节肢动物的其他成员如甲壳纲动物中是否也存在类似的蜕皮类固醇反应基因级联。此外，我们还为龙虾眼柄中蜕皮抑制激素（MIH）产生部位的蜕皮类固醇负反馈调节提供了进一步的证据。
