Huet F, Ruiz C, Richards G
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
Development. 1993 Jun;118(2):613-27. doi: 10.1242/dev.118.2.613.
The steroid hormone ecdysone orchestrates insect development by regulating gene networks. In Drosophila the most detailed description of ecdysone action is the sequential activation of early and late puffs in the polytene chromosomes of the late larval salivary gland. A number of these early puffs (2B5, 74EF and 75B) contain complex transcription units (Broad-Complex, E74 and E75 respectively) encoding families of regulatory proteins which are expressed in most if not all tissues. In vitro, transcripts of the different isoforms of these early genes as well as the ecdysone receptor (EcR) present varying dose response characteristics (Karim and Thummel, 1992, EMBO J. 11, 4083-4093). We have developed an in vivo approach using a reverse transcription-polymerase chain reaction assay (RT-PCR) so as to visualise these transcripts in the RNA extracted from a single salivary gland. Using one salivary gland lobe for developmental puff staging and the sister lobe for RT-PCR, we have obtained precise developmental profiles for these transcripts and have extended our study to other tissues and stages where puffing studies were not possible. In the salivary gland we have characterised three distinct ecdysone responses. For the mid and late third larval instar responses our results confirm and extend the conclusions of the in vitro studies concerning the temporal expression of the early gene isoforms. The relatively brief prepupal response contains elements in common with each of the larval responses and all three can be explained by the profiles of the respective ecdysone peaks. Interestingly EcR transcripts respond differently during each response. The analysis of different tissues of the same animal reveals subtle differences in the timing of the ecdysone response and isoform expression and suggests that this may reflect tissue differences in the ecdysone profiles. As these molecules have homologues in vertebrates, our analysis may have general implications for the organisation of hormonal responses in vivo.
类固醇激素蜕皮激素通过调控基因网络来协调昆虫的发育。在果蝇中，对蜕皮激素作用的最详细描述是其在晚期幼虫唾液腺多线染色体中对早期和晚期胀泡的顺序激活。这些早期胀泡中的许多（2B5、74EF和75B）都包含复杂的转录单元（分别为Broad-Complex、E74和E75），它们编码在大多数（如果不是所有）组织中表达的调节蛋白家族。在体外，这些早期基因不同异构体的转录本以及蜕皮激素受体（EcR）呈现出不同的剂量反应特性（卡里姆和萨默尔，1992年，《欧洲分子生物学组织杂志》11卷，4083 - 4093页）。我们开发了一种体内方法，使用逆转录 - 聚合酶链反应测定法（RT-PCR），以便在从单个唾液腺提取的RNA中观察这些转录本。利用一个唾液腺叶进行发育胀泡分期，另一个叶进行RT-PCR，我们获得了这些转录本精确的发育图谱，并将研究扩展到了无法进行胀泡研究的其他组织和阶段。在唾液腺中，我们鉴定出了三种不同的蜕皮激素反应。对于第三龄幼虫中期和后期的反应，我们的结果证实并扩展了体外研究中关于早期基因异构体时间表达的结论。相对短暂的蛹前期反应包含与每个幼虫反应共有的元素，并且所有这三种反应都可以用各自蜕皮激素峰值的图谱来解释。有趣的是，EcR转录本在每个反应期间的反应不同。对同一动物不同组织的分析揭示了蜕皮激素反应和异构体表达时间上的细微差异，并表明这可能反映了蜕皮激素图谱中的组织差异。由于这些分子在脊椎动物中有同源物，我们的分析可能对体内激素反应的组织具有普遍意义。
