Roy Anuradha, Shimizu Sakiko, Kiya Taketoshi, Mita Kazuei, Iwami Masafumi
Division of Life Sciences, Graduate school of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Zoolog Sci. 2012 May;29(5):333-9. doi: 10.2108/zsj.29.333.
The insect brain secretes prothoracicotropic hormone (PTTH), which stimulates the prothoracic gland to synthesize ecdysone. The active metabolite of ecdysone, 20-hydroxyecdysone (20E), works through ecdysone receptor (EcR) and ultraspiracle (USP) to initiate molting and metamorphosis by regulating downstream genes. Previously, we found that EcR was expressed in the PTTH-producing neurosecretory cells (PTPCs) in larval brain of the silkworm Bombyx mori, suggesting that PTPCs function as the master cells of development under the regulation of 20E. To gain a better understanding of the molecular mechanism of the 20E control of PTPCs, we performed a comprehensive screening of genes induced by 20E using DNA microarray with brains of day-2 fifth instar silkworm larvae. Forty-one genes showed greater than twofold changes caused by artificial application of 20E. A subsequent semiquantitative screening identified ten genes upregulated by 20E, four of which were novel or not previously identified as 20E-response genes. Developmental profiling determined that two genes, UP4 and UP5, were correlated with the endogenous ecdysteroid titer. Whole-mount in situ hybridization showed exclusive expression of these two genes in two pairs of cells in the larval brain in response to 20E-induction, suggesting that the cells are PTPCs. BLAST searches revealed that UP4 and UP5 are Bombyx homologs of vrille and tarsal-less, respectively. The present study identifies 20E-induced genes that may be involved in the ecdysone signal hierarchies underlying pupal-adult development and/or the 20E regulation of PTPCs.
昆虫脑部分泌促前胸腺激素（PTTH），该激素刺激前胸腺合成蜕皮激素。蜕皮激素的活性代谢物20-羟基蜕皮激素（20E）通过蜕皮激素受体（EcR）和超气门蛋白（USP）发挥作用，通过调节下游基因来启动蜕皮和变态过程。此前，我们发现EcR在家蚕幼虫脑内产生PTTH的神经分泌细胞（PTPCs）中表达，这表明PTPCs在20E的调控下作为发育的主控细胞发挥作用。为了更好地理解20E对PTPCs的分子调控机制，我们使用DNA微阵列技术，对五龄第二天家蚕幼虫的脑进行了20E诱导基因的全面筛选。41个基因在人工施加20E后出现了两倍以上的变化。随后的半定量筛选确定了10个被20E上调的基因，其中4个是新基因或之前未被鉴定为20E反应基因。发育图谱分析确定，两个基因UP4和UP5与内源性蜕皮甾体滴度相关。全组织原位杂交显示，这两个基因在20E诱导下，在幼虫脑内的两对细胞中特异性表达，表明这些细胞是PTPCs。BLAST搜索显示，UP4和UP5分别是果蝇生物钟基因vrille和跗节缺失基因tarsal-less在家蚕中的同源基因。本研究鉴定出了20E诱导的基因，这些基因可能参与了蛹-成虫发育过程中蜕皮激素信号层级，和/或20E对PTPCs的调控。
