T-box家族基因Doc和omb在果蝇翅芽发育过程中调控沟形成的作用机理

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. These subdivision are crucial for correct adult wing formation. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. In our recent paper (Development,2012),we demonstrated that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. The expression pattern of the Doc1, Doc2 and Doc3 (collectively Doc) genes appears indistinguishable during embryogenesis(Hamaguchi et al., 2004; Reim et al., 2003) and in the wing imaginal disc and are functionally redundant (Butler et al., 2003).In the wing pouch bending process, cells at the D/V boundary detach from the basal membrane, shorten and acquire a wedge-shaped morphology (Dominguez-Gimenez et al., 2007).This process is likely to contribute to the force that causes the doubling-up of the flat pouch epithelium (a model of D/V bending). Our data showed that Doc is required for proper hinge development by deepening the B/H folds of the wing disc. Doc expressing B/H fold cells have increased MMP levels, causing enhanced ECM degradation. This might facilitate the concerted migration of fold cells. Doc could contribute to the wing disc bending process by promoting the movement of B/H fold cells, possibly emulating its role in amnioserosa development (a model of B/H folds bending). To distinguish the two models of D/V bending and B/H folds bending, in this study, we focused on the three Doc genes and manipulated their expression level in B/H folds by a specific Gal4 line, Doc1-Gal4, which is only expressed in the B/H folds.This strategy would not affect the D/V bending, thus, would provide evidences to assay which model plays more important role during wing disc bending. Manipulation of gene expression is mainly performed by genetic tools such as transgenes and the Gal4-UAS system. The fold formation and wing disc bending are visualized by Phalloidin and DAPI staining either in plane or cross sections. Our results revealed the mutual antagonism between Doc/Vg and Doc/Hth that defines Doc expression at the two B/H folds.In our preliminary study, we found that another T-box gene, optomotor-blind (omb) also interacts with Doc and plays essential role in B/H folds and Notum/Hinge folds formation. Revealing the role of omb in folds formation would help to understand the general mechenism of folds formation and the fates differentiation during organs development.

果蝇3龄幼虫翅芽被几条深沟划分为背部、铰链、翅刀区域。这些区域划分对成虫翅的正确形成至关重要。这些区域细分和随后的变态发育的启动在分子机理水平上所知甚少。申请者（2012，Development）证明了T-box基因Doc促进沟的形成，这条沟不仅划分铰链和翅刀区域，而且通过改变细胞形态并把翅芽折叠起来促进翅的变态发育，提出了翅芽在铰链/翅刀沟处延伸折叠模型假说。本项目中，申请者拟聚焦在Doc基因的表达区域、也即是铰链/翅刀沟细胞内，操控Doc基因的表达，从而不会干扰翅芽背/腹边界处的折叠，进而区分翅芽到底是从背/腹边界（前人假说）还是从铰链/翅刀沟的地方启动折叠。申请者预实验还表明Doc与另外一个T-box基因omb有相互作用，并且omb在铰链/翅刀沟和背部/铰链沟的形成中也起重要作用。揭示Omb与Doc的互作关系及其在沟形成中的机制有助于理解器官发育中沟的形成及区域命运划分的普遍机理

本项目根据在以往研究基础上，进一步深入研究了T-box转录因子Omb和Doc在昆虫翅发育中的作用机理，发现了一些重要的新功能和机理。1、Omb和Iro-C的互补表达模式能够促进翅芽上形成沟从而划分翅铰链区和背板区。2、在翅前后隔间边界上由Omb缺失所诱导的沟形成需要Yki和JNK信号通路的协同作用。3、Omb和TBX2的过强表达能够诱导翅细胞的异常运动和入侵行为，而且不涉及到细胞外基质的变化。4、Omb的下游因子Sal在果蝇和飞蝗翅中的功能保守，都是促进翅生长的。5、Sal在翅的鳞片状和柱状两个细胞层中的不对称分布能够确保正确的翅细胞形貌发生。6、综述了TBX基因在昆虫附肢发育中的作用。上述这些基因功能的解析，为筛选害虫遗传学控制的靶标基因打下了良好基础，后续有利于利用纳米材料载体装载dsRNA调控害虫的关键靶标基因、从而控制害虫。

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