Notch信号通路参与家蚕胚胎发育分子机制的研究

Notch signaling, responsible for cell differentiation and cell migration, plays important roles in several developmental processes. Although Notch signaling mediates segmentation in some insects, it is still controversial whether the involvement of Notch signaling in segmentation is conserved in arthropods. In our previous study, it was found that Notch signaling took part in body segmentation and appendage development in silkworm, Bombyx mori. RNA interference (RNAi) against Notch signaling led to defects with fused appendages at the ventral midline due to cell migration and loss of ventral midline tissue. However, the molecular mechanism for involvement of Notch signaling in embryonic development of silkworm remains unknown. In this project, whole-mount in situ hybridization (WISH) is employed to examine the expression pattern of notch and delta gene on silkworm embryo. WISH and immunohistochemistry, following the Notch signaling RNAi, are used to investigate the expression of twi, en, dpp and cell apoptosis to study the loss of ventral midline tissue. The expression of Dfd and Scr is examined by WISH after RNAi to elucidate the mechanism of cell migration. This project is useful for elucidating the molecular mechanism for involvement of Notch signaling in embryonic development of silkworm, and resolving the key problem that the role and evolution of Notch signaling for segmentation in arthropods.

Notch信号通路是细胞分化和细胞迁移的重要信号，介导了一些昆虫的体节分节。该机制在节肢动物中是否保守，还存在争议。在前期研究中发现Notch信号参与家蚕(Bombyx mori)体节分节和附肢发育；Notch信号缺失后，由于细胞迁移和腹侧中线组织缺失，成对的附肢在腹侧中线融合。但Notch信号在家蚕胚胎发育中作用的机理尚不清楚。本项目利用整体原位杂交(Whole-mount in situ hybridization, WISH)技术研究notch、delta基因在胚胎的原位表达。对Notch信号通路基因进行RNA干扰(RNAi)后，利用WISH和免疫组化，检测twi、en、dpp和细胞凋亡以研究腹侧中线组织的缺失及其机理，检测Dfd和Scr以研究细胞迁移的机理。从而阐明Notch信号通路参与家蚕胚胎发育的分子机制，解决Notch信号通路在节肢动物体节分节中作用与进化这一关键问题。

Notch 信号通路是细胞分化和细胞迁移的重要信号，介导了一些昆虫的体节分节。该机制在节肢动物中是否保守，还存在争议。在前期研究中发现Notch 信号参与家蚕(Bombyx mori)体节分节和附肢发育；Notch 信号缺失后，由于细胞迁移和腹侧中线组织缺失，成对的附肢在腹侧中线融合。但Notch 信号在家蚕胚胎发育中作用的机理尚不清楚。项目利用WISH技术研究notch、delta基因在胚胎的原位表达。发现Notch信号通路不具有动态的周期性振荡式的表达图式，表明Notch信号通路在体节分节完成后调控体节自身的发育图式，反映了Notch信号通路参与昆虫体节分节在进化上的多样性。利用WISH检测twi、en、dpp在缺陷型的表达，发现en的表达成一条完整的条纹，而在腹侧中线处twi不再表达，表明 Notch信号通路被干扰后导致腹侧中线组织缺失，证明了 Notch信号通路在家蚕中外胚层的形成中发挥作用；而dpp基因在胚胎原位表达的位置及强度没有变化，表明与腹侧背侧化没有直接关系。发现Scr基因除在所有体节都表达，而Dfd基因在上颚和下颚体节的表达受到抑制；Scr双链RNA可减弱Bmnotch RNAi导致的腹侧对称融合的现象，且Dfd恢复在上颚和下颚体节的表达；表明Notch信号通路控制家蚕腹侧细胞迁移是通过调控Dfd和Scr实现，其他体节也因此获得了向腹侧中线横向移动的潜能。Bmemc RNAi得到胚动缺陷表型，确认了Bmemc基因在家蚕胚胎发育中的作用。项目从而从基因相互作用与调控的角度，阐明Notch 信号通路参与家蚕胚胎发育的分子机制，表明Notch信号通路在家蚕中外胚层的形成中的发挥作用，揭示Notch信号通路参与昆虫体节分节在进化上的多样性，解决Notch信号通路在节肢动物体节分节中作用与进化这一关键问题，为阐明节肢动物分节机制的起源与进化奠定基础。项目首次从Notch信号通路与Scr、Dfd相互作用角度研究胚胎发育中的细胞迁移问题，发现Notch信号通路控制家蚕腹侧细胞迁移是通过调控Dfd和Scr实现。同时首次发现emc在Notch信号通路中起到重要作用，且Bmemc参与了家蚕躯体模式决定。