Dissecting the role of Hedgehog signalling during development, regeneration and ageing in vivo using novel phosphorescent small molecules

本博士项目的目标是利用新型磷光化合物研究刺猬信号在文昌鱼从胚胎发育到成体再生的整个生命周期中的作用变化。主要目标包括：1。通过免疫组织化学和全挂载原位杂交来表征影响Hedgehog信号传导的化合物处理所产生的表型2。产生新的铱标记化合物，可以用来成像Hh信号通路。目前的成像方法（免疫荧光）有以下局限性：难以量化，大多数细胞和生物体自然产生自身荧光，这使得特定的荧光标记难以可视化，并且使用它们对活生物体成像存在问题。我们计划创造的新型铱标记化合物需要具备以下条件：水溶性和生物相容性（允许体内成像）。我们计划使用磷光阳离子铱（III）复合物，我们希望进一步开发它来创建一个调色板。制备具有光转化特性的新型铱标记化合物，以调节生物分子的时空活性。文文鱼是这个项目中使用的模式生物，因为它与脊椎动物有许多相似之处，但具有更简单的基因组和解剖结构（使刺猬信号通路更容易研究），它是半透明的（有助于实时成像），它再生良好，并且作为无脊椎脊索动物使它成为脊椎动物成体再生研究的一个伟大的伦理选择。由于Hedgehog信号通路与神经缺陷、骨骼缺陷、独眼、癌症和纤毛病有关，该项目具有临床应用价值。此外，再生的研究与伤口愈合密切相关，因此可以帮助我们理解刺猬信号在再生和伤口愈合中的作用。

The goal of this PhD project is to study the changing roles of Hedgehog signalling during amphioxus' entire lifespan, from embryonic development to adult regeneration, using novel phosphorescent compounds. The primary aims include: 1. to characterise phenotypes resulting from treatment of chemical compounds affecting Hedgehog signalling via immunohistochemistry and whole mount in situ hybridisation 2. to generate novel iridium tagged compounds that can be used to image the Hh signalling pathway. The current way of imaging (immunofluorescence) has the following limitations: it is hard to quantify, most cells and organisms naturally autofluoresce which makes specific fluorescent labels hard to visualise, and they are issues with using them to image live organisms. The novel iridium tagged compound we plan to create needs to be the following: water soluble and biocompatible (to allow in vivo imaging). We are planning on using a phosphorescent cationic iridium(III) complex and we hope to develop this further to create a colour palette.3. to generate novel iridium tagged compounds with photoconvertible properties in order to modulate spatial and temporal activity of the biomolecules. Amphioxus is the model organism used for this project due to the following: it shares many similarities with vertebrates but has a simpler genome and anatomy (making the Hedgehog signalling pathway easier to study), it is semi-translucent (aids live imaging), it regenerates well and being an invertebrate chordate makes it a great ethical alternative to adult regeneration research in vertebrates. This project has clinical applications due to the Hedgehog signalling pathway having links to neural defects, skeletal defects, cyclopia, cancer and ciliopathies. In addition, the study of regeneration is closely linked to wound healing so can help our understanding of hedgehog signalling in both regeneration and wound healing.