Evolution and functionality of Wnt genes in the fruit fly Drosophila melanogaster

Wnt信号调节动物发育过程中的许多生物过程，包括细胞命运规范、分裂和模式。在后生动物中有13个Wnt配体亚科，其中7个在果蝇果蝇中有代表。大多数后生动物物种都保留了大量的wnt。尽管一些Wnt基因在果蝇中似乎有重叠的作用，但人们认为它们在Wnt环境中协同作用，在某些情况下它们发挥独特的功能。然而，到目前为止，Wnt蛋白功能多样化的分子基础及其与受体和其他蛋白的个体相互作用仍然未知。在这个项目中，我将使用一种新的重新整合系统来识别Wnt基因之间功能分化的蛋白质序列。CRISPR/Cas9系统将应用于基因组工程，删除特定的基因组序列，同时使用靶向载体重新引入外源DNA。该系统为直接分析和比较Wnt配体的功能提供了一个绝佳的机会，从而测试这些配体是否可以挽救彼此的功能，从而了解它们差异的分子基础。因此，我将测试在果蝇基因丢失后，wg和Wnt6是否可以相互取代其表型。接下来，我将分析Wnt10和Wnt9在胚胎发生过程中是否可以相互挽救对方的功能。由于一个Wnt基因不太可能在功能上完全取代另一个Wnt基因，我将重新引入不同Wnt基因的嵌合配体，以确定负责两种Wnt配体多样化的结构域。因此，我将分别引入嵌合的wg-Wnt6和Wnt9-Wnt10基因，并对其进行检测，以提高抢救效果。在不太可能的情况下，Wg和Wnt6或Wnt9和Wnt10完全功能兼容，我将使用下一个最接近的Wnt配体Wnt8及其嵌合配体。本项目将确定Wnt配体之间功能差异的分子基础，这将有助于阐明Wnt配体如何单独和联合调节发育过程。这些发现可能为Wnt信号在发育和退行性疾病（包括各种类型的癌症）中的作用提供进一步的见解。

Wnt signalling regulates many biological processes during animal development, including cell fate specification, division and patterning. There are thirteen subfamilies of Wnt ligands in metazoans, seven of which are represented in the fruit fly Drosophila melanogaster. Most metazoan species have retained a large number of Wnts. Although some Wnt genes appear to have overlapping roles in Drosophila, it is thought they act cooperatively in Wnt landscapes and in some contexts they perform unique functions. To date, however, the molecular basis for the functional diversification of Wnt proteins and their individual interactions with receptors and other proteins remains unknown. For the project, I will use a novel reintegration system to identify the protein sequences responsible for functional divergence between Wnt genes. The CRISPR/Cas9 system will be applied for genomic engineering to delete specific genomic sequences and to simultaneously reintroduce exogenous DNA using targeting vectors. This system offers a remarkable opportunity to directly analyse and compare functions of Wnt ligands and hence test if the ligands can rescue each others functions to understand the molecular basis of their differences. Therefore, I will test if wg and Wnt6 can be replaced by each other in regards to their phenotypes after a gene loss in Drosophila. Next, I will analyse if Wnt10 and Wnt9 can rescue each others functions during embryogenesis. Since it is unlikely that one Wnt gene can completely and functionally replace another Wnt gene, I will reintroduce chimeric ligands of different Wnt genes to identify domains responsible for the diversification of two Wnt ligands. Thus, I will introduce chimeric wg-Wnt6 and Wnt9-Wnt10 genes, respectively, and assay for improved rescue. In the unlikely event that Wg and Wnt6 or Wnt9 and Wnt10 are fully functionally compatible, I will use the next closest related Wnt ligand Wnt8, and chimeric ligands thereof. This project will identify the molecular basis of functional differences among Wnt ligands, which will help to clarify how Wnt ligands regulate developmental processes individually and in combination. The findings may provide further insights into the role of Wnt signalling in developmental and degenerative diseases, including various types of cancer.