Appl1/Akt介导的信号通路调控爪蛙胰腺、胃和十二指肠前体细胞存活的分子机制研究

全面认识胚胎发育期间器官原基的维持、分化和成熟对体外诱导胚胎干细胞形成成熟的功能性细胞至关重要。接头蛋白APPL1主要位于一种独特的早期内涵体上参与多种信号通路的调控，但也可在细胞核中发挥作用。我们的研究表明Appl1/Akt2信号通路对爪蛙胰腺、胃及十二指肠前体细胞的存活必不可少，但该通路中与Appl1相互作用的受体、Appl1/Akt2下游靶蛋白以及Appl1/Akt2所介导的细胞外信号分子等均尚不清楚。另外，我们的前期研究提示，除了Appl1/Akt2通路，Appl1可能还通过其它方式调控这些细胞的存活。本项目拟在原来工作的基础上，用胚胎学、分子遗传学以及生物化学的方法系统地揭示调控热带爪蛙胰腺、胃及十二指肠前体细胞存活的Appl1/Akt2信号通路分子机制，全面认识Appl1功能，为体外诱导胚胎干细胞形成成熟的胰岛β等功能性细胞提供依据。

Comprehensive understanding of the maintenance, differentiation, and maturation of organ primordial progenitor cells in vivo is essential for the directed induction of embryonic stem (ES) cells into various mature functional cells. Adaptor protein APPL1 mainly functions via a kind of unique early endosomes named APPL1 endosomes to mediate trafficking of several signaling pathways. It may also play a role in nuclei. Our study revealed that Appl1 is essential for the survival of Xenopus pancreas, stomach, and duodenum progenitor (PSDP) cells, which functions mainly, but not exclusive, via Akt2 signaling pathway. It remains to be investigated what are the Appl1 interacting receptors, what are the survival signals, and what are the Appl1/Akt2 downstream target proteins in this scenario. Here, we propose three aims to investigate how Appl1/Akt2 controls the survival of PSDP cells in Xenopus tropicalis. In Aim 1, we will define the dynamic subcellular localization of Appl1 protein in developing X.tropicalis PSDP cells at high resolution. In Aim 2, we will screen Appl1 and Akt2 interacting proteins in X.tropicalis PSDP cells via CoIP and mass spectrometry. In Aim 3, we will define the cellular repertoire of Appl1/Akt2 pathway via a number of in vivo and in vitro biochemistry and functional assays. We hope that our study will give hints to direct the generation of mature functional cells, such as islet β cells, from ES cells in vitro.