转录因子Mnx1调控爪蛙背胰前体细胞发育的分子机制研究

Mnx1是背部胰腺原基形成的关键转录因子。用基因芯片筛选的方法，我们发现视黄酸的下游激活基因mnx1在爪蛙原肠胚期即有表达，尾芽胚期特异表达于背部胰腺原基。进一步研究发现视黄酸和经典Wnt信号通路可激活mnx1的表达，另外，mnx1过表达和基因敲除均能造成背部胰腺前体细胞的缺失。由此，我们提出mnx1的适量表达保证了背部胰腺前体细胞的形成。目前对Mnx1的研究结果主要来源于模式动物胚胎表型分析的数据，但视黄酸和Wnt信号通路如何激活mnx1的表达，其内源性相互作用蛋白及下游调控基因仍有待发现。本课题在目前工作基础上，拟用遗传标记的方法精确追踪早期mnx1阳性细胞的发育命运；同时用胚胎学、分子遗传学以及生物化学的方法系统地揭示mnx1调控背部胰腺前体细胞形成的分子机制，为发现新的胰腺发育的调控因子，更为设计体外诱导胚胎干细胞形成真正成熟的胰岛β细胞的实验方案提供更全面的理论依据。

Mnx1 is a key transcriptional factor for dorsal pancreas bud formation. In an attempt to screen for retinoic acid (RA) regulated genes in Xenopus endoderm by microarray, we identified an RA regulated gene mnx1 that showed expression as early as gastrula stages and later in dorsal pancreatic buds of tailbud stage embryos. Our unpublished data indicate that mnx1 expression can be up-regulated by RA and canonical Wnt signal pathways. As either overexpression or knockout of Mnx1 resulted in the same phenotype, i.e. an inhibition of pancreatic dorsal bud specification, we proposed that strictly-regulated mnx1 expression level is critical for dorsal pancreatic bud specification. So far, the studies on Mnx1function are largely descriptive with little mechanistic studies. It remains to be defined how RA and canonical Wnt signal pathways activate the expression of mnx1, which proteins interact with Mnx1 in vivo, and what downstream genes Mnx1 controls. Based on our current data, on one hand, we will make a genetic lineage tracing of mnx1 positive cells. On the other hand, we plan to analyse mnx1 promoter by using luciferase reporter assay and validate the obtained data with EMSA and ChIP. We will identify Mnx1 interacting proteins and its downstream genes by CoIP, mass spectrometry and ChIP-Seq. The proposed studies will lead to identification of novel regulators for pancreas development and hold great potential to develop strategies to direct embryonic stem cells into islet beta cells.