eyeSC - The core retinal determination network sequentially patterns intestinal stem cells and their daughter cells in the adult Drosophila midgut.

成体器官和组织的大小和功能是由多能自我更新的干细胞维持的。在成年果蝇黑腹果蝇中，肠道干细胞（ISC）维持中肠稳态。ISC主要产生成肠细胞（enteroblast， EB），致力于向上皮性肠细胞（epithelial enterocytes， EC）分化。分化EB最近被证明具有谱系可塑性、能动性并调节其数量以维持中肠稳态。然而，很少知道细胞内的遗传网络使这些新的特性在谱系规范。在初步实验中，我们发现pax6同源蛋白在肠ISC和EB中表达。随后的数据挖掘显示，果蝇视网膜决定网络（RDN）的大部分基因在中肠中表达。RDN基因是众所周知的协调幼虫眼影像盘的逐步模式。利用我们的追踪方法ReDDM在中肠祖细胞中进行特异性操作，不同RDN基因的缺失会阻碍祖细胞在从ISC到终末EB分化的各个形态阶段的成熟。因此，我们假设RDN基因在中肠祖细胞的模式中具有类似的功能，正如我们从眼盘发育中所知道的那样。在此，我们建议对成年果蝇中肠中的RDN基因进行深入研究，以阐明其确切的表达时间和功能。首先，我们将通过基因重组设计转基因荧光标记的RDN基因转录报告蝇，并将其定位到不同的祖细胞阶段。特定阶段的报告者将被FACS分类并进行RNAseq分析。为了将RDN基因与活化阶段特异性细胞内遗传网络联系起来，我们将进行基因集富集和KEGG途径分析。在第二个子项目中，我们将深入分析RDN基因在中肠生理和病理中的功能。利用我们实验室开发和建立的新工具，如细胞类型特异性ReDDM追踪，遗传标记和报告器，我们将通过挖掘第一个子项目的数据来剖析RDN基因功能，并结合发现。我们的初步功能数据表明祖细胞分化、存活、运动和增殖发生了变化；在结直肠癌（CRC）中所有的过程都不受控制。有趣的是，一项计算机分析揭示了腺癌样本中RDN基因的上调及其对患者生存的影响，这就是为什么我们将使用两种已建立的果蝇CRC模型来研究RDN基因在肿瘤发生早期阶段的功能。综上所述，我们将研究RDN基因在苍蝇肠道干细胞向终末分化前体分化过程中的表达和功能。RDN基因在动物物种中的高度功能保守性提高了我们在果蝇中的观察结果与加深对哺乳动物肠道生理和病理过程的理解相关的可能性。

Adult organs and tissues are maintained in size and function by multipotent self-renewing stem cells. In the adult fruit fly, Drosophila melanogaster, intestinal stem cells (ISC) maintain midgut homeostasis. ISC mainly produce enteroblasts (EB), committed to differentiate to epithelial enterocytes (EC). Differentiating EB were recently shown to bear lineage plasticity, motility and adjust their numbers to maintain midgut homeostasis. However, little is known about intracellular genetic networks enabling these new properties upon lineage specification.In preliminary experiments, we found the Pax6-homologue eyeless expressed in intestinal ISC and EB. Subsequent data mining revealed that the majority of genes from the Drosophila retinal determination network (RDN) is expressed in the midgut. RDN genes are well known orchestrating the stepwise patterning of larval eye imaginal discs. Manipulated specifically in midgut progenitors using our tracing method ReDDM, loss of different RDN genes stalls progenitor maturation at various morphological stages from ISC to terminal EB differentiation. Thus, we hypothesize a similar function of RDN genes in patterning midgut progenitors as known from eye disc development. Here, we propose a thorough investigation of RDN genes in the adult Drosophila midgut to elaborate their exact expression timing and function. Initially, we will engineer transgenic fluorophore tagged transcriptional reporter flies for RDN genes with genetic recombineering and map them to different progenitor stages. Stage specific reporters will be FACS sorted and subjected to RNAseq analysis. To connect RDN genes with activated stage specific intracellular genetic networks, we will perform e.g. gene set enrichment- and KEGG pathway analysis. In the second subproject, we will perform an in depth analysis of RDN gene functions in midgut physiology and pathology. Applying new tools like cell-type specific ReDDM tracing, genetic markers and reporters developed and established in our lab, we will dissect RDN gene functions incorporating findings by mining the data from the first subproject as well. Our preliminary functional data suggests changes in progenitor differentiation, survival, motility and proliferation; processes all deregulated in colorectal cancer (CRC). Interestingly, an in silico analysis revealed uniform RDN gene upregulation in adenocarcinoma samples and effects on patient survival, which is why we will address RDN gene function in early steps of tumorigenesis using two established Drosophila CRC models. Taken together, we will investigate RDN gene expression and function during differentiation from intestinal stem cell to terminally differentiating precursors in the fly gut. The high degree of functional conservation of RDN genes across animal species raises the probability that our observations in Drosophila gain relevance to deepen understanding of physiological and pathological processes in the mammalian intestine as well.