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

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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.