Final Report Summary - GUTENCODE (Decoding the transcriptional networks controlling the adult Drosophila midgut compartmentalization) The average adult human gut is approximately 10 meters in length, with a surface area of approximately 200 m2. The inner lining functions primarily to digest and absorb nutrients, but as it comes into direct contact with the external environment, it is constantly faced with a wide variety of pathogens. Despite its importance, our understanding of this complex organ has been limited by the scarce attention it has historically received from scientists beyond the field of gastroenterology per se. However, recent years have seen an increased awareness from both the public and the scientists alike regarding its importance. This is not only true in mammals but also in insects for which very little is known about gut structure and function. Since the insect gut is an essential passage for several human parasites, as well as the main target of insecticides, a better knowledge of this organ will have applied outcomes. In both structure and function, the Drosophila gut bears many similarities with the human gastrointestinal tract. It consists of a simple epithelial tube surrounded by visceral muscles, nerves, and tracheae. Like mammals, the Drosophila gut is renewed by stem cells and longitudinally compartmentalized. Using morphometric, histological and genetic approaches, we have recently generated a detailed atlas of the Drosophila adult midgut. Our study showed that the adult midgut can be divided into 6 regions and 14 sub-regions revealing an underlying complexity that was hitherto not appreciated. Our project aimed to identify gene regulatory networks underlying the Drosophila midgut compartmentalization. We initially searched for new genes that could explain the differential properties of stem cells along the anterior-posterior axis at the origin of this compartmentalization. This led us to the identification of a new gene, Sox21a, encoding a transcription factor which orchestrates the differentiation of stem cells in various enterocyte types. Taking in consideration the importance of this gene and the observation that loss-of-function mutations in these genes cause intestinal tumors, we focused most of our attention on the role of this gene.The adult fly midgut contains multipotent intestinal stem cells (ISCs) that differentiate into either an absorptive enterocyte or a secretory enteroendocrine cell, through an intermediate but already committed phase, called enteroblast. We identified that the Drosophila Sox transcription factor, Sox21a, controls the differentiation of ISCs with marked differences between the anterior and the posterior midgut. This transcription factor is responsible for the differentiation program mediated by the Jak/Stat signaling in both normal and regenerating midguts following damage. We observed that the loss of Sox21a blocks stem cells at the enteroblast state, leading to the formation of tumors that invade the lumen of the midgut. Sox21a-mutant derived tumors are especially aggressive in the anterior midgut, as macroscopic tumors can be found in all old Sox21a mutant flies. We could show that in absence of Sox21a, progenitor cells cannot differentiate and stimulate the proliferation of surrounding stem cells, thereby promoting a hyperplastic growth. This effect is specific of the anterior midgut as it was not observed in the posterior part of the digestive tract. An increase in some secreted factors (JAK-STAT ligands, epidermal growth factors) was found to be functionally important for the non-cell autonomous growth effect. Our study provides a model of tumor in which a failure in the differentiation program leads to tumor formation and aggressiveness.Thus, our study leads to the identification of a new gene involved in stem cell differentiation in the midgut with differential effect along the midgut. It also provides a model of cancer as mutation in Sox21a induces spontaneous tumors in the anterior part of the gut. Using genetic tools, we have deciphered the cause of these tumors. Our study shows that the blockage of differentiation is sufficient to induce the release of mitogenic factors that induces stem cell proliferation and the formation of a tumor by affecting the surrounding cells through the release of Reactive Oxygen Species.This project has led to the discovery of an important aspect of Drosophila midgut compartmentalization by identifying the mechanisms underlying intestinal epithelium differentiation in different cell type of the gut. Our findings are relevant to certain human intestinal pathologies. Due to the conserved functions of SOX transcription factors in Drosophila and mammals, this project is expected to impact the study of human intestinal development and cancer.