Fluorescent monitoring of enteroendocrine cell development and function

The project will investigate how intestinal endocrine cells sense food intake, using fluorescence imaging techniques in live tissue. It will address the questions of how enteroendocrine cells are formed and how they detect changes in food intake and metabolism.
Enteroendocrine cells (EECs) are located in the intestinal epithelium and convert nutrient signals arriving in the gut lumen into chemical hormonal signals, which in turn regulate intestinal function, insulin release and food intake. Like the absorptive enterocytes, EECs are continuously formed from stem cells in intestinal crypt, and have a life span of about 5 days. The hormones they produce exhibit a longitudinal gradient along the gut, with some hormones exhibit preferential expression in the proximal gut, and others in the distal gut.
In recent years, we have generated a number of transgenic mouse models in which enteroendocrine cells are labelled by fluorescent markers, cre-recombinase, rtTA or fluorescent sensors of intracellular cAMP or calcium. These models enable the identification and purification of specific populations of EEC as well as dynamic monitoring of their intracellular signalling pathways in primary culture. In recent years we have exploited the combination of fluorescent imaging, electrophysiology and transcriptomic analysis to dissect how EECs detect a range of ingested nutrients. By assaying hormone release from mixed primary culture and intact tissue pieces mounted in Ussing chambers, we correlated cellular activity with hormone secretion. We identified a range of nutrient transporters, metabolic pathways and G-protein coupled receptors that variously result in the detection of digested carbohydrates, fat and protein. Transcriptomic analysis further revealed that several different EEC types, once thought to represent distinct populations, are actually highly related, and likely reflect a single cell type with a variable hormonal signature.
In the current project we will extend these techniques to the study of mouse and human EECs in organoid culture. Organoids will be grown from intestinal crypts from different regions of the mouse or human gut, and maintained in primary culture. Using this system, organoids can be maintained in culture indefinitely. Genes under specific hormonal promoters will be transduced into organoids using lentivirus or other methods, to introduce fluorescent markers and sensors specifically into the EEC population. Using the methodologies described above, we will address the following questions:
-What specifies the hormonal signature of an EEC? Is it determined by the position of the gastrointestinal tract from which the crypts originated? Can it be influenced by external factors such as nutrients, cytokines or other metabolic factors, and can it be changed during the lifetime of a single EEC (as monitored by time lapse imaging of fluorescent markers driven by hormonal promoters in organoids)?
-Do EECs in organoid culture exhibit similar secretory responsiveness to stimuli as EEC cell lines, L-cells in mixed primary culture, and L-cells in intact tissue pieces?
-Which nutrient detection machinery and intracellular signaling pathways are implicated in the detection by human EECs of nutrients, hormones and neurotransmitters, and are the pathways similar to those found in mice?