The pancreas is a secretory organ composed of exocrine and endocrine compartments. During development, endocrine cells delaminate from the pancreatic epithelium to associate with the local vasculature where they will secrete hormones such as insulin into the blood stream. The exocrine pancreas is composed of ductal cells which form a network of tubes to secrete and transport fluid carrying the digestive enzymes secreted from acini located at the terminal ends of ductal branches. Unlike many branched epithelia, the pancreas does not exhibit a stereotypical branching pattern. The ductal network develops from a mesh of interconnected lumens which are eventually pruned and give rise to a final tree-like structure optimized for the most economical delivery of enzymes and fluid to the digestive tract. In silico modelling suggests that fluid flow plays a role in resolving the final structure of the ductal network during development, indicating that physical forces may play a role in this self-organization Recent work in the adult human pancreas has shown that the cells of the small ducts in the most distal parts of the ductal network do not express the same transcripts as the proximal large ducts.
The pancreas derives this structure and function from the differentiation and self-organization of progenitors into terminally differentiated cells which, together with mesenchymal cells and vasculature, contribute to the tissue niche of the organ. Despite the importance of this process in development and disease, little is known about how pancreatic progenitors balance differentiation with morphogenesis. The goal of this project was to uncover niche components that influence the differentiation of pancreas progenitors, and understand how identity and morphogenesis are mediated by niche-driven changes in gene expression. This remains a challenging process to understand due to limited accessibility of the embryonic pancreas. Therefore, human sphere and organoid models represent a valuable tool to address this question and were used together with expression profiling and manipulation of the extracellular environment to understand this relationship during pancreas development.
Time-course bulk RNA sequencing of human pancreas progenitor spheres at different days of culture revealed the sequential processes happening as the cells form their niche, and then start proliferating and forming lumen. Notably, at the stage of lumen expansion, we observed an upregulation of genes associated with ductal epithelia, such as CFTR, MUC1, MUC6, and CA2 in tandem with increased expression of genes encoding proteins for ion and fluid secretion. This suggested hydraulics may act to integrate ductal differentiation with luminogenesis, which is consistent with in silico modelling and the secretory function of the pancreatic epithelia. Indeed, driving chloride ion secretion with the CFTR activator forskolin resulted in inflation of the sphere lumen and increased the expression of the ductal genes identified above. Induction of CFTR and MUC1 can also be achieved by inflating the lumen in a CFTR-independent manner using prostaglandin E2. This revealed the changes in gene expression were not due to a small feedback loop under the control of CFTR, and maybe due to morphological changes related to lumen inflation. Importantly, it was revealed that the induction of Cftr expression upon lumen inflation also occurred in pancreas explants isolated from embryonic mice, which suggests the relationship between lumen inflation and ductal identity is conserved between mouse and human.
Datamining of single cell RNA sequencing of adult and fetal pancreas samples identified novel marker genes for progenitor, acinar, and large- and small-ducts of the human fetal pancreas. Comparison of these marker genes with gene expression patterns of pancreas progenitor spheres revealed a shift to a small-duct-like identity when the lumen is inflated. This shift seemed to be dependent on inflation of the lumen rather than cAMP signalling, as it is not observed in pancreas progenitors grown in 2D and treated with forskolin.
The above experiments suggest a link between lumen inflation and small duct identity but the exact mechanism remains unclear. Lumen inflation is likely driven by an increase in hydrostatic pressure that occurs downstream of changes in osmotic pressure due to ion channel activation. Independent manipulation of osmotic pressure, hydraulic pressure, tissue stretching, and fluid shear stress will be valuable to decipher the mechanisms of ductal gene regulation. Taken together these results support the hypothesis that differential gene expression in ducts of different sizes is regulated by mechanical forces in the pancreas, and 3D sphere culture represents a powerful model to investigate these processes in finer detail.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:92816 |
| Date | 05 August 2024 |
| Creators | Lewis, Allison Christina |
| Contributors | Dahmann, Christian, Grapin-Botton, Anne, Rovira, Mertixell, Technische Universitat Dresden, Max Planck Institute of Molecular Cell Biology and Genetics |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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