Epithelial polarity as a fate determinant of multipotent endocrine progenitors during human pancreas development

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A fundamental unanswered question in developmental biology is how different cell fates are induced from the same multipotent progenitors during the formation of complex organs that are composed of many different cell types. We use the developing pancreas as a model system to study this central general question and has recently discovered that apical-basal polarization of epithelial cells plays a critical role for steering pancreatic cell fate specification. Based on preliminary experiments, we hypothesize that epithelial polarity is the decisive cue for the commitment of multipotent endocrine progenitors in the pancreas to differentiate to either glucagon-producing alpha cells or insulin-producing beta cells. To directly test this hypothesis, we will use CRISPR/Cas9 technology to introduce fluorescent reporters for the endocrine progenitor marker NGN3 and a polarity marker into the same human embryonic stem cell (hESC) line. These double-reporter hESCs will be directed to differentiate in vitro toward pancreatic endocrine cells. Using a novel lineage-tracing approach based on confocal live-imaging, we will determine the correlation between progenitor cell polarization and eventual alpha or beta cell fate. The host group’s previous investigations indicate that apical-basal polarization affects endocrine commitment by repression of the Notch signaling pathway. We will dissect the molecular mechanisms that link the polarity complex to Notch signaling and cell fate decisions in multipotent progenitors by a combination of experimental approaches such as RNA interference and protein co-immunoprecipitation. The expected results will not only shed light on unknown details of basic biological processes and of human pancreas development in particular, but will also help design more refined protocols for the in vitro generation of beta cells from hESCs, thus contributing to the development of cutting-edge cellular therapies for diabetes.