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Using the precision mouse genetic tool MADM to elucidate the role of EGFR in directing beta cell differentiation and pancreatic morphogenesis

Periodic Reporting for period 1 - MADMpancreas (Using the precision mouse genetic tool MADM to elucidate the role of EGFR in directing beta cell differentiation and pancreatic morphogenesis)

Reporting period: 2019-06-01 to 2021-05-31

The pancreas houses two physiologically distinct compartments within one organ. The hormone-producing endocrine cells of the islets and the tubular ductal system of the exocrine pancreas share a common developmental progenitor. Recent work from the Semb group implicated Egfr signaling as a critical commitment event that both transcriptionally primes cells for endocrine differentiation–by upregulating Neurog3 expression in metastable progenitors—while also facilitating islet morphogenesis—by antagonizing cellular polarity and inducing delamination from the bipotent trunk epithelium. A central objective of this project was to understand how Egfr signaling is regulated in the developing pancreas. Specifically, we wanted to test the hypothesis that quantitative differences in pathway activation explain why metastable Neurog3-expressing cells commit to endocrinogenesis in a seemingly stochastic and temporally dispersed manner. We further test if such quantitative differences facilitate selection of the different hormone producing fates. To quantitatively manipulate Egfr levels at single-cell resolution, this project employs the mouse genetic tool Mosaic Analysis with Double Markers (MADM).
To test the hypothesis that pre-existing heterogeneity for Egfr pathway components predisposes pancreatic progenitors to endocrine commitment, the fellow established a collaboration with Bob Coffey of Vanderbilt University to examine the newly developed Egfr-EmGFP fusion protein mouse reporter line. Overall low expression levels were detected in the developing pancreatic epithelium, especially when compared with other embryonic epithelia like the gut, and no obvious cell to cell heterogeneity was observed in the bipotent trunk epithelium. To interrogate the role of Egfr signaling in commiting bipotent progenitors to the endocrine fate and to specifically test if Egfr is subsequently required for differentiation to the insulin-expressing beta cell fate, we conditionally ablated Egfr in the Ngn3 lineage. No differences in the ratio of hormone cell types were detected. While we found little evidence that Egfr signaling influences differentiation outcomes, we did find genetic evidence that quantitative differences in Egfr signaling might influence progenitor allocation to pattern the organ. Ongoing experiments using the Egfr-MADM system will test if Egfr levels direct progenitor cell position in the pancreatic bud.

We successfully adapted the MADM system to the embryonic pancreas and achieved mosaic labeling that is compatible with single cell tracking in time-lapse imaging experiments. One key feature of this system is that it generates uniparental disomy of the rearranged chromosome. An unexpected but significant finding of this project was that imprinting of chromosome 11 strongly influences the growth of pancreatic progenitors. Cells with uniparental disomy (UPD) expanded at significantly increased or decreased rates compared to cells with normal biparental heterodisomy. This finding was unexpected as published studies of this MADM line in several brain compartments revealed no evidence of any imprinted phenotypes.
The scientific findings from this project will be of particular interest to other developmental biologists and experts in mouse genetics who seek to understand how morphogenesis and differentiation are integrated and mutually influencing processes. The rich dataset of 4D scanning confocal movies can be exploited to extract the principles of tissue organization at a cellular scale using AI approaches. Because this study also uncovered new findings about the regulation of pancreatic progenitor expansion, it is also of interest to translationally focused investigators seeking to optimize in vitro directed differentiation protocols to manufacture islet-like tissue as a transplant therapy for type 1 diabetes.