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.
