Apicobasal polarity is known to affect epithelial morphogenesis and cell differentiation, but it remains unknown how these processes are mechanistically orchestrated. We find that ligand-specific EGFR signalling via PI(3)K and Rac1 autonomously modulates apicobasal polarity to enforce the sequential control of morphogenesis and cell differentiation. Initially, EGF controls pancreatic tubulogenesis by negatively regulating apical polarity induction. Subsequently, betacellulin, working via inhibition of atypical protein kinase C (aPKC), causes apical domain constriction within neurogenin3+ endocrine progenitors, which results in reduced Notch signalling, increased neurogenin3 expression, and β-cell differentiation. Notably, the ligand-specific EGFR output is not driven at the ligand level, but seems to have evolved in response to stage-specific epithelial influences. The EGFR-mediated control of β-cell differentiation via apical polarity is also conserved in human neurogenin3+ cells. We provide insight into how ligand-specific EGFR signalling coordinates epithelial morphogenesis and cell differentiation via apical polarity dynamics.
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We thank K. Schachter for careful reading and editing of the manuscript and help with western blots, A. Lundqvist, J. Larsen, D. Klüver Hansen, K. Stohlmann and A. Stiehm for technical assistance, and G. Karemore, S. Heilmann and A. L. Jackson for help with statistical analysis. We are grateful to the DanStem FCCF and the FACS Core at Lund SCC for FACS assistance, CFIM for use of microscopes and the transgenic core facility at the University of Copenhagen for oocyte injections. We thank Novo Nordisk A/S for access to their proprietary pancreatic endoderm and endocrine progenitor (PE and EP) differentiation protocols as well as for sponsoring the establishment of the human NGN3-reporter line. We thank X. Varelas at Boston University School of Medicine for the Crb3 antibody. This work was supported by the Swedish Foundation for Strategic Research, the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) as part of the Beta Cell Biology Consortium (1 UO1 DK089570-01), the Juvenile Diabetes Research Foundation, the Novo Nordisk Foundation, the Danish Strategic Research Council, the Lundbeck Foundation (R100-A9422) and the Danish Council for Independent Research (ID: DFF—1331-00310A).
Integrated supplementary information
Time-lapse recording of Ngn3RG1/Muc1mCherry pancreatic explants of the conversion of Ngn3Low to Ngn3High cells revealed that Ngn3Low cells (marked with a dot) are fully polarized and located in close proximity to the primitive duct lumen. Concomitant with apical domain size reduction and increased Ngn3 expression (Ngn3High), the cell body moves to a basal position within the epithelium.
Time-lapse recording of Ngn3RG1/Muc1mCherry pancreatic explants in the WT revealed that the time to convert Ngn3Low cells (RFP +) into Ngn3High cells (RFP + /GFP +) was ≈ 4 h (from birth until the cell (marked with a dot) turns on GFP). Quantifications can be found in Supplementary Fig. 3p. Statistic source data can be found in Supplementary Table 3.
Time-lapse recording of Ngn3RG1/Muc1mCherry pancreatic explants in the presence of LY revealed that the time to convert Ngn3Low cells (RFP +) into Ngn3High cells (RFP + /GFP +) was ≈ 8 h (from birth until the cell (marked with a dot) turns on GFP). Quantifications can be found in Supplementary Fig. 3p. Statistic source data can be found in Supplementary Table 3.
Immunofluorescence stainings with an antibody against the apical marker EZRIN revealed that the epithelial cells facing the media were polarized. EZRIN (white), DAPI (blue) & GFP (green).
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Developmental Cell (2019)