Acini are essential for the secretory function of multiple exocrine organs. During acini formation, a cluster of epithelial end bud progenitors must undergo secretory program initiation, maturation and cell polarization to form a unit of single layered secretory cells enclosing a central lumen. Yet, how this complex process is regulated remains unknown. Using the developing salivary gland as a model, we reveal the EGF receptor ERBB3 as a master driver of acinus formation. We discovered ERBB3 to be enriched in end buds, and through conditional deletion of epithelial Erbb3, to be essential in multiple steps of secretory cell program initiation, cell maturation, expansion and polarization. Moreover, we demonstrate that addition of a single factor, ERBB3 ligand neuregulin-1, to isolated epithelia ex vivo is sufficient to recapitulate the complex outcomes of in vivo acinus formation in multiple exocrine organs. Finally, we reveal a new ERBB3-mTOR1/2 signaling link that governs the full formation of the secretory acinus.

Mouse embryonic stem cells (mESCs), an in vitro model for naive pluripotent differentiation into primed pluripotent epiblast-like cells (EpiLCs), have been used to reveal transcriptional and epigenetic control of early embryonic development. However, the control and significance of morphological changes during lineage specification remain less defined. We show with quantitative time-lapse imaging marked changes in morphology and actin architectures during differentiation that depend on activity of the Arp2/3 complex, an actin filament nucleator. Inhibiting Arp2/3 complex activity pharmacologically or genetically does not block exit from naive pluripotency but attenuates increases in EpiLC markers compared with controls. We found that loss of Arp2/3 complex activity delays entry into the intermediate formative pluripotent state, resulting in globally defective lineage specification as indicated by RNA-sequencing with marked effects on TBX3-depedendent transcriptional programs. Further, we identified two previously unreported indicators of pluripotent status; MRTF and FHL2, which have inverse actin-dependent nuclear translocation as competing SRF co-factors that is dependent on Arp2/3 complex but not formin activity. These data reveal a previously unrecognized role for Arp2/3 complex-dependent actin remodeling in mESC differentiation, timing of formative pluripotency, and TBX3-dependent lineage specification, as well as newly identified MRTF and FHL2 nuclear shuttling. Moreover, our current findings on a role for Arp2/3 complex activity in mESC differentiation compared with the established role for formin activity in epithelial to mesenchymal transition indicate that distinct actin nucleators regulate distinct modes of epithelial plasticity.

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. In Chapters 2 and 3, we utilize a combination of single-cell RNA-sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing, to profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time. We find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating towards a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population towards the alpha or beta cell lineages. Within the mesenchyme, we identify previously underappreciated cellular heterogeneity and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. In Chapter 4, we further characterize the pancreatic mesothelium, identifying novel markers and secreted factors expressed within this population. Knockout of the secreted factor specifically expressed by the mesothelium, Fgf9, reveals a hypoplastic pancreas by late gestation. We find a disrupted ratio of epithelial and mesenchymal cells that suggests Fgf9 regulates the epithelial-mesenchymal interactions critical for pancreatic development. In Chapter 5, we extend our single-cell RNA-sequencing approach to adult pancreatic homeostasis and disease, describing multiple subtypes of mesenchymal and mesothelial populations in both conditions. Comparison of diseased and healthy pancreata reveal shifts in mesenchymal cell types, highlighting populations and transcriptional targets that may regulate the development of disease. In summary, this work reveals transcriptional and cellular heterogeneity of the developing, adult, and diseased pancreas, and identifies lineage relationships among novel populations within both the epithelial and mesenchymal compartments.