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A complex interplay of intrinsic factors and extrinsic signalling pathways controls both cell lineage commitment and maintenance of cell identity. Loss of defined cellular states is the cause of many different cancers, including pancreatic cancer. Recent findings suggest a clinical role for the conserved SLIT/ROBO signalling pathway in pancreatic cancer. However, whilst this pathway has been extensively studied in many processes, a role for Slit and Robo genes in pancreas cell identity and plasticity has not been established yet. Here, we identify Slit/Robo signalling as a key regulator of pancreatic progenitor identity. We find that Robo1 and Robo2 are required for preserving pancreatic cell identity shortly after fate induction and, subsequently, for expansion of the pancreatic progenitor pool in the mouse. Furthermore, we show that Robo receptors control the expression of Tead transcription factors as well as its downstream transcriptional activity. Our work identifies an interplay between Slit/Robo pathway and Tead intrinsic regulators, functioning as gatekeeper of pancreatic cell identity.

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Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal, endothelial) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, while the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirement for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as Pancreas Embryonic Cell Atlas).

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Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key development time points in the mouse embryo. We used transgenic mouse models and antibodies to visualize the three main cellular components within the developing pancreas, including epithelial, mesenchymal and endothelial cell populations. We demonstrated the utility of the approach by providing volumetric data, 3D distribution of distinct progenitor populations and quantification of relative cellular abundance within the tissue. Lastly, our image data were combined in an open source online repository (referred to as Pancreas Embryonic Cell Atlas). This image dataset will serve the scientific community by enabling further investigation on pancreas organogenesis but also for devising strategies for the in vitro generation of transplantable pancreatic tissue for regenerative therapies.

SummarySingle cell-based studies have revealed tremendous cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degree of plasticity during organogenesis1–5.The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including liver, pancreas, gallbladder, and extra-hepatic bile ducts6, 7. Experimental manipulation of various developmental signals in the mouse embryo underscored an important cellular plasticity in this embryonic territory6, 8. This is also reflected in the existence of human genetic syndromes as well as congenital or environmentally-caused human malformations featuring multiorgan phenotypes in liver, pancreas and gallbladder6, 8. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary, and pancreatic structures are not yet established. Here, we combine computational modelling approaches with genetic lineage tracing to assess the tissue dynamics accompanying the ontogeny of the hepato-pancreato-biliary organ system. We show that a long-term multipotent progenitor domain persists at the border between liver and pancreas, even after pancreatic fate is specified, contributing to the formation of several organ derivatives, including the liver. Moreover, using single-cell RNA sequencing we define a specialized niche that possibly supports such long-term cell fate plasticity.