ARTICLE https://doi.org/10.1038/s41467-020-17968-x OPEN Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during foregut organogenesis Lu Han 1,6, Praneet Chaturvedi1,6, Keishi Kishimoto1,2,3,6, Hiroyuki Koike 4,6, Talia Nasr1, Kentaro Iwasawa4, Kirsten Giesbrecht4, Phillip C. Witcher1, Alexandra Eicher1, Lauren Haines1, Yarim Lee1, John M. Shannon5, ✉ Mitsuru Morimoto 2,3, James M. Wells 1, Takanori Takebe 4 & Aaron M. Zorn 1,3 1234567890():,; Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal sig- naling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm- derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ ZornLab-singlecell. 1 Center for Stem Cell and Organoid Medicine (CuSTOM), Perinatal Institute, Division of Developmental Biology, Cincinnati Children’s Hospital, Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH 45229, USA. 2 Laboratory for Lung Development, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe 650-0047, Japan. 3 CuSTOM-RIKEN BDR Collaborative Laboratory, Cincinnati Children’s Hospital, Cincinnati, OH, USA. 4 CuSTOM, Division of Gastroenterology, Cincinnati Children’s Hospital, Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH 45229, USA. 5 Division of Pulmonary Biology, Cincinnati Children’s Hospital, Department of Pediatrics, University of Cincinnati, College of Medicine, ✉ Cincinnati, OH 45229, USA. 6These authors contributed equally: Lu Han, Praneet Chaturvedi, Keishi Kishimoto, Hiroyuki Koike. email: Aaron.zorn@cchmc. org NATURE COMMUNICATIONS | (2020) 11:4158 | https://doi.org/10.1038/s41467-020-17968-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17968-x n early fetal development, between embryonic day (E) 8.5 and roadmap of the reciprocal endoderm–mesoderm inductive E9.5 in mouse, equivalent to 17–23 days of human gestation, a interactions that coordinate organogenesis. We validate key pre- I fi series of inductive tissue interactions between the de nitive dictions with mouse genetics showing that differential hedgehog endoderm (DE) and the surrounding splanchnic mesoderm (SM) signaling from the epithelium patterns the SM into gut tube progressively patterns the naive foregut tube into different pro- mesenchyme versus mesenchyme of the liver. Leveraging the genitor domains. These domains further develop into distinct signaling roadmap, we generate different subtypes of human SM visceral organs including the trachea, lung, esophagus, stomach, from hPSCs, which previously have been elusive. liver, pancreas, and proximal small intestine1,2. The DE gives rise to the epithelial lining and parenchyma of the respiratory and digestive organs, while the SM gives rise to the mesenchymal Results tissues such as smooth muscle, fibroblasts, and mesentery sur- Single-cell transcriptomes define diversity in the foregut.To rounding the visceral organs1,2. This foregut patterning defines comprehensively define lineage diversification during foregut the landscape of the thoracic and abdominal cavities, setting the organogenesis, we performed single-cell RNA sequencing relative position of different organs. Disruptions in this process (scRNA-seq) of the mouse embryonic foregut at three time points can lead to life threatening congenital birth defects. that span the period of early patterning and lineage induction: A critical inductive role for the mesenchyme in gut tube E8.5 (5–10 somites; s), E9.0 (12–15 s), and E9.5 (25–30 s) (Fig. 1a, organogenesis was first established in the 1960s, when it was b). We microdissected the foregut between the posterior pharynx shown that SM transplanted from different anterior–posterior (A- and the midgut, pooling tissue from 15 to 20 embryos for each P) regions of the embryo could instruct the adjacent epithelium to time point. At E9.5, we isolated anterior and posterior regions adopt the organ identity consistent with the original SM position3. separately, containing lung/esophagus and liver/pancreas pri- Since that time we have learned much about the mesoderm- mordia, respectively. A total of 31,268 single-cell transcriptomes derived paracrine signals in endoderm organogenesis1,2. However, passed quality control measures with an average read depth of most studies to date have focused on individual organ lineages or 3178 transcripts/cell. Cells were clustered based on the expression individual signaling pathways, and thus we still lack a compre- of highly variable genes across the population and visualized hensive understanding of the temporally dynamic combinatorial using uniform manifold approximation projection (UMAP) and signaling in the foregut microenvironment that orchestrates t-distributed stochastic neighbor embedding (tSNE) (Fig. 1c; organogenesis. Moreover, several fundamental questions about the Supplementary Fig. 1). This identified 24 cell clusters that could mesoderm have remained unanswered over the decades. How be grouped into nine major cell lineages based on well-known many types of SM are there, and does each fetal organ primordia marker genes: DE, SM, cardiac, other mesoderm (somatic and have its own specific mesenchyme? How are the SM and DE paraxial), endothelium, blood, ectoderm, neural, and extra- lineages coordinated during organogenesis? What role if any does embryonic (Supplementary Fig. 1). DE clusters (4448 cells) were the endoderm have in regionalization of the mesoderm? characterized by co-expression of Foxa1/2, Cdh1, and/or Epcam, Initial specification and patterning of the embryonic mesoderm whereas SM (10,097 cells) was defined by co-expression of Foxf1 and endoderm occurs during gastrulation, from E6.25 to E8.0 in (Fig. 1d), Vim, and/or Pdgfra as well as low or absent expression the mouse, as these germ layers progressively emerge from the of cardiac and other mesoderm specific transcripts. primitive streak. The lateral plate mesoderm emerges from the Focusing on lineage diversification in the DE and SM, we streak after the extra-embryonic mesoderm, and is followed by selected these cells in silico for further analysis. We defined 11 the intermediate, paraxial, and axial mesoderm4,5. Concomitantly major DE clusters consisting of 26 stage-specific sub-clusters DE cells also delaminate from the streak and migrate along the (E9.5, 12 clusters; E9.0, 8 clusters; E8.5, 6 clusters) and 13 major outer surface of the mesoderm eventually intercalating into the SM groups comprised of 36 stage-specific sub-clusters (E9.5, 17 overlaying visceral endoderm. By E8.0, morphogenetic processes clusters; E9.0, 12 clusters; E8.5, 7 clusters) (Fig. 1e, f, begin to transform the bi-layered sheet of endoderm and meso- Supplementary Figs. 2, 3, Supplementary Data 1). We annotated derm into a tube structure as the anterior DE folds over to form clusters by comparing their distinguishing genes with published the foregut diverticulum and the adjacent lateral plate mesoderm expression patterns of over 160 genes in the Mouse Genome containing cardiac progenitors migrates towards the ventral Informatics (MGI) database20. These data provide a comprehen- midline6. The lateral plate mesoderm further splits into an outer sive single-cell resolution view of early foregut organogenesis and somatic mesoderm layer next to the ectoderm which gives rise to can be explored at URL https://research.cchmc.org/ZornLab- the limbs and body wall, and an inner splanchnic mesoderm singlecell. layer, which surrounds the epithelial gut tube7,8. The first mole- Our annotations identified all the major DE organ lineages cular indication of regional identity in the SM is the differential at E9.5 including: Tbx1+ pharynx, two Nkx2-1/Foxa2+ respira- expression of Hox genes along the A-P axis of the embryo9. tory clusters (trachea and lung), two Sox2+ esophagus clusters However, in contrast to heart development, where cell diversifi- (one of which is likely dorsal based on Mnx1 expression), two cation has been well studied10–12, the molecular mechanism Sox2/Osr1+ stomach clusters, two Alb/Prox1/Afp+ hepatic governing the foregut SM regionalization are obscure, particularly clusters (c1_hepatoblasts and c10_ early hepatocytes with higher during the critical 24 h when the foregut DE subdivides into Alb/HNF4a expression), Sox17/Pdx1+ ventral hepatopancreatic distinct organ primordia. duct, Mnx1/Pdx1+ dorsal pancreas, and Cdx2+ duodenum Recently, single-cell transcriptomics have begun to examine (Fig. 1e). Consistent with our dissections we did not detect any organogenesis at an unprecedented resolution13–16, however,