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SnapShot: GI Tract Development Efficiently differentiate human embryonic stem or induced pluripotent stem cells to the endoderm lineage with STEMdiff™ Patrick S. McGrath and James M. Wells Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA Pancreatic lineage allocation Endoderm formation Gastrulation Mesendoderm e7.5 Mesoderm Foregut endoderm Tcf2 (Hnf1β) Hnf6 (Onecut1) Foxa2 (Hnf3β) Hlxb9 (Hb9) Hhex Sox17 Ectoderm Use the STEMdiff™ Pancreatic Progenitor Kit (Catalog #05120) to generate multipotent PDX-1+/NKX6.1+ pancreatic progenitor cells that are capable of downstream maturation to both endocrine and exocrine cells. Endoderm 200 μm Endoderm patterning Posterior Anterior e8.5 8–10 somites Pancreatic progenitor Pdx1 Nkx2.2 Nkx6.1 Ptf1a (P48) Hlxb9 (Hb9) Posterior Anterior Wnt / FGF4 / BMP Wnt-antagonists 300 μm Organ specification Anterior foregut Use the STEMdiff™ Definitive Endoderm Kit (Catalog #05110) to generate multipotent definitive endoderm cells that are capable of differentiating downstream toward hepatic, intestinal, pancreatic and pulmonary cells. Posterior foregut Midgut Hindgut e9.0 ~15 somites Endocrine progenitor Neurog3 NeuroD Ia1 Isl1 Pax6 Rfx6 Exocrine progenitor Ptf1a Hnf6 Hnf1β STEMCELL Technologies is committed to making sure your research works. As Scientists Helping Scientists, we support our customers by creating novel products of consistently high quality and by providing unparalleled scientific support. 500 μm Organ buds Dorsal pancreas Duodenum Stomach Thyroid Liver Lungs e9.5 ~25 somites Small intestine Ventral pancreas Large intestine Biliary Sox2 Pou3f4 (Brn4) Pax6 MafB Pax4 MafB Pdx1 Nkx2.2 Nkx2.2 Pdx1 Ins PP Pax4 Pax6 Learn more at www.stemcell.com/GItract_stemdiff ? For legend and references visit www.stemcell.com/wallchart_GItract Cdx Pdx1 Hoxa2 500 μm Proliferation/differentiation Hoxb1 Tbx1 Hex1 Thyroid Hoxa3, Hoxb4 Nkx2.1 Lung Biliary tree Hex1 Sox17 Hoxc5 Hoxc6 Ptf1a Stomach Small intestine >e13.5 50–60 somites Esophagus Liver Pancreas Gall bladder Hoxb6 Hoxb8,Hoxc9 Hoxb9 Hoxa/d13 Sst Cpa Elastase Colon α Glucagon Arx MafB Brn4 β Insulin Pdx1 Mnx1 Nkx2.2 Nkx6.1 NeuroD MafA γ δ ε Pancreatic Somatostatin Ghrelin polypeptide Pdx1 -Pax4 Arx -NKX2.2 Exocrine cell Ptf1a Gata4 Mist1 Amylase Duct Hnf6 Hnf1β Sox9 Foxa2 DOCUMENT #27018 | VERSION 1.0.0 SnapShot: GI Tract Development Patrick S. McGrath and James M. Wells Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA The endoderm germ layer contributes to the respiratory and gastrointestinal (GI) lineages during development, giving rise to an array of specialized epithelial cell types lining organs, including the thyroid, thymus, lungs, liver, biliary system, pancreas, and intestines. This SnapShot timelines and summarizes key stages following gastrulation, including endoderm patterning, organ specification, and organogenesis. A lineage tree of the developing endocrine pancreas is outlined to further illustrate this process. Timeline of Endoderm Formation, Patterning, and Organogenesis During development in mice (left), the blastula gives rise to the three germ layers (ectoderm, mesoderm, and definitive endoderm) through the process of gastrulation (middle), which occurs between embryonic day 5 and 7.5 (e5–e7.5). After gastrulation, the two-dimensional sheet of definitive endoderm is patterned along the anterior-posterior (A-P) axis and undergoes morphogenesis to form a three-dimensional gut tube that is surrounded by a primitive mesenchyme (e8.5). A-P patterning of the endoderm occurs through reciprocal signaling with the mesenchyme involving growth factors such as Wnts, Fgfs, and Bmps. At this stage in development, these factors largely act to promote posterior fate and repress anterior fate. The anterior endoderm gives rise to the foregut (thyroid, lungs, esophagus, liver, stomach, pancreas), while the midgut and hindgut give rise to the small and large intestines, respectively. The first evidence of organ specification occurs in the early gut tube by the expression transcription factors that begin to demarcate specific organ domains, including the respiratory tract (Nkx2.1), liver (Hhex), stomach (Sox2 and Pdx1), extrahepatic biliary system (Sox17), pancreas (Pdx1 and Ptf1a), duodenum (Pdx1 and Cdx2), and intestine (Cdx2). The spatially restricted expression of these transcription factors predicts where organs will begin to form starting around e9.5. By e13.5, the organs of the respiratory and GI tracts are formed and undergoing growth and differentiation into specialized lineages. Pancreatic Lineage Allocation: A Transcription Factor Map Temporal lineage formation of the pancreas involves the expression of unique sets of transcription factors that mark and often direct cell fate decisions (right). All developing cell lineages of the pancreas (acinar, duct, and endocrine) arise from the foregut endoderm, which expresses markers such as Foxa2, Hnf6, and Hlxb9. The pancreatic endoderm becomes specified when the gut tube begins to express Pdx1 and Ptf1a in dorsal and ventral domains of the tube (e8.5–9.0). Morphogenesis of the pancreas initiates with an endodermal thickening (e9.0) and evagination of dorsal and ventral pancreatic buds (e9.5–e10.0) into the surrounding mesenchyme, forming an expanding pool of multipotent pancreatic progenitor cells. The lineage allocation and maturation of specific pancreatic cell subtypes are mediated by a network of signaling pathways and transcription factors. Commitment of progenitor cells to the endocrine lineage occurs following transient expression of Neurog3 and its downstream targets Neurod, Rfx6, and Pax6, whereas exocrine-committed cells express high levels of Ptf1a and carboxypeptidase A (CpA). Allocation of the separate endocrine lineages involves the combinatorial actions of multiple transcription factors. For example, development of mature β cells requires Pdx1, NeuroD, Nx6.1, and MafA. The ductal lineage involves a different set of factors, including Hnf1β and Hnf6. Each pancreatic cell lineage is portrayed with a subset of defining transcription factors throughout development. References Jørgensen, M.C., Ahnfelt-Rønne, J., Hald, J., Madsen, O.D., Serup, P., and Hecksher-Sørensen, J. (2007). Endocr. Rev. 28, 685–705. Pagliuca, F.W., and Melton, D.A. (2013). Development 140, 2472–2483. Pan, F.C., and Wright, C. (2011). Dev. Dyn. 240, 530–565. Zorn, A.M., and Wells, J.M. (2009). Annu. Rev. Cell Dev. Biol. 25, 221–251. 176.e1 Cell 161, March 26, 2015 ©2015 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2015.03.014