We found that WNT/FGF require BMP activity to initiate posterior gene expression, consistent with the known role of BMP as a posteriorizing factor11C13

We found that WNT/FGF require BMP activity to initiate posterior gene expression, consistent with the known role of BMP as a posteriorizing factor11C13. antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust system for elucidating AN7973 the mechanisms underlying human stomach development and disease. is then patterned along the anterior-to-posterior (ACP) axis and transformed into a gut tube consisting of Sox2+ foregut in the anterior and Cdx2+ mid-hindgut in the posterior (Fig. 1a). We previously demonstrated that WNT3A and FGF4 synergize to induce the morphogenesis of gut tube-like structures expressing the posterior marker CDX26,10. To generate foregut, from which the stomach derives, we aimed to stimulate gut tube morphogenesis with WNT and FGF while inhibiting their ability to promote posterior fate. We found that WNT/FGF require BMP activity to initiate posterior gene expression, consistent with the known role of BMP as a posteriorizing factor11C13. Specifically, inhibiting BMP signaling with the antagonist Noggin resulted in repression of the posterior marker CDX2, activation of the foregut marker SOX2 and AN7973 assembly of three-dimensional foregut spheroids (Fig. 1bCd and Extended Data Fig. 1). Foregut spheroid morphogenesis was a robust process using both hESC and hiPSC lines (Fig. 1cCd and Extended Data Fig. 2). Thus, we identified a new epistatic relationship between WNT, FGF and BMP in which all three pathways cooperate to promote a mid-hindgut fate, but WNT and FGF act separately from BMP to drive morphogenesis of AN7973 gut tube structures. Open in a separate window Figure 1 Generation of three-dimensional posterior foregut spheroidsa, Sox2 marks foregut endoderm and Cdx2 marks mid/hindgut endoderm in E8.5 (14 somite stage) mouse embryo. bCc, qPCR analysis (b) and wholemount immunostaining (c) for patterning markers in hPSC-DE cultures exposed to three days in media alone (control) or with the indicated growth factors/antagonists. WNT3A and FGF4 induced CDX2 expression whereas the BMP antagonist noggin repressed CDX2 and induced high levels of the foregut marker SOX2. Results are normalized to expression in Control (stage-matched, no growth factor-treated) endoderm. *, p<0.05 compared to control. **, p<0.005 compared to WNT/FGF; two-tailed students t-test; are posterior patterning of the foregut and specification of the fundic and antral domains of the stomach. To direct spheroids into a posterior foregut fate (indicated by co-expression of Sox2 and Hnf1; Fig. 1e), we focused on retinoic acid (RA) signaling given its AN7973 role in development of posterior foregut-derived organs14C16. Exposing DE to RA for 24 hours on the final day (d5-6) of the patterning/spheroid generation stage resulted in the formation of SOX2/HNF1+ posterior foregut spheroids (Fig. 1fCg and Extended Data Fig. 3). the posterior foregut undergoes morphogenesis and is subdivided into the Sox2+/Pdx1? fundus, Sox2/Pdx1+ antrum, Pdx1/Ptf1+ pancreas, and Pdx1/Cdx2+ duodenum (Fig. 2b). To promote three-dimensional growth and morphogenesis, we transferred posterior foregut spheroids to a semisolid matrix and found that an additional 72 hours of RA (d6-9) caused a >100-fold increase in mRNA levels while maintaining high expression (Fig. 2cCd), indicating specification into antrum. Importantly, the RA treatment did not promote a pancreatic fate8, since expression of the pancreas-specific marker culture system used to direct the differentiation of hPSCs into three-dimensional gastric organoids. b, Defining molecular domains of the posterior foregut in E10.5 mouse embryos with Sox2, Pdx1 and Cdx2; Sox2/Pdx1, antrum (a); Sox2, fundus (f); Pdx1, dorsal and ventral pancreas (dp and vp); Pdx1/Cdx2, duodenum (d). c, Posterior foregut spheroids exposed Rabbit Polyclonal to Thyroid Hormone Receptor beta for three days to RA (2 M) exhibited >100-fold induction of compared to control spheroids, measured by qPCR. *, p<0.05; two-tailed students t-test; at day 6 (posterior foregut endoderm), AN7973 followed by induction of at day 9 (presumptive antrum). Day 9 antral spheroids had a 500-fold increase in and a 10,000-fold increase in relative to day 3 DE. *, p<0.05; two-tailed students t-test; was not significantly increased. e, Stereomicrographs showing morphological changes during growth of gastric organoids. By four weeks, the.