To study gene expression during endodermal organogenesis, we sought to identify genes expressed in restricted domains during organogenesis. For gene expression analysis, six morphologically distinct endodermal domains were dissected at E11.5: the esophageal region; the lung and distal tracheal region; the stomach region; the hepatic region; the dorsal and ventral pancreatic region; and the intestinal region. Through flow cytometric separation using EpCAM expression to distinguish endoderm from surrounding mesenchyme, pure populations of endoderm progenitors from the esophageal, lung, stomach, pancreatic, and intestinal regions were isolated. Expression of Liv2 was used to isolate a pure population of hepatic endoderm progenitors.

Understand the mechanism by which the endoderm is divided into organ domains during embryogenesis.

Six early endodermal organ domains were dissected at E11.5 to obtain progenitors for: esophagus, lung and distal trachea, stomach, liver, dorsal and ventral pancreas, and intestine. Surrounding mesenchyme was separated from endoderm by flow cytometry using anti-EpCAM; anti-Liv2 was used to purify hepatic endoderm progenitors. Definitive endoderm from E7.5, E8.25, and E8.75 were also profiled. Illumina MouseRef-8 v2.0 expression beadchip arrays were used on 3 biological replicates of each sample. Expression analysis was followed up by dynamic immunofluorescence analysis of key transcription factors.

A total of 93 transcription factors were found to vary between E11.5 endoderm organs. Myt1, Ngn3, Nkx2.2, Nkx6.1, and Ptf1a were exclusive to pancreas as were novel factors Rxrg and Tcf15. Other patterns observed included: Foxa3 and Gata6 in the 5 most posterior organs (lung, stomach, liver, pancreas, intestine); Hnf4a and Onecut2 in stomach. liver, pancreas, and intestine; Nr5a2 in liver, pancreas, and intestine; Onecut3 in stomach, liver, and pancreas; Cited1, Hhex, Prox1 in liver and pancreas. Several cell surface proteins expressed in a single organ domain were identified (pancreas:Car9, Chrna4, Chst2, Flrt2, Gfra3, Gja9, Gng4, Gpr43, P2rx1, Pcdh21, Sez6l2, Syp, Tmem27, Abcc8, Celsr3, Kcnk3, Lrp11, Slc38a5). Dlk1 (commercial antibody available) was expressed in pancreatic and liver endoderm. Additionally, a repressive interaction between Cdx2 and Sox2 was found to occur at the prospective stomach-intestine border, with the hepatic and pancreatic domains forming at this boundary, and Hlxb9 was revealed to have graded expression along the dorsal-ventral axis.

Expression profiling and immunofluorescence has provided information about genes that underlie endoderm organogenesis and characterization of associated anterior-posterior and dorsal-ventral patterning.

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