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O0042 WNT SIGNALING IN EARLY LIVER AND FOREGUT DEVELOPMENT

McLin, V. A.1; Zorn, A. M.2

Journal of Pediatric Gastroenterology and Nutrition: June 2004 - Volume 39 - Issue - p S23
ABSTRACTS: Oral Presentation Abstracts
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1Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center,2Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States

Submitted by: Valerie.Mclin@cchmc.org

Introduction: The Wnt signaling cascade is conserved in vertebrate development. It is also known to be reactivated in hepatocellular carcinoma, and nuclear betacatenin may be a prognostic factor in hepatoblastoma. As developmental pathways are often reactivated in disease processes such as tumorigenesis and regeneration, understanding the regulation of Wnts in early liver development may offer insight into the pathophysiology and potential treatments of liver disease. Both published as well as preliminary data have shown that Wnt antagonists are strongly expressed in the anterior endoderm, which gives rise to foregut and liver precursors. Our preliminary data suggests that Wnt antagonists are strongly expressed in the anterior endoderm at gastrula stage. Using a Xenopus model, the aim of this study is to demonstrate that inhibition of Wnt signaling in the anterior endoderm is necessary for normal foregut and liver development.

Methods: (1) To demonstrate that Wnt signaling needs to be repressed in the early embryo, gain of function experiments were performed to disrupt anterior endoderm formation: a) gastrulae were placed in a LiCl bath for 15′ to ectopically activate Wnt by inhibiting GSK3 function b) Wnt 8 DNA was injected in the presumptive anterior endoderm at the two cell stage. (2) Loss of function experiments were conducted to show that excessive Wnt inhibition leads to abnormal anterior endoderm: a) a dominant negative form of Wnt 8 RNA was injected in all blastomeres at the 4 cell stage b) mRNA for the Wnt antagonist Dikkhopf-1 was injected in all blastomeres at the 4 cell stage. All embryos were allowed to develop to the organ bud stage when they were fixed for whole mount in situ hybridization. DIG labeled antisense RNA probes for each major organ derived from the anterior endoderm (AE) were used to look at the effect Wnt activation or inhibition on AE structures. The probes used were Hex and FOR (Farnesoid-like Orphan Receptor) for liver, Nkx2.1 for lung, and XlhBox8 for pancreas.

Results: (1) Ectopic Wnt activation at gastrula stage yielded microcephalic embryos which displayed decreased liver tissue by in situ compared to age matched controls. (2) Inhibition of Wnt signaling by injection of Wnt antagonists in 4 cell embryos yielded embryos with enlarged head structures, together with a protuberant AE, and an expanded liver and foregut by in situ.

Conclusion: Wnt signaling needs to be repressed in the early specification of the anterior endoderm for liver and other foregut organs to form normally. Based on these data, we hypothesize that liver and foregut development requires a series a Wnt free window in the anterior endoderm at gastrula stage.

© 2004 Lippincott Williams & Wilkins, Inc.