The purpose of this grant application is to explore new findings from our work as well as new models recently developed in our lab, in order to understand the mechanisms controlling assembly and formation of liver tissue. Gene array analysis of organoid cultures, composed of reassembled hepatic cellular elements, led to better understanding of the mechanisms by which dexamethasone, HGF and EGF regulate hepatic tissue assembly. We also found, unexpectedly, that in cultures maintained in the absence of dexamethasone HGF and EGF there is high proliferative activity, presumably driven by novel mitogen(s) not predicted from the literature derived from hepatocyte cultures or from whole animal, liver regeneration, models. The focused list of these potential mitogens identified from the gene array analysis will be studied in detail until the signaling molecules regulating this process are determined. After this is accomplished, the role of these mitogens in liver early embryogenesis, overall tissue repair and early carcinogenesis will be studied. We will also pursue further analysis of the role of HGF in liver embryology by utilizing an imported colony of Met -/+ mice, heterozygous for deletion of the HGF receptor (cMet). The livers of Met -/- embryos will be carried out alive further and beyond the day of embryonic lethality and will be studied as embryonic implants in the peritoneal cavity. We have recently developed this model and found out that it allows development and organization of all epithelial elements seen in mature liver including hepatocyte plates and bile ductules, even though mature portal triads do not appear. These studies will finally determine whether embryonic defects seen in livers of HGF -/- and Met -/- mice are primary to the genetic deletions or secondary to the placental anomalies seen in these embryos. The pathways leading to development of biliary ductules in wild type and Met -/- embryo livers after embryonic implantation will also allow determination of the importance of HGF as a key factor for biliary development, an item which has been often postulated but never proven. In addition to mitogenic signals, liver tissue assembly and repair also depends on the capacity of cellular elements to function as tissue restricted facultative stem cells for each other. Biliary cells can transdifferentiate to hepatocytes via the """"""""oval cell"""""""" pathway. We have now shown that hepatocytes can transdifferentiate to biliary epithelium, in culture and in the whole animal. This relationship between hepatocytes and biliary epithelium establishes a new paradigm of organ stability in which mature, fully differentiated, cell types can function as """"""""stem cells"""""""" for each other The signaling pathways and molecular determinants (growth and transcription factors) regulating this process will be studied in cultures, whole animals and embryonic implants. The above studies will provide insights and better understanding of the dynamics of liver tissue formation and provide paradigms with general applicability to other models of formation and maintenance of complex tissues.
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