Several questions regarding liver development and regeneration have been difficult to address because of the slow rate of replication of adult hepatocytes, and the inability to examine an individual cell for its differentiated functions at more than one time point. Transgenic mouse lines have been generated which express the E coli beta-galactosidase (beta-gal) gene from the relatively liver-specific human alpha1- antitrypsin (hAAT) promoter. Intriguingly, only 10% of hepatocytes from these animals contain beta-gal protein, in clusters of cells which bear no relationship to the liver architecture. One goal of this project is to determine if expressing cells are derived from a single clone, which would enable one to use patterns of expression observed in adult and developing animals to predict the location and frequency of hepatocyte progenitor cells. Clonality of beta-gal expression will be determined in vivo by transplanting transgenic hepatocytes which express an hAAT-beta-gal transgene in some hepatocytes, and an L-FABP-hGH gene in all hepatocytes, into the liver of a congenic mouse. Hepatocytes will be stimulated to divide in recipient animals in vivo by induction of liver damage. Recipient livers will be examined for the size and frequency of clusters of hGH-positive hepatocytes, and for their homogeneity of expression (or lack thereof) of beta-gal. This transplant technique will also be used to quantitate the proliferative potential of hepatocytes in vivo. The relative location of hepatocytes within the liver architecture at various times after transplantation will be measured to determine if there is a slow migration of cells from the periportal to the pericentral region. The final focus of this proposal will be to try to identify the biochemical factors which lead to the existence of subsets of hepatocytes which express overlapping but non-identical sets of liver-specific proteins. Hepatocytes which express the beta-gal protein from the hAAT promoter will be separated from those that do no by using fluorescence activated cell sorting, and compared biochemically. These studies may yield key insights into how a cell becomes committed to express or not express a liver-specific function.
