The cells that will give rise to the mammalian liver are first distinguishable in the embryo as a thickening of the epithelium of the ventral foregut in the region that is in contact with the precardiac mesoderm. Although these cells can be identified morphologically, the genetic and epigenetic events important in early liver development have not been fully elucidated. The work described here represents a multifaceted approach aimed at defining the molecular genetic events required for the commitment of endoderm cells to the hepatic epithelial cell lineage and the differentiation of these committed precursors into hepatocytes and bile duct epithelial cells. This work will provide a more complete understanding of the biology of liver stem cells in the embryo and will serve as a comparison for putative stem cells in the adult liver.
Specific Aim 1 : Since it is believed that transcriptional controls determine cell fate, the initial experiments will focus on determining the pattern of gene expression during the differentiation of hepatic stem cells along the hepatocyte or bile ductular lineage. They will use a newly established murine hepatic endoderm cell line, isolated from the hepatic diverticulum at day 9.5 of gestation, that has been designated HBC-3. Our findings in t his aim will form he basis for specific tests for gene function during hepatocyte or bile duct differentiation in Specific Aims 2 and 3.
Specific Aim 2 contains experiments designed to determine whether overexpression of transcription factors that are expressed during hepatocyte or bile ductular differentiation is sufficient to induce differentiation of HBC-3 cells.
Specific Aim 3 contains experiments that will utilize gene targeting technology to determine whether putative liver regulatory genes are essential for differentiation of HBC-3 cells along the hepatocyte or bile ductular lineage.
Specific Aim 4 contains experiments designed to determine how early in development can embryonic liver stem cells be isolated that possess the capacity to differentiate into liver derivatives? Additional experiments will attempt to identify the inductive signals involved in the establishment and differentiation of the hepatic epithelial cell lineage. The approaches used for these experiments include: (A) Microdissection of e8.0-9.5 embryos and isolation of embryonic tissues that gives rise to liver. Tissues will be isolated from metallothionine Lac Z (MT-Lac Z) embryos so that the cells will be biochemically marked when transplanted into the liver of albumin Urokinase plasminogen activator (alb uPA) mouse liver to test their differentiation capacity. (B) Coculture of prehepatic and hepatic endoderm with embryonic mesoderm from different regions of the embryo to define the specific mesodermal-endodermal interactions required for the establishment of the hepatic epithelial lineage in mammals.""""""""
