Characterization of foetal hepatic cells during rat liver development

Abstract

During embryonic development the embryonic/foetal liver is the site where hepatogenesis and hematopoiesis take place. Hepatoblasts have been partly characterized in several previous studies. However, their characterization in the developing liver has not been previously studied. Therefore, the purpose of the current work was to characterize, in a rat model, hepatoblasts in vitro and in vivo during liver development. We consider the progress from early developmental stage, 10 days of gestation (E10) when the liver first starts to develop, to adulthood.Albumin and alpha-fetoprotein (AFP) are the main hepatic markers and are the earliest synthetic products of hepatoblasts during liver development. In this study, we established a reliable method for the first time using a sensitive radioactive biosynthetic labelling, to analyse the albumin and AFP synthesis and secretion capacity of endodermal cells derived from ventral foregut region (E10). It seems that the whole program controlling the regulation of gene expression, synthesis and secretion of albumin and AFP already acts at the earliest developmental stage, when specification of hepatic endoderm appears. The present study shows that explanted endodermal cells from ventral endoderm can express HNF4-alpha, Prox1, beta-catenin, BMP-4, Foxa2, and GATA-4.In the second part of this work we were interested in the development of the liver after it was clearly identifiable as a separate organ (from E12 to adulthood). We demonstrated that during the embryonic and foetal stages about 50% of liver cells are engaged in both albumin and AFP gene expression. In addition the ratio of albumin and AFP producing cells to proliferating cells increases during embryonic stage. At 18 days of gestation the ratio of albumin and AFP producing cells to proliferating cells reaches its maximum. Quantitatively we found that at 18 days of gestation, albumin and alpha-fetoprotein mRNA reaches a maximum and a high rate of synthesis and secretion of albumin and AFP was observed. Additionally, it was observed that at the embryonic stage (from E12 up to E16) albumin and alpha-fetoprotein were synthesized and secreted at different rates. From 18 days of gestation to birth the kinetics of synthesis and secretion of albumin is similar to the kinetics in mature hepatocytes.In the rat embryonic (E12 and E14) and foetal (E18) liver three cell populations were identified. Two of these had a unipotent character, developing into either hepatic lineage (Prox1 positive cells/CK-19 negative cells) or into intrahepatic bile duct lineage (Prox1 negative/CK19 positive cells). The third population retained its bipotent character (CK19 and prox1 positive cells), being able to differentiate into hepatic or bile duct epithelial cells. At this stage CK- 7 was also first detected.Lastly hematopoiesis in the embryonic liver was investigated. A high expression of genes coding for factors which regulate hematopoiesis, such as GM-SCF, G-CSF, SCF and Epo, was observed at 12 and 14 days of gestation.