Organogenesis and Histologic Development of the Liver

Introduction

The liver is the largest internal organ of the body, compromising approximately 6% to 7% of the total weight of an adult. The organ is unique in that it receives dual supply, including venous inflow via the portal vein—predominantly from the intestines, pancreas, and spleen—and arterial inflow from the aorta via the hepatic artery. Together these sources provide the delivery of nutrients, hormones, toxins, and oxygen to hepatic tissues ( Fig. 88.1 ). The parenchyma of the liver is dominated by bipolar hepatocytes, organized into linear plates that are exposed to these blood elements on their basolateral surface that abuts a plexus of sinusoidal capillaries. Although hepatocytes are the predominant cellular element of the liver, their function, structure, and differentiation are supported by interactions with other cellular components including cholangiocytes, stellate cells, Kupffer cells, Ito cells, endothelial cells, and hematopoietic elements. An intercellular matrix provides structural integrity and supports intracellular communications between these cellular elements.

The hepatocytes perform various secretory and metabolic functions, including but not limited to detoxification of drugs and toxins, synthesis of key serum proteins (e.g., albumin, clotting factors, complement, apolipoproteins), synthesis and metabolism of dietary lipids, glucose homeostasis, and bile production. Distortion of the normal architecture of the liver either by chronic disease states or congenital malformations can have significant impact on the ability of the liver to perform these complex functions. As expected, the developmental steps necessary to ensure proper hepatic organization and function involve multiple and complex communications between the cellular and matrix components of the organ during organogenesis of the liver. Investigators continue to discover new intracellular signals (specific growth factors and transcription factors) that appear to combine to initiate and propagate the transformation of fetal endodermal tissue into differentiated hepatic cellular elements and a functioning organ. This chapter will attempt to summarize some of the experiments using molecular genetics, molecular biology techniques, and tissue explant culture techniques that have shed light on our understanding of early steps in hepatic development.

Early Embryogenesis: An Overview

Extensive investigations have helped to clarify many of the steps involved with the early development of the liver and the specific messengers and tissue signals that orchestrate this process, as well as directing the maturation of the hepatocytes. ^, Specific signaling molecules, hormones, growth factors, transcriptional factors, and intracellular matrix interactions have been shown to contribute to the induction of pluripotential primitive tissues into cells committed to a hepatic fate. In addition, these molecules are critical to the maturation of the cells into specialized epithelium and contribute to the proper organization of these cells into the correct configuration constituting the fully functioning organ. Much of this work has been derived from observations in genetically altered mice, zebrafish, and tissue culture systems. During the third to fourth week of gestation, a bud of proliferating endodermal tissue is observed originating from the ventral foregut constituting the hepatic diverticulum. The progenitor cells of this region are derived from three domains that fuse to form a single prehepatic domain adjacent to the cardiogenic mesoderm. Cells of this endodermal region become committed to hepatic fate by a process referred to as hepatic specification. The primitive cells of this bud, referred to as hepatoblasts, appear to be bipotential, with the ability to differentiate into either mature hepatocytes or cholangiocytes. These cells are in contact with embryonic cardiac mesoderm and abut the septum transversum mesenchyme. ^, Following inductive signaling from adjacent mesoderm, liver bud morphogenesis proceeds by conversion of cubital foregut epithelium to columnar hepatoblasts that subsequently change to pseudostratified epithelium mediated by transcription factor Hhex; they then migrate into adjacent mesenchyme. The hepatoblasts migrate as cords into the septum transversum, closely associated with primitive sinusoidal endothelial cells. The migration process is mediated by a loss of contact between hepatoblasts as a result of downregulation of E-cadherin as well as concomitant extracellular matrix remodeling of the basal membrane, mediated in part by mesenchyme-derived Mmp2 and hepatocyte-expressed Mmp14. As the process progresses, the sinusoidal structure is established, initially lacking the fenestrations observed at the latter stages of maturity. The undifferentiated hepatoblasts have few organelles at this stage, with a high nuclear to cytoplasmic ratio, scant rough endoplasmic reticulum, and few lysosomes. Intercellular communication appears to be mediated via cell surface adhesions with other hepatoblasts and surrounding mesenchymal cells. The anterior vitelline vessels of the yolk sac are the initial source of sinusoidal blood. Early synthesis and secretion of α-fetoprotein, albumin, transthyretin, transferrin, and α-1-antitrypsin can be observed from these immature prehepatic cells. ^,

Late Embryogenesis: An Overview

Early in the second month of gestation, the hepatoblasts gradually begin to differentiate into mature hepatocytes with the necessary intercellular components (rough endoplasmic reticulum and Golgi apparatus) to conduct their multiple synthetic and metabolic functions. These cells also acquire polarity with the increased production of specific membrane-associated proteins and transmembrane transporters. This process creates a basolateral hepatocyte domain with clusters of membrane-associated receptors and transporters for protein secretion in association with sinusoidal vessels, and an apical hepatocyte domain constituting of the bile canaliculi with transporters related to bile secretion. Both these surfaces develop microvillus architecture, presumably to optimize surface area for extracellular contact. Other hepatoblasts are thought to develop into cholangiocytes that organize into the hepatic biliary system. ^, The intrahepatic bile ducts are formed from periportal hepatoblasts forming the ductal plate. Differentiation and maturation of the intrahepatic ducts occurs via interactions with periportal connective tissue, glucocorticoid hormones, and basal laminar components. Intrahepatic hematopoiesis increases at this stage and appears to progress with interactions between maturing hepatocytes and undifferentiated mesenchymal elements. These hematopoietic precursors release interleukin 6, stimulating hepatocyte differentiation, which is also mediated by Hnf4α , an important transcriptional regulator of hepatocyte maturation. ^,

Specific Interactions Promoting Hepatogenesis

The initial induction of the ventral foregut to commit to a hepatic fate has been shown, in embryo tissue transplant studies in the chick, to be a function of interactions of this endodermal region with cardiac mesoderm ( Table 88.1 ). The growth factors found to mediate this process produced by cardiac mesoderm are fibroblast growth factors (FGFs) FGF-1 and FGF-2. Further studies using purified FGFs and FGF inhibitors confirmed this interaction. The specificity of FGF for this region appears to be related to the fact that FGF migration is limited by high-affinity interactions with extracellular matrix. ^, In fact, further explanted embryonic tissue studies demonstrate that without this induction stimulus the fate of the ventral foregut that normally gives rise to the hepatic diverticulum will default to a pancreatic fate. Specifically, FGF stimulation of the ventral foregut endoderm inhibits pancreatic genes and induces liver genes in this bipotential precursor cell population.

The induction stimulus provided by FGF is not sufficient to stimulate hepatocyte differentiation. Hepatocyte differentiation appears to be a function of a second induction stimulus from another mesoderm-derived tissue, the septum transversum mesenchyme. The signal proteins for this interaction appear to be bone morphogenetic proteins (BMPs). Specifically, BMP-2, BMP-4, and BMP-7 are produced by the septum transversum mesenchyme cells. Further studies using a BMP signal inhibitor, Noggin, demonstrated that BMP as well as FGF were needed to achieve hepatoblast induction from ventral gut endoderm. These mechanisms controlling the initiation of liver development are well conserved among various organisms, as demonstrated in studies in chick and zebrafish models. ^{, ,} The stimulatory effects of FGFs are focused to the prehepatic endodermal tissue by a network of transcription factors expressed in embryonic gut tissue. One such molecule is hepatocyte nuclear factor (HNF) 3Β. These transcription factors appear to be important mediators of hepatocyte differentiation that bind to specific hepatic gene enhancer regions promoting gene expression and cellular differentiation.

The structure of the liver is organized into bipolar hepatocytes in linear plates interfacing with sinusoids on their basolateral surface and bile canaliculi at their apical pole. The molecular mechanisms responsible for hepatocyte polarity have been studied in vivo using HepG2, MDCK, and WIF-B cell lines as well as sandwich cultures of primary hepatocytes. ^{, ,} Despite these investigations, the mechanisms responsible for the establishment of hepatocyte polarity during development are as yet unknown.

The importance of the interaction between the septum transversum mesenchyme and the developing prehepatic endoderm cannot be overemphasized. The septum transversum mesenchyme is the source of BMP signaling, leading to induction of hepatoblast differentiation and propagation of the hepatocyte maturation process. Extracellular matrix components of the septum transversum mesenchyme aid in the regulation of differentiation by binding and concentrating signaling molecules. ^, In addition, extracellular matrix components can directly mediate intracellular communication through interactions with integrins, focal adhesion kinase, and other signaling molecules. ^,

Membrane trafficking pathways and intracellular trafficking contribute to hepatocyte maturation and polarity regulated by liver kinase B1. This molecule mediates its effects through adenosine monophosphate (AMP) activated protein kinase.

Hepatocyte growth factor (HGF) is a potent hepatocyte proliferation stimulant that affects cell migration as well as differentiation. ^, Retinoic acid is one additional factor that indirectly stimulates hepatoblast proliferation and stellate cell formation by inducing production of trophic factors by mesodermal cells, as demonstrated in chick embryo studies. In addition, retinoic acid signaling has been characterized as an early regulator of liver asymmetry in zebrafish, leading to differential morphogenesis of the right and left liver lobes, but whether this contributes to liver symmetry in mammals is unknown. These are but a few of the recognized components of the septum transversum mesenchyme and its extracellular matrix that are part of an integrated and diverse signaling process ensuring integrity of the hepatocyte maturation process and the overall hepatic structural organization.