Embryonic development of the liver†

The combined use of genetic manipulation in mice and advanced culturing techniques, along with the availability of model genetic systems, underlies the recent expansion in our understanding of the fundamental processes that govern the development of the liver. Although gaps in our knowledge still exist, many of the molecules and pathways that act during the earliest stages of hepatic development have been identified. This knowledge has been applied to control the differentiation of embryonic stem and hepatic progenitor cells into hepatocytes and cholangiocytes, raising the prospect of generating quantities of hepatic cells in culture that are suitable for therapeutic uses. mRNA, messenger RNA; E, embryonic day; FGF, fibroblast growth factor; BMP, bone morphogenetic protein. The principal functional cell type of the liver is the hepatocyte, which accounts for 78% of parenchymal volume in the rat liver.1 The hepatocyte is a polarized epithelial cell that exhibits both endocrine and exocrine properties. Biliary epithelial cells (cholangiocytes), sinusoidal endothelial cells, hepatic stellate cells (Ito cells), Kupffer cells, and pit cells (liver-specific natural killer cells) represent the majority of non-hepatocyte cell types in the liver. Although most research has traditionally focused on the hepatocyte, it is now clear that non-hepatocyte cell types make crucial contributions to liver function and pathology, and efforts to understand their embryonic origins and the mechanisms underlying their development have intensified.2 In addition, there is mounting evidence to support the proposal that some nonparenchymal cells have a direct impact on the development of hepatocytes.3-5 For example, hepatoblasts purified from fetal mouse liver have been shown to require nonparenchymal cells for proliferation and expression of hepatocyte messenger RNA (mRNA).4 To understand how the liver is generated during embryogenesis, it is first necessary to define the lineage relationships and the morphogenic movements of the cells that contribute to the liver's function. Several decades of work in numerous vertebrate model systems have firmly established that hepatocytes and bile duct cells originate from a common precursor, the hepatoblast, that derives from the definitive endoderm (Fig. 1).6-13 At around embryonic day (E) 7.0 in the mouse, definitive endoderm emerges from the primitive streak to displace the extra-embryonic endoderm of the yolk sac. Shortly after this, the endoderm invaginates to form a portal at the anterior region of the developing embryo that will ultimately define the foregut of the mouse. By approximately E8.0, in mouse embryos containing seven somite pairs, the ventral wall of the foregut endoderm is positioned adjacent to the developing heart, and signals from the heart induce the underlying endoderm to initiate its development toward a hepatic fate.6, 9, 14, 15 The endoderm responds to this induction by generating the primary liver bud that can be identified as an anatomical outgrowth from the ventral wall of the foregut by E8.5 to E9.0 in mouse embryos containing 10 to 12 somite pairs. Recently, lineage tracing experiments in cultured mouse embryos have accurately defined the position of endodermal cells that give rise to the hepatic progenitors prior to their interaction with the heart.16 Two domains of endodermal cells were identified when E8.5 embryos containing one to three somite pairs were labeled: a lateral endodermal cluster that migrates toward the midline as the foregut is generated, and ventral midline endodermal lip cells that grow caudally to generate the midline of the ventral aspect of the gut. By E9.5, in mouse embryos containing approximately 15 to 20 somite pairs, the basement membrane surrounding the liver bud is lost, and cells delaminate from the bud and invade the surrounding septum transversum mesenchyme as cords of hepatoblasts (see Fig. 1; Fig. 2A-B). Expression of a number of proteins has been used to identify cells undergoing this phase of hepatic development, including expression of the transcription factors HNF4α and GATA4, which identify the migrating hepatic cells and the septum transversum mesenchyme, respectively (see Fig. 2). Although the molecular mechanisms that control the onset of delamination and migration of the hepatoblasts are not well defined, the process does require the transcription factor prospero-related homeobox 1 (Prox1).17 Examination of Prox1−/− embryos revealed that hepatoblasts failed to migrate and invade the septum transversum mesenchyme, and the mutant cells remained clustered and surrounded by basement membrane. The Prox1−/− hepatoblasts also expressed abnormally high levels of the cell adhesion molecule E-cadherin, which may contribute to the inability of these cells to fully delaminate.17 Surprisingly, the Prox1−/− embryos did generate liver lobes that were devoid of hepatocytes.17 This finding implies that the mesenchymal component of the liver contributes a significant portion of the morphogenetic cues required for at least some aspects of hepatic architecture. Overview of early stages of liver development in mouse embryos. At E8.0, in embryos containing fewer than six to seven somites, a ventral portion of the definitive endoderm (blue area) is positioned close to the developing heart (red area) and in the vicinity of mesenchyme that will contribute to the septum transversum (green area). In response to inductive cues from these tissues, including BMPs and FGFs, the ventral endoderm is specified to adopt a hepatic fate. By E8.5, in embryos containing 8 to 10 somites, the specified hepatic endoderm forms the liver bud (blue area), which expresses several liver mRNAs, including Albumin. Endothelial cells (white area) surround the bud and are necessary for its expansion. By E9.5, the nascent hepatoblasts delaminate from the endoderm and cords of hepatoblasts invade the septum transversum mesenchyme, which is the source of stellate cells as well as sinusoidal endothelial cells that begin to form vessels (white area). Transcription factors with roles in specific aspects of early hepatic development discussed in the text are shown below. He, heart; S, septum transversum; VE, ventral endoderm; FGF, fibroblast growth factor; BMP, bone morphogenetic protein; E, endothelial cells; Lb, liver bud; H, hepatoblasts. Expression of HNF4α and GATA4 identifies migrating hepatoblasts and septum transversum mesenchyme, respectively. (A,C) HNF4α and (B,D) GATA4 were identified in transverse sections through the developing liver of (A,B) E9.5 and (C,D) E10.5 mouse embryos by immunohistochemistry. HNF4α is detected within the hepatoblasts of the liver bud (brown nuclear staining) (solid arrow), but not the septum transversum mesenchyme (brown nuclear staining) (open arrow). In contrast, at E9.5 GATA4 is detected within the mesenchyme but not the heptoblasts. At E10.5, GATA4 is identified within the endothelial cells (arrowhead) lining newly formed blood vessels and sinusoids, while HNF4α is restricted to the hepatoblasts (arrow). Boxed areas are shown in a higher resolution within the insets. Generation of the liver bud is also accompanied by development of the hepatic vasculature, which forms through a combination of angiogenesis and vasculogenesis.5, 18-20 As soon as the primary liver bud emerges from the endoderm, angioblasts or endothelial cells surround it and separate it from the septum transversum mesenchyme (see Fig. 1).5, 21 Studies of Flk1−/− mouse embryos, which lack mature endothelial cells and blood vessels, revealed that although initiation of hepatic development had occurred in these embryos, the liver bud failed to expand, and there was no evidence of hepatoblasts invading the septum transversum mesenchyme.5 These results suggest not only that the hepatic vasculature develops in concert with the hepatoblasts, but that endothelial cells have an integral role in controlling growth of the hepatic primordium. In the adult mammal, the afferent blood vessels of the liver consist of branches of the hepatic artery and portal veins, and the efferent vessels consist of centrilobular veins. These two systems are connected by a network of small capillaries called sinusoids, which are separated from the basal surface of the hepatocytes by the space of Disse, which also contains retinoid-storing hepatic stellate cells. The sinusoidal capillaries consist of phagocytic Kupffer cells, which scavenge spent cell debris from the circulation, and fenestrated endothelial cells that are highly specialized to facilitate selective transport between hepatocytes and the blood.22 Although Kupffer cells are believed to develop from bone marrow–derived monocytes in adults, their presence in the fetal liver precedes bone marrow development, and they may originate from the yolk sac.23 The sinusoidal capillaries and portal veins are among the first hepatic vessels to develop, with centrilobular veins and portal arteries forming later.19 Descriptive studies of both human and mouse development are consistent with the proposal that sinusoids develop through vasculogenesis of vessels that originate within the mesenchyme of the septum transversum.5, 19, 20, 23, 24 In chick embryos, the septum transversum develops later than seen in mammals; however, lineage-tracing experiments have shown that the liver mesothelium may supplant the septum transversum as the origin of the hepatic sinusoids during avian development.18 Hepatic stellate cells also appear to originate from the septum transversum mesenchymal cells, and many transcription factors with roles in stellate cell activation (e.g., the forkhead box factor FoxM1 and the LIM homeobox protein Lhx2) are expressed in both cell types.24-28 A concerted effort has been made over the last decade to identify the factors that regulate the onset of hepatogenesis. Although experiments involving the culture of explanted chick/quail tissues had identified the heart and septum transversum mesenchyme as important sources of signals for development of the liver, nothing was known about the identity of the signaling molecules.6, 15 Advances were made when an assay was developed that reconstituted hepatic specification in vitro.9 In this assay, expression of hepatic mRNA within cultured ventral endoderm, isolated from four to six somite stage (E8.0) mouse embryos, was shown to be dependent upon the presence of cardiogenic mesoderm.9 Using this assay, Jung et al. demonstrated that fibroblast growth factors (FGF) 1 and 2, but not FGF8, could substitute for cardiogenic mesoderm to induce the onset of hepatic development in primary ventral endoderm In addition, of a of signaling in the culture could cardiogenic hepatic expression within the ventral endoderm only is the presence of for induction of hepatic but is the of work by et al. demonstrated that when 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the liver, it is highly that could soon a