358Y. Yokouchi

causing a lack of the fundamental data now neededto initiate molecular studies.

The development of liver in the chick embryo issimilar to that of the mammalian embryo. Therefore,any knowledge about chick liver development will beapplicable to humans and have medical relevance.To improve the status of the chick embryo as a prac-tical model for analyzing liver development, we per-formed four fundamental studies: (i) constructed anatlas of the developing chick liver; (ii) identi ed dif-ferentiation marker genes in the developing chickembryo; (iii) established germ-layer speci c electro-poration; and (iv) established organ culture from thedeveloping chick liver.

Using our chick model system, we were able todemonstrate the functions of candidate genes withina shorter period and in a more cost-effective mannerthan is feasible for the mouse embryo. In order toprove the rapidity and effectiveness of this modelsystem, we performed pilot experiments on threegenes. With the aid of only three graduate students,we were able to demonstrate the functions of thesegenes in hepatogenesis within three years.

In parallel with the establishment of the system, weexamined the expression patterns of genes knownto be involved in chick embryo development in orderto identify any that were also involved in liver develop-ment. To date we have found 16 ‘development’ genesthat are also expressed in the developing chick liver(GELD, genes expressed in liver development).The embryonic liver is composed of three tissues,namely liver bud from foregut endoderm, septumtransversum mesenchyme and endothelial cells ofprimary sinusoids. The liver develops from epithelio–mesenchymal interactions between these embryonictissues. Based on tissue speci city of expression,we classi ed the GELD into three subclasses: liverendoderm-speci c (3 genes), septum transversumspeci c (9 genes), and endothelial layer-speci c(4 genes). These genes appear to play speci cfunctions in each embryonic tissue.

The concentrated investment of research funds onthe characterization of genes expressed in liverdevelopment will reveal an outline of the epithelio–mesenchymal interactions involved in hepatogenesisand lead to the establishment of basic technologyfor tissue-engineering in the near future.

Genes involved in the early hepatogenesis in the mouse embryo

Studies conducted in the last two decades haveelucidated the molecular mechanisms controllingthe early steps of vertebrate liver development. The

combination of traditional embryology and advancedgenetics applied to the study of the mouse embryohelped in the identi cation of soluble signaling mole-cules and transcription factors that are essential forhepatogenesis. In this section, I would like to reviewthe developmental functions of the identi ed hepa-togenic genes in the mouse embryo.The structure and development of the liver

In the last three decades, knowledge regarding liverdevelopment has been gained from studies usingrodent embryos. The development of the liver pro-ceeds as follows. At the earliest stage of liver devel-opment, the proliferated diverticulum (liver bud) isobserved from the ventral foregut endoderm at the14–20 somite stage (Le Douarin 1975). At this stage,the liver bud is separated from the septum transver-sum (ST) by the basement membrane (Medlock &Harr 1983). Following this stage, the liver bud cellsdelaminate from the epithelium of the foregut; this isaccompanied by the destruction of the basementmembrane and the migration of these cells into thesurrounding septum transversum (Le Douarin 1975;Medlock & Haar 1983).

These endodermal cells composing the liver budare known as hepatoblasts, which possess the bi-potency to differentiate into hepatic parenchymalcells and biliary epithelial cells (Blouin et al. 1995;Rogler 1997; Spagnoli et al. 1998).

The migration of these hepatoblasts into the ST isaccompanied by the vascularization of the primarysinusoidal endothelial cells (Medlock & Haar 1983;Enzan et al. 1997).

Induction of the hepatic endoderm by broblast growth factor from cardiac mesoderm

Experimental embryological studies using chickembryos validated that hepatic induction comprisestwo distinct developmental steps (Le Douarin 1975).During the primary step at the 5–7 somite stage, thecardiac mesoderm induces the ventral part of theforegut endoderm to differentiate into the hepaticendoderm. During the secondary step at the 20–22somite stage, the secondary stimuli from the septumtransversum mesenchyme (STM) induce the hepato-blasts to proliferate and completely differentiate intomature hepatocytes (Le Douarin 1975).

Recently, it has been con rmed that similar induc-tion processes exist in the mouse embryo. Analysisusing tissue explants and reverse transcription–polymerase chain reaction (RT–PCR) for detectinghepatic differentiation markers demonstrated that the