Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha

Abstract

HNF-4alpha is a transcription factor of the nuclear hormone receptor family that is expressed in the hepatic diverticulum at the onset of liver development. Mouse embryos lacking HNF-4alpha fail to complete gastrulation due to dysfunction of the visceral endoderm. This early embryonic lethality has so far prevented any analyses of the contribution of HNF-4alpha toward liver development and hepatocyte differentiation. However, we have shown that complementation of HNF-4alpha(-/-) embryos with a tetraploid embryo-derived wild-type visceral endoderm rescues this early developmental arrest and allows HNF-4alpha(-/-) embryos to proceed normally through midgestation stages of development. Examination of these rescued embryos revealed that HNF-4alpha was dispensable for specification and early development of the liver. However, HNF-4alpha(-/-) fetal livers failed to express a large array of genes whose expression in differentiated hepatocytes is essential for a functional hepatic parenchyma, including genes encoding several apolipoproteins, metabolic proteins, and serum factors. In addition, we have demonstrated that HNF-4alpha is essential for expression of the transcription factors HNF-1alpha and PXR within the fetal liver. We therefore conclude that HNF-4alpha is both essential for hepatocyte differentiation during mammalian liver development and also crucial for metabolic regulation and liver function.

Figure 1

Hepatoblasts derived from HNF-4α^−/− ES cells contribute toward the developing liver. (A) Fetal livers were isolated from wild-type (CD-1), ROSA26 (ROSA), or chimeric (ROSA: HNF-4α^−/−) embryos at E11.5 and stained for β-galactosidase expression. (B) Photomicrograph showing a 5 μm section through the chimeric fetal liver shown in A. The section was counterstained with eosin to show HNF-4α^−/− hepatoblasts (h).

Figure 2

Hepatoblasts are present in embryos generated from HNF-4α^−/− ES cells by tetraploid aggregation. (A) Embryos were generated from HNF-4α^−/− ES cells (red) by aggregating the cells with HNF-4α^+/+ embryos (blue) made tetraploid by electrofusion. Embryos continued their development in utero after transfer of the aggregates to psuedopregnant surrogate mothers. Fetal tissues generated by this procedure arise from HNF-4α^−/− ES cells; the extraembryonic tissues, including the visceral endoderm, are tetraploid embryo derived. The presence of HNF-4α^+/+ visceral endoderm allows HNF-4α^−/− embryos to proceed through midgestation stages of development. (B) The genotype of embryos generated from HNF-4α^+/+ (lane 1), HNF-4α^+/− (lanes 11,12), and HNF-4α^−/− ES cells (lanes 2–10) could be determined by PCR using primers which specifically amplified DNA fragments from the HPRT, bacterial neo, or HNF-4α (HNF-4) genes. (C–F) Dark-field micrographs showing in situ hybridization analyses that were performed on 5 μm paraffin sections of E10.5 embryos generated from HNF-4α^+/− (C,D) and HNF-4α^−/− (E,F) ES cells. In all embryos, HNF-3α mRNAs (C,E) could be detected in gut endoderm (e) and hepatoblasts (h), whereas albumin mRNAs (D,F) were detected only in hepatoblasts.

Figure 3

Whole mount micrographs showing wild-type CD-1, HNF-4α^+/−, and HNF-4α^−/− ES cell derived fetuses and livers after 12 days gestation. Embryos were removed and photographed under a dissecting microscope at 15× magnification. Livers were dissected and photographed at 70× magnification.

Figure 4

Light micrographs and electron micrographs comparing HNF-4α^+/− and HNF-4α^−/− fetal livers. (A,B) Sections (1 μm) through livers from HNF-4α^−/− or HNF-4α^+/− ES cell-derived mice at E12. Sections were stained with toluidine blue and photographed using a 100× objective. Both HNF-4α^+/− and HNF-4α^−/− livers contained hepatoblasts (h), nucleated fetal blood cells (b), and endothelial cells (e) surrounding sinusoidal spaces. (C,D) Electron micrographs of the same fetal livers at 3000× magnification. Both dark (dh) and light (lh) hepatoblasts can be identified in HNF-4α^+/− and HNF-4α^−/− livers. The light hepatoblasts are believed to be in a less differentiated state containing a high nuclear (n) to cytoplasmic ratio and few organelles. In contrast, the more differentiated dark hepatoblasts have a relatively small nucleus and contain many organelles including endoplasmic reticulum (er) and mitochondria (m).

Figure 5

HNF-4α is essential for normal hepatocyte differentiation. Steady-state mRNA levels from several genes normally expressed in differentiated hepatocytes was measured in livers isolated from HNF-4α^+/+ (lane 2), HNF-4α^+/− (lanes 5,6), and HNF-4α^−/− (lanes 3,4) ES-cell-derived embryos at day 12 of gestation by RT–PCR. All samples contained comparable amounts of cDNA, as HPRT primers amplified a similar level of product in all cases. HPRT primers did not amplify product in the absence of cDNA (lane 1). apoAI, apoAII, apoB, apoCIII, apoCII, and albumin, aldolase B, pAH, LFABP, TFN, RBP, and Epo mRNA levels were similar in HNF-4^+/+ and HNF-4^+/− livers. In HNF-4α^−/− livers the relative level of the same mRNAs was decreased dramatically and in some cases undetectable.

Figure 6

HNF-1α and PXR lie downstream of HNF-4α during hepatogenesis. Steady-state mRNA levels of several transcription factors acting in the fetal liver were measured in E12 HNF-4α^+/+ (lane 2), HNF-4α^+/− (lanes 5,6), and HNF-4α^−/− (lanes 3,4) livers by RT–PCR. Primers recognizing HNF-3α, -3β, 3γ, HNF-1β, GATA-4, HNF-6, c/EBP-β and -γ, RAR-α, -β and -γ, RXR-α, and COUP-TFII all amplified similar levels of product in all fetal livers. However, HNF-1α and PXR steady-state mRNA levels (arrowed) were greatly decreased in HNF-4α^−/− fetal livers.

Figure 7

Schematic depicting key points during hepatic development and the stage at which specific transcription factors first act. During gastrulation, at ∼7.0 days of gestation in the mouse, the definitive endoderm (yellow) is generated from the primitive ectoderm. Both ventral and dorsal aspects of the definitive embryonic endoderm express HNF-3 and are competent to adopt a hepatic fate. By the four- to six-somite stage a ventral portion of the embryonic endoderm (blue) lies close to the developing heart. During hepatic specification, FGFs (fgf's) from the developing cardiac mesoderm (red) induce the underlying endoderm to commit to a hepatic fate. In response to this signal the endodermal cells proliferate, expand into the surrounding septum transversum, and adopt a stellate morphology. At this point these cells are considered hepatoblasts. The hepatoblasts continue to proliferate and begin to differentiate to become mature hepatocyte in a process that requires HNF-4α.