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The forkhead box m1 (Foxm1) transcription factor is essential for initiation of carcinogen-induced liver tumors; however, whether FoxM1 constitutes a therapeutic target for liver cancer treatment remains unknown. In this [...]

Conditional deletion of the mouse Forkhead Box (Fox) m1b targeted allele in adult hepatocytes (Foxm1, previously called HFH-11B, Trident, Win, or MPP2) demonstrated that the Foxm1b transcription factor is essential for hepatocyte mitosis during liver regeneration. To determine the role of Foxm1b in liver development, we have generated Foxm1b -/- mice that deleted the Foxm1b exons encoding the winged helix DNA binding and transcriptional activation domains. Here. we show that all of the Foxm1b -/- embryos died in utero by 18.5 days postcoitum (dpc). Embryonic Foxm1b -/- livers displayed a 75% reduction in the number of hepatoblasts, resulting from diminished DNA replication and a failure to enter mitosis causing a polyploid phenotype. Reduced hepatoblast mitosis was associated with decreased protein levels of the Polo-like kinase 1 and Aurora B kinase, which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis. Diminished proliferation of Foxm1b -/- hepatoblasts contributed to abnormal liver development with significant reduction in the number of large hepatic veins compared to embryonic wild-type (WT) liver. Furthermore, embryonic Foxm1b -/- livers did not develop intrahepatic bile ducts, and these presumptive biliary hepatoblasts failed to express either biliary cytokeratins or nuclear levels of hepatocyte nuclear factor 1 [beta]. These results suggest that Foxmlb is critical for hepatoblast precursor cells to differentiate toward biliary epithelial cell lineage Finally, we used a hepatoblast-specific Cre recombinase transgene to mediate deletion of the Foxm1b fl/fl allele in the developing liver, and these embryos died in utero and exhibited diminished hepatoblast proliferation with similar abnormalities in liver morphogenesis, suggesting that the defect in liver development contributed to embryonic lethality. Keywords: Winged helix DNA binding domain; Mouse liver development; Intrahepatic bile ducts; Aurora B kinase; Polo-like kinase; Mitosis; Forkhead Box

Previously, we showed that newborn forkhead box (Fox)f1(+/-) mice with diminished pulmonary FoxF1 levels died of severe lung hemorrhage and exhibited abnormal formation of alveolar sacs and capillaries. Another group recently reported that Foxf1(+/-) mouse embryos displayed a number of organ and skeletal defects including fusion of lung lobes. However, identification of pulmonary genes whose altered expression was associated with the lobular fusion defect in Foxf1(+/-) lungs remains uncharacterized. The present study was conducted to determine the nature of the malformations leading to lung fusions in the FoxF1 embryos and to identify potential signaling pathways influenced by FoxF1 haploinsufficiency. We show that Foxf1(+/-) embryos exhibit defects in formation and branching of primary lung buds, which causes fusion of the right lung lobes and vessels. The severity of the Foxf1(+/-) lung fusions was correlated with decreased levels of FoxF1 mRNA. In situ hybridization studies demonstrated that the defective primary lung-bud development in early Foxf1(+/-) embryos was associated with fewer pulmonary mesenchymal-epithelial interfaces. Defects in branching morphogenesis in the Foxf1(+/-) embryos were associated with altered expression of the fibroblast growth factor-10, bone morphogenetic protein-4, and the Gli3 transcription factor, which are known to influence primary lung-bud development. fibroblast growth factor-10; Gli3; bone morphogenetic protein-4; Patched; winged-helix transcription factor; defective lung-bud formation

HNF-3/forkhead homologue 4 (HFH-4), a transcription factor of the winged helix/forkhead family, is expressed in various tissues including lung, brain, oviduct, testis, and embryonic kidney. In order to test whether the temporospatial expression of HFH-4 influences lung morphogenesis, HFH-4 was expressed in lungs of transgenic mice under control of the surfactant protein C (SP-C) promoter. The morphology of the lungs from SP-C/HFH-4 embryos (day 18 postconception) was distinctly abnormal, and the severity of the alterations correlated with the level of transgene expression as detected by in situ hybridization. At high levels of expression, HFH-4 altered epithelial cell differentiation and inhibited branching morphogenesis. Atypical cuboidal or columnar cells lined the lung periphery of SP-C/HFH-4 transgenic mice. The atypical epithelial cells seen in the SP-C/HFH-4 mice expressed thyroid transcription factor-1 and hepatocyte nuclear factor 3(beta) (HNF-3(beta)). However, surfactant proteins SP-B, SP-C, and Clara cell secretory protein, normally produced by nonciliated epithelial cells in lung parenchyma were lacking. (beta)-Tubulin IV, a marker of ciliated cells, stained the atypical columnar cells produced by expression of high levels of the SP-C/HFH-4 transgene. Ectopic expression of HFH-4 in developing mouse lung altered epithelial cell differentiation and morphology, restricting the expression of markers typical of nonciliated cells of the distal lung parenchyma.

Murine hepatocyte nuclear factor-3[Beta] (HNF-3[Beta]) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3[Beta] also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial, and pancreatic acinar cells. We have previously identified a liver-enriched transcription factor, HNF-6, which is required for HNF-3[Beta] promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes. In this study we used the yeast one-hybrid system to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein. Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3[Beta] or transthyretin (TTR) promoter regions. We used interspecific backcross analysis to determine that murine Hnf6 gene is located in the middle of mouse chromosome 9. In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3[Beta], are coexpressed in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3[Beta]'s developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells. The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube. We also found that HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain. At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3[Beta] transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium. HNF-6, but not HNF-3[Beta], expression continues in the pancreatic ductal epithelium, whereas only HNF-3[Beta] becomes restricted to the endocrine cells of the islets of Langerhans. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

The regulation of the Clara cell 10 kilodalton protein (CC10) gene expression by interferon-gamma was analyzed in Clara cell lines that express CC10 and hepatocute nuclear factor 3beta (HNF-3beta). In vivo and in vitro administration of recombinant murine IFN-gamma in HFN-3beta- and CC10-expressing Clara cell lines increased the expression of CC10 messenger RNA. The effects of INF-gamma on CC10 gene expression also indicated the presence of a regulatory pathway between the proinflammatory cytokine and Clara cells during pulmonary inflammation.

Molecular cloning of a new hepatocyte nuclear factor 3 (HNF-3) forkhead (fkh) homolog 8 (HFH-8) and amino acid sequence analysis of HFH-8, and previously defined HFH-1 and -4 proteins show that the expression of the proteins of HNF-3 /fkh family of transcription factors is seen in various cell types in the adult lung. The HFH-8 and HNF-3 proteins transactivate the DNA binding site in the surfactant protein B (SPB) promoter region and identify the SPB-f2 sequence in the binding site. HFH-8 and HNF-3 alpha proteins regulate SPB promoter functions in the different cell types.