Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates

Hepatocyte nuclear factor-4α (HNF4α) presents in multiple isoforms generated using alternative promoter (P1 and P2) and splicing. Neither conservation of tissue distribution of HNF4α isoforms, nor presence of alternative promoter usage is known. In this study, to detect the expression of HNF4α in some species of animals, we have applied monoclonal antibodies against P1 (K9218) and P2 (H6939) promoter-driven and P1/P2 promoter-driven H1415 HNF4α for immunohistochemistry and western blot analysis. Antibody K9218 was observed in the hepatocytes, proximal tubules of the kidney, and epithelial cells in the mucosa of the small intestine and colon of rats, chicken, and tortoise, whereas antibody H6939 signal were detected in the stomach, pancreas, bile duct, and pancreatic duct of human and rats. The signal for antibody K9218 was recognized in tissues of a wide range of mammals, bird, reptile, amphibian, and fish as well. Antibody H1415 displayed a positive reaction in hepatocytes and intestinal epithelial cells in chicken and tortoise, whereas the bile duct, mucosal epithelial cells in the stomach, or pancreas in these animals were negative. Western blotting showed the binding of the antibody with HNF4α protein from each animal. The sequence of human HNF4α was 100% identical to murine and rat HNF4α, 88.9% to chicken, 77.8% to Xenopus HNF4α, and 81.5% to medaka. However, the specific part of human and invertebrate Drosophila HNF4 shares only 14.8% sequence identity. This antibody is useful for detecting HNF4α isoforms in a wide range of vertebrates, and suggests many insights into animal evolution.