Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells

Abstract

Confocal scanning laser microscopy has been used to make three-dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno-labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double-fluorescence emission requires an image processing step for the correction of 'contamination' effects due to the overlap between fluorescein and Texas Red emission spectra. At the pre-natal period (day 20 of gestation) each type of intermediate filament labelling is only present in a certain cellular category, C18 cytokeratin in hepatocytes and vimentin in mesenchymal cells. However, at the earliest developmental stages (day 12 of gestation), vimentin and cytokeratin seem to be found in the same type of cells, probably mesenchymal cells. Some striking developmental changes, associated with the differentiation of the liver parenchyma, are observed for both C18 cytokeratin and vimentin. In earlier foetal stages, C18 filaments are scarce, hazily labelled and randomly distributed inside the hepatocytic cytoplasm. Late during foetal development (days 18-20 of gestation), hepatocytic cytokeratin filaments are abundant, well individualized and sharply labelled. The hepatocytes are arranged in a muralium duplex architecture (two-cell-thick sheets) and the labelling intensity measured in the hepatocytic cytoplasm at the basal pole is double that measured at the sinusoidal pole, while, in the adult, hepatocytes are arranged in a muralium simplex architecture (one-cell-thick sheets) and cytokeratin filaments have a symmetrical distribution in relation to the nuclear region.