REGULATION OF NEUROFILAMENT-L, NEUROFILAMENT-M AND NEUROFILAMENT-H GENE-EXPRESSION DURING RETINOIC ACID-INDUCED NEURAL DIFFERENTIATION OF P19 EMBRYONAL CARCINOMA-CELLS

We have investigated the regulation of neurofilament gene expression d uring retinoic acid (RA)-induced neural differentiation of P19 embryon al carcinoma (EC) cells. Western blot analysis demonstrated that P19 E C cells contain significant levels of NF-L protein in the insoluble fr action but undetectable levels of NF-M and NF-H protein in either the insoluble or total cell fractions. However, immunocytochemical detecti on of NF-L protein in P19 EC cells showed diffuse staining within the majority of cells, rather than association with intermediate filament- like structures or staining within a subpopulation of differentiated n eurons. Detectable levels of both NF-L and NF-M mRNA were present in P 19 EC cells whereas NF-H mRNA remained below levels of detection, even by RT-PCR analysis. When RA-treated aggregates of P19 cells were cult ured under conditions permissive for neurite outgrowth, we observed a significant increase in the amount of detectable NF-L protein localize d within morphologically distinct neurons. Differentiation was also ac companied by the appearance of both the NF-M and NF-H subunits. Northe rn analysis revealed that this differentiation was accompanied by coin cident increase in the steady-state levels of the mRNA for all three s ubunits and that the temporal pattern of increase was similar to what has been observed in the fetal and neonatal brain. The increase in NF- L and NF-M mRNA levels were accompanied by a concomitant increase in t he rate of transcription, however, our results suggest that additional post-transcriptional mechanisms may be involved in regulating NF gene expression during the differentiation of pluripotent P19 cells. (C) 1 997 Elsevier Science B.V.