RESUMEN
We have examined the ultrastructural aspect of neuronal precursors derived from 14-day-old rat embryos during their development under various culture conditions. Cells maintained in serum-free medium which have developed for 1 week in vitro present ultrastructural features of young neurons. They contain many free ribosomes and microtubules, but few other organelles and incompletely developed Golgi apparatus. In the presence of basic fibroblast growth factor (bFGF), besides cells remaining in aggregates and displaying morphological features of undifferentiated cells, dispersed neuroblasts underwent accelerated ultrastructural maturation. They present well-developed Golgi apparatus, axodendritic synapses and dense-core vesicles already after 3 days in culture. By contrast, in the presence of astroglial-conditioned medium a more homogeneous population developed showing ultrastructural features of relatively mature neurons. However, the neuronal precursors acquired the most mature ultrastructural aspect when they were cocultured with astroglial cells. The neuronal cell bodies contain highly developed Golgi complexes, well-differentiated ergastoplasm and Niss1 body formations, while in the complex neurite network much more numerous mature synapses with clear and dense-core vesicles are visible. These observations indicate that a combination of soluble factors and membrane-bound factors is essential for extensive ultrastructural development of neuronal precursors in vitro. Another finding was that in these cultured neurons neurofilaments (NF) were never seen, while NF protein subunits were found. These data suggest that the polymerization of the three NF subunits into intermediate filaments might need particular cellular factors which probably do not exist under our in vitro conditions.
Asunto(s)
Astrocitos/fisiología , Corteza Cerebral/embriología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neuronas/ultraestructura , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Aparato de Golgi/ultraestructura , Proteínas de Neurofilamentos/metabolismo , Orgánulos/ultraestructura , Ratas , Médula Espinal/metabolismo , Médula Espinal/ultraestructuraRESUMEN
Basic fibroblast growth factor (bFGF) is a well-characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain-derived oligodendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c-fos in OL. The role of protein kinase C (PKC) in mediating bFGF-stimulated proliferation as well as c-fos expression in OL was investigated. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated c-fos expression but did not trigger cell proliferation. When PKC was down-regulated by pretreatment of OL with PMA for 20 h, the bFGF-mediated stimulations of OL proliferation and c-fos mRNA expression were still observed, whereas the induction of c-fos mRNA by PMA was totally inhibited. These data demonstrate that the bFGF mitogenic signaling pathway in OLs does not require PKC. On the other hand, bFGF was found to stimulate specifically the phosphorylation of a limited number of PKC substrates in oligodendroglial cells, including the MARCKS protein. The bFGF-dependent phosphorylation of MARCKS protein was totally inhibited when PKC was first down-regulated, indicating that the phosphorylation of this protein is PKC dependent. Tryptic digestion of the phosphorylated MARCKS protein revealed that bFGF stimulated specifically the phosphorylation of the MARCKS protein on a single phosphopeptide. We provide evidence that bFGF also stimulated fatty acylation of the MARCKS protein, which might explain the observed specific bFGF-dependent phosphorylation of this protein in OL. We propose that bFGF-dependent fatty acylation and phosphorylation of the MARCKS protein are not essential for the transduction of the bFGF mitogenic signal but are probably linked to differentiation processes elicited by bFGF on OL.