RESUMEN
IIIG9 is the regulatory subunit 32 of protein phosphatase 1 (PPP1R32), a key phosphatase in the regulation of ciliary movement. IIIG9 localization is restricted to cilia in the trachea, fallopian tube, and testicle, suggesting its involvement in the polarization of ciliary epithelium. In the adult brain, IIIG9 mRNA has only been detected in ciliated ependymal cells that cover the ventricular walls. In this work, we prepared a polyclonal antibody against rat IIIG9 and used this antibody to show for the first time the ciliary localization of this protein in adult ependymal cells. We demonstrated IIIG9 localization at the apical border of the ventricular wall of 17-day-old embryonic (E17) and 1-day-old postnatal (PN1) brains and at the level of ependymal cilia at 10- and 20-day-old postnatal (PN10-20) using temporospatial distribution analysis and comparing the localization with a ciliary marker. Spectral confocal and super-resolution Structured Illumination Microscopy (SIM) analysis allowed us to demonstrate that IIIG9 shows a punctate pattern that is preferentially located at the borders of ependymal cilia in situ and in cultures of ependymocytes obtained from adult rat brains. Finally, by immunogold ultrastructural analysis, we showed that IIIG9 is preferentially located between the axoneme and the ciliary membrane. Taken together, our data allow us to conclude that IIIG9 is localized in the cilia of adult ependymal cells and that its expression is correlated with the process of ependymal differentiation and with the maturation of radial glia. Similarly, its particular localization within ependymal cilia suggests a role of this protein in the regulation of ciliary movement.
Asunto(s)
Diferenciación Celular/fisiología , Cilios/metabolismo , Epéndimo/metabolismo , Células Ependimogliales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Epéndimo/citología , Células Ependimogliales/citología , Femenino , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for beta III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons.
Asunto(s)
Epéndimo/citología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ventrículos Laterales/citología , Neuronas/citología , Factores de Edad , Animales , Bovinos , Diferenciación Celular/fisiología , División Celular , Células Cultivadas , Epéndimo/metabolismo , Epéndimo/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ventrículos Laterales/fisiología , Ventrículos Laterales/ultraestructura , Microscopía Electrónica de Rastreo , Neuroglía/citología , Neuroglía/metabolismo , Neuroglía/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , ARN Mensajero/análisis , Receptor IGF Tipo 1/antagonistas & inhibidoresRESUMEN
By using one polyclonal antiserum raised against bovine Reissner's fiber and seven monoclonal antibodies raised against bovine Reissner's fiber and against immunopurified bovine subcommissural organ glycoproteins, we have investigated two freshwater planarian species (Girardia tigrina, Schmidtea mediterranea) by light- and electron-microscopic immunocytochemistry. ELISA probes showed that the monoclonal antibodies recognized different, nonoverlapping, unrepeated, proteinaceous epitopes present in the same compounds of bovine Reissner's fiber. Cells immunoreactive to the polyclonal and monoclonal antibodies were found in the dorsal and ventral integument of both planarian species. Labeled cuboid epidermal cells bore cilia and displayed several types of secretory granules; they were covered by a film of immunoreactive material. Studies on adjacent thin and semithin sections revealed coexistence of label in the same regions and in the same cells when two different monoclonal antibodies were used. These results indicate that a secretory substance immunologically similar to the secretion of the vertebrate subcommissural organ is present in primitive tripoblasts such as planarians, suggesting that these secretions are ancient and well conserved in phylogeny.