RESUMEN
Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1(-/-), but not Robo2(-/-), animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1(-/-) mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1(-/-)/Slit2(-/-) mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1(-/-), Robo2(-/-) and Slit1(-/-)/Slit2(-/-), but not in Slit1(-/-) mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.
Asunto(s)
Corteza Cerebral/citología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Interneuronas/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores Inmunológicos/fisiología , Transducción de Señal , Animales , Biomarcadores , Calbindinas , Técnicas de Cultivo de Célula , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , ADN Complementario , Electroporación , Embrión de Mamíferos , Células Madre Embrionarias/química , Células Madre Embrionarias/metabolismo , Marcadores Genéticos , Vectores Genéticos , Biblioteca Genómica , Inmunohistoquímica , Integrasas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interneuronas/citología , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Selección Genética , Telencéfalo/citología , Transfección , Proteínas RoundaboutRESUMEN
The role of neurotrophins in thalamic axon growth was studied by culturing embryonic rat thalamus on collagen-coated substrate or fixed cortical slices in the presence of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Both BDNF and NT-3 promoted axonal growth, but the axonal growth-promoting activity depended on culture substrates. Axonal growth on collagen-coated membrane was accelerated by BDNF, but not by NT-3. In contrast, axonal outgrowth on fixed cortex was significantly enhanced by NT-3, but not by BDNF. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cultured thalamic cells demonstrated that culture substrates did not alter the expression of their receptors, trkB and trkC. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining further demonstrated that axonal growth promoted by neurotrophins was not due to reduction of cell death. Measurement of the developmental changes in BDNF and NT-3 levels revealed that, in contrast to the rapid elevation of BDNF after the arrival of thalamocortical axons to their target layer, the regulation of NT-3 protein accompanies the phase of their outgrowth in neocortex. These findings suggest that BDNF and NT-3 promote thalamic axon growth in different manners in terms of substrate dependency and developmental stage.
Asunto(s)
Axones/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Corteza Cerebral/citología , Neurotrofina 3/farmacología , Tálamo/citología , Acetilación , Factores de Edad , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Etiquetado Corte-Fin in Situ/métodos , Técnicas de Cultivo de Órganos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tálamo/crecimiento & desarrollo , Tubulina (Proteína)/metabolismoRESUMEN
The role was studied of ephrin-B3, a ligand of the Eph family of tyrosine kinase receptors, in the formation of cortical connectivity. In situ hybridization and immunohistochemistry showed that EphA4, a receptor of ephrin-B3, was expressed in the lateral thalamus (visual and somaotosensory thalamus) of the developing rat brain, but not in the medial thalamic nuclei which project to the limbic cortex. Correspondingly, ephrin-B3 was expressed strongly in the developing limbic cortex including amygdala, entorhinal cortex and hippocampus. To examine the action of ephrin-B3 on thalamic axons, either lateral or medial thalamic explants were cultured on membranes obtained from ephrin-B3-expressing COS cells. Axonal growth was inhibited for cells from the lateral thalamus but not from the medial thalamus. These results suggest that ephrin-B3 contributes to regional specificity by suppressing axonal growth of lateral thalamic neurons.