RESUMEN
BACKGROUND: Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs) lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain. PRINCIPAL FINDINGS: As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited. CONCLUSIONS: Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical neurogenesis. The results also suggest that HDACs may regulate the developmental switch from neurogenesis to astrogliogenesis that occurs in late gestation.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Encéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/metabolismo , Neuronas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Astrocitos/citología , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Cromatina/metabolismo , Ácidos Hidroxámicos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Modelos Biológicos , Transducción de SeñalRESUMEN
Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest.
Asunto(s)
Sistema Nervioso/embriología , Animales , Animales Recién Nacidos , Tipificación del Cuerpo , Muerte Celular , Femenino , Ganglios Espinales/citología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Técnicas de Cultivo de Órganos , Embarazo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semaforina-3A/deficiencia , Semaforina-3A/genética , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Nervios Espinales/citología , Nervios Espinales/embriología , Nervios Espinales/metabolismoRESUMEN
Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.