RESUMO
LEC1, LEC2, FUS3 and ABI3 (collectively abbreviated LEC/ABI3 here) are required for embryo maturation and have apparent roles in repressing post-germinative development. lec mutant embryos exhibit some heterochronic characteristics, as exemplified by the development of true leaf-like cotyledons during embryogenesis. Although the roles of LEC/ABI3 as positive regulators of embryo maturation have been extensively studied, their roles in the negative regulation of post-germinative development have not been explored in detail. Based on microarray analyses, we chose PYK10, which encodes an endoplasmic reticulum (ER)-body-localized protein, as a molecular marker of post-germinative development. lec/abi3 embryos exhibited PYK10 misexpression and the formation of 'constitutive' ER-bodies, which develop specifically during the seedling stage, confirming the heterochronic nature of these mutants at both the gene expression and cellular levels. The PYK10 reporter expression in lec1 embryos started as early as the globular-heart transition stage. The onset of PYK10 promoter-enhanced green fluorescent protein (EGFP) reporter expression occurred in a stochastic, cell-by-cell manner in both developing lec/abi3 embryos and germinating wild-type seedlings. Additionally, clustered EGFP-positive cells were frequently found along cell files, probably representing the transmission of the expression state via cell division. These observations, together with the results of the experiments using PYK10-EGFP/PYK10-CFP double reporter transgenic lines and the analyses of H3K27me3 levels in the PYK10 chromatin, suggested the involvement of epigenetic mechanisms in repressing post-germinative genes during embryogenesis and derepressing these genes upon the transition to post-germinative development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/embriologia , Epigênese Genética/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , beta-Glucosidase/genética , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cotilédone/citologia , Cotilédone/embriologia , Cotilédone/genética , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes Reporter , Germinação/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/citologia , Folhas de Planta/embriologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Plântula/citologia , Plântula/embriologia , Plântula/genética , Sementes/citologia , Sementes/embriologia , Sementes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta-Glucosidase/metabolismoRESUMO
While a mother's excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.
Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Etanol/toxicidade , Neurônios/efeitos dos fármacos , Fosfolipase D/antagonistas & inibidores , 1-Butanol/farmacologia , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/enzimologia , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Etanol/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurônios/enzimologia , Ácidos Fosfatídicos/antagonistas & inibidores , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfolipase D/genética , Fosfolipase D/metabolismo , Regulação para CimaRESUMO
Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.