RESUMO
Homeoprotein transcription factors play fundamental roles in development, ranging from embryonic polarity to cell differentiation and migration. Research in recent years has underscored the physiological importance of homeoprotein intercellular transfer in eye field development, axon guidance and retino-tectal patterning, and visual cortex plasticity. Here, we have used the embryonic chick neural tube to investigate a possible role for homeoprotein Pax6 transfer in oligodendrocyte precursor cell (OPC) migration. We report the extracellular expression of Pax6 and the effects of gain and loss of extracellular Pax6 activity on OPCs. Open book cultures with recombinant Pax6 protein or Pax6 blocking antibodies, as well as in ovo gene transfer experiments involving expression of secreted Pax6 protein or secreted Pax6 antibodies, provide converging evidences that OPC migration is promoted by extracellular Pax6. The paracrine effect of Pax6 on OPC migration is thus a new example of direct non-cell autonomous homeoprotein activity.
Assuntos
Movimento Celular/genética , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Tubo Neural/embriologia , Oligodendroglia/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Comunicação Parácrina , Proteínas Repressoras/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Espaço Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo , Tubo Neural/fisiologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/farmacologia , Comunicação Parácrina/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Especificidade por Substrato , Distribuição TecidualRESUMO
Oligodendrocytes are the myelin-forming cells of the vertebrate CNS. Little is known about the molecular control of region-specific oligodendrocyte development. Here, we show that oligodendrogenesis in the mouse rostral hindbrain, which is organized in a metameric series of rhombomere-derived (rd) territories, follows a rhombomere-specific pattern, with extensive production of oligodendrocytes in the pontine territory (r4d) and delayed and reduced oligodendrocyte production in the prepontine region (r2d, r3d). We demonstrate that segmental organization of oligodendrocytes is controlled by Hox genes, namely Hoxa2 and Hoxb2. Specifically, Hoxa2 loss of function induced a dorsoventral enlargement of the Olig2/Nkx2.2-expressing oligodendrocyte progenitor domain, whereas conditional Hoxa2 overexpression in the Olig2(+) domain inhibited oligodendrogenesis throughout the brain. In contrast, Hoxb2 deletion resulted in a reduction of the pontine oligodendrogenic domain. Compound Hoxa2(-/-)/Hoxb2(-/-) mutant mice displayed the phenotype of Hoxb2(-/-) mutants in territories coexpressing Hoxa2 and Hoxb2 (rd3, rd4), indicating that Hoxb2 antagonizes Hoxa2 during rostral hindbrain oligodendrogenesis. This study provides the first in vivo evidence that Hox genes determine oligodendrocyte regional identity in the mammalian brain.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Oligodendroglia/metabolismo , Rombencéfalo/metabolismo , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Rombencéfalo/embriologia , Fatores de Transcrição/metabolismoRESUMO
The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/deficiência , Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Células-Tronco Neurais/fisiologia , Animais , Apoptose , Núcleo Celular/metabolismo , Proliferação de Células/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Embrião de Mamíferos/efeitos da radiação , Feminino , Instabilidade Genômica/efeitos da radiação , Ventrículos Laterais/metabolismo , Ventrículos Laterais/patologia , Ventrículos Laterais/efeitos da radiação , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos da radiação , Neuroglia/fisiologia , Neuroglia/efeitos da radiação , Gravidez , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Estatísticas não ParamétricasRESUMO
Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.
Assuntos
Encéfalo/citologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Neurônios/citologia , Células-Tronco/citologia , Animais , Apoptose , Encéfalo/embriologia , Proliferação de Células , Aberrações Cromossômicas , Reparo do DNA , Desenvolvimento Embrionário , Anemia de Fanconi , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Feminino , Camundongos , Camundongos Knockout , GravidezRESUMO
Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs, or treatment with recombinant BMP4, with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin, a BMP antagonist, demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore, BECs may provide a feedback mechanism to control the proliferation of NSPCs.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Encéfalo/metabolismo , Proliferação de Células , Células Endoteliais/metabolismo , Neurônios/fisiologia , Células-Tronco/metabolismo , Animais , Encéfalo/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/citologia , Células-Tronco/citologiaRESUMO
The aim of this study was to investigate acute variations in antioxidant defense systems in the intestinal mucosa after abdominal radiation exposure and the role played by radiation-induced inflammation in these variations. Antioxidant defense systems of mouse small intestinal mucosa were studied at 6 h and 4 days after abdominal radiation exposure. Superoxide dismutases, glutathione peroxidases, catalase, metallothioneins and thioredoxins were followed in terms of mRNA expression, protein expression and enzyme activities. Dexamethasone was administered to investigate the relationship between variations in mucosal antioxidant capacity and radiation-induced inflammation. Six hours after exposure, only mitochondrial-associated antioxidant systems were induced (the superoxide dismutase and thioredoxin 2). Four days after exposure, during the inflammatory phase, superoxide dismutases were decreased and modulations of the second line of the antioxidant network were also observed: Catalase was decreased and glutathione peroxidases and metallothioneins were induced. Dexamethasone treatment modulated only glutathione peroxidase expression and did not influence either metallothionein or superoxide dismutase expression. Our findings provide direct in vivo evidence that antioxidant mechanisms of the small intestinal mucosa were not markedly mobilized during the very acute tissue radiation response. During the radiation-induced acute inflammatory response, the antioxidant capacity appeared to be dependent on inflammatory status to a certain extent.
Assuntos
Antioxidantes/metabolismo , Enterite/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos da radiação , Intestino Delgado/metabolismo , Intestino Delgado/efeitos da radiação , Lesões por Radiação/metabolismo , Animais , Relação Dose-Resposta à Radiação , Enterite/etiologia , Enterite/patologia , Exposição Ambiental/efeitos adversos , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos da radiação , Doses de Radiação , Lesões por Radiação/etiologia , Lesões por Radiação/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos da radiaçãoRESUMO
Wolcott-Rallison syndrome (WRS) is a rare autosomal-recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, multiple epiphyseal dysplasia and growth retardation, and other variable multisystemic clinical manifestations. Based on genetic studies of two inbred families, we previously identified the gene responsible for this disorder as EIF2AK3, the pancreatic eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Here, we have studied 12 families with WRS, totalling 18 cases. With the exception of one case, all patients carried EIF2AK3 mutations resulting in truncated or missense versions of the protein. Exclusion of EIF2AK3 mutations in the one patient case was confirmed by both linkage and sequence data. The activities of missense versions of EIF2AK3 were characterized in vivo and in vitro and found to have a complete lack of activity in four mutant proteins and residual kinase activity in one. Remarkably, the onset of diabetes was relatively late (30 months) in the patient expressing the partially defective EIF2AK3 mutant and in the patient with no EIF2AK3 involvement (18 months) compared with other patients (<6 months). The patient with no EIF2AK3 involvement did not have any of the other variable clinical manifestations associated with WRS, which supports the idea that the genetic heterogeneity between this variant form of WRS and EIF2AK3 WRS correlates with some clinical heterogeneity.
Assuntos
Diabetes Mellitus Tipo 1/genética , Nanismo/genética , Heterogeneidade Genética , Mutação , Osteocondrodisplasias/genética , eIF-2 Quinase/genética , Adolescente , Adulto , Criança , Pré-Escolar , Consanguinidade , Genes Recessivos , Humanos , Lactente , Mutação de Sentido Incorreto , Linhagem , SíndromeRESUMO
PURPOSE: Following ionizing radiation exposure, intestinal crypt regeneration is possible but it is still not known if regenerating crypts give rise to differentiated functional epithelial cells on villi. The aim of this study was to demonstrate that irradiated progeny of enterocytic precursor cells are capable of proliferation and subsequent differentiation using the HT-29?cell line. MATERIALS AND METHODS: Cells were cultured, irradiated (5 Gy or 10 Gy) and incubated in the presence or absence of butyrate (5 mM). Cell numbers, cell cycle parameters, alkaline phosphatase (ALP) activity, occludin labelling and gene expression were determined at different times post-exposure. RESULTS: Butyrate-induced inhibition of cell growth and arrest in G0 phase was comparable in both sham and irradiated cells in addition to similar development of ALP activity and expression. Cells also formed a monolayer with tight junctions post-irradiation. Butyrate-stimulated modulation of integrin expression during differentiation was unchanged after radiation exposure. Genes known to be implicated in differentiation mechanisms, i.e., growth and transcription factors (vascular Epidermal Growth Factor, v-EGF ; Activating Transcription Factor 4, ATF4), cell cycle genes (Cyclin-Dependent Kinase Inhibitor 1A, CDKN1A/p21(Cip1/waf1)), were studied. Most responded similarly to the differentiation stimulus whether irradiated or not. CONCLUSION: These results demonstrate that irradiated HT-29 cells still respond to butyrate to form a differentiated, functional epithelium.
Assuntos
Diferenciação Celular/efeitos da radiação , Enterócitos/efeitos da radiação , Fosfatase Alcalina/metabolismo , Butiratos/farmacologia , Enterócitos/citologia , Fase G1/efeitos dos fármacos , Células HT29 , Humanos , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
PURPOSE: We investigated the DNA damage response (DDR) of fetal neural stem and progenitor cells (NSPC), since exposure to ionizing radiation can severely impair the brain development. MATERIAL AND METHODS: We compared apoptosis induction in the dorsal telencephalon and the lateral ganglionic eminences (LGE) of mouse embryos after an in utero irradiation. We used two thymidine analogs, together with the physical position of nuclei within brain structures, to determine the fate of irradiated NSPC. RESULTS: NSPC did not activate an apparent protein 21(p21)- dependent G1/S checkpoint within the LGE as their counterparts within the dorsal telencephalon. However, the levels of radiation-induced apoptosis differed between the two telencephalic regions, due to the high radiation sensitivity of intermediate progenitors of the LGE. Besides radial glia cells, that function as neural stem cells, were more resistant and were reoriented toward self-renewing within hours following irradiation. CONCLUSIONS: The lack of the p21-dependent-cell cycle arrest at the G1/S transition appears to be a general feature of NSPC in the developing brain. However, we found variation of radiation-response in function of the types of NSPC. Factors involved in DDR and those involved in the regulation of neurogenesis are intricately linked in determining the cell fate after irradiations.
Assuntos
Encéfalo/citologia , Encéfalo/embriologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Encéfalo/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Camundongos , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neuroglia/efeitos da radiação , Telencéfalo/citologia , Telencéfalo/metabolismo , Telencéfalo/efeitos da radiação , Fatores de TempoRESUMO
We characterized the in vivo importance of the homologous recombination factor RAD54 for the developing mouse brain cortex in normal conditions or after ionizing radiation exposure. Contrary to numerous homologous recombination genes, Rad54 disruption did not impact the cortical development without exogenous stress, but it dramatically enhanced the radiation sensitivity of neural stem and progenitor cells. This resulted in the death of all cells irradiated during S or G2, whereas the viability of cells irradiated in G1 or G0 was not affected by Rad54 disruption. Apoptosis occurred after long arrests at intra-S and G2/M checkpoints. This concerned every type of neural stem and progenitor cells, showing that the importance of Rad54 for radiation response was linked to the cell cycle phase at the time of irradiation and not to the differentiation state. In the developing brain, RAD54-dependent homologous recombination appeared absolutely required for the repair of damages induced by ionizing radiation during S and G2 phases, but not for the repair of endogenous damages in normal conditions. Altogether our data support the existence of RAD54-dependent and -independent homologous recombination pathways.
Assuntos
Reparo do DNA/genética , Recombinação Homóloga , Células-Tronco Neurais/metabolismo , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/efeitos da radiação , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Dano ao DNA/genética , DNA Helicases/deficiência , DNA Helicases/metabolismo , Reparo do DNA/efeitos da radiação , Feminino , Recombinação Homóloga/efeitos da radiação , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos da radiação , Neuroglia/citologia , Neuroglia/metabolismo , Neuroglia/efeitos da radiação , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Gravidez , Fatores de TempoRESUMO
BACKGROUND: Endothelins (ET) have diuretic and natriuretic actions via ETB receptors that are found in most renal tubular segments, although the thin limbs have not been studied. Data also suggest that dysfunction of the renal ET system may be important in the pathogenesis of hypertension. The present study was aimed at determining the presence and nature of ET receptors in the thin limbs of Henle's loop and their ability to activate a Ca2+-dependent signaling pathway, as well as whether ET-induced Ca2+ signals are altered in hypertension. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Fura 2 fluoreselected strains of Lyon rats with low-normal (LL), normal (LN), and high (LH) blood pressure. RESULTS: In SD rats, ET induced Ca2+ signals in DTL of long-looped nephrons, but not in DTL of short loops, or in ascending thin limbs. Ca2+ increases were abolished by BQ123, an antagonist of the ETA receptor, but not by BQ788, an antagonist of the ETB subtype. Endothelin-3 and sarafotoxin 6c, two ETB receptor agonists, were both inactive. RT-PCR showed the presence of both ETA and ETB receptor mRNA. Ca2+ signals measured scence measurements of [Ca2+]i were made to characterize ET receptors in descending thin limbs (DTL) of Sprague-Dawley rats, spontaneously hypertensive (SH) rats, and control Wistar-Kyoto (WKY) rats, and the three in DTL of WKY LL and LN rats were similar to those in Sprague-Dawley rats, but were significantly diminished (LH) or abolished (SH) in hypertensive rats. CONCLUSION: A functional ETA receptor activating a Ca2+-dependent pathway is expressed in DTL. This ETA-induced calcium signaling is impaired in two strains of genetically hypertensive rats.