RESUMEN
Fibroblast growth factor (FGF) signaling and PAX6 transcription are required for neuroectoderm specification of human embryonic stem cells (hESCs). In this study, we asked how FGF signaling leads to PAX6 transcription and neuroectoderm specification from hESCs. Under a chemically defined medium, FGF inhibition blocked phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) with a significant reduction of PAX6-expressing neuroepithelia, indicating that FGF regulates neural induction through ERK1/2 activation. Activation of FGF-ERK1/2 pathway was necessary for the activity of poly(ADP-ribose) polymerase-1 (PARP-1), a conserved nuclear protein catalyzing polymerization of ADP-ribose units. Pharmacological inhibition and genetic ablation of PARP-1 inhibited neural induction from hESCs, suggesting that FGF-ERK1/2 signal pathway regulates neuroectoderm specification through regulating PARP-1 activity. Furthermore, FGF-ERK1/2-PARP-1 cascade regulated the expression of PAX6, a transcription determinant of human neuroectoderm. Together, we propose that FGF regulates hESC neural specification through the ERK1/2-PARP-1 signaling pathway.
Asunto(s)
Proteínas del Ojo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Sistema de Señalización de MAP Quinasas , Placa Neural/citología , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Butadienos/farmacología , Diferenciación Celular , Células Cultivadas , Medios de Cultivo/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Activación Enzimática , Proteínas del Ojo/genética , Factores de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Placa Neural/efectos de los fármacos , Placa Neural/metabolismo , Nitrilos/farmacología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Fenantrenos/farmacología , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Pirroles/farmacología , Proteínas Represoras/genéticaRESUMEN
Inhibition of bone morphogenetic protein (BMP) signaling is required for vertebrate neural induction, and fibroblast growth factors (FGFs) may affect neural induction through phosphorylation at the linker region of Smad1, thus regulating BMP signaling. Here we show that human embryonic stem cells efficiently convert to neuroepithelial cells in the absence of BMP antagonists, or even when exposed to high concentrations of exogenous BMP4. Molecular and functional analyses revealed multiple levels of endogenous BMP signaling inhibition that may account for the efficient neural differentiation. Blocking FGF signaling inhibited neural induction, but did not alter the phosphorylation of the linker region of Smad1, suggesting that FGF enhances human neural specification independently of BMP signaling.
Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/metabolismo , Proteínas del Ojo/biosíntesis , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/biosíntesis , Humanos , Inmunohistoquímica , Neuronas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/biosíntesis , Fosforilación , Proteínas Represoras/biosíntesis , Transducción de Señal/efectos de los fármacos , Proteína Smad1/antagonistas & inhibidores , Proteína Smad1/metabolismoRESUMEN
The transcriptional regulation of neuroectoderm (NE) specification is unknown. Here we show that Pax6 is uniformly expressed in early NE cells of human fetuses and those differentiated from human embryonic stem cells (hESCs). This is in contrast to the later expression of Pax6 in restricted mouse brain regions. Knockdown of Pax6 blocks NE specification from hESCs. Overexpression of either Pax6a or Pax6b, but not Pax6triangle upPD, triggers hESC differentiation. However, only Pax6a converts hESCs to NE. In contrast, neither loss nor gain of function of Pax6 affects mouse NE specification. Both Pax6a and Pax6b bind to pluripotent gene promoters but only Pax6a binds to NE genes during human NE specification. These findings indicate that Pax6 is a transcriptional determinant of the human NE and suggest that Pax6a and Pax6b coordinate with each other in determining the transition from pluripotency to the NE fate in human by differentially targeting pluripotent and NE genes.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Placa Neural/citología , Placa Neural/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Humanos , Técnicas In Vitro , Ratones , Ratones SCID , Modelos Biológicos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Teratoma/patologíaRESUMEN
Brain fatty acid binding protein (Fabp7), which is important in early nervous system development, is expressed in astrocytes and neuronal cell precursors in mature brain. We report here that levels of Fabp7 mRNA in adult murine brain change over a 24 hour period. Unlike Fabp5, a fatty acid binding protein that is expressed widely in various cell types within brain, RNA analysis revealed that Fabp7 mRNA levels were elevated during the light period and lower during dark in brain regions involved in sleep and activity mechanisms. This pattern of Fabp7 mRNA expression was confirmed using in situ hybridization and found to occur throughout the entire brain. Changes in the intracellular distribution of Fabp7 mRNA were also evident over a 24 hour period. Diurnal changes in Fabp7, however, were not found in postnatal day 6 brain, when astrocytes are not yet mature. In contrast, granule cell precursors of the subgranular zone of adult hippocampus did undergo diurnal changes in Fabp7 expression. These changes paralleled oscillations in Fabp7 mRNA throughout the brain suggesting that cell-coordinated signals likely control brain-wide Fabp7 mRNA expression. Immunoblots revealed that Fabp7 protein levels also underwent diurnal changes in abundance, with peak levels occurring in the dark period. Of clock or clock-regulated genes, the synchronized, global cycling pattern of Fabp7 expression is unique and implicates glial cells in the response or modulation of activity and/or circadian rhythms.
Asunto(s)
Astrocitos/metabolismo , Ritmo Circadiano , Proteínas de Unión a Ácidos Grasos/genética , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/genética , Células Madre/metabolismo , Animales , Astrocitos/citología , Proteína de Unión a los Ácidos Grasos 7 , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Neuronas , ARN Mensajero/análisisRESUMEN
Understanding neuroectoderm formation and subsequent diversification to functional neural subtypes remains elusive. We show here that human embryonic stem cells (hESCs) differentiate to primitive neuroectoderm after 8-10 days. At this stage, cells uniformly exhibit columnar morphology and express neural markers, including anterior but not posterior homeodomain proteins. The anterior identity of these cells develops regardless of morphogens present during initial neuroectoderm specification. This anterior phenotype can be maintained or transformed to a caudal fate with specific morphogens over the next week, when cells become definitive neuroepithelia, marked by neural tube-like structures with distinct adhesion molecule expression, Sox1 expression, and a resistance to additional patterning signals. Thus, primitive neuroepithelia represents the earliest neural cells that possess the potential to differentiate to regionally specific neural progenitors. This finding offers insights into early human brain development and lays a foundation for generating neural cells with correct positional and transmitter profiles. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Diferenciación Celular , Ectodermo/citología , Células Madre Embrionarias/citología , Neuronas/citología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Epiteliales/citología , Perfilación de la Expresión Génica , Humanos , Macaca mulatta , Modelos Biológicos , Neuronas/metabolismo , Factores de TiempoRESUMEN
In the mammalian brain, sleep and wakefulness are associated with widespread changes in gene expression. Sleep in fruit flies shares many features with mammalian sleep, but it is currently unknown to what extent behavioral states affect gene expression in Drosophila. To find out, we performed a comprehensive microarray analysis of gene expression in spontaneously awake, sleep-deprived and sleeping flies. Fly heads were collected at 4 am, after 8 h of spontaneous sleep or sleep deprivation, and at 4 pm, after 8 h of spontaneous wakefulness. As in rats, we found that behavioral state and time of day affect Drosophila gene expression to a comparable extent. As in rats, transcripts with higher expression in wakefulness and in sleep belong to different functional categories, and in several cases these groups overlap with those previously identified in rats. Wakefulness-related genes code for transcription factors and for proteins involved in the stress response, immune response, glutamatergic transmission, and carbohydrate metabolism. Sleep-related transcripts include the glial gene anachronism and several genes involved in lipid metabolism. Finally, the expression of many wakefulness-related and sleep-related Drosophila transcripts is also modulated by the time of day, suggesting an interaction at the molecular level between circadian and homeostatic mechanism of sleep regulation.