RESUMEN
Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.
Asunto(s)
Neoplasias Encefálicas/genética , Células Endoteliales/metabolismo , Glioblastoma/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Patológica/genética , Receptores Notch/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas de Unión al Calcio , Movimiento Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Genes Reporteros , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Fisiológica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Notch/genética , Retina/citología , Retina/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacologíaAsunto(s)
Melanoma/metabolismo , Melanoma/patología , Proteínas de la Membrana/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factor de Transcripción AP-2/metabolismo , Biomarcadores de Tumor/metabolismo , Humanos , Inmunohistoquímica , Invasividad Neoplásica , PronósticoRESUMEN
BACKGROUND: Different approaches have been developed to dissect the interplay between transcription factors (TFs) and their cis-acting sequences on DNA in order to identify TF target genes. Here we used a combination of computational and experimental approaches to identify novel direct targets of TFAP2A, a key TF for a variety of physiological and pathological cellular processes. Gene expression profiles of HeLa cells either silenced for TFAP2A by RNA interference or not were previously compared and a set of differentially expressed genes was revealed. RESULTS: The regulatory regions of 494 TFAP2A-modulated genes were analyzed for the presence of TFAP2A binding sites, employing the canonical TFAP2A Positional Weight Matrix (PWM) reported in Jaspar http://jaspar.genereg.net/. 264 genes containing at least 2 high score TFAP2A binding sites were identified, showing a central role in "Cellular Movement" and "Cellular Development". In an attempt to identify TFs that could cooperate with TFAP2A, a statistically significant enrichment for SP1 binding sites was found for TFAP2A-activated but not repressed genes. The direct binding of TFAP2A or SP1 to a random subset of TFAP2A-modulated genes was demonstrated by Chromatin ImmunoPrecipitation (ChIP) assay and the TFAP2A-driven regulation of DCBLD2/ESDN/CLCP1 gene studied in details. CONCLUSIONS: We proved that our computational approaches applied to microarray selected genes are valid tools to identify functional TF binding sites in gene regulatory regions as confirmed by experimental validations. In addition, we demonstrated a fine-tuned regulation of DCBLD2/ESDN transcription by TFAP2A.
