RESUMEN
The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Retina/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción SOXE/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
Increasingly, studies reveal novel functions for cell cycle proteins during development. Here, we investigated the role of E2F4 in eye development. E2F4-deficient mouse embryos exhibit severe early eye patterning defects, which are evident from embryonic day 11.5 and characterized by aberrant shape of the optic cup, coloboma as well as abnormal eye pigmentation. Loss of E2F4 is associated with proximal-distal patterning defects in the optic vesicle. These defects are characterized by the expansion of optic stalk marker gene expression to the optic cup and reduced expression of ventral optic cup markers. These defects are associated with a split of Shh expression domain at the ventral midline of the forebrain and expansion of the Shh activity into the ventral optic cup. Despite these patterning defects, early neuronal differentiation and Shh expression in the retina are not affected by E2F4 deletion. Overall, the results of our studies show a novel role of E2F4 in the early eye development.
Asunto(s)
Tipificación del Cuerpo/genética , Factor de Transcripción E2F4/genética , Ojo/embriología , Animales , Diferenciación Celular/genética , Factor de Transcripción E2F4/metabolismo , Embrión de Mamíferos , Ojo/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Neurogénesis/genética , Células Ganglionares de la Retina/citologíaRESUMEN
The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.
Asunto(s)
Células Endoteliales/metabolismo , Proteínas Hedgehog/metabolismo , Epitelio Pigmentado Ocular/embriología , Esclerótica/embriología , Transducción de Señal , Animales , Biomarcadores/metabolismo , Coroides/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas de Homeodominio/metabolismo , Hipopigmentación/patología , Factores de Transcripción de Tipo Kruppel , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Órbita/metabolismo , Epitelio Pigmentado Ocular/anomalías , Epitelio Pigmentado Ocular/ultraestructura , Retina/embriología , Retina/patología , Esclerótica/anomalías , Esclerótica/ultraestructura , Transactivadores/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.
Asunto(s)
Ciclo Celular/fisiología , Factor de Transcripción E2F4/fisiología , Telencéfalo/embriología , Telencéfalo/metabolismo , Animales , Células Cultivadas , Factor de Transcripción E2F4/deficiencia , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Telencéfalo/citología , Telencéfalo/crecimiento & desarrolloRESUMEN
The Sonic hedgehog (Shh) signaling pathway plays a key role in the development of the vertebrate central nervous system, including the eye. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that differentially activate and repress the expression of specific downstream target genes. In this study, we investigated the roles of the three vertebrate Glis in mediating midline Shh signaling in early ocular development. We examined the ocular phenotypes of Shh and Gli combination mutant mouse embryos and monitored proximodistal and dorsoventral patterning by the expression of specific eye development regulatory genes using in situ hybridization. We show that midline Shh signaling relieves the repressor activity of Gli3 adjacent to the midline and then promotes eye pattern formation through the nonredundant activities of all three Gli proteins. Gli3, in particular, is required to specify the dorsal optic stalk and to define the boundary between the optic stalk and the optic cup.