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1.
Proc Natl Acad Sci U S A ; 114(25): E4934-E4943, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28584103

RESUMEN

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2-/-;Ascl1-/- mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+/Ctip2+ layer V neurons, and precocious differentiation of normally late-born, Satb2+ layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1+/Ctip2+ deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deep-layer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Axones/metabolismo , Diferenciación Celular/fisiología , Femenino , Masculino , Ratones , Neurogénesis/fisiología , Neuronas/metabolismo , Proteínas Represoras/metabolismo
2.
J Neurosci ; 33(1): 259-72, 2013 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-23283339

RESUMEN

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.


Asunto(s)
Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Hipotálamo Medio/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Linaje de la Célula , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Hipotálamo Medio/embriología , Hipotálamo Medio/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Transducción de Señal , Factores de Transcripción/genética
3.
Dev Biol ; 363(1): 95-105, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22206757

RESUMEN

The growth of new blood vessels by angiogenesis and their stabilization by the recruitment of perivascular mural cells are thought to be two sequential, yet independent events. Here we identify molecular links between both processes through the ßPix and integrin α(v)ß(8) proteins. Bubblehead (bbh) mutants with a genetic mutation in ßPix show defective vascular stabilization. ßPix is a guanine nucleotide exchange factor and scaffold protein that binds many proteins including Git1, which bridges ßPix to integrins at focal adhesions. Here we show that the ability of ßPix to stabilize vessels requires Git1 binding residues. Knockdown of Git1 leads to a hemorrhage phenotype similar to loss of integrin α(v), integrin ß(8) or ßPix, suggesting that vascular stabilization through ßPix involves interactions with integrins. Furthermore, double loss of function of ßPix and integrin α(v) shows enhanced hemorrhage rates. Not only is vascular stability impaired in these embryos, but we also uncover a novel role of both ßPix and integrin α(v)ß(8) in cerebral angiogenesis. Downregulation of either ßPix or integrin α(v)ß(8) results in fewer and morphologically abnormal cerebral arteries penetrating the hindbrain. We show that this is coupled with a significant reduction in endothelial cell proliferation in bbh mutants or integrin α(v)ß(8) morphants. These data suggest that a complex involving ßPix, GIT1 and integrin α(v)ß(8) may regulate vascular stability, cerebral angiogenesis and endothelial cell proliferation in the developing embryo.


Asunto(s)
Vasos Sanguíneos/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Integrinas/genética , Neovascularización Fisiológica/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Vasos Sanguíneos/embriología , Encéfalo/irrigación sanguínea , Encéfalo/embriología , Encéfalo/metabolismo , Proliferación Celular , Hemorragia Cerebral/embriología , Hemorragia Cerebral/genética , Circulación Cerebrovascular/genética , Células Endoteliales/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Inmunohistoquímica , Hibridación in Situ , Integrinas/metabolismo , Mutación , Neovascularización Fisiológica/fisiología , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Intercambio de Guanina Nucleótido Rho , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
4.
J Vis Exp ; (52)2011 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-21730943

RESUMEN

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos(1,2) that we have successfully adapted(3,4). It is largely based on the success of in ovo electroporation technologies that are commonly used in chick(5). Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described(6). Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells. To date, in utero electroporation has been most widely used for the analysis of neocortical development(1,2,3,4). More recent studies have targeted the embryonic retina(7,8,9) and thalamus(10,11,12). Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3(rd) ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.


Asunto(s)
ADN/administración & dosificación , Diencéfalo/fisiología , Electroporación/métodos , Técnicas de Transferencia de Gen , Retina/fisiología , Telencéfalo/fisiología , Animales , ADN/genética , Embrión de Mamíferos , Femenino , Ratones , Embarazo
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