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1.
EMBO Rep ; 19(11)2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30166336

RESUMEN

SOX transcription factors have important roles during astrocyte and oligodendrocyte development, but how glial genes are specified and activated in a sub-lineage-specific fashion remains unknown. Here, we define glial-specific gene expression in the developing spinal cord using single-cell RNA-sequencing. Moreover, by ChIP-seq analyses we show that these glial gene sets are extensively preselected already in multipotent neural precursor cells through prebinding by SOX3. In the subsequent lineage-restricted glial precursor cells, astrocyte genes become additionally targeted by SOX9 at DNA regions strongly enriched for Nfi binding motifs. Oligodendrocyte genes instead are prebound by SOX9 only, at sites which during oligodendrocyte maturation are targeted by SOX10. Interestingly, reporter gene assays and functional studies in the spinal cord reveal that SOX3 binding represses the synergistic activation of astrocyte genes by SOX9 and NFIA, whereas oligodendrocyte genes are activated in a combinatorial manner by SOX9 and SOX10. These genome-wide studies demonstrate how sequentially expressed SOX proteins act on lineage-specific regulatory DNA elements to coordinate glial gene expression both in a temporal and in a sub-lineage-specific fashion.


Asunto(s)
Astrocitos/fisiología , Oligodendroglía/fisiología , Factor de Transcripción SOX9/genética , Factores de Transcripción SOXB1/genética , Médula Espinal/citología , Animales , Diferenciación Celular/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Ratones , Células-Madre Neurales , Neuroglía/citología , Neuroglía/fisiología , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXB1/metabolismo , Médula Espinal/crecimiento & desarrollo
2.
Nucleic Acids Res ; 46(W1): W163-W170, 2018 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-29893885

RESUMEN

The new web resource EviNet provides an easily run interface to network enrichment analysis for exploration of novel, experimentally defined gene sets. The major advantages of this analysis are (i) applicability to any genes found in the global network rather than only to those with pathway/ontology term annotations, (ii) ability to connect genes via different molecular mechanisms rather than within one high-throughput platform, and (iii) statistical power sufficient to detect enrichment of very small sets, down to individual genes. The users' gene sets are either defined prior to upload or derived interactively from an uploaded file by differential expression criteria. The pathways and networks used in the analysis can be chosen from the collection menu. The calculation is typically done within seconds or minutes and the stable URL is provided immediately. The results are presented in both visual (network graphs) and tabular formats using jQuery libraries. Uploaded data and analysis results are kept in separated project directories not accessible by other users. EviNet is available at https://www.evinet.org/.


Asunto(s)
Genes , Programas Informáticos , Animales , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Internet , Ratones , Transcriptoma
3.
Development ; 143(14): 2616-28, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27287799

RESUMEN

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.


Asunto(s)
Linaje de la Célula , Células Enterocromafines/citología , Células Enteroendocrinas/citología , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Serotonina/biosíntesis , Factores de Transcripción/metabolismo , Envejecimiento/metabolismo , Animales , Biomarcadores/metabolismo , Linaje de la Célula/genética , Colon/metabolismo , Duodeno/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/química , Ratones Endogámicos C57BL , Modelos Biológicos , Mutación/genética , Reacción en Cadena de la Polimerasa , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Células Madre/citología , Factores de Transcripción/química , Proteínas de Pez Cebra
4.
Proc Natl Acad Sci U S A ; 113(30): E4387-96, 2016 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-27407143

RESUMEN

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.


Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Proteínas con Homeodominio LIM/genética , Mesencéfalo/metabolismo , Mitocondrias/metabolismo , Factores de Transcripción/genética , Animales , Supervivencia Celular/genética , Daño del ADN , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Proteínas con Homeodominio LIM/deficiencia , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Estrés Oxidativo , Agregación Patológica de Proteínas , Factores de Transcripción/deficiencia , alfa-Sinucleína/metabolismo
5.
Dev Biol ; 429(1): 132-146, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28688895

RESUMEN

Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.


Asunto(s)
Organogénesis , Proteínas Represoras/metabolismo , Alelos , Animales , Tipificación del Cuerpo/genética , Embrión de Mamíferos/metabolismo , Exones/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Homocigoto , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Animales , Tubo Neural/embriología , Tubo Neural/metabolismo , Organogénesis/genética , Mutación Puntual/genética , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética
6.
Proc Natl Acad Sci U S A ; 110(18): 7330-5, 2013 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-23589857

RESUMEN

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.


Asunto(s)
Redes Reguladoras de Genes/genética , Neuronas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/genética , Animales , Sitios de Unión , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/farmacología , Pollos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Genoma/genética , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/efectos de los fármacos , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Ratones , Modelos Biológicos , Neuronas/efectos de los fármacos , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Retinoides/farmacología , Transducción de Señal/efectos de los fármacos
7.
Development ; 139(14): 2625-34, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22696295

RESUMEN

The transcription factors Foxa1 and Foxa2 promote the specification of midbrain dopaminergic (mDA) neurons and the floor plate. Whether their role is direct has remained unclear as they also regulate the expression of Shh, which has similar roles. We characterized the Foxa2 cis-regulatory network by chromatin immunoprecipitation followed by high-throughput sequencing of mDA progenitors. This identified 9160 high-quality Foxa2 binding sites associated with 5409 genes, providing mechanistic insights into Foxa2-mediated positive and negative regulatory events. Foxa2 regulates directly and positively key determinants of mDA neurons, including Lmx1a, Lmx1b, Msx1 and Ferd3l, while negatively inhibiting transcription factors expressed in ventrolateral midbrain such as Helt, Tle4, Otx1, Sox1 and Tal2. Furthermore, Foxa2 negatively regulates extrinsic and intrinsic components of the Shh signaling pathway, possibly by binding to the same enhancer regions of co-regulated genes as Gli1. Foxa2 also regulates the expression of floor plate factors that control axon trajectories around the midline of the embryo, thereby contributing to the axon guidance function of the floor plate. Finally, this study identified multiple Foxa2-regulated enhancers that are active in the floor plate of the midbrain or along the length of the embryo in mouse and chick. This work represents the first comprehensive characterization of Foxa2 targets in mDA progenitors and provides a framework for elaborating gene regulatory networks in a functionally important progenitor population.


Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Mesencéfalo/citología , Células Madre/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Electroporación , Genotipo , Factor Nuclear 3-beta del Hepatocito/genética , Inmunohistoquímica , Hibridación in Situ , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Factor de Transcripción MSX1/genética , Factor de Transcripción MSX1/metabolismo , Ratones , Ratones Mutantes , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Proteínas Represoras , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
8.
Stem Cells ; 32(3): 609-22, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24549637

RESUMEN

An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type.


Asunto(s)
Neuronas Adrenérgicas/citología , Linaje de la Célula , Células Madre Embrionarias/citología , Neuronas Motoras/citología , Factores de Transcripción/metabolismo , Neuronas Adrenérgicas/metabolismo , Animales , Línea Celular , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Ingeniería Genética , Genoma/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Neuronas Motoras/metabolismo , Transducción de Señal
10.
Development ; 138(17): 3711-21, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21775418

RESUMEN

During neural tube development, Shh signaling through Gli transcription factors is necessary to establish five distinct ventral progenitor domains that give rise to unique classes of neurons and glia that arise in specific positions along the dorsoventral axis. These cells are generated from progenitors that display distinct transcription factor gene expression profiles in specific domains in the ventricular zone. However, the molecular genetic mechanisms that control the differential spatiotemporal transcriptional responses of progenitor target genes to graded Shh-Gli signaling remain unclear. The current study demonstrates a role for Tcf/Lef repressor activity in this process. We show that Tcf3 and Tcf7L2 (Tcf4) are required for proper ventral patterning and function by independently regulating two Shh-Gli target genes, Nkx2.2 and Olig2, which are initially induced in a common pool of progenitors that ultimately segregate into unique territories giving rise to distinct progeny. Genetic and functional studies in vivo show that Tcf transcriptional repressors selectively elevate the strength and duration of Gli activity necessary to induce Nkx2.2, but have no effect on Olig2, and thereby contribute to the establishment of their distinct expression domains in cooperation with graded Shh signaling. Together, our data reveal a Shh-Gli-independent transcriptional input that is required to shape the precise spatial and temporal response to extracellular morphogen signaling information during lineage segregation in the CNS.


Asunto(s)
Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Elementos de Facilitación Genéticos/fisiología , Proteínas de Homeodominio/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Sistema Nervioso Central/citología , Embrión de Pollo , Inmunoprecipitación de Cromatina , Electroporación , Elementos de Facilitación Genéticos/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Hibridación in Situ , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Reacción en Cadena de la Polimerasa , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Factor de Transcripción 4 , Factores de Transcripción/genética , Proteínas de Pez Cebra
11.
Development ; 138(16): 3399-408, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21752929

RESUMEN

The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b. Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons (RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the differentiation of the DN lineage.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Linaje de la Célula , Dopamina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas con Homeodominio LIM , Mesencéfalo/citología , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Factores de Transcripción/genética
12.
Development ; 137(24): 4249-60, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21068056

RESUMEN

The transcription factors Nkx2.2 and Nkx2.9 have been proposed to execute partially overlapping functions in neuronal patterning of the ventral spinal cord in response to graded sonic hedgehog signaling. The present report shows that in mice lacking both Nkx2 proteins, the presumptive progenitor cells in the p3 domain of the neural tube convert to motor neurons (MN) and never acquire the fate of V3 interneurons. This result supports the concept that Nkx2 transcription factors are required to establish V3 progenitor cells by repressing the early MN lineage-specific program, including genes like Olig2. Nkx2.2 and Nkx2.9 proteins also perform an additional, hitherto unknown, function in the development of non-neuronal floor plate cells. Here, we demonstrate that loss of both Nkx2 genes results in an anatomically smaller and functionally impaired floor plate causing severe defects in axonal pathfinding of commissural neurons. Defective floor plates were also seen in Nkx2.2(+/-);Nkx2.9(-/-) compound mutants and even in single Nkx2.9(-/-) mutants, suggesting that floor plate development is sensitive to dose and/or timing of Nkx2 expression. Interestingly, adult Nkx2.2(+/-);Nkx2.9(-/-) compound-mutant mice exhibit abnormal locomotion, including a permanent or intermittent hopping gait. Drug-induced locomotor-like activity in spinal cords of mutant neonates is also affected, demonstrating increased variability of left-right and flexor-extensor coordination. Our data argue that the Nkx2.2 and Nkx2.9 transcription factors contribute crucially to the formation of neuronal networks that function as central pattern generators for locomotor activity in the spinal cord. As both factors affect floor plate development, control of commissural axon trajectories might be the underlying mechanism.


Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Homeodominio/metabolismo , Tubo Neural/embriología , Médula Espinal/embriología , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo/genética , Embrión de Mamíferos/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Mutantes , Células Madre/citología , Factores de Transcripción/genética , Proteínas de Pez Cebra
13.
Development ; 137(3): 437-45, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20081190

RESUMEN

Homeodomain (HD) transcription factors and components of the Notch pathway [Delta1 (Dll1), Jagged1 (Jag1) and the Fringe (Fng) proteins] are expressed in distinct progenitor domains along the dorsoventral (DV) axis of the developing spinal cord. However, the internal relationship between these two regulatory pathways has not been established. In this report we show that HD proteins act upstream of Notch signalling. Thus, HD proteins control the spatial distribution of Notch ligands and Fng proteins, whereas perturbation of the Notch pathway does not affect the regional expression of HD proteins. Loss of Dll1 or Jag1 leads to a domain-specific increase of neuronal differentiation but does not affect the establishment of progenitor domain boundaries. Moreover, gain-of-function experiments indicate that the ability of Dll1 and Jag1 to activate Notch is limited to progenitors endogenously expressing the respective ligand. Fng proteins enhance Dll1-activated Notch signalling and block Notch activation mediated by Jag1. This finding, combined with the overlapping expression of Fng with Dll1 but not with Jag1, is likely to explain the domain-specific activity of the Notch ligands. This outcome is opposite to the local regulation of Notch activity in most other systems, including the Drosophila wing, where Fng co-localizes with Jagged/Serrate rather than Dll/Delta, which facilitates Notch signalling at regional boundaries instead of within domains. The regulation of Notch activation in the spinal cord therefore appears to endow specific progenitor populations with a domain-wide autonomy in the control of neurogenesis and prevents any inadequate activation of Notch across progenitor domain boundaries.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Neurogénesis , Receptores Notch/metabolismo , Animales , Tipificación del Cuerpo/genética , Embrión de Pollo , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Ratones , Médula Espinal , Factores de Transcripción
14.
Development ; 137(23): 4051-60, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21062862

RESUMEN

The deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.


Asunto(s)
Tipificación del Cuerpo , Retroalimentación Fisiológica , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Neuronas/metabolismo , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas de Homeodominio/genética , Ratones , Ratones Mutantes Neurológicos , Modelos Biológicos , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuronas/citología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Factores de Tiempo
15.
J Cell Sci ; 123(Pt 17): 2931-42, 2010 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-20720151

RESUMEN

In Notch signaling, cell-bound ligands activate Notch receptors on juxtaposed cells, but the relationship between ligand endocytosis, ubiquitylation and ligand-receptor interaction remains poorly understood. To study the specific role of ligand-receptor interaction, we identified a missense mutant of the Notch ligand Jagged1 (Nodder, Ndr) that failed to interact with Notch receptors, but retained a cellular distribution that was similar to wild-type Jagged1 (Jagged1(WT)) in the absence of active Notch signaling. Both Jagged1(WT) and Jagged1(Ndr) interacted with the E3 ubiquitin ligase Mind bomb, but only Jagged1(WT) showed enhanced ubiquitylation after co-culture with cells expressing Notch receptor. Cells expressing Jagged1(WT), but not Jagged1(Ndr), trans-endocytosed the Notch extracellular domain (NECD) into the ligand-expressing cell, and NECD colocalized with Jagged1(WT) in early endosomes, multivesicular bodies and lysosomes, suggesting that NECD is routed through the endocytic degradation pathway. When coexpressed in the same cell, Jagged1(Ndr) did not exert a dominant-negative effect over Jagged1(WT) in terms of receptor activation. Finally, in Jag1(Ndr/Ndr) mice, the ligand was largely accumulated at the cell surface, indicating that engagement of the Notch receptor is important for ligand internalization in vivo. In conclusion, the interaction-dead Jagged1(Ndr) ligand provides new insights into the specific role of receptor-ligand interaction in the intracellular trafficking of Notch ligands.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Endocitosis , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Ligandos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proteínas Serrate-Jagged , Transducción de Señal , Transfección , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
16.
Proc Natl Acad Sci U S A ; 106(18): 7613-8, 2009 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-19383789

RESUMEN

Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%-95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.


Asunto(s)
Dopamina/metabolismo , Células Madre Embrionarias/fisiología , Proteínas de Homeodominio/biosíntesis , Mesencéfalo/citología , Neurogénesis , Neuronas/citología , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Proteínas de Homeodominio/genética , Humanos , Proteínas con Homeodominio LIM , Ratones , Enfermedad de Parkinson/cirugía , Ratas , Ratas Sprague-Dawley , Factores de Transcripción
17.
Nat Commun ; 13(1): 3046, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35650213

RESUMEN

Stem cell therapies for Parkinson's disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.


Asunto(s)
Enfermedad de Parkinson , Células Madre Pluripotentes , Animales , Diferenciación Celular , Neuronas Dopaminérgicas , Humanos , Mesencéfalo , Enfermedad de Parkinson/terapia , Ratas , Tretinoina/farmacología
18.
Dev Cell ; 10(2): 187-97, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16459298

RESUMEN

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at approximately E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu(-/-) embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu(-/-) fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu(+/-) mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.


Asunto(s)
Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Animales , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/metabolismo , Síndrome del Nevo Basocelular/patología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Femenino , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Mutantes , Sistema Nervioso/embriología , Receptores Patched , Fenotipo , Embarazo , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Piel/metabolismo , Piel/patología , Receptor Smoothened , Proteína con Dedos de Zinc GLI1
19.
J Cell Sci ; 122(Pt 18): 3351-7, 2009 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-19706676

RESUMEN

Dimerization is recognized as a crucial step in the activation of many plasma membrane receptors. However, a growing number of receptors pre-exist as dimers in the absence of ligand, indicating that, although necessary, dimerization is not always sufficient for signaling. The p75 neurotrophin receptor (p75(NTR)) forms disulfide-linked dimers at the cell surface independently of ligand binding through Cys257 in its transmembrane domain. Here, we show that crosslinking of p75(NTR) dimers by cysteine-scanning mutagenesis results in constitutive, ligand-independent activity in several pathways that are normally engaged upon neurotrophin stimulation of native receptors. The activity profiles of different disulfide-crosslinked p75(NTR) mutants were similar but not identical, suggesting that different configurations of p75(NTR) dimers might be endowed with different functions. Interestingly, crosslinked p75(NTR) mutants did not mimic the effects of the myelin inhibitors Nogo or MAG, suggesting the existence of ligand-specific activation mechanisms. Together, these results support a conformational model of p75(NTR) activation by neurotrophins, and reveal a genetic approach to generate gain-of-function receptor variants with distinct functional profiles.


Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Disulfuros/metabolismo , Multimerización de Proteína , Receptor de Factor de Crecimiento Nervioso/química , Receptor de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Células COS , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Chlorocebus aethiops , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ligandos , Datos de Secuencia Molecular , Proteínas Mutantes/efectos de los fármacos , Proteínas Mutantes/metabolismo , FN-kappa B/metabolismo , Factores de Crecimiento Nervioso/farmacología , Multimerización de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/metabolismo
20.
Proc Natl Acad Sci U S A ; 105(9): 3368-73, 2008 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-18299578

RESUMEN

Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1alpha (HIF-1alpha) becomes stabilized and directly activates transcription of downstream genes. In addition to this "canonical" response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1alpha can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N(1945) and N(2012)) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo. FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1alpha to its target promoters and derepresses HIF-1alpha function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1alpha, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1alpha. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Receptor Cross-Talk , Receptores Notch/metabolismo , Transducción de Señal , Animales , Línea Celular , Embrión de Pollo , Humanos , Hidroxilación , Ratones , Oxigenasas de Función Mixta , Desarrollo de Músculos , Proteínas Proto-Oncogénicas/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor Notch3 , Receptor Notch4 , Proteínas Represoras/farmacología , Factores de Transcripción/farmacología , Transfección
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