RESUMEN
Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.
Asunto(s)
Encéfalo , Microglía , Axones , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Macrófagos/fisiología , Microglía/patología , MorfogénesisRESUMEN
In response to repulsive cues, axonal growth cones can quickly retract. This requires the prompt activity of contractile actomyosin, which is formed by the non-muscle myosin II (NMII) bound to actin filaments. NMII is a molecular motor that provides the necessary mechanical force at the expense of ATP. Here, we report that this process is energetically coupled to glycolysis and is independent of cellular ATP levels. Induction of axonal retraction requires simultaneous generation of ATP by glycolysis, as shown by chemical inhibition and genetic knock-down of GAPDH. Co-immunoprecipitation and proximal-ligation assay showed that actomyosin associates with ATP-generating glycolytic enzymes and that this association is strongly enhanced during retraction. Using microfluidics, we confirmed that the energetic coupling between glycolysis and actomyosin necessary for axonal retraction is localized to the growth cone and near axonal shaft. These results indicate a tight coupling between on-demand energy production by glycolysis and energy consumption by actomyosin contraction suggesting a function of glycolysis in axonal guidance.
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Actomiosina , Axones , Glucólisis , Citoesqueleto de Actina , Actomiosina/metabolismo , Adenosina Trifosfato , Axones/fisiologíaRESUMEN
Sensory inputs are conveyed to distinct primary areas of the neocortex through specific thalamocortical axons (TCA). While TCA have the ability to reorient postnatally to rescue embryonic mistargeting and target proper modality-specific areas, how this remarkable adaptive process is regulated remains largely unknown. Here, using a mutant mouse model with a shifted TCA trajectory during embryogenesis, we demonstrated that TCA rewiring occurs during a short postnatal time window, preceded by a prenatal apoptosis of thalamic neurons-two processes that together lead to the formation of properly innervated albeit reduced primary sensory areas. We furthermore showed that preterm birth, through serotonin modulation, impairs early postnatal TCA plasticity, as well as the subsequent delineation of cortical area boundary. Our study defines a birth and serotonin-sensitive period that enables concerted adaptations of TCA to primary cortical areas with major implications for our understanding of brain wiring in physiological and preterm conditions.
Asunto(s)
Neocórtex , Nacimiento Prematuro , Recién Nacido , Ratones , Animales , Humanos , Embarazo , Femenino , Neuronas/fisiología , Serotonina , Corteza Cerebral/fisiología , Recien Nacido Prematuro , Axones/fisiología , Tálamo/fisiologíaRESUMEN
Cortical wiring relies on guidepost cells and activity-dependent processes that are thought to act sequentially. Here, we show that the construction of layer 1 (L1), a main site of top-down integration, is regulated by crosstalk between transient Cajal-Retzius cells (CRc) and spontaneous activity of the thalamus, a main driver of bottom-up information. While activity was known to regulate CRc migration and elimination, we found that prenatal spontaneous thalamic activity and NMDA receptors selectively control CRc early density, without affecting their demise. CRc density, in turn, regulates the distribution of upper layer interneurons and excitatory synapses, thereby drastically impairing the apical dendrite activity of output pyramidal neurons. In contrast, postnatal sensory-evoked activity had a limited impact on L1 and selectively perturbed basal dendrites synaptogenesis. Collectively, our study highlights a remarkable interplay between thalamic activity and CRc in L1 functional wiring, with major implications for our understanding of cortical development.
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Interneuronas , Células Piramidales , Dendritas/fisiología , Interneuronas/fisiología , Neuronas/fisiología , TálamoRESUMEN
The striatum controls behaviors via the activity of direct and indirect pathway projection neurons (dSPN and iSPN) that are intermingled in all compartments. While such cellular mosaic ensures the balanced activity of the two pathways, its developmental origin and pattern remains largely unknown. Here, we show that both SPN populations are specified embryonically and intermix progressively through multidirectional iSPN migration. Using conditional mutant mice, we found that inactivation of the dSPN-specific transcription factor Ebf1 impairs selective dSPN properties, including axon pathfinding, while molecular and functional features of iSPN were preserved. Ebf1 mutation disrupted iSPN/dSPN intermixing, resulting in an uneven distribution. Such architectural defect was selective of the matrix compartment, highlighting that intermixing is a parallel process to compartment formation. Our study reveals while iSPN/dSPN specification is largely independent, their intermingling emerges from an active migration of iSPN, thereby providing a novel framework for the building of striatal architecture.
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Neostriado/fisiología , Neuronas/fisiología , Animales , Diferenciación Celular , Movimiento Celular , Embrión de Mamíferos/fisiología , Eliminación de Gen , Ratones Endogámicos C57BL , Neostriado/embriología , Neuronas/citología , Transactivadores/deficiencia , Transactivadores/metabolismoRESUMEN
Trio, a member of the Dbl family of guanine nucleotide exchange factors, activates Rac1 downstream of netrin 1/DCC signalling in axon outgrowth and guidance. Although it has been proposed that Trio also activates RhoA, the putative upstream factors remain unknown. Here, we show that Slit2 induces Trio-dependent RhoA activation, revealing a crosstalk between Slit and Trio/RhoA signalling. Consistently, we found that RhoA activity is hindered in vivo in Trio mutant mouse embryos. We next studied the development of the ventral telencephalon and thalamocortical axons, which have been previously shown to be controlled by Slit2. Remarkably, this analysis revealed that Trio knockout (KO) mice show phenotypes that bear strong similarities to the ones that have been reported in Slit2 KO mice in both guidepost corridor cells and thalamocortical axon pathfinding in the ventral telencephalon. Taken together, our results show that Trio induces RhoA activation downstream of Slit2, and support a functional role in ensuring the proper positioning of both guidepost cells and a major axonal tract. Our study indicates a novel role for Trio in Slit2 signalling and forebrain wiring, highlighting its role in multiple guidance pathways as well as in biological functions of importance for a factor involved in human brain disorders.
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Tipificación del Cuerpo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Telencéfalo/embriología , Telencéfalo/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Orientación del Axón , Axones/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Conos de Crecimiento/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones Noqueados , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tálamo/embriología , Tálamo/metabolismoRESUMEN
In mammals, thalamic axons are guided internally toward their neocortical target by corridor (Co) neurons that act as axonal guideposts. The existence of Co-like neurons in non-mammalian species, in which thalamic axons do not grow internally, raised the possibility that Co cells might have an ancestral role. Here, we investigated the contribution of corridor (Co) cells to mature brain circuits using a combination of genetic fate-mapping and assays in mice. We unexpectedly found that Co neurons contribute to striatal-like projection neurons in the central extended amygdala. In particular, Co-like neurons participate in specific nuclei of the bed nucleus of the stria terminalis, which plays essential roles in anxiety circuits. Our study shows that Co neurons possess an evolutionary conserved role in anxiety circuits independently from an acquired guidepost function. It furthermore highlights that neurons can have multiple sequential functions during brain wiring and supports a general role of tangential migration in the building of subpallial circuits.
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Vías Aferentes/fisiología , Orientación del Axón/genética , Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Tegmento Pontino , Tálamo , Animales , Animales Recién Nacidos , Toxina del Cólera/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Embrión de Mamíferos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tegmento Pontino/citología , Tegmento Pontino/embriología , Tegmento Pontino/crecimiento & desarrollo , Embarazo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Tálamo/citología , Tálamo/embriología , Tálamo/crecimiento & desarrollo , Factor Nuclear Tiroideo 1/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The neocortex undergoes extensive developmental growth, but how its architecture adapts to expansion remains largely unknown. Here, we investigated how early born Cajal-Retzius (CR) neurons, which regulate the assembly of cortical circuits, maintain a dense superficial distribution in the growing neocortex. We found that CR cell density is sustained by an activity-dependent importation of olfactory CR cells, which migrate into the neocortex after they have acted as axonal guidepost cells in the olfactory system. Furthermore, using mouse genetics, we showed that CR cell density severely affects the architecture of layer 1, a key site of input integration for neocortical networks, leading to an excitation/inhibition ratio imbalance. Our study reveals that neurons reenter migration several days after their initial positioning, thereby performing sequential developmental roles in olfactory cortex and neocortex. This atypical process is essential to regulate CR cell density during growth, which in turn ensures the correct wiring of neocortical circuitry. VIDEO ABSTRACT.
Asunto(s)
Recuento de Células , Neocórtex/embriología , Neuronas/fisiología , Bulbo Olfatorio/embriología , Corteza Olfatoria/embriología , Animales , Axones , Movimiento Celular , Interneuronas/fisiología , Ratones , Bulbo Olfatorio/citologíaRESUMEN
BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.
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Axones/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Proteínas Supresoras de Tumor/farmacología , Animales , Axones/clasificación , Axones/fisiología , Técnicas de Cultivo de Célula/instrumentación , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Hipocampo/citología , Humanos , Laminina/farmacología , Ratones , Técnicas Analíticas Microfluídicas , Proteínas del Tejido Nervioso/administración & dosificación , Netrina-1 , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Tálamo/citologíaRESUMEN
Sensory perception relies on the formation of stereotyped maps inside the brain. This feature is particularly well illustrated in the mammalian neocortex, which is subdivided into distinct cortical sensory areas that comprise topological maps, such as the somatosensory homunculus in humans or the barrel field of the large whiskers in rodents. How somatosensory maps are formed and relayed into the neocortex remain essential questions in developmental neuroscience. Here, we will present our current knowledge on whisker map transfer in the mouse model, with the goal of linking embryonic and postnatal studies into a comprehensive framework.
Asunto(s)
Modelos Neurológicos , Neocórtex/fisiología , Corteza Somatosensorial/fisiología , Tálamo/fisiología , Animales , Mapeo Encefálico , Ratones , Neocórtex/anatomía & histología , Neocórtex/embriología , Vías Nerviosas/anatomía & histología , Vías Nerviosas/embriología , Vías Nerviosas/fisiología , Corteza Somatosensorial/anatomía & histología , Corteza Somatosensorial/embriología , Tálamo/anatomía & histología , Tálamo/embriología , Vibrisas/inervación , Vibrisas/fisiologíaRESUMEN
Sensory maps, such as the representation of mouse facial whiskers, are conveyed throughout the nervous system by topographic axonal projections that preserve neighboring relationships between adjacent neurons. In particular, the map transfer to the neocortex is ensured by thalamocortical axons (TCAs), whose terminals are topographically organized in response to intrinsic cortical signals. However, TCAs already show a topographic order early in development, as they navigate toward their target. Here, we show that this preordering of TCAs is required for the transfer of the whisker map to the neocortex. Using Ebf1 conditional inactivation that specifically perturbs the development of an intermediate target, the basal ganglia, we scrambled TCA topography en route to the neocortex without affecting the thalamus or neocortex. Notably, embryonic somatosensory TCAs were shifted toward the visual cortex and showed a substantial intermixing along their trajectory. Somatosensory TCAs rewired postnatally to reach the somatosensory cortex but failed to form a topographic anatomical or functional map. Our study reveals that sensory map transfer relies not only on positional information in the projecting and target structures but also on preordering of axons along their trajectory, thereby opening novel perspectives on brain wiring.
Asunto(s)
Neocórtex/embriología , Corteza Somatosensorial/embriología , Tálamo/embriología , Vibrisas/embriología , Animales , Axones/metabolismo , Mapeo Encefálico , Ratones , Neocórtex/citología , Neocórtex/metabolismo , Corteza Somatosensorial/citología , Corteza Somatosensorial/metabolismo , Tálamo/citología , Tálamo/metabolismo , Transactivadores/metabolismo , Vibrisas/citología , Vibrisas/metabolismoRESUMEN
Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.
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Corteza Cerebral/fisiología , Vías Nerviosas/fisiología , Tálamo/fisiología , Factores de Edad , Animales , Axones/fisiología , Tipificación del Cuerpo/genética , Calbindina 2 , Corteza Cerebral/citología , Contactina 2/metabolismo , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Glicoproteínas/genética , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular , Complejo de Antígeno L1 de Leucocito/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Factores del Dominio POU/genética , Proteínas Represoras/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Semaforinas , Proteínas de Dominio T Box , Tálamo/citología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Wnt3A/genética , Proteínas tau/genéticaRESUMEN
How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.
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Axones/fisiología , Conos de Crecimiento/fisiología , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tálamo/embriología , Tálamo/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Células COS , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Chlorocebus aethiops , Efrina-A5/genética , Efrina-A5/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Netrina-1 , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Proteínas Supresoras de Tumor/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Proteínas RoundaboutRESUMEN
Thalamocortical projections convey visual, somatosensory and auditory information to the cerebral cortex. A recent report in Neural Development shows how a forward genetic screen has enabled the identification of novel mutations affecting specific decision points of thalamocortical axon pathfinding.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Genes del Desarrollo , Mutación , Animales , Axones/fisiología , Ratones , Ratones Transgénicos , Tálamo/fisiologíaRESUMEN
Mouse metanephric kidney development begins with the induction of the ureteric bud (UB) from the caudal portion of the Wolffian duct by metanephric mesenchymal signals. While the UB undergoes branching morphogenesis to generate the entire urinary collecting system and the ureter, factors secreted by the UB tips induce surrounding mesenchymal cells to convert into epithelia and form the nephrons, the functional units of the kidney. Epithelial branching morphogenesis and nephrogenesis are therefore tightly orchestrated; defects in either of these processes lead to severe kidney phenotypes ranging from hypoplasia to complete aplasia. However, the underlying regulatory networks have been only partially elucidated. Here, we identify the transcription factor vHNF1 (HNF1beta) as a crucial regulator of these early developmental events. Initially involved in timing outgrowth of the UB and subsequent branching, vHNF1 is also required for nephric duct epithelial maintenance, Müllerian duct formation and early nephrogenesis. Mosaic analyses further suggest a cell-autonomous requirement for vHNF1 in the acquisition of a specialized tip domain and branching morphogenesis. vHNF1 exerts these intricate functions at least in part through the direct control of key regulatory molecules involved in different aspects of early kidney development. Notably, vHNF1 acting directly upstream of Wnt9b appears to orchestrate Wnt signaling action in the mesenchymal-epithelial transitions underlying the initiation of nephrogenesis. These results demonstrate that vHNF1 is an essential transcriptional regulator that, in addition to the known later functions in normal duct morphogenesis, plays a crucial role during the earliest stages of urogenital development and provide novel insights into the regulatory circuits controlling events.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-beta del Hepatocito/metabolismo , Riñón/embriología , Organogénesis/fisiología , Uréter/embriología , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Riñón/metabolismo , Ratones , Organogénesis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uréter/metabolismoRESUMEN
Mouse liver induction occurs via the acquisition of ventral endoderm competence to respond to inductive signals from adjacent mesoderm, followed by hepatic specification. Little is known about the regulatory circuit involved in these processes. Through the analysis of vHnf1 (Hnf1b)-deficient embryos, generated by tetraploid embryo complementation, we demonstrate that lack of vHNF1 leads to defective hepatic bud formation and abnormal gut regionalization. Thickening of the ventral hepatic endoderm and expression of known hepatic genes do not occur. At earlier stages, hepatic specification of vHnf1-/- ventral endoderm is disrupted. More importantly, mutant ventral endoderm cultured in vitro loses its responsiveness to inductive FGF signals and fails to induce the hepatic-specification genes albumin and transthyretin. Analysis of liver induction in zebrafish indicates a conserved role of vHNF1 in vertebrates. Our results reveal the crucial role of vHNF1 at the earliest steps of liver induction: the acquisition of endoderm competence and the hepatic specification.
Asunto(s)
Endodermo/embriología , Factor Nuclear 1-beta del Hepatocito/fisiología , Hepatocitos/citología , Hígado/embriología , Mesodermo/embriología , Proteínas de Pez Cebra/fisiología , Animales , Diferenciación Celular/fisiología , Endodermo/fisiología , Femenino , Factores de Crecimiento de Fibroblastos/fisiología , Factor Nuclear 1-beta del Hepatocito/genética , Hepatocitos/fisiología , Hígado/citología , Hígado/fisiología , Masculino , Mesodermo/fisiología , Ratones , Especificidad de la Especie , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Generation of pancreatic precursor cells in the endoderm is controlled by a network of transcription factors. Hepatocyte nuclear factor-6 (HNF6) is a key player in this network, because it controls the initiation of the expression of pancreatic and duodenal homeobox 1 (Pdx1), the earliest marker of pancreatic precursor cells. To further characterize this network, we have investigated how the expression of HNF6 is controlled in mouse endoderm, by using in vitro and in vivo protein-DNA interaction techniques combined with endoderm electroporation, transgenesis, and gene inactivation in embryos. We delineated Hnf6 regulatory regions that confer expression of a reporter gene in the embryonic endoderm but not in extraembryonic visceral endoderm. HNF6 expression in the embryonic endoderm was found to depend on an intronic enhancer. This enhancer contains functional binding sites for the tissue-specific factors of the forkhead box A and HNF1 families. Among the latter, variant HNF1 (vHNF1)/TCF2, which is expressed before HNF6 in the endoderm, was found to be critical for HNF6 expression. Therefore, the sequential activation of vHNF1, HNF6, and Pdx1 in the endoderm appears to control the generation of pancreatic precursors. This cascade may be used to benchmark in vitro differentiation of pancreatic precursor cells from embryonic stem cells, for cell therapy of diabetes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-beta del Hepatocito/metabolismo , Factor Nuclear 6 del Hepatocito/genética , Factor Nuclear 6 del Hepatocito/metabolismo , Páncreas/citología , Páncreas/embriología , Animales , Quimera , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Intrones/genética , Ratones , Regiones Promotoras Genéticas/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Mutations in the HNF1beta gene, encoding the dimeric POU-homeodomain transcription factor HNF1beta (TCF2 or vHNF1), cause various phenotypes including maturity onset diabetes of the young 5 (MODY5), and abnormalities in kidney, pancreas and genital tract development. To gain insight into the molecular mechanisms underlying these phenotypes and into the structure of HNF1beta, we functionally characterized eight disease-causing mutations predicted to produce protein truncations, amino acids substitutions or frameshift deletions in different domains of the protein. Truncated mutations, retaining the dimerization domain, displayed defective nuclear localization and weak dominant-negative activity when co-expressed with the wild-type protein. A frameshift mutation located within the C-terminal QSP-rich domain partially reduced transcriptional activity, whereas selective deletion of this domain abolished transactivation. All five missense mutations, which concern POU-specific and homeodomain residues, were correctly expressed and localized to the nucleus. Although having different effects on DNA-binding capacity, which ranged from complete loss to a mild reduction, these mutations exhibited a severe reduction in their transactivation capacity. The transcriptional impairment of those mutants, whose DNA-binding activity was weakly or not affected, correlated with the loss of association with one of the histone-acetyltransferases CBP or PCAF. In contrast to wild-type HNF1beta, whose transactivation potential depends on the synergistic action of CBP and PCAF, the activity of these mutants was not increased by the synergistic action of these two coactivators or by treatment with the specific histone-deacetylase inhibitor TSA. Our findings suggest that the complex syndrome associated with HNF1beta-MODY5 mutations arise from either defective DNA-binding or transactivation function through impaired coactivator recruitment.