RESUMEN
Inhibitory interneurons regulate cortical circuit activity, and their dysfunction has been implicated in autism spectrum disorder (ASD). 16p11.2 microdeletions are genetically linked to 1% of ASD cases. However, few studies investigate the effects of this microdeletion on interneuron development. Using ventral telencephalic organoids derived from human induced pluripotent stem cells, we have investigated the effect of this microdeletion on organoid size, progenitor proliferation and organisation into neural rosettes, ganglionic eminence marker expression at early developmental timepoints, and expression of the neuronal marker NEUN at later stages. At early stages, deletion organoids exhibited greater variations in size with concomitant increases in relative neural rosette area and the expression of the ventral telencephalic marker COUPTFII, with increased variability in these properties. Cell cycle analysis revealed an increase in total cell cycle length caused primarily by an elongated G1 phase, the duration of which also varied more than normal. At later stages, deletion organoids increased their NEUN expression. We propose that 16p11.2 microdeletions increase developmental variability and may contribute to ASD aetiology by lengthening the cell cycle of ventral progenitors, promoting premature differentiation into interneurons.
Asunto(s)
Trastorno del Espectro Autista , Células Madre Pluripotentes Inducidas , Humanos , Trastorno del Espectro Autista/metabolismo , Telencéfalo , Neuronas/metabolismo , Interneuronas/metabolismo , OrganoidesRESUMEN
The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6, which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size.
Asunto(s)
Ciclo Celular/fisiología , Corteza Cerebral/embriología , Quinasa 6 Dependiente de la Ciclina/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Neuroglía/metabolismo , Proteína Gli3 con Dedos de Zinc/metabolismo , Animales , Corteza Cerebral/citología , Quinasa 6 Dependiente de la Ciclina/genética , Femenino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuroglía/citología , Proteína Gli3 con Dedos de Zinc/genéticaRESUMEN
Dmrt5 (Dmrta2) and Dmrt3 are key regulators of cortical patterning and progenitor proliferation and differentiation. In this study, we show an altered apical to intermediate progenitor transition, with a delay in SP neurogenesis and premature birth of Ctip2+ cortical neurons in Dmrt5-/- mice. In addition to the cortical progenitors, DMRT5 protein appears present in postmitotic subplate (SP) and marginal zone neurons together with some migrating cortical neurons. We observed the altered split of preplate and the reduced SP and disturbed radial migration of cortical neurons into cortical plate in Dmrt5-/- brains and demonstrated an increase in the proportion of multipolar cells in primary neuronal cultures from Dmrt5-/- embryonic brains. Dmrt5 affects cortical development with specific time sensitivity that we described in two conditional mice with slightly different deletion time. We only observed a transient SP phenotype at E15.5, but not by E18.5 after early (Dmrt5lox/lox;Emx1Cre), but not late (Dmrt5lox/lox;NestinCre) deletion of Dmrt5. SP was less disturbed in Dmrt5lox/lox;Emx1Cre and Dmrt3-/- brains than in Dmrt5-/- and affects dorsomedial cortex more than lateral and caudal cortex. Our study demonstrates a novel function of Dmrt5 in the regulation of early SP formation and radial cortical neuron migration. SUMMARY STATEMENT: Our study demonstrates a novel function of Dmrt5 in regulating marginal zone and subplate formation and migration of cortical neurons to cortical plate.
Asunto(s)
Movimiento Celular/genética , Neocórtex/embriología , Neuronas/metabolismo , Factores de Transcripción/genética , Animales , Proliferación Celular/genética , Corteza Cerebral/citología , Corteza Cerebral/embriología , Embrión de Mamíferos , Ratones , Ratones Noqueados , Mitosis/genética , Neocórtex/citología , Neuronas/citología , Cultivo Primario de CélulasRESUMEN
Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.
Asunto(s)
Proteínas de Homeodominio/fisiología , Células-Madre Neurales/fisiología , Neuronas/fisiología , Telencéfalo/embriología , Factores de Transcripción/fisiología , Animales , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Telencéfalo/metabolismo , Factores de Transcripción/genéticaRESUMEN
A key step in the development of the cerebral cortex is a patterning process, which subdivides the telencephalon into several molecularly distinct domains and is critical for cortical arealization. This process is dependent on a complex network of interactions between signaling molecules of the Fgf and Wnt gene families and the Gli3 transcription factor gene, but a better knowledge of the molecular basis of the interplay between these factors is required to gain a deeper understanding of the genetic circuitry underlying telencephalic patterning. Using DNA-binding and reporter gene assays, we here investigate the possibility that Gli3 and these signaling molecules interact by directly regulating each other's expression. We show that Fgf signaling is required for Wnt8b enhancer activity in the cortical hem, whereas Wnt/ß-catenin signaling represses Fgf17 forebrain enhancer activity. In contrast, Fgf and Wnt/ß-catenin signaling cooperate to regulate Gli3 expression. Taken together, these findings indicate that mutual interactions between Gli3, Wnt8b, and Fgf17 are crucial elements of the balance between these factors thereby conferring robustness to the patterning process. Hence, our study provides a framework for understanding the genetic circuitry underlying telencephalic patterning and how defects in this process can affect the formation of cortical areas.
Asunto(s)
Factores de Crecimiento de Fibroblastos/fisiología , Proteínas del Tejido Nervioso/fisiología , Telencéfalo/fisiología , Proteínas Wnt/fisiología , Proteína Gli3 con Dedos de Zinc/fisiología , Animales , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Embarazo , Prosencéfalo/metabolismo , Prosencéfalo/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Telencéfalo/embriología , Telencéfalo/metabolismo , Tálamo/embriología , Tálamo/fisiología , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiología , Proteína Gli3 con Dedos de Zinc/genéticaRESUMEN
A unique population of cells, called "lot cells," circumscribes the path of the lateral olfactory tract (LOT) in the rodent brain and acts to restrict its position at the lateral margin of the telencephalon. Lot cells were believed to originate in the dorsal pallium (DP). We show that Lhx2 null mice that lack a DP show a significant increase in the number of mGluR1/lot cells in the piriform cortex, indicating a non-DP origin of these cells. Since lot cells present common developmental features with Cajal-Retzius (CR) cells, we analyzed Wnt3a- and Dbx1-reporter mouse lines and found that mGluR1/lot cells are not generated in the cortical hem, ventral pallium, or septum, the best characterized sources of CR cells. Finally, we identified a novel origin for the lot cells by combining in utero electroporation assays and histochemical characterization. We show that mGluR1/lot cells are specifically generated in the lateral thalamic eminence and that they express mitral cell markers, although a minority of them express ΔNp73 instead. We conclude that most mGluR1/lot cells are prospective mitral cells migrating to the accessory olfactory bulb (OB), whereas mGluR1+, ΔNp73+ cells are CR cells that migrate through the LOT to the piriform cortex and the OB.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Células Madre/fisiología , Tálamo/citología , Tálamo/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Embrión de Mamíferos , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Embarazo , Receptores de Glutamato Metabotrópico/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismoRESUMEN
Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3(-/-) corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3(-/-) VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development.
Asunto(s)
Tipificación del Cuerpo/genética , Corteza Cerebral/metabolismo , Proteínas de Unión al ADN/genética , Telencéfalo/metabolismo , Tálamo/metabolismo , Factores de Transcripción/genética , Animales , Embrión de Mamíferos , Homocigoto , Inmunohistoquímica , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Mutación , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas , Neuronas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Factores de Transcripción del Factor Regulador X , Telencéfalo/embriología , Telencéfalo/patología , Tálamo/embriología , Tálamo/patología , Proteína Gli3 con Dedos de ZincRESUMEN
The corticothalamic and thalamocortical tracts play essential roles in the communication between the cortex and thalamus. During development, axons forming these tracts have to follow a complex path to reach their target areas. While much attention has been paid to the mechanisms regulating their passage through the ventral telencephalon, very little is known about how the developing cortex contributes to corticothalamic/thalamocortical tract formation. Gli3 encodes a zinc finger transcription factor widely expressed in telencephalic progenitors which has important roles in corticothalamic and thalamocortical pathfinding. Here, we conditionally inactivated Gli3 in dorsal telencephalic progenitors to determine its role in corticothalamic tract formation. In Emx1Cre;Gli3(fl/fl) mutants, only a few corticothalamic axons enter the striatum in a restricted dorsal domain. This restricted entry correlates with a medial expansion of the piriform cortex. Transplantation experiments showed that the expanded piriform cortex repels corticofugal axons. Moreover, expression of Sema5B, a chemorepellent for corticofugal axons produced by the piriform cortex, is similarly expanded. Finally, time course analysis revealed an expansion of the ventral pallial progenitor domain which gives rise to the piriform cortex. Hence, control of lateral cortical development by Gli3 at the progenitor level is crucial for corticothalamic pathfinding.
Asunto(s)
Axones/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Corteza Piriforme/embriología , Corteza Piriforme/fisiopatología , Tálamo/embriología , Tálamo/fisiopatología , Animales , Axones/patología , Cuerpo Estriado/embriología , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Inmunohistoquímica , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel/genética , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/embriología , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Corteza Piriforme/patología , Semaforinas/metabolismo , Tálamo/patología , Técnicas de Cultivo de Tejidos , Proteína Gli3 con Dedos de ZincRESUMEN
The corpus callosum (CC) represents the major forebrain commissure connecting the 2 cerebral hemispheres. Midline crossing of callosal axons is controlled by several glial and neuronal guideposts specifically located along the callosal path, but it remains unknown how these cells acquire their position. Here, we show that the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn) displays agenesis of the CC and mislocation of the glial and neuronal guidepost cells. Using transplantation experiments, we demonstrate that agenesis of the CC is primarily caused by midline defects. These defects originate during telencephalic patterning and involve an up-regulation of Slit2 expression and altered Fgf and Wnt/ß-catenin signaling. Mutations in sprouty1/2 which mimic the changes in these signaling pathways cause a disorganization of midline guideposts and CC agenesis. Moreover, a partial recovery of midline abnormalities in Pdn/Pdn;Slit2(-/-) embryos mutants confirms the functional importance of correct Slit2 expression levels for callosal development. Hence, Gli3 controlled restriction of Fgf and Wnt/ß-catenin signaling and of Slit2 expression is crucial for positioning midline guideposts and callosal development.
Asunto(s)
Cuerpo Calloso/crecimiento & desarrollo , Factores de Transcripción de Tipo Kruppel/fisiología , Proteínas del Tejido Nervioso/fisiología , Telencéfalo/crecimiento & desarrollo , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/fisiopatología , Animales , Encéfalo/crecimiento & desarrollo , Análisis por Conglomerados , Cuerpo Calloso/embriología , Femenino , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/fisiología , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Mutación/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Polidactilia/genética , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Telencéfalo/embriología , Regulación hacia Arriba/fisiología , Vía de Señalización Wnt/fisiología , Proteína Gli3 con Dedos de Zinc , beta Catenina/fisiologíaRESUMEN
The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.
Asunto(s)
Cuerpo Calloso , Proteínas de Unión al ADN , Factor 8 de Crecimiento de Fibroblastos , Factores de Transcripción de Tipo Kruppel , Proteínas del Tejido Nervioso , Neuronas , Factores de Transcripción , Animales , Axones/metabolismo , Axones/fisiología , Cuerpo Calloso/crecimiento & desarrollo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Mutantes , Morfogénesis/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteína Gli3 con Dedos de ZincRESUMEN
The corpus callosum (CC) is the largest commissure in the forebrain and mediates the transfer of sensory, motor and cognitive information between the cerebral hemispheres. During CC development, a number of strategically located glial and neuronal guidepost structures serve to guide callosal axons across the midline at the corticoseptal boundary (CSB). Correct positioning of these guideposts requires the Gli3 gene, mutations of which result in callosal defects in humans and mice. However, as Gli3 is widely expressed during critical stages of forebrain development, the precise temporal and spatial requirements for Gli3 function in callosal development remain unclear. Here, we used a conditional mouse mutant approach to inactivate Gli3 in specific regions of the developing telencephalon in order to delineate the domain(s) in which Gli3 is required for normal development of the corpus callosum. Inactivation of Gli3 in the septum or in the medial ganglionic eminence had no effect on CC formation, however Gli3 inactivation in the developing cerebral cortex led to the formation of a severely hypoplastic CC at E18.5 due to a severe disorganization of midline guideposts. Glial wedge cells translocate prematurely and Slit1/2 are ectopically expressed in the septum. These changes coincide with altered Fgf and Wnt/ß-catenin signalling during CSB formation. Collectively, these data demonstrate a crucial role for Gli3 in cortical progenitors to control CC formation and indicate how defects in CSB formation affect the positioning of callosal guidepost cells.
Asunto(s)
Cuerpo Calloso/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Movimiento Celular , Cruzamientos Genéticos , Femenino , Inmunohistoquímica/métodos , Hibridación in Situ , Masculino , Ratones , Mutación , Transducción de Señal , Factores de Tiempo , Transgenes , Proteína Gli3 con Dedos de ZincRESUMEN
The formation of a functional cortical circuitry requires the coordinated growth of cortical axons to their target areas. While the mechanisms guiding cortical axons to their targets have extensively been studied, very little is known about the processes which promote their growth in vivo. Gli3 encodes a zinc finger transcription factor which is expressed in cortical progenitor cells and has crucial roles in cortical development. Here, we characterize the Gli3 compound mutant Gli3(Xt/Pdn), which largely lacks Neurofilament(+) fibers in the rostral and intermediate neocortex. DiI labeling and Golli-τGFP immunofluorescence indicate that Gli3(Xt/Pdn) cortical neurons form short and stunted axons. Using transplantation experiments we demonstrate that this axon growth defect is primarily caused by a nonpermissive cortical environment. Furthermore, in Emx1Cre;Gli3(Pdn/fl) conditional mutants, which mimic the reduction of Gli3 expression in the dorsal telencephalon of Gli3(Xt/Pdn) embryos, the growth of cortical axons is not impaired, suggesting that Gli3 controls this process early in telencephalic development. In contrast to cortical plate neurons, Gli3(Xt/Pdn) embryos largely lack subplate (SP) neurons which normally pioneer cortical projections. Collectively, these findings show that Gli3 specifies a cortical environment permissive to the growth of cortical axons at the progenitor level by controlling the formation of SP neurons.
Asunto(s)
Axones/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Neocórtex/embriología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Animales , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos C57BL , Mutación , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/genética , Proteína Gli3 con Dedos de ZincRESUMEN
Regional patterning of the cerebral cortex is initiated by morphogens secreted by patterning centers that establish graded expression of transcription factors within cortical progenitors. Here, we show that Dmrt5 is expressed in cortical progenitors in a high-caudomedial to low-rostrolateral gradient. In its absence, the cortex is strongly reduced and exhibits severe abnormalities, including agenesis of the hippocampus and choroid plexus and defects in commissural and thalamocortical tracts. Loss of Dmrt5 results in decreased Wnt and Bmp in one of the major telencephalic patterning centers, the dorsomedial telencephalon, and in a reduction of Cajal-Retzius cells. Expression of the dorsal midline signaling center-dependent transcription factors is downregulated, including Emx2, which promotes caudomedial fates, while the rostral determinant Pax6, which is inhibited by midline signals, is upregulated. Consistently, Dmrt5(-/-) brains exhibit patterning defects with a dramatic reduction of the caudomedial cortex. Dmrt5 is increased upon the activation of Wnt signaling and downregulated in Gli3(xt/xt) mutants. We conclude that Dmrt5 is a novel Wnt-dependent transcription factor required for early cortical development and that it may regulate initial cortical patterning by promoting dorsal midline signaling center formation and thereby helping to establish the graded expression of the other transcription regulators of cortical identity.
Asunto(s)
Corteza Cerebral/embriología , Factores de Transcripción/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Corteza Cerebral/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción/genética , Proteínas Wnt/metabolismoRESUMEN
Early development of the hippocampus, which is essential for spatial memory and learning, is controlled by secreted signaling molecules of the Wnt gene family and by Wnt/ß-catenin signaling. Despite its importance, little is known, however, about Wnt-regulated genes during hippocampal development. Here, we used the Gli3 mutant mouse extra-toes (Xt(J)), in which Wnt gene expression in the forebrain is severely affected, as a tool in a microarray analyses to identify potential Wnt target genes. This approach revealed 53 candidate genes with restricted or graded expression patterns in the dorsomedial telencephalon. We identified conserved Tcf/Lef-binding sites in telencephalon-specific enhancers of several of these genes, including Dmrt3, Gli3, Nfia, and Wnt8b. Binding of Lef1 to these sites was confirmed using electrophoretic mobility shift assays. Mutations in these Tcf/Lef-binding sites disrupted or reduced enhancer activity in vivo. Moreover, ectopic activation of Wnt/ß-catenin signaling in an ex vivo explant system led to increased telencephalic expression of these genes. Finally, conditional inactivation of Gli3 results in defective hippocampal growth. Collectively, these data strongly suggest that we have identified a set of direct Wnt target genes in the developing hippocampus and provide inside into the genetic hierarchy underlying Wnt-regulated hippocampal development.
Asunto(s)
Hipocampo/embriología , Hipocampo/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Distribución Tisular , Factores de Transcripción/metabolismo , Proteína Gli3 con Dedos de ZincRESUMEN
Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.
Asunto(s)
Cilios , Proteínas Hedgehog , Monoéster Fosfórico Hidrolasas , Telencéfalo , Cilios/enzimología , Cilios/genética , Cilios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Organoides/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Telencéfalo/enzimología , Telencéfalo/metabolismoRESUMEN
Previous studies have defined a requirement for Sonic hedgehog (Shh) signaling in patterning the ventral telencephalon, a major source of the neuronal diversity found in the mature telencephalon. The zinc finger transcription factor Gli3 is a critical component of the Shh signaling pathway and its loss causes major defects in telencephalic development. Gli3 is expressed in a graded manner along the dorsoventral axis of the telencephalon but it is unknown whether Gli3 expression levels are important for dorsoventral telencephalic patterning. To address this, we used the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn). We show that in Pdn/Pdn embryos, the telencephalic expression of Gli3 remains graded, but Gli3 mRNA and protein levels are reduced, resulting in an upregulation of Shh expression and signaling. These changes mainly affect the development of the lateral ganglionic eminence (LGE), with some disorganization of the medial ganglionic eminence mantle zone. The pallial/subpallial boundary is shifted dorsally and the production of postmitotic neurons is reduced. Moreover, LGE pioneer neurons that guide corticofugal axons into the LGE do not form properly, delaying the entry of corticofugal axons into the ventral telencephalon. Pdn/Pdn mutants also show severe pathfinding defects of thalamocortical axons in the ventral telencephalon. Transplantation experiments demonstrate that the intrinsic ability of the Pdn ventral telencephalon to guide thalamocortical axons is compromised. We conclude that correct Gli3 levels are particularly important for the LGE's growth, patterning, and development of axon guidance capabilities.
Asunto(s)
Axones/metabolismo , Tipificación del Cuerpo/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Telencéfalo/crecimiento & desarrollo , Animales , Western Blotting , Inmunohistoquímica , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telencéfalo/metabolismo , Proteína Gli3 con Dedos de ZincRESUMEN
Wnt signalling proteins regulate many aspects of animal development. We have investigated the function of mouse Wnt8b during forebrain development. Wnt8b is expressed in a highly restricted pattern including the prospective hippocampus and hypothalamus. Mutant mice lacking Wnt8b are viable and healthy. The size and morphology of the hippocampus appeared normal in mutant embryos and adults, and we found no evidence of hypothalamic defects in mutants. Wnt8b is also expressed in the neurogenic region of the adult dentate gyrus, however, cell proliferation was unchanged in Wnt8b(-/-) mutants. Mutant embryos did, however, display altered levels of expression of other Wnt genes normally expressed in forebrain. The spatial expression patterns of other Wnt genes and the overall level of canonical Wnt activity were indistinguishable from wild-types. Thus, loss of Wnt8b does not give rise to an overt morphological phenotype, but does affect expression levels of other Wnts in developing forebrain.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/embriología , Morfogénesis/genética , Transducción de Señal/fisiología , Proteínas Wnt/deficiencia , Animales , Southern Blotting , Bromodesoxiuridina , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Hipocampo/anatomía & histología , Hipocampo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Morfogénesis/fisiología , Tamaño de los Órganos , Transducción de Señal/genéticaRESUMEN
The primary cilium, a microtubule based organelle protruding from the cell surface and acting as an antenna in multiple signaling pathways, takes center stage in the formation of the cerebral cortex, the part of the brain that performs highly complex neural tasks and confers humans with their unique cognitive capabilities. These activities require dozens of different types of neurons that are interconnected in complex ways. Due to this complexity, corticogenesis has been regarded as one of the most complex developmental processes and cortical malformations underlie a number of neurodevelopmental disorders such as intellectual disability, autism spectrum disorders, and epilepsy. Cortical development involves several steps controlled by cell-cell signaling. In fact, recent findings have implicated cilia in diverse processes such as neurogenesis, neuronal migration, axon pathfinding, and circuit formation in the developing cortex. Here, we will review recent advances on the multiple roles of cilia during cortex formation and will discuss the implications for a better understanding of the disease mechanisms underlying neurodevelopmental disorders.
RESUMEN
The hypothalamus is a region of the diencephalon with particularly complex patterning. Sonic hedgehog (Shh), encoding a protein with key developmental roles, shows a peculiar and dynamic diencephalic expression pattern. Here, we use transgenic strategies and in vitro experiments to test the hypothesis that Shh expressed in the diencephalic neuroepithelium (neural Shh) coordinates tissue growth and patterning in the hypothalamus. Our results show that neural Shh coordinates anteroposterior and dorsoventral patterning in the hypothalamus and in the diencephalon-telencephalon junction. Neural Shh also coordinates mediolateral hypothalamic patterning, since it is necessary for the lateral hypothalamus to attain proper size and is required for the specification of hypocretin/orexin cells. Finally, neural Shh is necessary to maintain expression of differentiation markers including survival factor Foxb1.
Asunto(s)
Tipificación del Cuerpo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/metabolismo , Hipotálamo/citología , Células Neuroepiteliales/fisiología , Transducción de Señal/fisiología , Factores de Edad , Análisis de Varianza , Animales , Apoptosis/genética , Apoptosis/fisiología , Tipificación del Cuerpo/genética , Bromodesoxiuridina/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular , Embrión de Mamíferos , Exones/genética , Factores de Transcripción Forkhead/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Hedgehog/genética , Hipotálamo/embriología , Etiquetado Corte-Fin in Situ/métodos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/embriología , Transducción de Señal/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The role of the mitochondrial calcium uniporter (MCU) gene (Mcu) in cellular energy homeostasis and generation of electrical brain rhythms is widely unknown. We investigated this issue in mice and rats using Mcu-knockout and -knockdown strategies in vivo and in situ and determined the effects of these genetic manipulations on hippocampal gamma oscillations (30-70 Hz) and sharp wave-ripples. These physiological network states require precise neurotransmission between pyramidal cells and inhibitory interneurons, support spike-timing and synaptic plasticity and are associated with perception, attention and memory. Absence of the MCU resulted in (i) gamma oscillations with decreased power (by >40%) and lower synchrony, including less precise neural action potential generation ('spiking'), (ii) sharp waves with decreased incidence (by about 22%) and decreased fast ripple frequency (by about 3%) and (iii) lack of activity-dependent pyruvate dehydrogenase dephosphorylation. However, compensatory adaptation in gene expression related to mitochondrial function and glucose metabolism was not detected. These data suggest that the neuronal MCU is crucial for the generation of network rhythms, most likely by influences on oxidative phosphorylation and perhaps by controlling cytoplasmic Ca2+ homeostasis. This work contributes to an increased understanding of mitochondrial Ca2+ uptake in cortical information processing underlying cognition and behaviour.