RESUMO
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.
Assuntos
Ciclo Celular/fisiologia , Córtex Cerebral/embriologia , Quinase 6 Dependente de Ciclina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neuroglia/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Córtex Cerebral/citologia , Quinase 6 Dependente de Ciclina/genética , Feminino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Proteína Gli3 com Dedos de Zinco/genéticaRESUMO
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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Células-Tronco/fisiologia , Tálamo/citologia , Tálamo/metabolismo , Animais , Movimento Celular , Células Cultivadas , Embrião de Mamíferos , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Gravidez , Receptores de Glutamato Metabotrópico/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismoRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Telencéfalo/fisiologia , Proteínas Wnt/fisiologia , Proteína Gli3 com Dedos de Zinco/fisiologia , Animais , Feminino , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Gravidez , Prosencéfalo/metabolismo , Prosencéfalo/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Telencéfalo/embriologia , Telencéfalo/metabolismo , Tálamo/embriologia , Tálamo/fisiologia , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Proteína Gli3 com Dedos de Zinco/genéticaRESUMO
Formation of the lateral olfactory tract (LOT) and innervation of the piriform cortex represent fundamental steps to allow the transmission of olfactory information to the cerebral cortex. Several transcription factors, including the zinc finger transcription factor Gli3, influence LOT formation by controlling the development of mitral cells from which LOT axons emanate and/or by specifying the environment through which these axons navigate. Gli3 null and hypomorphic mutants display severe defects throughout the territory covered by the developing lateral olfactory tract, making it difficult to identify specific roles for Gli3 in its development. Here, we used Emx1Cre;Gli3fl/fl conditional mutants to investigate LOT formation and colonization of the olfactory cortex in embryos in which loss of Gli3 function is restricted to the dorsal telencephalon. These mutants form an olfactory bulb like structure which does not protrude from the telencephalic surface. Nevertheless, mitral cells are formed and their axons enter the piriform cortex though the LOT is shifted medially. Mitral axons also innervate a larger target area consistent with an enlargement of the piriform cortex and form aberrant projections into the deeper layers of the piriform cortex. No obvious differences were found in the expression patterns of key guidance cues. However, we found that an expansion of the piriform cortex temporally coincides with the arrival of LOT axons, suggesting that Gli3 affects LOT positioning and target area innervation through controlling the development of the piriform cortex.
Assuntos
Córtex Cerebral/metabolismo , Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Bulbo Olfatório/embriologia , Bulbo Olfatório/metabolismo , Organogênese/genética , Animais , Axônios/metabolismo , Feminino , Camundongos , Camundongos Knockout , Mutação , Córtex Piriforme/metabolismo , Telencéfalo/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
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.
Assuntos
Padronização Corporal/genética , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/genética , Telencéfalo/metabolismo , Tálamo/metabolismo , Fatores de Transcrição/genética , Animais , Embrião de Mamíferos , Homozigoto , Imuno-Histoquímica , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fatores de Transcrição de Fator Regulador X , Telencéfalo/embriologia , Telencéfalo/patologia , Tálamo/embriologia , Tálamo/patologia , Proteína Gli3 com Dedos de ZincoRESUMO
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.
Assuntos
Axônios/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Córtex Piriforme/embriologia , Córtex Piriforme/fisiopatologia , Tálamo/embriologia , Tálamo/fisiopatologia , Animais , Axônios/patologia , Corpo Estriado/embriologia , Corpo Estriado/patologia , Corpo Estriado/fisiopatologia , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Vias Neurais/embriologia , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Córtex Piriforme/patologia , Semaforinas/metabolismo , Tálamo/patologia , Técnicas de Cultura de Tecidos , Proteína Gli3 com Dedos de ZincoRESUMO
Lizards are soil surface animals that represent an important link between invertebrates and higher predators. Being part of wild fauna, they can be affected by contamination from anthropic activities and in particular, pesticides and chemical substances of various nature that reach the soil surface directly or through fall out. Among these substances, heavy metals such as cadmium may exert particularly marked toxic effect on both adult and embryos. In lizards, recent studies show that cadmium may cause developmental defects, including alteration of eye development, with appearance of unilateral microphthalmia and retinal folding. In the present study, the effects of cadmium incubation on retinal development were investigated demonstrating that cadmium interferes with cell cycle regulation by increasing proliferation. An increased expression of Otx2 and Pax6 genes, markers of retinal differentiation, was also found. However, the cellular localization of Pax6 and Otx2 transcripts did not change in treated embryos: in the early stages of retinogenesis, the two genes were expressed in all retinal cells; in the differentiated retina, Otx2 remained in the cellular bodies of retinal cells forming the nuclear and the ganglion layers, whereas Pax6 was expressed only in the cells of the inner nuclear and the ganglion layers. Data suggest that the increased expression of Pax6 and Otx2 could be ascribed to the hyperproliferation of retinal cells rather than to an effective gene overexpression.
Assuntos
Cádmio/toxicidade , Retina/crescimento & desenvolvimento , Poluentes do Solo/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lagartos/embriologia , Neurônios/efeitos dos fármacos , RNA Mensageiro/biossíntese , Retina/efeitos dos fármacos , Retina/embriologiaRESUMO
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.
Assuntos
Corpo Caloso/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Telencéfalo/crescimento & desenvolvimento , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/fisiopatologia , Animais , Encéfalo/crescimento & desenvolvimento , Análise por Conglomerados , Corpo Caloso/embriologia , Feminino , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Mutação/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Polidactilia/genética , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Telencéfalo/embriologia , Regulação para Cima/fisiologia , Via de Sinalização Wnt/fisiologia , Proteína Gli3 com Dedos de Zinco , beta Catenina/fisiologiaRESUMO
The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6âº/⺠and Pax6â»/â» cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6's modulation of cortical progenitor cell cycles is regional and direct.
Assuntos
Padronização Corporal/genética , Córtex Cerebral/citologia , Quinase 6 Dependente de Ciclina/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/metabolismo , Células-Tronco/fisiologia , Animais , Bromodesoxiuridina , Ciclo Celular/genética , Proliferação de Células , Imunoprecipitação da Cromatina , Quinase 6 Dependente de Ciclina/genética , Embrião de Mamíferos , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição PAX6 , Fator de Transcrição PAX7/genética , Fatores de Transcrição Box Pareados/genética , Fosforilação , Ligação Proteica/genética , Proteínas Repressoras/genética , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Corpo Caloso/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Movimento Celular , Cruzamentos Genéticos , Feminino , Imuno-Histoquímica/métodos , Hibridização In Situ , Masculino , Camundongos , Mutação , Transdução de Sinais , Fatores de Tempo , Transgenes , Proteína Gli3 com Dedos de ZincoRESUMO
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.
Assuntos
Axônios/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Neocórtex/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Animais , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/genética , Proteína Gli3 com Dedos de ZincoRESUMO
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.
Assuntos
Corpo Caloso , Proteínas de Ligação a DNA , Fator 8 de Crescimento de Fibroblasto , Fatores de Transcrição Kruppel-Like , Proteínas do Tecido Nervoso , Neurônios , Fatores de Transcrição , Animais , Axônios/metabolismo , Axônios/fisiologia , Corpo Caloso/crescimento & desenvolvimento , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Mutantes , Morfogênese/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteína Gli3 com Dedos de ZincoRESUMO
Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors.
Assuntos
Axônios/fisiologia , Córtex Cerebral/embriologia , Prosencéfalo/embriologia , Tálamo/embriologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Diferenciação Celular/genética , Córtex Cerebral/metabolismo , Feminino , Deleção de Genes , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Vias Neurais/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Gravidez , Prosencéfalo/metabolismo , Tálamo/metabolismoRESUMO
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.
Assuntos
Hipocampo/embriologia , Hipocampo/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
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.
Assuntos
Axônios/metabolismo , Padronização Corporal/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Telencéfalo/crescimento & desenvolvimento , Animais , Western Blotting , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telencéfalo/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain.
Assuntos
Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Cílios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Córtex Cerebral/ultraestrutura , Cílios/ultraestrutura , Epêndima/metabolismo , Epêndima/ultraestrutura , Feminino , Fatores de Transcrição Kruppel-Like/genética , Ventrículos Laterais/anormalidades , Ventrículos Laterais/metabolismo , Ventrículos Laterais/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/ultraestrutura , Peptídeo Hidrolases/metabolismo , Prosencéfalo/anormalidades , Prosencéfalo/metabolismo , Prosencéfalo/ultraestrutura , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
The layered organization of the cerebral cortex develops in an inside-out pattern, a process which is controlled by the secreted protein reelin. Here we report on cortical lamination in the Gli3 hypomorphic mouse mutant Xt(J)/Pdn which lacks the cortical hem, a major source of reelin(+) Cajal Retzius cells in the cerebral cortex. Unlike other previously described mouse mutants with hem defects, cortical lamination is disturbed in Xt(J)/Pdn animals. Surprisingly, these layering defects occur in the presence of reelin(+) cells which are probably derived from an expanded Dbx1(+) progenitor pool in the mutant. However, while these reelin(+) neurons and also Calretinin(+) cells are initially evenly distributed over the cortical surface they form clusters later during development suggesting a novel role for Gli3 in maintaining the proper arrangement of these cells in the marginal zone. Moreover, the radial glial network is disturbed in the regions of these clusters. In addition, the differentiation of subplate cells is affected which serve as a framework for developing a properly laminated cortex.
Assuntos
Córtex Cerebral/anatomia & histologia , Córtex Cerebral/embriologia , Embrião de Mamíferos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteína Axina , Biomarcadores/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Proteína Reelina , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
During corticogenesis, the cerebral cortex develops a laminated structure which is essential for its function. Early born neurons of the preplate and its derivatives, the marginal zone (MZ) and the subplate (SP), serve as a framework during the cortical lamination process. Here, I report on defects in the generation and specification of these early born cortical neurons in extra-toes (Xt(J)) mice which are defective for the Gli3 zinc finger transcription factor. The Gli3 mutation dramatically disrupts early steps in the cortical lamination process. The MZ, SP and the cortical plate (CP) do not form layers but cortical neurons are arranged in clusters. These defects start to become evident at E12.5 when the cortex forms several protrusions and the ventricular zone becomes undulated. At this stage, cortical progenitor cells start to loose their apical/basal cell polarity correlating with an ectopic expression of Wnt7b in the ventricular zone. In addition, the cellular composition of the preplate is severely altered. Cajal-Retzius cells are reduced in numbers while early born Calretinin(+) neurons are overproduced. These results show that multiple aspects of corticogenesis including the organization of the venticular zone, the apical/basal cell polarity of cortical progenitors and the differentiation of early born cortical neurons are affected in the Gli3 mutant.
Assuntos
Diferenciação Celular , Córtex Cerebral/embriologia , Indução Embrionária , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Animais , Linhagem da Célula , Polaridade Celular , Córtex Cerebral/crescimento & desenvolvimento , Desenvolvimento Embrionário , Glicoproteínas/análise , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos , Morfogênese , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/análise , Células-Tronco , Proteínas Wnt/análise , Proteína Gli3 com Dedos de ZincoRESUMO
Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.
Assuntos
Orelha Interna/embriologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Vertebrados/embriologia , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Embrião de Galinha , Galinhas , Anormalidades Congênitas/genética , Fator 10 de Crescimento de Fibroblastos , Fator 3 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/deficiência , Fatores de Crescimento de Fibroblastos/genética , Deleção de Genes , Camundongos/embriologia , Camundongos Knockout , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Xenopus/embriologia , Peixe-Zebra/embriologiaRESUMO
Regionalization of the neural plate and the early neural tube is controlled by several signaling centers that direct the generation of molecularly distinct domains. In the developing telencephalon, the anterior neural ridge (ANR) and the roof and floor plate act as such organizing centers via the production of Fgfs, Bmps/Wnts, and Shh, respectively. It remains largely unknown, however, how the combination of these different signals is used to coordinate the generation of different telencephalic territories. In the present study, we report on telencephalic development in Pdn mutant mice, which carry an integration of a retrotransposon in the Gli3 locus. Homozygous mutant animals are characterized by a partial dorsal-to-ventral transformation of the telencephalon and by an increased size of the septum. On a molecular level, these alterations correlate with a reduction and/or loss of Bmp/Wnt expression and a concomitant expansion of Fgf8 transcription. Finally, we provide evidence that the ectopic activation of Fgf signaling in the dorsal telencephalon provides an explanation for the ventralization of the Gli3 mutant telencephalon as application of Fgf8-soaked beads to dorsal telencephalic explants led to the specific induction and repression of ventral marker and dorsal marker genes, respectively.