RESUMO
Appropriate growth and synaptic integration of GABAergic inhibitory interneurons are essential for functional neural circuits in the brain. Here, we demonstrate that disruption of primary cilia function following the selective loss of ciliary GTPase Arl13b in interneurons impairs interneuronal morphology and synaptic connectivity, leading to altered excitatory/inhibitory activity balance. The altered morphology and connectivity of cilia mutant interneurons and the functional deficits are rescued by either chemogenetic activation of ciliary G-protein-coupled receptor (GPCR) signaling or the selective induction of Sstr3, a ciliary GPCR, in Arl13b-deficient cilia. Our results thus define a specific requirement for primary cilia-mediated GPCR signaling in interneuronal connectivity and inhibitory circuit formation.
Assuntos
Interneurônios/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Potenciais Sinápticos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Células Cultivadas , Cílios/metabolismo , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Neurogênese , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Sinapses/fisiologiaRESUMO
Primary cilia are essential conveyors of signals underlying major cell functions. Cerebral cortical progenitors and neurons have a primary cilium. The significance of cilia function for brain development and function is evident in the plethora of developmental brain disorders associated with human ciliopathies. Nevertheless, the role of primary cilia function in corticogenesis remains largely unknown. Here we delineate the functions of primary cilia in the construction of cerebral cortex and their relevance to ciliopathies, using an shRNA library targeting ciliopathy genes known to cause brain disorders, but whose roles in brain development are unclear. We used the library to query how ciliopathy genes affect distinct stages of mouse cortical development, in particular neural progenitor development, neuronal migration, neuronal differentiation and early neuronal connectivity. Our results define the developmental functions of ciliopathy genes and delineate disrupted developmental events that are integrally related to the emergence of brain abnormalities in ciliopathies.
Assuntos
Encefalopatias/genética , Córtex Cerebral/embriologia , Cílios/genética , Animais , Feminino , Biblioteca Gênica , Humanos , Camundongos Endogâmicos C57BL , Neurogênese , Gravidez , RNA Interferente PequenoRESUMO
The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Cílios/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Neuroglia/ultraestrutura , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Anormalidades Múltiplas , Animais , Axonema/ultraestrutura , Divisão Celular , Polaridade Celular , Doenças Cerebelares/enzimologia , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Cerebelo/anormalidades , Córtex Cerebral/anormalidades , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Ventrículos Cerebrais/anormalidades , Cílios/fisiologia , Epitélio/ultraestrutura , Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Humanos , Doenças Renais Císticas/enzimologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/ultraestrutura , Neurogênese/genética , Neuroglia/fisiologia , Retina/anormalidades , Retina/enzimologia , Retina/patologia , Telencéfalo/embriologia , Telencéfalo/ultraestruturaRESUMO
Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia, we show that migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron's migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may in part underlie the neurological defects in Joubert syndrome patients.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Cílios/fisiologia , Interneurônios/fisiologia , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Anormalidades Múltiplas , Animais , Movimento Celular/fisiologia , Doenças Cerebelares/etiologia , Doenças Cerebelares/patologia , Doenças Cerebelares/fisiopatologia , Cerebelo/anormalidades , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Anormalidades do Olho/etiologia , Anormalidades do Olho/patologia , Anormalidades do Olho/fisiopatologia , Humanos , Doenças Renais Císticas/etiologia , Doenças Renais Císticas/patologia , Doenças Renais Císticas/fisiopatologia , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Retina/anormalidades , Retina/patologia , Retina/fisiopatologiaRESUMO
The emergence of functional neuronal connectivity in the developing cerebral cortex depends on 1) neural progenitor differentiation, which leads to the generation of appropriate number and types of neurons, and 2) neuronal migration, which enables the appropriate positioning of neurons so that the correct patterns of functional synaptic connectivity between neurons can emerge. In this review, we discuss 1) currently available methods to study neural progenitor development and differentiation in the developing cerebral cortex and emerging technologies in this regard, 2) assays to study the migration of descendents of progenitors (i.e., neurons) in vitro and in vivo, and 3) the use of these assays to probe the molecular control of these events in the developing brain and evaluation of gene functions disrupted in human neurodevelopmental disorders.
Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/fisiologia , Neurônios/fisiologia , Organogênese/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Humanos , Neurogênese/fisiologia , Neurônios/citologia , Células-Tronco/citologiaRESUMO
Establishment of polarity is an essential process during proliferation, migration and differentiation in neurons, and the signaling pathways leading to polarization of the cytoskeleton are topics of intense research. One key marker for cell polarity is the centrosome, also known as the microtubule-organizing center. Recent discoveries have shown that the position of the centrosome is precisely regulated during neurogenesis, migration and differentiation, leading to the segregation of cell fate factors, efficient nucleokinesis and directed neurite outgrowth, respectively. Here, we review recent advances in the understanding of this interesting organelle and propose a model whereby centrosome position, determined by extracellular factors, directs multiple aspects of neuronal development.
Assuntos
Diferenciação Celular/fisiologia , Centrossomo/fisiologia , Neurônios/fisiologia , Fuso Acromático/fisiologia , Animais , Movimento Celular/fisiologia , Humanos , Neurônios/citologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
In comparison with other migratory cells, neurons exhibit a unique, highly polarized morphology and a distinctive pattern of movement. This migration consists of a repeating of three distinct phases: neurite outgrowth, movement of the centrosome into the leading process, and translocation of the nucleus towards the centrosome. The direction of movement is under the control of extracellular guidance cues, but mechanisms by which these determine neuronal polarity, centrosome position, and neuronal movement are not well understood. We found that in primary olfactory bulb neuronal precursors, Slit-mediated repolarization consisted of growth of a new process from the previous trailing edge, then reorientation of the centrosome followed by nuclear translocation in the reverse direction. Inhibition of cell polarity factors GSK3beta or PKCzeta resulted in impaired centrosome reorientation and process stabilization. Our findings suggest that activation of cell polarity signaling and positioning of the centrosome ahead of the nucleus are important steps in repolarization in response to guidance cues.
Assuntos
Centrossomo/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicoproteínas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Polaridade Celular/fisiologia , Sinais (Psicologia) , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurônios/citologia , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Bulbo Olfatório/metabolismo , Técnicas de Cultura de Órgãos , Células-Tronco/citologiaRESUMO
The ability of the mature mammalian nervous system to continually produce neuronal precursors is of considerable importance, as manipulation of this process might one day permit the replacement of cells lost as a result of injury or disease. In mammals, the anterior subventricular zone (SVZa) region is one of the primary sites of adult neurogenesis. Here we show that doublecortin (DCX), a widely used marker for newly generated neurons, when deleted in mice results in a severe morphological defect in the rostral migratory stream and delayed neuronal migration that is independent of direction or responsiveness to Slit chemorepulsion. DCX is required for nuclear translocation and maintenance of bipolar morphology during migration of these cells. Our data identifies a critical function for DCX in the movement of newly generated neurons in the adult brain.
Assuntos
Movimento Celular/fisiologia , Proliferação de Células , Proteínas Associadas aos Microtúbulos/fisiologia , Neurônios/metabolismo , Neuropeptídeos/fisiologia , Prosencéfalo/metabolismo , Células-Tronco/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Diferenciação Celular/genética , Forma Celular/genética , Quimiotaxia/genética , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurônios/citologia , Neuropeptídeos/genética , Fenótipo , Prosencéfalo/citologia , Transporte Proteico/genética , Células-Tronco/citologiaRESUMO
During brain development, neurons migrate great distances from proliferative zones to generate the cortical gray matter. A series of studies has identified genes that are critical for migration and targeting of neurons to specific brain regions. These genes encode three basic groups of proteins and produce three distinct phenotypes. The first group encodes cytoskeletal molecules and produces graded and dosage-dependent effects, with a significant amount of functional redundancy. This group also appears to play important roles during the initiation and ongoing progression of neuronal movement. The second group encodes signaling molecules for which homozygous mutations lead to an inverted cortex. In addition, this group is responsible for movement of neurons through anatomic boundaries to specific cortical layers. The third group encodes enzymatic regulators of glycosylation and appears to delineate where neuronal migration will arrest. There is significant cross-talk among these different groups of molecules, suggesting possible points of pathway convergence.
Assuntos
Movimento Celular , Córtex Cerebral/embriologia , Neurônios/fisiologia , Animais , Movimento Celular/genética , Córtex Cerebral/citologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Glicosilação , Mutação/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The centrosome plays diverse roles throughout the cellular mitotic cycle and in post-mitotic cells. Analysis of centrosome position and dynamics in living murine cells has been limited due to a lack of adequate reporters and currently requires either cell fixation/immunostaining or transfection with centrosome reporters. Here we describe the generation and characterization of a transgenic mouse line that constitutively expresses green fluorescent protein-labeled Centrin-2 (GFP-CETN2). The phenotype of the mouse is indistinguishable from wild-type and it displays a single pair of fluorescent centrioles in cells of every organ and time point examined. This model will be helpful for visualizing the centrosome in multiple experimental conditions.