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1.
PLoS Biol ; 21(8): e3002212, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37540708

RESUMO

The mature mammalian cortex is composed of 6 architecturally and functionally distinct layers. Two key steps in the assembly of this layered structure are the initial establishment of the glial scaffold and the subsequent migration of postmitotic neurons to their final position. These processes involve the precise and timely regulation of adhesion and detachment of neural cells from their substrates. Although much is known about the roles of adhesive substrates during neuronal migration and the formation of the glial scaffold, less is understood about how these signals are interpreted and integrated within these neural cells. Here, we provide in vivo evidence that Cas proteins, a family of cytoplasmic adaptors, serve a functional and redundant role during cortical lamination. Cas triple conditional knock-out (Cas TcKO) mice display severe cortical phenotypes that feature cobblestone malformations. Molecular epistasis and genetic experiments suggest that Cas proteins act downstream of transmembrane Dystroglycan and ß1-Integrin in a radial glial cell-autonomous manner. Overall, these data establish a new and essential role for Cas adaptor proteins during the formation of cortical circuits and reveal a signaling axis controlling cortical scaffold formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Distroglicanas , Integrina beta1 , Neuroglia , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Córtex Cerebral/metabolismo , Distroglicanas/genética , Distroglicanas/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Neuroglia/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia
2.
Heliyon ; 9(4): e15282, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37089315

RESUMO

Integrin Adhesion Complexes (IACs) serve as links between the cytoskeleton and extracellular environment, acting as mechanosensing and signaling hubs. As such, IACs participate in many aspects of cellular motility, tissue morphogenesis, anchorage-dependent growth and cell survival. Focal Adhesion Kinase (FAK) has emerged as a critical organizer of IAC signaling events due to its early recruitment and diverse substrates, and thus has become a genetic and therapeutic target. Here we present the design and characterization of simple, reversible, and scalable Bimolecular Complementation sensors to monitor FAK phosphorylation in living cells. These probes provide novel means to quantify IAC signaling, expanding on the currently available toolkit for interrogating FAK phosphorylation during diverse cellular processes.

3.
Cell Rep Methods ; 2(8): 100276, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-36046623

RESUMO

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.


Assuntos
Astrócitos , Encefalopatias , Humanos , Astrócitos/metabolismo , Encéfalo/metabolismo , Estudos Longitudinais , Encefalopatias/metabolismo , Inflamação/genética
4.
J Cell Sci ; 134(20)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34515305

RESUMO

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here, we build on previous knowledge to develop a simple adeno-associated virus (AAV)-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This expression by Boolean exclusion (ExBoX) system encodes for a gene of interest that is turned on by a particular recombinase (Cre or FlpO) and turned off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems.


Assuntos
Neurônios , Recombinases , Animais , Expressão Gênica , Camundongos , Recombinases/genética
5.
Sci Rep ; 8(1): 5996, 2018 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-29662228

RESUMO

Development of complex neural circuits like the peripheral somatosensory system requires intricate mechanisms to ensure axons make proper connections. While much is known about ligand-receptor pairs required for dorsal root ganglion (DRG) axon guidance, very little is known about the cytoplasmic effectors that mediate cellular responses triggered by these guidance cues. Here we show that members of the Cas family of cytoplasmic signaling adaptors are highly phosphorylated in central projections of the DRG as they enter the spinal cord. Furthermore, we provide genetic evidence that Cas proteins regulate fasciculation of DRG sensory projections. These data establish an evolutionarily conserved requirement for Cas adaptor proteins during peripheral nervous system axon pathfinding. They also provide insight into the interplay between axonal fasciculation and adhesion to the substrate.


Assuntos
Fasciculação Axônica , Proteína Substrato Associada a Crk/metabolismo , Gânglios Espinais/crescimento & desenvolvimento , Animais , Proteína Substrato Associada a Crk/análise , Proteína Substrato Associada a Crk/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fosforilação , RNA Mensageiro/análise , RNA Mensageiro/genética , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura
6.
Sci Rep ; 8(1): 680, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330522

RESUMO

During mammalian cerebellar development, postnatal granule cell progenitors proliferate in the outer part of the External Granule Layer (EGL). Postmitotic granule progenitors migrate tangentially in the inner EGL before switching to migrate radially inward, past the Purkinje cell layer, to achieve their final position in the mature Granule Cell Layer (GCL). Here, we show that the RacGAP ß-chimaerin is expressed by a small population of late-born, premigratory granule cells. ß-chimaerin deficiency causes a subset of granule cells to become arrested in the EGL, where they differentiate and form ectopic neuronal clusters. These clusters of granule cells are able to recruit aberrantly projecting mossy fibers. Collectively, these data suggest a role for ß-chimaerin as an intracellular mediator of Cerebellar Granule Cell radial migration.


Assuntos
Cerebelo/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Movimento Celular , Proliferação de Células , Cerebelo/química , Cerebelo/citologia , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Neurônios/metabolismo
7.
Annu Rev Cell Dev Biol ; 31: 779-805, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26436703

RESUMO

The assembly of functional neural circuits requires the combined action of progressive and regressive events. Regressive events encompass a variety of inhibitory developmental processes, including axon and dendrite pruning, which facilitate the removal of exuberant neuronal connections. Most axon pruning involves the removal of axons that had already made synaptic connections; thus, axon pruning is tightly associated with synapse elimination. In many instances, these developmental processes are regulated by the interplay between neurons and glial cells that act instructively during neural remodeling. Owing to the importance of axon and dendritic pruning, these remodeling events require precise spatial and temporal control, and this is achieved by a range of distinct molecular mechanisms. Disruption of these mechanisms results in abnormal pruning, which has been linked to brain dysfunction. Therefore, understanding the mechanisms of axon and dendritic pruning will be instrumental in advancing our knowledge of neural disease and mental disorders.


Assuntos
Axônios/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Animais , Humanos , Neuroglia/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia
8.
Neuron ; 81(4): 779-86, 2014 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-24559672

RESUMO

Stratification of retinal neuronal cell bodies and lamination of their processes provide a scaffold upon which neural circuits can be built. However, the molecular mechanisms that direct retinal ganglion cells (RGCs) to resolve into a single-cell retinal ganglion cell layer (GCL) are not well understood. The extracellular matrix protein laminin conveys spatial information that instructs the migration, process outgrowth, and reorganization of GCL cells. Here, we show that the ß1-Integrin laminin receptor is required for RGC positioning and reorganization into a single-cell GCL layer. ß1-Integrin signaling within migrating GCL cells requires Cas signaling-adaptor proteins, and in the absence of ß1-Integrin or Cas function retinal neurons form ectopic cell clusters beyond the inner-limiting membrane (ILM), phenocopying laminin mutants. These data reveal an essential role for Cas adaptor proteins in ß1-Integrin-mediated signaling events critical for the formation of the single-cell GCL in the mammalian retina.


Assuntos
Proteína Substrato Associada a Crk/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/fisiologia , Animais , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo
9.
Cell ; 149(7): 1594-606, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22726444

RESUMO

Axon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) ß2-Chimaerin (ß2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds ß2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. ß2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/ß2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling.


Assuntos
Axônios/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas de Neoplasias/metabolismo , Neuropeptídeos/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Giro Denteado/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses , Proteínas rac1 de Ligação ao GTP
10.
Dev Biol ; 305(1): 52-62, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17321515

RESUMO

The partitioning of the ventral neural tube into five distinct neuronal progenitor domains is dependent on the morphogenic action of the secreted protein Sonic hedgehog (Shh). The prevailing model stipulates that Class I genes are repressed and Class II genes are activated by high levels of Shh signaling and that sharp progenitor domain boundaries are established by the mutual repression of complementary pairs of Class I and Class II transcription factors. While core elements of this model are supported by experimental evidence, a number of issues remain unresolved. Foremost of these is a more thorough understanding of the mechanism by which Class I genes are regulated. In this study, we describe the consequences of Shh misexpression on Class I and Class II gene expression in the hindbrain of ShhP1 embryos. We observed that an ectopic source of Shh in the otic vesicle of ShhP1 embryos ventralized the adjacent hindbrain by inducing, rather than repressing, the expression of several Class I genes (Pax6, Dbx1, Dbx2). The Shh dependent activation of Class I genes was mediated, in part, by Gli2. These results bear significance on the model of ventral neural tube patterning as they suggest a dual role for Shh in the regulation of Class I genes, whereby low levels of Shh signaling initiate Class I gene transcription, while higher levels restrict the domains of Class I gene expression to intermediate positions of the neural tube through the activation of Class II transcriptional regulators.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Rombencéfalo/embriologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Proteína Gli2 com Dedos de Zinco
11.
Genes Dev ; 19(13): 1612-23, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15961523

RESUMO

The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types.


Assuntos
Orelha Interna/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Morfogênese , Transativadores/fisiologia , Animais , Sequência de Bases , Proteínas do Citoesqueleto/metabolismo , Primers do DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Transativadores/metabolismo , Proteínas Wnt , Proteína Wnt1 , beta Catenina
12.
J Cell Biol ; 169(2): 309-20, 2005 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-15837799

RESUMO

Canonical Wnt signaling instructively promotes sensory neurogenesis in early neural crest stem cells (eNCSCs) (Lee, H.Y., M. Kleber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler, and L. Sommer. 2004. Science. 303:1020-1023). However, during normal development Wnt signaling induces a sensory fate only in a subpopulation of eNCSCs while other cells maintain their stem cell features, despite the presence of Wnt activity. Hence, factors counteracting Wnt signaling must exist. Here, we show that bone morphogenic protein (BMP) signaling antagonizes the sensory fate-inducing activity of Wnt/beta-catenin. Intriguingly, Wnt and BMP act synergistically to suppress differentiation and to maintain NCSC marker expression and multipotency. Similar to NCSCs in vivo, NCSCs maintained in culture alter their responsiveness to instructive growth factors with time. Thus, stem cell development is regulated by combinatorial growth factor activities that interact with changing cell-intrinsic cues.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Crista Neural/embriologia , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Camundongos , Crista Neural/citologia , Células-Tronco Pluripotentes/citologia , Ratos , Transativadores/metabolismo , Proteínas Wnt , beta Catenina
13.
Genes Dev ; 16(18): 2365-78, 2002 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12231626

RESUMO

Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh(-/-) embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia failed to develop in the absence of Shh. The origin of the inner ear defects in Shh(-/-) embryos could be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. We further show that several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, our data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh.


Assuntos
Cóclea/embriologia , Transativadores/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteínas de Ligação a DNA/genética , Orelha Interna/anormalidades , Orelha Interna/embriologia , Gânglios/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Fator de Transcrição PAX2 , Transdução de Sinais , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/genética , Tretinoína/farmacologia
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