RESUMO
Semaphorins are a large family of axon guidance molecules that are known primarily as ligands for plexins and neuropilins. Although class-6 semaphorins are transmembrane proteins, they have been implicated as ligands in different aspects of neural development, including neural crest cell migration, axon guidance and cerebellar development. However, the specific spatial and temporal expression of semaphorin 6B (Sema6B) in chick commissural neurons suggested a receptor role in axon guidance at the spinal cord midline. Indeed, in the absence of Sema6B, post-crossing commissural axons lacked an instructive signal directing them rostrally along the contralateral floorplate border, resulting in stalling at the exit site or even caudal turns. Truncated Sema6B lacking the intracellular domain was unable to rescue the loss-of-function phenotype, confirming a receptor function of Sema6B. In support of this, we demonstrate that Sema6B binds to floorplate-derived plexin A2 (PlxnA2) for navigation at the midline, whereas a cis-interaction between PlxnA2 and Sema6B on pre-crossing commissural axons may regulate the responsiveness of axons to floorplate-derived cues.
Assuntos
Axônios/fisiologia , Movimento Celular/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Semaforinas/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Análise de Variância , Animais , Axônios/metabolismo , Embrião de Galinha , Imuno-Histoquímica , Interferência de RNARESUMO
Many genes have several, sometimes divergent functions during development. Therefore, timing of gene knockdown for functional analysis during development has to be done with precise temporal control, as loss of a gene's function at early stages prevents its analysis later in development. RNAi, in combination with the accessibility of chicken embryos, is an effective approach for temporally controlled analysis of gene function during neural development. Here, we describe novel plasmid vectors that contain cell type-specific promoters/enhancers to drive the expression of a fluorescent marker, followed directly by a miR30-RNAi transcript for gene silencing. These vectors allow for direct tracing of cells experiencing gene silencing by the bright fluorescence. The level of knockdown is sufficient to reproduce the expected pathfinding defects upon perturbation of genes with known axon guidance functions. Mixing different vectors prior to electroporation enables the simultaneous knockdown of multiple genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a fast and precise manner. The advancements of the in ovo RNAi technique that we describe will not only markedly enhance functional gene analysis in the chicken, but also could be adapted to other organisms in developmental studies.
Assuntos
Técnicas de Silenciamento de Genes , Neurônios/metabolismo , Interferência de RNA , Medula Espinal/embriologia , Animais , Embrião de Galinha , Vetores Genéticos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , MicroRNAs/metabolismo , Tubo Neural/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Axon navigation depends on the interactions between guidance molecules along the trajectory and specific receptors on the growth cone. However, our in vitro and in vivo studies on the role of Endoglycan demonstrate that in addition to specific guidance cue - receptor interactions, axon guidance depends on fine-tuning of cell-cell adhesion. Endoglycan, a sialomucin, plays a role in axon guidance in the central nervous system of chicken embryos, but it is neither an axon guidance cue nor a receptor. Rather, Endoglycan acts as a negative regulator of molecular interactions based on evidence from in vitro experiments demonstrating reduced adhesion of growth cones. In the absence of Endoglycan, commissural axons fail to properly navigate the midline of the spinal cord. Taken together, our in vivo and in vitro results support the hypothesis that Endoglycan acts as a negative regulator of cell-cell adhesion in commissural axon guidance.
Assuntos
Orientação de Axônios/fisiologia , Cones de Crescimento/fisiologia , Mucinas/farmacologia , Animais , Axônios/fisiologia , Adesão Celular/efeitos dos fármacos , Embrião de Galinha , Células HEK293 , Humanos , Interferência de RNA , Medula Espinal/embriologiaRESUMO
Repulsive guidance molecule (RGM) a is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein that has been implicated in chemorepulsive axon guidance. Although RGMa binds the transmembrane receptor Neogenin, the developmental events controlled by the RGMa-Neogenin interactions in vivo remain largely unknown. We have cloned full-length RGMa from Xenopus borealis for the first time and identified two homologous genes referred to as RGMa1 and RGMa2. Here we show RGMa1 overexpression at 2-cell-stage resulted in cell death, which lead to an early embryonic lethal phenotype of the embryos. Time-lapse photomicroscopy revealed that embryos began to show initial morphological defects from approximately 5 h post-fertilization (hpf) which was then followed by extensive blastomere cell death at approximately 11 hpf. This phenotype was rescued by simultaneous knock down of RGMa using translation blocking anti-sense morpholinos. Knock down of the RGMa1 receptor Neogenin in RGMa1 overexpressing embryos was also able to rescue the phenotype. Together these results indicated that RGMa1 was signalling through Neogenin to induce cell death in the early embryo. While previous studies have suggested that Neogenin is a dependence receptor that induces cell death in the absence of RGM, we have instead shown that Neogenin-RGM interactions induce cell death in the early embryo. The roles of RGMa1 and Neogenin appear to be context specific so that their co-ordinated and regulated expressions are essential for normal development of the vertebrate embryo.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Xenopus/genética , Animais , Morte Celular , Clonagem Molecular , Embrião não Mamífero/citologia , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Neogenin is a multifunctional transmembrane receptor belonging to the immunoglobulin superfamily. It displays identical secondary structure to deleted in colorectal cancer (DCC), a netrin receptor that is involved in axon guidance and cell survival. Like DCC, neogenin is able to transduce signals elicited by netrin. These neogenin-netrin interactions have been implicated in tissue morphogenesis, angiogenesis, myoblast differentiation and most recently in axon guidance. Neogenin is also a receptor for repulsive guidance molecule, a glycosylphosphatidylinositol-linked protein involved in neuronal differentiation, apoptosis and repulsive axon guidance. Numerous studies have been started to elucidate the in vivo functions of neogenin, and its role in multiple aspects of development and homeostasis.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Animais , Axônios/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Ligação Proteica , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
To maintain tissue integrity during epithelial morphogenesis, adherens junctions (AJs) must resist the mechanical stresses exerted by dynamic tissue movements. Junctional stability is dependent on actomyosin contractility within the actin ring. Here we describe a novel function for the axon guidance receptor, Neogenin, as a key component of the actin nucleation machinery governing junctional stability. Loss of Neogenin perturbs AJs and attenuates junctional tension. Neogenin promotes actin nucleation at AJs by recruiting the Wave regulatory complex (WRC) and Arp2/3. A direct interaction between the Neogenin WIRS domain and the WRC is crucial for the spatially restricted recruitment of the WRC to the junction. Thus, we provide the first example of a functional WIRS-WRC interaction in epithelia. We further show that Neogenin regulates cadherin recycling at the AJ. In summary, we identify Neogenin as a pivotal component of the AJ, where it influences both cadherin dynamics and junctional tension.
Assuntos
Junções Aderentes/metabolismo , Proteínas de Membrana/fisiologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Actinas/fisiologia , Células CACO-2 , Caderinas/análise , Caderinas/genética , Caderinas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/fisiologia , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Upon reaching their intermediate target, the floorplate, commissural axons acquire responsiveness to repulsive guidance cues, allowing the axons to exit the midline and adopt a contralateral, longitudinal trajectory. The molecular mechanisms that regulate this switch from attraction to repulsion remain poorly defined. Here, we show that the heparan sulfate proteoglycan Glypican1 (GPC1) is required as a coreceptor for the Shh-dependent induction of Hedgehog-interacting protein (Hhip) in commissural neurons. In turn, Hhip is required for postcrossing axons to respond to a repulsive anteroposterior Shh gradient. Thus, Shh is a cue with dual function. In precrossing axons it acts as an attractive guidance molecule in a transcription-independent manner. At the same time, Shh binds to GPC1 to induce the expression of its own receptor, Hhip, which mediates the repulsive response of postcrossing axons to Shh. Our study characterizes a molecular mechanism by which navigating axons switch their responsiveness at intermediate targets.
Assuntos
Axônios/fisiologia , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glipicanas/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal/genética , Células COS , Proteínas de Transporte/metabolismo , Embrião de Galinha , Chlorocebus aethiops , Lateralidade Funcional/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Glipicanas/genética , Proteínas de Fluorescência Verde/genética , Imunoprecipitação , Glicoproteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação/genética , Tubo Neural/citologia , Tubo Neural/embriologia , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
As indicated by their name, morphogens were first identified for their role in the formation of tissues early in development. Secreted from a source, they spread through the tissue to form gradients by which they affect the differentiation of precursor cells in a concentration-dependent manner. In this context, the antagonistic roles of the morphogens of the Wnt family and Sonic hedgehog (Shh) in the specification of cell types along the dorso-ventral axis of the neural tube have been studied in detail. However, more recently, morphogens have been demonstrated to act well beyond the early stages of nervous system development, as additional roles of morphogen gradients in vertebrate neural circuit formation have been identified. Both Wnt and Shh affect neural circuit formation at several stages by their influence on neurite extension, axon pathfinding and synapse formation. In this review, we will summarize the mechanisms of morphogen function during axon guidance in the vertebrate nervous system.
RESUMO
Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development(1,2). These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development. Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors(3) harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript(4) (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown(3). Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations(5), this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development(6). This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.
Assuntos
Carbocianinas/química , Eletroporação/métodos , Técnicas de Silenciamento de Genes/métodos , MicroRNAs/genética , Tubo Neural/fisiologia , Plasmídeos/genética , Animais , Carbocianinas/administração & dosagem , Embrião de Galinha , DNA/administração & dosagem , DNA/genética , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , MicroRNAs/administração & dosagem , Fenótipo , Plasmídeos/administração & dosagemRESUMO
Gradients of secreted morphogens, such as Sonic hedgehog (Shh), Wnt, and TGFß/Bmp, have classically been shown to control many aspects of early development by regulating cell proliferation and determining cell fate. However, recent studies demonstrate that these molecules also play important and evolutionarily conserved roles in later aspects of neural development. Depending on the context, these molecules can elicit gene transcription in the nucleus, or alternatively can provide instructive signals at the growth cone that induce local and rapid changes in cytoskeletal organization. Shh can activate different cellular transduction pathways via its binding to alternative coreceptor complexes or simply by adaptation of its "classical" signaling pathway. However, in most of its activities during neural development, Shh does not act alone but rather in concert with other morphogens, particularly the Wnts. This review provides an overview of the mechanisms by which Shh signaling acts in concert with Wnts to mediate a myriad of cellular processes that are required for neural circuit formation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Hedgehog/metabolismo , Tubo Neural/embriologia , Neurogênese/fisiologia , Medula Espinal/embriologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Transdução de Sinais/fisiologiaRESUMO
In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory.