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1.
Development ; 141(19): 3709-20, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25209245

RESUMEN

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.


Asunto(s)
Axones/fisiología , Movimiento Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Semaforinas/metabolismo , Médula Espinal/citología , Médula Espinal/embriología , Análisis de Varianza , Animales , Axones/metabolismo , Embrión de Pollo , Inmunohistoquímica , Interferencia de ARN
2.
J Cell Sci ; 127(Pt 24): 5288-302, 2014 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-25335893

RESUMEN

Synaptic cell adhesion molecules (SynCAMs) are crucial for synapse formation and plasticity. However, we have previously demonstrated that SynCAMs are also required during earlier stages of neural circuit formation because SynCAM1 and SynCAM2 (also known as CADM1 and CADM2, respectively) are important for the guidance of post-crossing commissural axons. In contrast to the exclusively homophilic cis-interactions reported by previous studies, our previous in vivo results suggested the existence of heterophilic cis-interactions between SynCAM1 and SynCAM2. Indeed, as we show here, the presence of homophilic and heterophilic cis-interactions modulates the interaction of SynCAMs with trans-binding partners, as observed previously for other immunoglobulin superfamily cell adhesion molecules. These in vitro findings are in agreement with results from in vivo studies, which demonstrate a role for SynCAMs in the formation of sensory neural circuits in the chicken embryo. In the absence of SynCAMs, selective axon-axon interactions are perturbed resulting in aberrant pathfinding of sensory axons.


Asunto(s)
Axones/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Células Receptoras Sensoriales/metabolismo , Sinapsis/metabolismo , Animales , Axones/ultraestructura , Adhesión Celular , Embrión de Pollo , Ganglios Espinales/citología , Ganglios Espinales/ultraestructura , Técnicas de Silenciamiento del Gen , Sustancia Gris/metabolismo , Conos de Crecimiento/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Neuritas/metabolismo , Neuronas Aferentes/metabolismo , Unión Proteica , Células Receptoras Sensoriales/ultraestructura , Médula Espinal/metabolismo
3.
Methods Mol Biol ; 2047: 439-456, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31552670

RESUMEN

Despite the development of brain organoids and neural cultures derived from iPSCs (induced pluripotent stem cells), brain development can only be studied in an animal. The mouse is the most commonly used vertebrate model for the analysis of gene function because of the well-established genetic tools that are available for loss-of-function studies. However, studies of gene function during development can be problematic in mammals. Many genes are active during different stages of development. Absence of gene function during early development may cause aberrant neurogenesis or even embryonic lethality and thus prevent analysis of later stages of development. To avoid these problems, precise temporal control of gene silencing is required.In contrast to mammals, oviparous animals are accessible for experimental manipulations during embryonic development. The combination of accessibility and RNAi- or Crispr/Cas9-based gene silencing makes the chicken embryo a powerful model for developmental studies. Depending on the time window during which gene silencing is attempted, chicken embryos can be used in ovo or ex ovo in a domed dish for easier access during later stages of development. Both techniques allow for precise temporal control of gene silencing during embryonic development.


Asunto(s)
Encéfalo/embriología , ADN/administración & dosificación , Animales , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Embrión de Pollo , Electroporación , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Interferencia de ARN
4.
Methods Mol Biol ; 1082: 253-66, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24048939

RESUMEN

The mouse is the most commonly used vertebrate model for the analysis of gene function because of the well-established genetic tools that are available for loss-of-function studies. However, studies of gene function during development can be problematic in mammals. Many genes are active during different stages of development. Absence of gene function during early development may cause embryonic lethality and thus prevent analysis of later stages of development. To avoid these problems, precise temporal control of gene silencing is required. In contrast to mammals, oviparous animals are accessible for experimental manipulations during embryonic development. The combination of accessibility and RNAi-based gene silencing makes the chicken embryo a powerful model for developmental studies. Depending on the time window during which gene silencing is attempted, chicken embryos can be used for RNAi in ovo or cultured in a domed dish for easier access during ex ovo RNAi. Both techniques allow for precise temporal control of gene silencing during embryonic development.


Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Ingeniería Genética/métodos , Interferencia de ARN , Animales , Encéfalo/citología , Embrión de Pollo , Electroporación , Inyecciones , Tubo Neural/embriología , Tubo Neural/metabolismo , Óvulo/metabolismo , Fenotipo , Técnicas de Cultivo de Tejidos
5.
Methods Mol Biol ; 1101: 353-68, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24233790

RESUMEN

When studying gene function in vivo during development, gene expression has to be controlled in a precise temporal and spatial manner. Technologies based on RNA interference (RNAi) are well suited for such studies, as they allow for the efficient silencing of a gene of interest. In contrast to challenging and laborious approaches in mammalian systems, the use of RNAi in combination with oviparous animal models allows temporal control of gene silencing in a fast and precise manner. We have developed approaches using RNAi in the chicken embryo to analyze gene function during neural tube development. Here we describe the construction of plasmids that direct the expression of one or two artificial microRNAs (miRNAs) to knock down expression of endogenous protein/s of interest upon electroporation into the spinal cord. The miRNA cassette is directly linked to a fluorescent protein reporter, for the direct visualization of transfected cells. The transcripts are under the control of different promoters/enhancers which drive expression in genetically defined cell subpopulations in the neural tube. Mixing multiple RNAi vectors allows combinatorial knockdowns of two or more genes in different cell types of the spinal cord, thus permitting the analysis of complex cellular and molecular interactions in a fast and precise manner. The technique that we describe can easily be applied to other cell types in the neural tube, or even adapted to other organisms in developmental studies.


Asunto(s)
Técnicas de Silenciamiento del Gen , MicroARNs/genética , Tubo Neural/fisiología , Plásmidos/genética , Animales , Secuencia de Bases , Embrión de Pollo , Clonación Molecular , Cartilla de ADN/genética , Electroporación , Escherichia coli , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Tubo Neural/citología , Fenotipo , Reacción en Cadena de la Polimerasa , Interferencia de ARN , Transfección
6.
Neural Dev ; 6: 22, 2011 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-21542908

RESUMEN

BACKGROUND: Long-distance axonal growth relies on the precise interplay of guidance cues and cell adhesion molecules. While guidance cues provide positional and directional information for the advancing growth cone, cell adhesion molecules are essential in enabling axonal advancement. Such a dependence on adhesion as well as guidance molecules can be well observed in dorsal commissural interneurons, which follow a highly stereotypical growth and guidance pattern. The mechanisms and molecules involved in the attraction and outgrowth towards the ventral midline, the axon crossing towards the contralateral side, the rostral turning after midline crossing as well as the guidance along the longitudinal axis have been intensely studied. However, little is known about molecules that provide the basis for commissural axon growth along the anterior-posterior axis. RESULTS: MDGA2, a recently discovered cell adhesion molecule of the IgCAM superfamily, is highly expressed in dorsolaterally located (dI1) spinal interneurons. Functional studies inactivating MDGA2 by RNA interference (RNAi) or function-blocking antibodies demonstrate that either treatment results in a lack of commissural axon growth along the longitudinal axis. Moreover, results from RNAi experiments targeting the contralateral side together with binding studies suggest that homophilic MDGA2 interactions between ipsilaterally projecting axons and post-crossing commissural axons may be the basis of axonal growth along the longitudinal axis. CONCLUSIONS: Directed axonal growth of dorsal commissural interneurons requires an elaborate mixture of instructive (guidance) and permissive (outgrowth supporting) molecules. While Wnt and Sonic hedgehog (Shh) signalling pathways have been shown to specify the growth direction of post-crossing commissural axons, our study now provides evidence that homophilic MDGA2 interactions are essential for axonal extension along the longitudinal axis. Interestingly, so far each part of the complex axonal trajectory of commissural axons uses its own set of guidance and growth-promoting molecules, possibly explaining why such a high number of molecules influencing the growth pattern of commissural interneurons has been identified.


Asunto(s)
Axones/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células Receptoras Sensoriales/citología , Aminoácidos , Animales , Anticuerpos/farmacología , Axones/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular Transformada , Embrión de Pollo , Lateralidad Funcional , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Ganglios Espinales , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Conos de Crecimiento/metabolismo , Humanos , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/inmunología , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Médula Espinal/citología , Transfección/métodos
7.
PLoS One ; 6(5): e19887, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21655271

RESUMEN

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.


Asunto(s)
Axones/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Médula Espinal/citología , Animales , Axones/metabolismo , Femenino , Proteínas Activadoras de GTPasa/genética , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Unión Proteica/genética , Unión Proteica/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Roundabout
8.
Neural Dev ; 2: 28, 2007 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-18088409

RESUMEN

BACKGROUND: During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS. RESULTS: Downregulation of Sema6A in boundary cap cells by in ovo RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point. CONCLUSION: Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.


Asunto(s)
Tipificación del Cuerpo/genética , Sistema Nervioso Central/embriología , Neuroglía/metabolismo , Sistema Nervioso Periférico/embriología , Semaforinas/metabolismo , Animales , Células COS , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/genética , Movimiento Celular/genética , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Embrión de Pollo , Chlorocebus aethiops , Regulación hacia Abajo/genética , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Neuroglía/citología , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/metabolismo , Interferencia de ARN , Semaforinas/genética , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Raíces Nerviosas Espinales/citología , Raíces Nerviosas Espinales/embriología , Raíces Nerviosas Espinales/metabolismo
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