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Andermatt I.,Institute of Molecular Life science and Neuroscience Center Zurich | Wilson N.H.,Institute of Molecular Life science and Neuroscience Center Zurich | Bergmann T.,Institute of Molecular Life science and Neuroscience Center Zurich | Mauti O.,Institute of Molecular Life science and Neuroscience Center Zurich | And 3 more authors.
Development (Cambridge) | Year: 2014

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. © 2014. Published by The Company of Biologists Ltd. Source


Andermatt I.,Institute of Molecular Life science and Neuroscience Center Zurich | Wilson N.,Institute of Molecular Life science and Neuroscience Center Zurich | Stoeckli E.T.,Institute of Molecular Life science and Neuroscience Center Zurich
Methods in Molecular Biology | Year: 2014

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. © Springer Science+Business Media, LLC 2014. Source

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