Nicole H. Wilson

Sonic Hedgehog Regulates Its Own Receptor on Postcrossing Commissural Axons in a Glypican1-Dependent Manner

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.

Neogenin and RGMa Control Neural Tube Closure and Neuroepithelial Morphology by Regulating Cell Polarity

In humans, neural tube closure defects occur in 1:1000 pregnancies. The design of new strategies for the prevention of such common defects would benefit from an improved understanding of the molecular events underlying neurulation. Neural fold elevation is a key morphological process that acts during neurulation to drive neural tube closure. However, to date, the molecular pathways underpinning neural fold elevation have not been elucidated. Here, we use morpholino knock-down technology to demonstrate that Repulsive Guidance Molecule (RGMa)-Neogenin interactions are essential for effective neural fold elevation during Xenopus neurulation and that loss of these molecules results in disrupted neural tube closure. We demonstrate that Neogenin and RGMa are required for establishing the morphology of deep layer cells in the neural plate throughout neurulation. We also show that loss of Neogenin severely disrupts the microtubule network within the deep layer cells suggesting that Neogenin-dependent microtubule organization within the deep cells is essential for radial intercalation with the overlying superficial cell layer, thereby driving neural fold elevation. In addition, we show that sustained Neogenin activity is also necessary for the establishment of the apicobasally polarized pseudostratified neuroepithelium of the neural tube. Therefore, our study identifies a novel signaling pathway essential for radial intercalation and epithelialization during neural fold elevation and neural tube morphogenesis.

Sonic hedgehog and Wnt: antagonists in morphogenesis but collaborators in axon guidance

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.

Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development

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 genes 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.

Neogenin recruitment of the WAVE regulatory complex maintains adherens junction stability and tension

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.

In Ovo Electroporation of miRNA-Based-Plasmids to Investigate Gene Function in the Developing Neural Tube

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.

Netrin-1 is required for efficient neural tube closure

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell–cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin‐1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin‐1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin‐1 results in delayed neural fold apposition and neural tube closure. We further show that netrin‐1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin‐RGMa interactions, neogenin‐netrin‐1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin‐1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure.

Introductory glycosylation analysis using SDS-PAGE and peptide mass fingerprinting.

Glycosylation is extremely complex, with the potential for a protein to have oligosaccharides attached at multiple sites, and for each site of glycosylation to have multiple structures attached to it. Structural information on the oligosaccharides bound to either asparagine residues (N-linked) or serine and threonine residues (O-linked) requires sensitive, specialised, and complex techniques and equipment. We show here, however, that a large amount of information regarding the glycosylation of glycoproteins can be obtained with common protein techniques such as 1D SDS-PAGE and peptide mass fingerprinting (PMF). Enzymatic deglycosylation in combination with SDS-PAGE and PMF analysis can determine the relative percentage of N-linked carbohydrate on the glycosylated protein, as well as attachment sites of the oligosaccharides.

Endoglycan plays a role in axon guidance and neuronal migration by negatively regulating cell-cell adhesion

Cell migration and axon guidance are important steps in the formation of neural circuits. Both steps depend on the interactions between cell surface receptors and molecules on cells along the pathway. In addition to cell-cell adhesion, these molecular interactions provide guidance information. The fine-tuning of cell-cell adhesion is an important aspect of cell migration, axon guidance, and synapse formation. Here, we show that Endoglycan, a sialomucin, plays a role in axon guidance and cell migration in the central nervous system. In the absence of Endoglycan, commissural axons failed to properly navigate the midline of the spinal cord. In the developing cerebellum, a lack of Endoglycan prevented migration of Purkinje cells and resulted in a stunted growth of the cerebellar lobes. Taken together, these results support the hypothesis that Endoglycan acts as a ‘lubricant’, a negative regulator of cell-cell adhesion, in both commissural axon guidance and Purkinje cell migration.

In ovo electroporation of miRNA plasmids to silence genes in a temporally and spatially controlled manner

The ability to spatially and temporally control gene expression during development is crucial for the elucidation of gene function in vivo. The use of RNA interference (RNAi)-based technologies in combination with oviparous animal models allows for efficient, precise gene silencing. 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 endogenous target protein/s upon electroporation into the spinal cord. The miRNA cassette is directly linked to a fluorescent protein reporter, which allows the faithful visualization of transfected cells. Different promoters/enhancers drive transcript 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, thus permitting the rapid analysis of complex cellular and molecular interactions.