The planar cell polarity protein Vangl2 bidirectionally regulates dendritic branching in cultured hippocampal neurons
© Hagiwara et al.; licensee BioMed Central Ltd. 2014
Received: 14 August 2014
Accepted: 23 October 2014
Published: 12 November 2014
Van Gogh-like (Vangl) 2 is a planar cell polarity (PCP) protein that regulates the induction of polarized cellular and tissue morphology during animal development. In the nervous system, the core PCP signaling proteins have been identified to regulate neuronal maturation. In axonal growth cones, the antagonistic interaction of PCP components makes the tips of filopodia sensitive to guidance cues. However, the molecular mechanism by which the PCP signaling regulates spine and dendritic development remains obscure.
Here we explored the finding that a loss of function of Vangl2 results in a significant reduction in spine density and complexity of dendritic branching. In spite of a previous report, in which the Vangl2 C-terminal TSV motif was shown to be required for the interaction with PSD-95 and the C-terminal intracellular domain was shown to associate with N-cadherin, overexpression of deletion mutants (Vangl2-ΔTSV and Vangl2-ΔC) had little effect on spine density. However, when an N-terminal region deletion mutant was overexpressed, spine density was slightly down-regulated. Intriguingly, the deletion mutants had a more potent effect on dendritic branching, such that the deletion of the N-terminal region reduced dendritic branching, whereas deletion of the C-terminal region increased it.
Based on these results, Vangl2, a core PCP signaling pathway component, appears to have a functional role in neural complex formation. Especially in the case of dendritic branching, Vangl2 serves as a molecular hub to regulate neural morphology in opposite directions.
KeywordsPlanar cell polarity signal Van Gogh-like protein Dendritic spine Sholl analysis
One of the most complex and elaborate structures in the brain is the dendritic branches, which are required for precise processing of information coming from a large number of presynaptic inputs -. Studies using invertebrate and vertebrate systems have revealed that some planar cell polarity (PCP) components affect dendritic morphology and development -. Van Gogh (Vang), also known as Strabismus (Stbm) was originally identified in Drosophila as one of the core PCP proteins, mutations in which caused significant misorientation of organized epithelial structures such as hairs on the wing cells, bristles on the legs, and ommatidia of eyes . Vang/Stbm is evolutionarily conserved in many species, and a mammalian homolog Van Gogh-like (Vangl) 2 has been shown to be expressed in developing and mature mouse brain, at least at the mRNA level ,.
In a previous report, we showed that Vangl2 is tightly associated with the postsynaptic density (PSD) fraction and forms a protein complex with PSD-95 and NMDA receptors . Vangl2 directly binds to the third PDZ domain of PSD-95 via its C-terminal TSV motif. This interaction may be required for localization of Vangl2 to synaptic spines . Furthermore, Vangl2 directly binds to N-cadherin, and this interaction may regulate spine formation . However, it is still largely unknown how Vangl2 regulates postsynaptic morphology including spine formation and dendritic branching.
Here, we show that Vangl2 knockdown by shRNA caused significant reductions of spine density and the complexity of dendritic branching. Concerning dendritic complexity, the branching was down- and up-regulated by overexpression of deletion mutants for N- and C-terminal regions of Vangl2, respectively. These results suggest that Vangl2 plays pivotal roles in postsynaptic functions, and may serve as a molecular hub to regulate neural morphology in opposite directions, at least for dendritic branching.
Results and discussion
Vanglis required for spine and dendritic development in primary cultured neurons
Effect of Vangldeletion mutants on spinogenesis
Spine density (number / 10Δm)
Density(mean ± SEM)
7.08 ± 0.99
6.27 ± 1.03
5.24 ± 0.27
6.21 ± 0.88
4.70 ± 0.56
Bidirectional regulation of dendritic branching with Vanglmutants
We have two hypotheses for how Vangl2 mediates regulation of dendritic branching. First, Vangl2 binds to either an accelerator at the N-terminus (AC-Vangl2) or an inhibitor at the C-terminus (Vangl2-IN). During development, the AC-Vangl2 complex may be prevalent, and the relatively dominant AC-Vangl2-mediated pathway facilitates dendritic branching. With the expression of full length Vangl2 or Vangl2-ΔTSV, the ratio between the AC-Vangl2 and Vangl2-IN complexes remains unchanged, thus having no effect on dendritic branching. However, the expression of Vangl2-ΔN increases the presence of Vangl2-IN, enhancing the inhibitory pathway. On the other hand, expression of Vangl2-ΔC increases the level of the AC-Vangl2 complex, and the enhanced accelerator-mediated signal further facilitates branching. Our second hypothesis is that Vangl2 has an inhibitor at the N-terminus and an accelerator at the C-terminus. Similar to the first hypothesis, expression of Vangl2 or Vangl2-ΔTSV keeps the balance. However, if overexpressed Vangl2-ΔC acts as a dominant active signal to endogenous Vangl2, the inhibitor should be captured by overexpressed Vangl2-ΔC and the accelerator signal on Vangl2 is relatively enhanced. On the other hand, overexpressed Vangl2-ΔN works in a dominant negative fashion, and the inhibitor signal on Vangl2 may suppress branching. Currently, it is unclear which model is the case. Thus, to reveal the molecular mechanism underlying Vangl2-mediated bidirectional regulation of neural development, we need to identify the binding partners of the N- and C-terminal regions of Vangl2 in future research.
Planar cell polarity has been highly scrutinized in Drosophila. In recent years, evidence has accumulated suggesting that the PCP pathway is highly conserved and regulates polarized cellular and tissue morphology. Here, we show a requirement of Vangl2 for spine and dendritic development. N- and C-terminal intracellular domain deletion mutants regulated the dendritic branching bidirectionally, suggesting that Vangl2 is a molecular hub for neural development.
Materials and methods
Expression vectors for Vangl2 were constructed in pCAII-HA by standard molecular biological methods. Constructs for Vangl2 knockdown were created using the pSUPER vector system (shRNA, pSUPER-neo + GFP, Oligoengine). Sequence of constructs was as follows: control shRNA (5'-GAAACGGAAAGCAGGTACG-3'), rat Vangl2 shRNA (5'-GGGAGAAACAACAACGGTG-3', ).
We used the following primary antibodies: anti-PSD-95 (1:500, Thermo Scientific), anti-Vangl2 (1:100, ), anti-GFP (1:500, Life Technologies), anti-HA (1:400, Roche Applied Science), anti-tubulin (1:1000, Oncogene), and anti-actin (1:500, Merck Millipore). To detect the immunofluorescence signal, Alexa Fluor 488- or 568-labeled secondary antibodies (1:500, Molecular Probes) were used. For western blotting, HRP-linked secondary antibodies (1:20005000, GE Healthcare) were used.
Hippocampal primary culture, transfection, and immunohistochemistry
The following procedures were reviewed and approved by the animal welfare committees at the University of Yamanashi. Primary cultures of hippocampal neurons were prepared from eighteen day old pregnant Wistar rats as described previously ,. Neurons were transfected with shRNA constructs or various mutants of HA-Vangl2 with pmEGFP-β-actin (kindly provided by Dr. H. Bito, University of Tokyo) or EGFP at 4 DIV using Lipofectamine 2000 (Life Technologies). Immunostaining was performed at 10 or 21 DIV. The transfected cells were fixed with 4% paraformaldehyde and treated with blocking solution containing 4% Block Ace (Snow Brand Milk Products), 2% normal goat serum, and 0.2% Triton X-100, in PBS. Then, cells were incubated with primary antibodies diluted in blocking solution for 1h, followed by secondary antibodies.
Image acquisition and quantification
Fluorescence images were acquired by confocal laser microscopy (Fluoview FV1000, Olympus) using a 60 oil immersion objective lens. For analysis of spine density, dendritic spines visualized by pmEGFP-β-actin were counted on the main dendritic shaft. For Sholl analysis, neurons labeled with EGFP were visualized in a series of images taken throughout the z aspect of each cell. The number of dendrites crossing each concentric circle with 10Δm differences in diameter around the cell soma was counted using the ImageJ plugin ShollAnalysis (v1.0, provided by the Ghosh Lab, University of California, San Diego). Acquisition of microscopy images and morphometric quantification were performed by investigators blind to the experimental condition.
AH designed and carried out the immunohistochemical analysis and drafted the manuscript. MY performed immunohistochemistry experiments, YH performed western blotting, and EI designed the nucleofection experiments with primary cultured neurons. TO directed the study and edited the manuscript. All authors read and approved the final manuscript.
Day in vitro
Planar cell polarity
Prickle binding domain
This work was supported by the University of Yamanashi.
We thank members in the Ohtsuka laboratory and A. Togawa-Nakatani for their technical assistance and T. Yoshioka for his technical support during the initial experiments.
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