Mutations in Wnt2 Alter Presynaptic Motor Neuron Morphology and Presynaptic Protein Localization at the Drosophila Neuromuscular Junction

Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. We sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins

Identification of genes affecting glutamate receptor expression at the Drosophila neuromuscular junction.

Identification of genes affecting glutamate receptor expression at the Drosophila neuromuscular junction

Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the <i>Drosophila</i> Neuromuscular Junction

<div><p>The <i>Drosophila</i> neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the <i>Drosophila</i> larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.</p></div

Kis localizes to the nucleus of motor neurons and muscles.

<p>(<b>A</b>) Confocal images of Kis-GFP expression in midline of third instar larval ventral nerve cord expressing RFP in all neurons using the <i>Elav^c155-Gal4</i> driver. Neurons are labeled in red (Elav), nuclei in blue (DAPI), and Kis in green (Kis-GFP). Note presence of Kis-GFP in neuron nuclei. Right panels show individual channels. Scale bar  = 10 µm. (<b>B</b>) Confocal images of Kis-GFP expression in multi-nucleated muscle cells of muscles 6 and 7 in third instar larval NMJs. Muscles are labeled with phalloidin (PHL, red), muscle nuclei in blue (DAPI), and Kis in green (Kis-GFP). Note presence of Kis-GFP in muscle nuclei. Right panels show individual channels. Scale bar  = 50 µm.</p

Kis positively regulates localization of postsynaptic glutamate receptors.

<p>High resolution confocal images of 6/7 NMJs from third instar larvae immunolabeled with α-HRP (magenta) and α-GluRIIA (<b>A</b>, green), α-GluRIIB (<b>B</b>, green), or α-GluRIIC (<b>C</b>, green). Scale bar  = 5 µm. Histograms show quantification of GluR cluster sizes (left histograms) and mean relative GluR fluorescence (right histograms) in genotypes listed.</p

Kis negatively affects growth of the presynaptic motor neuron but does not alter cytoskeletal proteins at the NMJ.

<p>(<b>A</b>) Confocal images of third instar larval NMJs, muscles 6 and 7, labeled with α-HRP (white) to detect presynaptic neuronal membranes and phalloidin (red). Scale bar  = 20 µm (<b>B</b>) Quantification of muscle 6 sizes in <i>w¹¹¹⁸</i>, <i>kis^k13416</i>, and <i>kis^LM27/kis^k13416</i>. (<b>C</b>) Confocal micrographs of 6/7 NMJs from third instar larvae immunolabeled with α-HRP (magenta) and α-acetylated tubulin (green). Scale bar  = 20 µm (<b>D</b>) Quantification of mean relative synaptic acetylated tubulin levels (top histogram) and mean relative muscle acetylated tubulin levels (bottom histogram). (<b>E</b>) Quantification of total boutons (left histogram) and branches (right histogram) per 6/7 NMJ.</p

Kis influences the apposition of postsynaptic GluRs with presynaptic active zones.

<p>(<b>A</b>) High resolution confocal images of third instar larval NMJs, muscles 6 and 7, immunolabeled with α-HRP (gray), α-Brp (magenta), and α-GluRIIC (green). Arrows indicate examples of GluRIIC clusters that are unapposed to an active zone as indicted by Brp immunlabeling. Scale bar  = 5 µm. (<b>B</b>) Quantification of the per cent of GluRIIC clusters that are unopposed to a Brp puncta.</p

Kis is important pre- and postsynaptically for NMJ development.

<p>(<b>A</b>) Confocal micrographs of third instar larval 6/7 NMJs immunolabled with α-HRP (magenta) and α-GluRIIB (green). Insets show high magnification image of a single terminal bouton. Scale bar  = 20 µm. (<b>B</b>) Quantification of GluRIIB cluster size in µm² in genotypes listed indicates that knockdown of <i>kis</i> in all cells or presynaptic neurons but not postsynaptic muscles results in a significant reduction in GluRIIB cluster size. (<b>C</b>) Quantification of the number of 6/7 NMJ boutons (left) and branches (right) in the genotypes listed.</p

Kis functions predominantly in motor neurons to influence larval behavior.

<p>(<b>A</b>) Quantification of the number of muscle contractions per 30 seconds from third instar larvae in genotypes listed. Muscle contractions were quantified as the number of full body, entire peristaltic waves (originating forward or backward) generated by each third instar larvae. The mean muscle contraction was determined from three consecutive trials per larvae performed for genotypes listed. (<b>B</b>) Quantification of crawling distance in cm/min from third instar larvae in genotypes listed. Manual analysis of 60 video frames per larvae. Three trials per larvae were carried out to calculate the average. The mean was used as a single data point in the analysis.</p