Neuronal expression of transgenic Cbp53E rescues type Ib axon branching defects.

<p>A) Representative maximum intensity projections of muscle 4 type Ib innervations from the indicated genotypes stained with anti-DLG (green) and anti-HRP (red). Scale bar is 10μm. B) Quantitation of the average branch number of type Ib innervations from the indicated genotypes. *0.01</p

<i>Drosophila</i> Cbp53E Regulates Axon Growth at the Neuromuscular Junction

<div><p>Calcium is a primary second messenger in all cells that functions in processes ranging from cellular proliferation to synaptic transmission. Proper regulation of calcium is achieved through numerous mechanisms involving channels, sensors, and buffers notably containing one or more EF-hand calcium binding domains. The <i>Drosophila</i> genome encodes only a single 6 EF-hand domain containing protein, Cbp53E, which is likely the prototypic member of a small family of related mammalian proteins that act as calcium buffers and calcium sensors. Like the mammalian homologs, Cbp53E is broadly though discretely expressed throughout the nervous system. Despite the importance of calcium in neuronal function and growth, nothing is known about Cbp53E’s function in neuronal development. To address this deficiency, we generated novel null alleles of <i>Drosophila</i> Cbp53E and examined neuronal development at the well-characterized larval neuromuscular junction. Loss of Cbp53E resulted in increases in axonal branching at both peptidergic and glutamatergic neuronal terminals. This overgrowth could be completely rescued by expression of exogenous Cbp53E. Overexpression of Cbp53E, however, only affected the growth of peptidergic neuronal processes. These findings indicate that Cbp53E plays a significant role in neuronal growth and suggest that it may function in both local synaptic and global cellular mechanisms.</p></div

Loss of Cbp53E increases axon branching of type II/III terminals.

<p>A) Representative maximum intensity projections of muscle 12 innervations. All synapses are labeled with anti-HRP (green) while type I synapses are eliminated from analysis with anti-DLG labelling (red). Arrows point to type II/III axons branches of the indicated genotypes. Scale bar is 10μm. B) Quantitation of the average branch number of type II/III innervations from the indicated genotypes. * 0.01</p

Expression of Cbp53E in the larval brain.

<p>A) Representative maximum intensity projection of a third instar larval brain from animals expressing UAS-mCD8GFP (green) from a D42-GAL4 motor neuron specific driver. Anti-Cbp53E staining (red) shows discrete expression patterns. Scale bar is 50μm. B) A single slice from the magnified region outlined in the merge panel in (A). Significant colocalization of anti-Cbp53E (red) and GFP (green) is seen in many cells (arrows). Scale bar is 10μm. C) Strong Cbp53E expression (green) can be seen in nerve fibers labeled with anti-HRP (red) as they exit the larval central nervous system (arrows). Scale bar is 20μm.</p

Generation of novel loss of function Cbp53E alleles.

<p>A) A Minos transposable element in the 3′ portion of the CBP53E locus was mobilized to generate 3 new alleles with reduced Cbp53E expression in the adult brain. <i>Cbp53E</i>^<i>Mi2</i> shows residual Cbp53E, while alleles <i>Cbp53E</i>^<i>Mi22</i> and <i>Cbp53E</i>^<i>Mi41</i> are complete nulls. Insets show the same image with increased detector gain settings. Scale bar is 100μm. B) Quantitative RT-PCR of Cbp53E mRNA levels normalized to GAPDH2 from the indicated genotypes and reported as the relative fold change to control levels. *** p<0.001.</p

Neuronal expression of transgenic Cbp53E rescues type II/III axon branching defects.

<p>A) Representative maximum intensity projections of muscle 12 innervations labeled with anti-HRP (green) and anti-DLG (red). Arrows point to type II/III axon branches of the indicated genotypes. Scale bar is 10μm. B) Quantitation of the average branch number of type II/III innervations from the indicated genotypes. *0.01</p

Expression of Cbp53E at the larval neuromuscular junction.

<p>A) Insulin peptide (purple) and Cbp53E (red) co-label type III boutons on muscle 12 of the NMJ. HRP (green) labels all neurons. B) A representative maximum intensity projection of strong Cbp53E staining (green) in type II boutons on muscles 6/7. C) A representative single slice image of weak Cbp53E staining (green) in type II boutons (arrow) on muscle 4. Cbp53E can also be seen in the nerve fibers around most muscles (arrowhead). Scale bars in all panels are 20μm.</p

Muscle expression of transgenic Cbp53E rescues type II/III axon branching defects.

<p>A) Representative maximum intensity projections of muscle 12 innervations labeled with anti-HRP (green) and anti-DLG (red). Arrows point to type II/III axon branches of the indicated genotypes. Scale bar is 10μm. B) Quantitation of the average branch number of type II/III innervations from the indicated genotypes. *0.01</p