Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation

<p>Abstract</p> <p>Background</p> <p>The sequestration of Muscleblind splicing regulators results in myotonic dystrophy. Previous work on Muscleblind has largely focused on its roles in muscle development and maintenance due to the skeletal and cardiac muscle degeneration phenotype observed in individuals with the disorder. However, a number of reported nervous system defects suggest that Muscleblind proteins function in other tissues as well.</p> <p>Results</p> <p>We have identified a mutation in the <it>Caenorhabditis elegans </it>homolog of Muscleblind, <it>mbl-1</it>, that is required for proper formation of neuromuscular junction (NMJ) synapses. <it>mbl-1 </it>mutants exhibit selective loss of the most distal NMJ synapses in a <it>C. elegans </it>motorneuron, DA9, visualized using the vesicle-associated protein RAB-3, as well as the active zone proteins SYD-2/liprin-α and UNC-10/Rim. The proximal NMJs appear to have normal pre- and postsynaptic specializations. Surprisingly, expressing a <it>mbl-1 </it>transgene in the presynaptic neuron is sufficient to rescue the synaptic defect, while muscle expression has no effect. Consistent with this result, <it>mbl-1 </it>is also expressed in neurons.</p> <p>Conclusions</p> <p>Based on these results, we conclude that in addition to its functions in muscle, the Muscleblind splice regulators also function in neurons to regulate synapse formation.</p

MTM-6 functions within the neuron and can be rescued by EGL-20 overexpression.

<p>A) Confocal images of DA9 axons were traced, straightened and aligned to produce an image montage. An image montage of the GFP::RAB-3 puncta from twenty animals for each strain was compiled for (B) wild-type, (C) <i>mtm-6 (ok330)</i>, (D) <i>mtm-6 (ok330); Ex[pmig-13::mtm-6a]</i>, (E) <i>mtm-6(ok330); Ex[plin-44::mtm-6a]</i>, (F) <i>mtm-6(ok330); Ex[pegl-20::mtm-6a]</i>. (G) Quantification of the GFP::RAB-3 puncta for <i>mtm-6(ok330)</i> and various rescue constructs. n>40, *** is p<0.001, n.s. is not significant, and error bars are SD.</p

Loss of <i>mtm-6</i> enhances the Wnt phenotype in DA9 in an <i>egl-20</i> dependent manner.

<p>(A) Schematic of DA9. GFP::RAB-3 puncta were quantified in the commissural region, the dorsal asynaptic region and the total dorsal region. <i>lin-44(n1792)</i> shows a stereotyped shift of GFP::RAB-3 into the dorsal asynaptic region of DA9 (B). <i>lin-44(n1792); mtm-6(ok330)</i> animals have GFP::RAB-3 puncta in the commissure (C,D). (E) Quantification of the number of commissural puncta to measure the enhancement of the Wnt phenotype by <i>mtm-6</i> and to measure the cell-autonomous rescue experiments. n = 40, **** is p<0.0001, n.s is not significant, and error bars are SD. Arrows in (A-D) mark the DA9 dorsal commissure turn. Scale bar is 10 µm.</p

Loss of <i>mtm-6</i> causes a reduction in the number of DA9 presynaptic puncta.

<p>(A) Schematic of DA9. The dendrite of DA9 extends anterior from a cell body in the ventral side of the animal. The axon of DA9 extends towards the posterior before extending a commissure to the dorsal nerve cord, where the axon turns and continues towards the anterior of the animal. Synapses form with a stereotyped number and location on to muscle and Ventral D neurons. (B) Quantification of the number of puncta formed in wild-type and <i>mtm-6 (ok330)</i> animals using GFP::RAB-3 to mark presynapses and UNC-10::GFP as a marker for active zones (n>60, **** is p<0.0001). (C-F) <i>mtm-6 (ok330)</i> animals show a reduced number of dorsal synaptic puncta when compared to wild-type animals for the GFP::RAB-3 and UNC-10::GFP markers. (C) Wild-type; GFP::RAB-3. (D) Wild-type; UNC-10::GFP. (E) <i>mtm-6(ok330)</i>; GFP::RAB-3. (F) <i>mtm-6(ok330)</i>; UNC-10::GFP. The UNC-10::GFP puncta on the ventral side of the animal correspond to the VA12 motor neuron. The dashed boxes mark the regions that are enlarged for the insets above each window. Error bars are SD. Scale bar is 10 µm.</p

MTM-6, a Phosphoinositide Phosphatase, is Required to Promote Synapse Formation in <i>Caenorhabditis elegans</i>

<div><p>Forming the proper number of synapses is crucial for normal neuronal development. We found that loss of function of the phosphoinositide phosphatase <i>mtm-6</i> results in a reduction in the number of synaptic puncta. The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action. MTM-6 shows relative specificity for EGL-20 over the other Wnt ligands. We suggest that the ability of MTM-6 to regulate EGL-20 secretion is a function of its expression pattern. We conclude that regulation of secretion of different Wnt ligands can use different components. Additionally, we present a novel neuronal function for MTM-6.</p></div

<i>egl-20</i> and <i>mtm-6</i> have similar reductions in synaptic puncta and act in a non-linear pathway.

<p>(A) Straightened DA9 neuron. Image montages of GFP::RAB-3 puncta for 20 animals were produced for (B) wild-type, (C) <i>mtm-6(ok330)</i>, (D) <i>egl-20(n585)</i>, (E) <i>mtm-6(ok330)</i>; <i>egl-20(n585)</i>, and <i>mtm-6(ok330)</i>; <i>egl-20(n585); Ex[Pmig-13::mtm-6a]</i>. (F) Linescans of mean fluorescence intensity with respect to distance of the aligned image montages. (H) Quantification of puncta number in single, double and triple mutants for different Wnt molecules. (I) Quantification of puncta number in the rescue of the different double and triple for different Wnt molecules. (J) Quantification of puncta number in the image montages (B-F) using ImageJ. For (H and I) n = 40, **** is p<0.0001, ** is p<0.01, * is p<0.05, n.s. is not significant, and error bars are SD. For (J) n = 20.</p

<i>mtm-6</i> is expressed in neurons.

<p>The expression pattern of <i>mtm-6</i> using the <i>WRM0629bC04</i> fosmid with the insertion of SL2::mCherry. Insets show head neurons (blue asterisk), commissures (yellow arrows), and non-neuronal cells (red asterisk) in the tail. Red arrows point to the ventral nerve cord neurons. Scale bars are 10 µm.</p