Glia promote synaptogenesis through an IQGAP PES-7 in C. elegans
Description
Synapses are essential for the function of the nervous system. Glia play an important role in regulating synaptic formation. To address how glia regulate synaptic development, we use cima-1 mutant C. elegans as an in vivo model. In this data set, we provided data that support 1) Rho GTPase CDC-42 and IQGAP PES-7 are required in presynaptic neurons for VCSC glia-induced presynaptic formation; 2) cdc-42 and pes-7 are also required for normal synaptogenesis during postembryonic developmental stages; 3)PES-7 activated by CDC-42 promotes presynaptic formation most likely through regulating F-actin assembly.
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Steps to reproduce
Plasmids and transformation Constructs were built with either the pSM vector (derivation of pPD49.26)(Shen and Bargmann, 2003), gateway system (Invitrogen), or L4440 for RNAi (Kamath and Ahringer, 2003). RNAi constructs were made by inserting target cDNA into the XmaI or NotI site of the L4440. All cdc-42 plasmids are made with the pSM (Shen and Bargmann, 2003). Endogenous and tissue specific pes-7 rescue plasmids were generated with gateway system (Basherudin and Curtis, 2006). PES-7 truncation constructs were made by fragment recombination (Gibson et al., 2009). Detailed information is described in Table S3. Transgenic strains were generated by microinjections as previously (Mello and Fire, 1995). We used the Phlh-17::mCherry (20ng/µl), Punc-122::GFP (20ng/µl) or Punc-122::RFP (20ng/µl) as coinjection markers. All transgenes and the corresponding concentrations of the plasmids are listed in Table S2. RNA interference RNAi screen was performed as described (Kamath and Ahringer, 2003). The empty vector L4440 was used as a negative control, and the dpy-7, a gene that suppresses cima-1(wy84) (Shao et al., 2013), was used as a positive control. Synchronized L4 animals were fed with RNAi bacteria strain until the F1 generation reaches adult day 1 for the synaptic phenotype scoring. FDU937 [cima-1(wy84);wyIs45(Pttx-3::GFP::RAB-3)] was used in the RNAi screen. Genes screened are listed in the Table S1. Confocal microscopy and imaging analysis Synchronized animals were anaesthetized with muscimol and mounted on 3% agarose pad for phenotyping or imaging. AIY ectopic synapses were those at the AIY zone 1 region indicated by dashed boxes in figures. Images presented in this study were obtained using either Perkin Elmer UltraVIEW VoX or Andor Dragonfly Spinning Disc Confocal Microscope with 40x objectives, 488nm laser for GFP and 561nm laser for mCherry. For fluorescent intensity quantification, all images were taken with Andor Dragonfly Spinning Disc Confocal Microscope with 40x objective. The Z-series of optical sections were acquired 0.5μm step size. Adobe photoshop CC was used to rotate and crop the image. We quantified the percentage of animals with ectopic synapses using a Nikon Ni-U fluorescent microscope. Animals were considered to have ectopic synapses when synaptic markers were detected in the AIY zone 1 region (Shao et al., 2013). At least three biological replicates were obtained for each quantification. The total number of animals, biological replicates and the number of extrachromosomal lines used are indicated in each bar of the graphs. Adult phenotypes were scored 24 hours after the larva L4 stage. For the larval phenotype, eggs collected within a two-hour window were cultured for 12, 21, 29, and 38 hours to reach the L1, L2, L3, and L4 stages.