Synapse location during growth depends on glia location

Abstract

Synaptic contacts are largely established during embryogenesis and are then maintained during growth. To identify molecules involved in this process, we conducted a forward genetic screen in C. elegans and identified cima-1. In cima-1 mutants, synaptic contacts are correctly established during embryogenesis, but ectopic synapses emerge during postdevelopmental growth. cima-1 encodes a solute carrier in the SLC17 family of transporters that includes sialin, a protein that when mutated in humans results in neurological disorders. cima-1 does not function in neurons but rather functions in the nearby epidermal cells to correctly position glia during postlarval growth. Our findings indicate that CIMA-1 antagonizes the FGF receptor (FGFR), and does so most likely by inhibiting FGFR's role in epidermal-glia adhesion rather than signaling. Our data suggest that epidermal-glia crosstalk, in this case mediated by a transporter and the FGF receptor, is vital to preserve embryonically derived circuit architecture during postdevelopmental growth.

Figure 1. cima-1 is required for maintenance of AIY presynaptic distribution during growth

(A) Schematic diagram of bilaterally symmetric AIYs (grey) in the C. elegans head (modified from WormAtlas with permission). Green marks presynaptic positions. There are three distinct anatomical regions along the AIY neurite: a segment proximal to AIY cell body that is devoid of synapses (Zone 1, dashed box); a dense presynaptic region at the dorsal turn of the AIY neurite (Zone 2); and a region with discrete presynaptic clusters at the distal part of the neurite (Zone 3) (Colón-Ramos et al., 2007; White et al., 1986). (B–E) The AIY presynaptic pattern in wild-type animals. Using synaptic vesicle associated GFP::RAB-3, we observe that AIY presynaptic pattern is already established in newly hatched larval L1 stage animals (B) and maintained in adults (C). A similar pattern was observed when we visualized synaptic vesicle protein SNG-1::YFP (D) and active zone protein GFP::SYD-1 (E) in adults and L1 larva (data not shown). (F–I) cima-1(wy84) mutant animals fail to maintain the AIY presynaptic pattern in adults. cima-1(wy84) mutant L1 animals display a wild-type presynaptic pattern as visualized with GFP::RAB-3 (F). Adult animals display an abnormal presynaptic pattern as visualized with presynaptic proteins GFP::RAB-3 (G), SNG-1::YFP (H) and GFP::SYD-1 (I). The ectopic presynaptic structure was confirmed with fluorescence electron microscopy (fEM) (Figure S1). In all images, dashed box corresponds to normally asynaptic Zone 1, and scale bars are 10µm. (Scale bar in B applies to B and F; scale bar in C applies to C–E and G–I). (J–K) Quantification of the AIY presynaptic pattern. Note that the length of ventral portion of the presynaptic region (presynaptic region in Zone 1 and Zone 2) is similar in cima-1(wy84) and wild-type animals at L1 stage, but becomes longer at L4 or adult stages (J). The ratio of the presynaptic length (the length of ventral presynaptic region divided by total presynaptic region as shown in K) is a metric that reflects the general pattern of AIY, and persists in wild-type animals during growth (black bars). Note how in cima-1(wy84) mutant animals this ratio becomes abnormally larger, particularly in post-developmental growth after the L4 stage (red bars). n≥34 for each group. Error bars are s.e.m., *: p<0.05, **: p<0.01, ***: p<0.001 by t-test comparison. See also Figure S1.

Figure 2. cima-1 (wy84) mutants have a posteriorly extended Zone 2 and ectopic presynaptic structures in Zone 1

(A) AIY (grey) forms synapses onto postsynaptic partner RIA (blue) in Zone 2 (Colón-Ramos et al., 2007; White et al., 1986); Schematic diagram modified from WormAtlas with permission. (B–C) Simultaneous visualization of synaptic vesicles in AIY (pseudocolor green) and postsynaptic GLR-1 sites in RIA (pseudocolor blue) in a wild-type animal. Note that in both L1 (B) and adults (C), RIA contacts AIY in Zone 2, and not in Zone 1. The arrow indicates the transition between Zone 2 and Zone 1, as determined by the position where RIA contacts AIY. (D) Three dimensional profile of AIY presynaptic CFP::RAB-3 (pseudocolor green) fluorescence intensity (arbitrary units) of (C). The arrow indicates the transition between Zone 2 and Zone 1. (E) Simultaneous visualization of GRASP GFP (which indicates contact between presynaptic neuron AIY and postsynaptic partner RIA) and mCherry::RAB-3 in a wild type animal. Note that the AIY:RIA contact indicated by GFP overlaps with presynaptic mCherry::RAB-3 at Zone 2 region. (F–G) The AIY presynaptic pattern and RIA morphology are wild type in newly hatched (L1 stage) cima-1(wy84) mutant animals (F). However, at the adult stage the RIA neurite is posteriorly extended (from AIY ventral turn indicated by yellow arrow to the posterior site indicated by white arrow) and ectopic presynapses are seen in Zone 1 (bracket) (G). (H) Three dimensional profile of AIY presynaptic GFP::RAB-3 fluorescence intensity (arbitrary units) of the image in (G). (I) Simultaneous visualization of GRASP GFP and mCherry::RAB-3 in a cima-1(wy84) animal. Note that presynaptic mCherry::RAB-3 extends beyond the AIY:RIA contact region (indicated by GRASP GFP signal). Ectopic presynaptic sites in AIY are bracketed. All arrows except the yellow in (G) indicate the end of Zone 2 and the beginning of Zone 1. The yellow arrow in (G) indicates where the end of Zone 2 and the beginning of Zone 1 should be. Scale bars are 10µm (scale bar in B applies to B and F; scale bar in C applies to C, G, E and I).

Figure 3. cima-1(wy84) is an allele of F45E4.11, which encodes a conserved SLC17 family transporter

(A) SNP mapping indicates that the genetic lesion corresponding to the cima-1(wy84) allele is between 7.40Mb and 7.83Mb on chromosome IV. Two overlapping fosmids in this region (WRM0612bA03 and WRM0615cC03) rescue the cima-1(wy84) AIY presynaptic defect. Those two fosmids overlap in a genomic area that includes just two genes: F45E4.11 and C08G9.1. Only F45E4.11 was able to rescue the AIY defect. Sequencing data indicates a missense mutation in the coding region that alters conserved G388 to E. (B) Quantification of the percentage of animals displaying the AIY presynaptic patterning defect in cima-1(wy84) mutants transformed with fosmid WRM0612bA03 (which includes gene F45E4.11) or with just gene F45E4.11. n≥86 for each category. Error bars are s.e.m., ***: p<0.001 by t-test. (C) A different F45E4.11 allele, gk902655, contains a nonsense mutation in the coding region (R476 to opal stop codon) and phenocopies the wy84 allele. (D, E) Knockdown of F45E4.11 by RNAi phenocopies the cima-1(wy84) presynaptic phenotype in AIY. Animals fed with bacteria transfected with control vector L4440 show normal AIY presynaptic distribution (D), while animals fed with bacteria expressing F45E4.11 dsRNA phenocopy the cima-1(wy84) AIY presynaptic phenotype (E). The scale bar is 10µm and applies to (C–E). In (C–E), Zone 1 region is highlighted with a dashed box. (F) A schematic diagram of the predicted cima-1 topology. The mutated G388 (asterisk) in cima-1(wy84) is located in ninth transmembrane domain. CIMA-1 is a member of SLC17 transporter family (See also Figure S2).

Figure 4. cima-1 is expressed and required in epidermal cells for maintenance of the AIY presynaptic pattern

(A) A larval animal displaying the endogenous cima-1 expression pattern as determined by rescuing construct CIMA-1(genomic)::SL2::GFP. The dashed box corresponds to the region where AIY is located (AIY not shown; this region is also similar to the region examined in (B and C)). Arrows point at CIMA-1 expressing epidermal cells. (B–D) Simultaneous visualization of cima-1(genomic)::SL2::GFP and body wall muscle reporter Pmyo-3::mcherry. Note the non-overlapping expressing pattern of cima-1 in epidermal cells and Pmyo-3::mcherry in muscles, both in the sagittal cross-section (B) and in the transverse cross-section (C). Dashed line in (B) indicates site of transverse cross-section image in (C). And (D) is the schematic drawing of (C), with muscle quadrants (“M”), epidermal cells (green) and pharynx (P) labeled. (E) Quantification of tissue-specific rescue. Expression of cima-1 cDNA in AIY (ttx-3 promoter) or pan-neuronally (rab-3 promoter) does not rescue the AIY presynaptic defect in cima-1(wy84) mutant animals. However, expression of cima-1 cDNA in epidermal cells (dpy-4) robustly rescues the AIY presynaptic defect (see also rescue with epidermal promoters rol-6, dpy-7 and col-19 in Figure S3I). n≥50 for each group. Error bars are s.e.m., n.s.: not significant, ***: p<0.001 by t-test. Scale bars are 10µm. See also Figure S3, S4 and S5.

Figure 5. cima-1 is required for maintenance of VCSC glial morphology during growth

(A) Relative position of epidermal cells, VCSC glia and AIY in C. elegans. A cross section of EM image of a wild-type animal in the Zone 2 region of AIY (from WormAtlas.org and WormImage.org)(White et al., 1986). VCSC glia (pseudocolored red in the micrograph) lie between the epidermal cells (purple) and AIY Zone 2 synapses (green, note vesicles and dense projections in the two AIY-neurite cross sections). The dashed box in the schematic of the worm head represents the region where images (and cartoons) in (B–Q) were obtained. (B–Q) Simultaneous visualization of AIY presynaptic sites (green) and VCSC glia (red) in wild type adult animal (B–D), cima-1(wy84) L4 animal (F–H), cima-1 (wy84) adult animal (J–L), or cima-1(wy84) adult animal rescued with Pdpy-4::cima-1 (N–P). Images (D, H, L and P) are as (C, G,K and O), but overlaid with DIC. Note that in cima-1(wy84) adult animals, VCSC glia abnormally distend to the Zone 1 region and overlap with AIY ectopic presynapses (dashed box; J–M). Both glia and AIY presynaptic defects are rescued by expressing cima-1 cDNA in epidermal cells (N–Q). In all images, white and black asterisk represent the location of the AIY cell body, and orange asterisks mark pharyngeal grinder. Note that in K and L, synapses are formed in Zone 1, past the orange asterisk. (E, I, M, and Q) are cartoons of (D, H, L and P) with pharynx in grey (see schematic in A). The hlh-17 promoter labels both dorsal and ventral glial cells. The dorsal glia not labeled in (K and L) is due to the mosaic retention of the transgenic marker. Scale bar in (B) corresponds to 10µm and applies to all images. See also Figure S6.

Figure 6. VCSC glia abnormally contact AIY and are required for formation of ectopic presynaptic sites in Zone 1 in cima-1(wy84)

(A–F) Simultaneous visualization of GRASP GFP signal (which indicates contact between AIY and VCSC glia) (A, D) and mCherry::RAB-3 (B, E) in wild-type (A–C) and cima-1 (wy84) (D–F) adult animals. Note that in cima-1 (wy84) mutants VCSC glia abnormally contact AIY in Zone 1 (indicated by GRASP GFP in dashed box), and that these sites correlate with sites of ectopic mCherry::RAB-3 in Zone 1. (G) In cima-1 (wy84) adult animals, AIY forms ectopic presynaptic sites in Zone 1 (dashed box) beyond Zone 2 (determined by the position of postsynaptic partner RIA in blue). Arrow indicates the end of Zone 2 and the beginning of Zone 1. (H) As in (G), but with VCSC genetically ablated through the cell-specific expression of caspases. Note suppression of ectopic presynaptic sites in Zone 1 (dashes box). (I) Quantification of the percentage of animals displaying ectopic presynaptic sites in Zone 1 in cima-1(wy84) adult mutants; in cima-1(wy84) adult mutants expressing caspases cell-specifically in VCSC glia, or in cima-1(wy84) mls-2(ns156) double mutants. The scale bar in (A) is 10µm and applies to (A–H). n≥37 for each genotype. Error bars represent 95% confidence interval. *** : p<0.001 between groups as determined by Fisher’s exact test. See also Figure S1.

Figure 7. egl-15/fgfr is required for ectopic synapse formation in cima-1(wy84) animals

(A–C) GFP::RAB-3 in cima-1(wy84) adult mutants (A), cima-1(wy84) egl-15(n484) double mutant (B) and cima-1(wy84) egl-15(ay1) double mutant (C). Note that alleles egl-15(n484) and egl-15(ay1), which specifically disrupt egl-15(5A) isoform, suppress cima-1(wy84) presynaptic distribution defect. (D–E) Simultaneous visualization of presynaptic sites in AIY (green) and glia (red) in cima-1(wy84) (D) and cima-1(wy84) egl-15(n484) double mutant adult animals (E). Note that egl-15(n484) allele suppresses both the AIY presynaptic defect and the glia morphology defect in cima-1(wy84). (F) Expression of egl-15(5A) cDNA in the epidermal cells (using the dpy-7 promoter) reverts the suppression of the cima-1(wy84) AIY presynaptic phenotype by egl-15(n484). (G) EGL-15 crackle antibody (a gift from M Stern), detects endogenous EGL-15, the 141kD band in wild type (lane 1), but not in egl-15(n1477) mutants (lane 2), which produced the C-terminus truncated EGL-15 (M Stern, personal communication). HA antibody specifically recognizes HA-tagged EGL-15(5A) expressed in epidermal cells (lane 4 and 5), but not in wild type animals without the transgene (lane 3). For comparison in lanes 4 and 5, the same HA-tagged EGL-15(5A) expressing transgenic line were used. Note that EGL-15(5A) protein levels are higher in cima-1(wy84) mutant animals (lane 5) as compared to wild type animals (lane 4). Actin and coinjection marker Punc-122::GFP were used as loading control. (H) Quantification of the EGL-15(5A)::HA protein levels from four independent blots. n≥150 for each group. Error bars are s.e.m., **: p<0.01 by student’s t-test. (I–J) AIY presynaptic GFP::RAB-3 (I) is mislocalized to Zone 1 region abnormally ensheathed by VCSC (J) upon over-expression of egl-15(5A) in epidermal cells by using the dpy-7 promoter (compare to Fig. 5C). (K) Quantification of the percentage of animals with the phenotype shown in (I). Error bars represent 95% confidence interval. ** : p<0.01 groups as determined by Fisher’s exact test. (L) A model for cima-1 and egl-15(5A) in epidermal cells (purple) regulating VCSC glia (red) morphology and AIY presynaptic distribution (green). In wild-type animals, cima-1 negatively regulates egl-15(5A), thereby reducing epidermal-glia adhesion and preventing glia extension during growth. This interaction contributes to maintaining wild type VCSC morphology, which in turn specifies correct synaptic distribution during growth (left cartoon). In animals with cima-1 loss-of-function, or animals in which egl-15(5A) is overexpressed, the interaction between the epidermal cells and VCSC is misregulated, resulting in VCSC glia distension posteriorly, ectopic contacts between glia and axons and ectopic presynaptic sites. See also Figure S7