1. Neuroscience

A muscle-epidermis-glia signaling axis sustains synaptic specificity during allometric growth in Caenorhabditis elegans

  1. Jiale Fan
  2. Tingting Ji
  3. Kai Wang
  4. Jichang Huang
  5. Mengqing Wang
  6. Laura Manning
  7. Xiaohua Dong
  8. Yanjun Shi
  9. Xumin Zhang
  10. Zhiyong Shao  Is a corresponding author
  11. Daniel A Colón-Ramos  Is a corresponding author
  1. Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, China
  2. State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, China
  3. Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, United States
  4. Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico, Puerto Rico
https://doi.org/10.7554/eLife.55890
Share this article
Cite this article
  1. Jiale Fan
  2. Tingting Ji
  3. Kai Wang
  4. Jichang Huang
  5. Mengqing Wang
  6. Laura Manning
  7. Xiaohua Dong
  8. Yanjun Shi
  9. Xumin Zhang
  10. Zhiyong Shao
  11. Daniel A Colón-Ramos
(2020)
A muscle-epidermis-glia signaling axis sustains synaptic specificity during allometric growth in Caenorhabditis elegans
eLife 9:e55890.
https://doi.org/10.7554/eLife.55890
  2. Download BibTeX
  3. Download .RIS

Abstract

Synaptic positions underlie precise circuit connectivity. Synaptic positions can be established during embryogenesis and sustained during growth. The mechanisms that sustain synaptic specificity during allometric growth are largely unknown. We performed forward genetic screens in C. elegans for regulators of this process and identified mig-17, a conserved ADAMTS metalloprotease. Proteomic mass spectrometry, cell biological and genetic studies demonstrate that MIG-17 is secreted from cells like muscles to regulate basement membrane proteins. In the nematode brain, the basement membrane does not directly contact synapses. Instead, muscle-derived basement membrane coats one side of the glia, while glia contact synapses on their other side. MIG-17 modifies the muscle-derived basement membrane to modulate epidermal-glial crosstalk and sustain glia location and morphology during growth. Glia position in turn sustains the synaptic pattern established during embryogenesis. Our findings uncover a muscle-epidermis-glia signaling axis that sustains synaptic specificity during the organism’s allometric growth.

Introduction

Proper nervous system architecture depends on establishing and maintaining precise connectivity between pre- and post-synaptic partners. Failure to maintain proper synaptic connectivity leads to impaired nervous system function and neurological disorders (Mariano et al., 2018). Remarkably, circuit architecture is largely maintained during growth even as tissues change in relative size and position to each other. The mechanisms that sustain synaptic connectivity during growth remain largely unknown.

Our understanding of correct synaptic connectivity primarily derives from developmental studies examining the precise positioning of synapses during their biogenesis (Kurshan and Shen, 2019; Park et al., 2018; Rawson et al., 2017). These studies indicate that precise connectivity during development occurs through orchestrated signaling across multiple tissues. While cell-cell recognition and signaling between synaptic partners are pivotal for synaptogenesis, non-neuronal cells are also critical in vivo to guide synaptic specificity (Colón-Ramos, 2009; Margeta and Shen, 2010; Sanes and Yamagata, 2009; Shimozono et al., 2019). For example, during development, guidepost cells such as glia instruct synaptic specificity by secreting positional cues to the extracellular matrix (ECM) (Ango et al., 2008; Colón-Ramos et al., 2007; Eroglu and Barres, 2010; Molofsky et al., 2014; Shen and Bargmann, 2003; Tsai et al., 2012; Ullian et al., 2001). Therefore, non-cell autonomous mechanisms, mediated through the ECM, can coordinate synaptic connectivity during development in vivo.

Less is known about the factors required for sustaining the synaptic pattern during post-embryonic growth. Multiple studies have identified mechanisms required for post-embryonic maintenance of synapses, but not synaptic positions. These studies on post-embryonic maintenance of synapses have resulted in the discovery of important regulators of synaptic stability, density and morphology (Burden et al., 2018; Cherra and Jin, 2016; Hasan and Singh, 2019; Lin and Koleske, 2010; Luo et al., 2014; Sytnyk et al., 2017), including roles for ECM components in the maintenance of synapses of both the peripheral and the central nervous system. In the peripheral nervous system (PNS), disrupting ADAMTS metalloproteases and basement membrane proteins impairs the post-embryonic maintenance of the morphology of neuron-muscle synapses (called neuromuscular junctions, or NMJs) (Cescon et al., 2018; Dear et al., 2016; Heikkinen et al., 2019; Kurshan et al., 2014; Qin et al., 2014; Singhal and Martin, 2011). Basement membrane proteins are also important for neuron-neuron synapses in the central nervous system (CNS) (Heikkinen et al., 2014). However, unlike NMJs in the PNS, most neuron-neuron synapses in the CNS are not in direct contact with the basement membrane (Heikkinen et al., 2014; Krishnaswamy et al., 2019). How the basement membrane sustains CNS neuron-neuron synapses, particularly during brain allometric growth, remains unknown.

Sustaining the relative synaptic positions during growth, and therefore embryonically derived synaptic specificity, is important for sustaining circuit integrity. As an animal grows, organs scale in different proportions relative to body size. This conserved principle is termed ‘allometry’ (Huxley, 1924; Huxley, 1936). For relevance to the brain, neocortical white matter and grey matter scale differently from each other, indicating that specific sub-structures of the brain scale allometrically to total brain size (de Jong et al., 2017). Presynaptic partners, postsynaptic partners and non-neuronal cells that provide positional cues also scale allometrically during growth. We do not know the underlying mechanisms that sustain embryonically-derived circuit architecture as different tissues disproportionately grow in size.

The nematode C. elegans provides a tractable genetic model to examine questions related to sustaining synaptic specificity during growth (Shao et al., 2013). After hatching from its egg, C. elegans grows an order of magnitude in length during post-embryonic growth (Knight et al., 2002). The architecture of the nervous system, which is established during embryogenesis, is largely preserved during this process (Bénard and Hobert, 2009). The use of cell-specific promoters in conjunction with in vivo probes permits visualizing and tracking synapses in single neurons of known identity during the lifetime of the organism (Colón-Ramos et al., 2007; Nonet, 1999).

In our prior work, we identified cima-1 as a gene required for sustaining the synaptic pattern during growth (Shao et al., 2013). In cima-1 mutants, synaptic contacts are correctly established during embryogenesis, but ectopic pre-synaptic sites emerge as the animals grow. cima-1 encodes a novel solute carrier in the SLC17 family of transporters that includes Sialin, a protein that when mutated in humans produces neurological disorders (Verheijen et al., 1999). However, cima-1 does not function in neurons. Instead, it functions in nearby epidermal cells to antagonize the FGF Receptor, likely by inhibiting its role in epidermal-glia adhesion (Figure 1). Thus, cima-1 functions in non-neuronal cells during post-embryonic growth to preserve the synaptic pattern (Shao et al., 2013).

Figure 1 with 1 supplement see all
Download asset Open asset
Synaptic allometry in AIY neurons.

(A–C) Distribution of AIY synapses in wild-type animals, and model. (A–B) Confocal micrograph images of AIY presynaptic sites labeled with the synaptic vesicle marker mCherry::RAB-3 (pseudo-colored green) in wild-type larval stage 1 (L1) animals (A) and adult animals (B). Note that although animals grow (scale bars in A and B both correspond to 10 μm), in wild-type animals the synaptic pattern is sustained from L1 to adults. Asterisks indicate the synaptic-rich Zone 2 and brackets indicate the asynaptic Zone 1 regions of AIY (see Figure 2A). (C) Graphical abstract of the findings of Shao et al. (2013). In wild-type animals, CIMA-1 acts in epidermal cells to suppress the epidermally derived FGF Receptor/EGL-15, which in turn maintains VCSC glia morphology, which likely mediates adhesion between the epidermal cell and glia. In cartoon, epidermal cells in beige, glia in red, AIY neuron in grey, synapses in green, Zone 2 region indicated by asterisk and stitches represent contact sites between the epidermis and glia. Also outlined in grey dashed lines, the position of the pharynx for reference. (D–F) As (A–C), but for cima-1(wy84) loss-of-function mutants. In cima-1 loss-of-function mutants, EGL-15(5A)/FGF Receptor protein levels are upregulated, and this promotes adhesion of epidermis to glia and causes glia position and morphology defects during growth (F). This in turn extends the glia-AIY contact site to the asynaptic Zone 1 region, causing ectopic synapse formation in Zone 1 (see also Figure 1—figure supplement 1C–F). Blue arrow in (F) represent the changes in glia position and morphology due to increased interaction with epidermal cells, and green arrow marks ectopic synapses in Zone 1 (brackets). (G–H) As in (A–B), but in cima-1(wy84);ola226 double mutants. Note that the cima-1 synaptic phenotype (E) is suppressed in the cima-1(wy84);ola226 double mutant (H). (I) Schematic model of the multi-tissue CIMA-1 regulation of synaptic allometry in AIY. The scale bars in (A) apply to (D and G), and scale bars in (B) apply to (E and H). Both are 10 μm.

To further determine the cellular and molecular mechanisms that regulate the synaptic pattern during growth, we performed suppressor forward genetic screens in the cima-1 mutant background, and identified mig-17, encoding a secreted ADAMTS metalloprotease (Nishiwaki et al., 2000). We find that the secreted mig-17 modulates muscle-derived basement membrane proteins. The synapses examined in this study are not in direct contact with the basement membrane. Instead, the basement membrane coats the side of glia facing the pseudocoleum, while glia contact synapses on their other side facing the nerve ring. We find that MIG-17 modifies the muscle-derived basement membrane to modulate epidermal-glial crosstalk and sustain glia location and morphology during growth. Glia location and morphology in turn sustains the presynaptic pattern as the animal grows. Therefore a muscle-epidermis-glia signaling axis, modulated by mig-17 and the basement membrane, regulates synaptic allometry during growth.

Results

Mutant allele ola226 suppresses synaptic allometry defects in cima-1 (wy84)

AIY interneurons are a pair of bilaterally symmetric neurons in the C. elegans nerve ring. AIYs display a stereotyped and specific pattern of presynaptic specializations (Colón-Ramos et al., 2007; White et al., 1986). This pattern is established during embryogenesis. Even though animals grow an order of magnitude in length from early embryogenesis to adulthood (from ~100 μm to ~1 mm) (Knight et al., 2002; Shibata et al., 2016), the AIY synaptic pattern is sustained during growth (Figure 1A–C and Shao et al., 2013). Here, we term this process of sustaining the synaptic pattern during growth ‘synaptic allometry’. Synaptic allometry requires coordination between different tissues to sustain the relative pre- and postsynaptic positions during growth (Shao et al., 2013). Which cell types are required, and how they signal to coordinately sustain synaptic allometry is not well understood.

Using forward genetic screens, we previously identified cima-1 as a gene required for sustaining the synaptic pattern during growth (Shao et al., 2013). In cima-1(wy84) mutants, the embryonic AIY synaptic pattern developed correctly (Figure 1D). However, during growth, synaptic positions were disrupted and ectopic presynaptic sites emerged in the Zone 1 region, a normally asynaptic region of the AIY neuron (Figure 1E–F and Shao et al., 2013). cima-1 encodes a solute carrier transporter required in epidermal cells to antagonize the FGF receptor and likely modulate epidermal-glia adhesion (Shao et al., 2013 and Figure 1I). cima-1(wy84) mutants result in defects in the ventral cephalic sheath cell (VCSC) glia position and morphology during growth (Figure 1—figure supplement 1A–B). Abnormal VCSC glia ectopically ensheath the normally asynaptic Zone 1 region of AIY, which causes ectopic presynaptic sites in Zone 1 that are not in apposition to AIY’s wild-type postsynaptic partner, the RIA neurons (Figure 1E–F,I, Figure 1—figure supplement 1C–F and Shao et al., 2013). Therefore, in cima-1 mutants, abnormal glia morphology and position during growth of the organism resulted in changes to the relationship between the glia and the neurite, which in turn disrupted the embryonically established synaptic pattern as the animal grew (Figure 1F and I). To identify molecules which cooperate with cima-1 to regulate synaptic allometry, we performed an unbiased EMS screen in cima-1(wy84) mutants for suppressors of defects in the synaptic pattern, and isolated allele ola226 (Figure 2).

Figure 2 with 1 supplement see all
Download asset Open asset
Mutant allele ola226 suppresses cima-1 (wy84) synaptic allometry defects in AIY.

(A) Cartoon diagram of the distribution of presynaptic sites in the AIY interneurons of the nematode C. elegans. The head of C. elegans (solid black lines) and the pharynx (dashed grey line) are outlined. A single AIY interneuron is depicted in gray, an oval represents the cell body and a solid gray line represents the neurite. Presynaptic puncta are green. The AIY neurites can be subdivided into three zones: an asynaptic region proximal to the cell body called Zone 1, a synapse-rich region called Zone 2 (asterisk) and a region with sparse synapses, called Zone 3. The red (b) and blue (a) dashed lines represent synaptic distribution and correspond to Zone 2 and 3 (respectively) in wild-type animals. The dotted box represents the region of the head imaged in B-D’. (B–D’’) Confocal micrograph images of AIY presynaptic sites labeled with the synaptic vesicle marker mCherry::RAB-3 (pseudo-colored green, B–D) and active zone protein GFP::SYD-1 (pseudo-colored red, (B’–D’) for wild type (B, B’, B’’), cima-1(wy84) mutants (C, C’, C’’) or cima-1(wy84);ola226 (D, D’, D’’). Merged images display co-localization of synaptic vesicle marker mCherry::RAB-3 and active zone protein GFP::SYD-1 in (B”–D”). Schematic diagrams of the observations are depicted in (B’’’–D’’’). Scale bar in (B) applies to all images, 10 μm. Asterisk: Zone 2 region; Arrows: ectopic synapses in Zone 1 region (see also Figure 1—figure supplement 1C–F). (E) Quantification of the percentage of animals displaying ectopic AIY presynaptic sites in the Zone 1 region for indicated genotypes. (F) Quantification of the ratio of ventral synaptic length (see red (b) to total synaptic region (sum of the length of blue (a) and red (b) in schematic in (A and B’’’–D’’’)). The total number of animals (N) and the number of times scored (n) are indicated in each bar for each genotype as N/n. Error bars represent SEM. Statistical analyses are based on one-way ANOVA by Tukey’s multiple comparison test, ****p<0.0001 as compared to wild type (if on top of bar graph), unless brackets are used between two compared genotypes.

Although the animal’s morphology and the guidance of AIY neurites are largely unaffected in cima-1(wy84);ola226 double mutants (Figure 2—figure supplement 1A–C), we found that ola226 suppressed the ectopic distribution of both the vesicular marker RAB-3 and the active zone marker SYD-1 in cima-1(wy84) (Figure 1H, and Figure 2A–D’’’). Young cima-1(wy84);ola226 animals displayed a wild-type pattern of presynaptic specializations (Figure 1G), suggesting that the ola226 allele does not generally affect synaptogenesis. Instead, the ola226 allele robustly suppresses the synaptic allometry defects observed in cima-1(wy84) mutants, as scored by the percentage of animals displaying ectopic presynaptic sites in the Zone 1 region and the relative presynaptic length in the neurite (93.9% of animals displayed ectopic presynaptic sites in cima-1(wy84) vs 54.6% in cima-1(wy84);ola226 double mutants, p<0.0001; Figure 2E–F). Together, these results indicate that the ola226 allele is specifically required for the suppression of the ectopic presynaptic specializations that form post-embryonically in the cima-1(wy84) mutants.

Mutant allele ola226 suppresses glia position and morphology defects in cima-1 mutants

The emergence of ectopic presynaptic sites in cima-1(wy84) mutants requires ventral cephalic sheath cell (VCSC) glia extension during growth (Shao et al., 2013). Therefore growth, and the size of the animal, affect the expressivity of the allometry phenotypes in cima-1(wy84) mutants. For example, shorter dpy mutants suppress cima-1(wy84) synaptic allometry defects, while the longer lon mutants enhance cima-1(wy84) synaptic allometry defects (Figure 2—figure supplement 1D–I’ and Shao et al., 2013). We examined the size of ola226 and cima-1(wy84);ola226 adult mutant animals and determined that it is indistinguishable from wild-type animals (Figure 2—figure supplement 1C), indicating that the effects of ola226 in the cima-1(wy84) phenotype is through mechanisms distinct from those regulating the general size of the animal during development.

Next, we examined if ola226 could alter VCSC glia morphology. We labeled VCSC glia with mCherry in wild type and the mutants, and quantified VCSC glia position and morphology (Figure 3). Consistent with and extending our previous observations, we observed that the VCSC glia in cima-1(wy84) mutants displayed defects in both position and morphology during growth. As cima-1 mutant animals grew, VCSC glia were posteriorly displaced, resulting in longer VCSC glia anterior processes (mean length of the VCSC glia anterior process: 113.35 μm in wild type, 127.53 μm in cima-1(wy84) mutants, p<0.0001. Figure 3B,C,F). cima-1 mutants glia endfeet also abnormally extended posteriorly (mean length of VCSC glia endfeet: 45.52 μm in wild type and 51.47 μm in cima-1(wy84) mutants, p<0.0001. Figure 3B,C,G). These two defects changed the positions of VCSC glia relative to the AIY neurite, resulting in ectopic presynaptic sites in cima-1 mutant animals (Figure 3B’, C’, H). The AIY ectopic presynaptic sites in cima-1 mutant animals are not in apposition to the normal postsynaptic RIA neurons (Figure 1—figure supplement 1C–F). Ablation of VCSC glia suppressed the ectopic presynaptic phenotype in cima-1 mutants (Shao et al., 2013), indicating the importance of glia for the emergence of these ectopic presynaptic sites that disrupt the embryonically derived pattern of synaptic connectivity.

Figure 3 with 1 supplement see all
Download asset Open asset
Glia morphology is affected in ola226 mutants.

(A) Cartoon diagram of the ventral and dorsal cephalic sheath cell glia (red) in the C. elegans head. The ventral cephalic sheath cell (VCSC) glia, located at the bottom half in the schematic, contacts the AIY synapses in the Zone 2 region. (B–E’) Confocal micrographs of the morphology of VCSC glia and the anterior process (red, labeled with Phlh-17::mCherry, (B–E), or VCSC glia cell body and endfeet (red) with the AIY presynaptic marker (green, GFP::RAB-3, (B’–E’) in adult wild type (B, B’), cima-1(wy84) mutants (C, C’), cima-1(wy84);ola226 mutants (D, D’), and ola226 mutants (E, E’). Brackets indicate the AIY Zone 1 region, and asterisks mark the AIY Zone 2 region (see Figure 2A). The animals imaged in B-E are not the same as B’-E’. (F–H) Quantification of phenotypes, including the length of glia anterior process (F, indicated in schematic A), the length of ventral endfeet (G, indicated in schematic A) and the percentage of animals displaying overlap between AIY synapses and VCSC glia in Zone 1 (H). The total number of animals (N) and the number of times scored (n) are indicated in each bar for each genotype as N/n. Statistical analyses are based on one-way ANOVA by Tukey’s multiple comparison test. Error bars represent SEM, N.S.: not significant as compared to wild type, ****p<0.0001 as compared to wild type (if on top of bar graph), unless brackets are used between two compared genotypes.

cima-1(wy84);ola226 double mutants suppressed VCSC glia position and endfeet morphology phenotypes (length of glia anterior process: 127.53 μm in cima-1(wy84) and 120.68 μm in cima-1(wy84);ola226, p<0.0001; length of VCSC glia endfeet: 51.47 μm in cima-1(wy84) and 45.19 μm in cima-17(wy84);ola226, p<0.0001. Figure 3D,F–G). In these double mutants, the suppression caused a reduction in the abnormal region of contact seen in cima-1(wy84) mutants for the AIY neuron and VCSC glia (88.70% in cima-1(wy84) and 33.67% in cima-1(wy84);ola226, p<0.0001. Figure 3D’, H). Consequently, ectopic presynaptic specializations that arise during growth in the AIY Zone 1 of cima-1 mutants were suppressed, resulting in a synaptic pattern similar to that observed for wild type animals (Figure 3B’, D’). Our findings suggest that ola226 is a genetic lesion that suppresses cima-1(wy84) ectopic presynaptic sites by regulating glia position and morphology during allometric growth.

To better understand the phenotype of ola226, we outcrossed cima-1(wy84) and examined the resulting VCSC glia and AIY synaptic phenotypes for just the ola226 mutants. We found that ola226 mutant animals do not display defects in the position of VCSC glia (length of glia anterior process: 113.35 μm in wild type and 113.68 μm in ola226, p=0.72. Figure 3E,F). However, ola226 mutants did display a modest but significant defect in VCSC glia morphology, with shorter posterior end-feet in ola226 animals as compared to wild-type animals (length of glia end-feet: 45.52 μm in wild type, 39.79 μm in ola226 p<0.0001. Figure 3E,G). ola226 mutants also displayed a concomitant defect in the position of AIY, as both the neurite and the soma were anteriorly displaced compared to wild type animals (Figure 3—figure supplement 1). This anterior displacement of VCSC glia and AIY are the opposite phenotype to that observed for cima-1(wy84) mutants, in which these cells are posteriorly displaced (Shao et al., 2013). Interestingly, unlike in cima-1(wy84) mutants, in the ola226 mutants the area of overlap between the glia and AIY was not affected (Figure 3E’, H). The distribution of presynaptic specializations in these animals was similar to that seen for wild type (Figure 3B’, E’), consistent with the importance of glia position in sustaining presynaptic positions.

These phenotypes demonstrate that it is not just glia morphology, glia position or even the position of the AIY neurite in the animal that regulates synaptic allometry. Rather, the relative position between the VCSC glia and the AIY neurons appears to drive presynaptic positions during growth. Our data underscore the role of glia as guideposts in sustaining the synaptic pattern during post-embryonic growth.

ola226 is a lesion in mig-17, which encodes an ADAMTS metalloprotease

To identify which gene is affected in the ola226 allele, we performed SNP mapping, whole genome sequencing and transgenic rescue experiments. The ola226 allele results from a G to A mutation at the end of first exon of the mig-17 gene and alters a conserved glutamic acid residue at position 19 to a lysine (Figure 4A). To test if ola226 is a loss-of-function allele of mig-17, we examined two additional loss-of-function mig-17 alleles, mig-17(k113) and mig-17(k174) (Nishiwaki, 1999; Nishiwaki et al., 2000). mig-17(k113) is a point mutation in the first intron of the gene and is predicted to affect correct splicing, while the mig-17(k174) allele results from a change in Q111 to a premature stop codon, producing a putative null allele (Figure 4A; Shibata et al., 2016). We found that just like ola226, both k113 and k174 alleles did not display phenotypes in the AIY presynaptic distribution on their own (Figure 4—figure supplement 1), yet robustly suppressed the ectopic presynaptic sites in cima-1(wy84) mutants (91.9% of animals displayed ectopic presynaptic sites in cima-1(wy84), 62.3% in cima-1(wy84);mig-17(k113), 29.9% in cima-1(wy84);mig-17(k174) and 45.7% in cima-1(wy84);mig-17(ola226), p<0.0001 for all double mutants as compared to cima-1(wy84); Figure 4B–F,H). Importantly, introducing a wild-type copy of the mig-17 genomic sequence results in robust rescue of the ola226 phenotype in cima-1(wy84);mig-17(ola226) double mutants (45.70% of animals displayed ectopic synapses in cima-1(wy84);mig-17(ola226) and 78.04% in cima-1(wy84);mig-17(ola226);Pmig-17::mig-17(genomic), p<0.0001; Figure 4G,H). Together our findings indicate that ola226 is a recessive loss-of-function allele of mig-17 which suppresses cima-1(wy84) defects in synaptic allometry by affecting glia positions during growth.

Figure 4 with 2 supplements see all
Download asset Open asset
ola226 is a lesion in the mig-17 gene.

(A) Schematic diagram of the mig-17 gene and corresponding protein domains coded by the exons (colored) and genetic lesions for the alleles used in this study. (B–G) Confocal micrographs of the AIY synaptic vesicle marker GFP::RAB-3 (green) in adult wild type (B), cima-1(wy84) (C), cima-1(wy84);mig-17(ola226) (D), cima-1(wy84);mig-17(k113) (E), cima-1(wy84);mig-17(k174) (F), and cima-1(wy84);mig-17(ola226) animals expressing a wild-type copy of the mig-17 gene (Pmig-17::mig-17(genomic)) (G). Brackets indicate the AIY Zone 1 region. Asterisks indicate the Zone 2 region. Scale bar in (B) applies to all images, 10 μm. (H) Quantification of the percentage of animals with ectopic synapses in the AIY Zone 1 region for the indicated genotypes. The total number of animals (N) and the number of times scored (n1) are indicated in each bar for each genotype and for the transgenic lines created, the number of transgenic lines (n2) examined (all using the convention N/n1/n2). Statistical analyses are based on one-way ANOVA by Tukey’s multiple comparison test. Error bars represent SEM, **p<0.01, ****p<0.0001 as compared to cima-1 (wy84) (if on top of bar graph), unless brackets are used between two compared genotypes.

MIG-17 is an ADAMTS metalloprotease best known for its post-embryonic roles in regulating distal tip cell migration during gonad development (Nishiwaki, 1999) and pharyngeal size and shape during growth (Shibata et al., 2016). ADAMTS proteins have also been shown to regulate the basement membrane to maintain synaptic morphology at neuromuscular junctions (NMJs) (Kurshan et al., 2014; Qin et al., 2014). Careful examination of the pharynx length and the synaptic allometry defects in AIY revealed that the AIY synaptic allometry phenotypes do not simply arise from a defect in pharynx length (Figure 4—figure supplement 2). Unlike the NMJs, the basement membrane is not in direct contact with synapses in the nerve ring, including the AIY synapses (White et al., 1986). Therefore, the basement membrane cannot signal directly to AIY synapses as it does to the NMJs (Kurshan et al., 2014; Qin et al., 2014). Instead, our collective findings suggest that MIG-17 modulates synaptic allometry in AIY through the modulation of VCSC glia position and morphology.

MIG-17 is expressed in muscles and neurons in the nerve ring

To examine how MIG-17 modulates synaptic allometry through the modulation of glia position and morphology, we next analyzed the expression pattern of mig-17 in the nerve ring region. We found that a mig-17 transcriptional GFP reporter was robustly expressed by body wall muscles as colabeled by Pmyo-3::mCherry (Figure 5A–A’’’ and consistent with Nishiwaki et al., 2000). We also observed that in the head region, the reporter was detected in the nervous system (Figure 5B–B’’’). We did not detect expression of MIG-17 in VCSC glial cells or in epidermal cells, where the MIG-17 genetic interactors CIMA-1 and EGL-15/FGFR are expressed (Figure 5C–D’’’; Shao et al., 2013).

Figure 5 with 1 supplement see all
Download asset Open asset
MIG-17 is a secreted molecule that regulates synaptic allometry.

(A–D”’) Confocal micrographs of adult animals expressing the transcriptional reporter mig-17(genomic)::SL2::GFP (green) with reporters that co-label body wall muscles (Pmyo-3::mCherry (A–A”’)), neurons (Prab-3::mCherry (B–B”’)), VCSC glia (Phlh-17::mCherry (C–C”’)), epidermal cells (Pdpy-4::mCherry (D–D”’)). Images (A’–D”’) correspond to a transverse cross-section of the confocal micrographs, specifically for the region corresponding to the dashed line in (A–D). The scale bar in (A) applies to (B, C, D), and in (A’) applies all transverse cross-section images, and both scale bars are 10 μm. (E) Quantification of the percentage of adult animals with ectopic synapses in the AIY Zone 1 region of the indicated genotypes and rescue experiments. The total number of animals (N) and the number of times scored (n1) are indicated in each bar for each genotype, as are, for the transgenic lines created, the number of transgenic lines (n2) examined (all using the convention N/n1/n2). See also Figure 5—figure supplement 1 for additional rescue experiments. Statistical analyses are based on one-way ANOVA by Tukey’s multiple comparison test. Error bars represent SEM, N.S.: not significant, ****p<0.0001 compared to the no-transgene control (if on top of bar graph), unless brackets are used between two compared genotypes.

To determine the mig-17 site of action, we expressed mig-17 in the two tissues that showed mig-17 expression: the nervous system (using the rab-3 promoter Nonet et al., 1997); and the body wall muscles using the myo-3 promoter Miller et al. (1983); Miller et al. (1986). We found robust rescue of the cima-1(wy84);mig-17(ola226) phenotype when mig-17 was expressed either in body wall muscles or in the nervous system (Figure 5E), consistent with MIG-17 being a secreted ADAMTS protease. Indeed, expression of MIG-17 from a number of different cell-specific promoters, including glia and epidermal cells in which we did not detect expression, all resulted in rescue (Figure 5—figure supplement 1). Together, our findings suggest that secreted MIG-17 modulates glia morphology and synaptic allometry.

MIG-17 requires its metalloprotease activity to promote the formation of ectopic presynaptic sites in cima-1(wy84) mutants

MIG-17 is an ADAMTS metalloprotease which remodels the basement membrane (Nishiwaki et al., 2000). To determine if MIG-17 acts through its canonical role of remodeling the basement membrane to regulate synaptic allometry, we first examined if its metalloprotease enzymatic activity was required for promoting the formation of ectopic synapses in cima-1(wy84) mutants. We engineered an E303A point mutation at the metalloprotease catalytic site (Nishiwaki et al., 2000) via CRISPR/cas-9 to generate the mig-17(shc8) allele (Figure 6A, CRISPR strategy outlined in Figure 6—figure supplement 1A is based on Dickinson et al., 2013; Nishiwaki et al., 2000). We observed that our engineered mig-17(shc8) allele behaved like other mig-17 loss-of-function alleles and suppressed ectopic synapses in cima-1(wy84) mutant animals (91.91% of animals displayed ectopic synapses in cima-1(wy84) vs 57.49% in cima-1(wy84);mig-17(shc8), p<0.0001, Figure 6B–E,H). Consistent with this result, we also found that a transgene with the E303A (mig-17(E303A)) lesion is incapable of rescuing the mig-17-induced suppression in mig-17(ola226);cima-1(wy84) mutants (Figure 6F–H). These findings indicate that MIG-17 metalloprotease enzymatic activity is required for promoting the formation of ectopic synapses in cima-1(wy84) mutants, and are consistent with a model whereby MIG-17 remodels the basement membrane to modulate synaptic allometry during growth.

Figure 6 with 1 supplement see all
Download asset Open asset
The metalloprotease activity of MIG-17 is required to suppress the formation of ectopic synapses in cima-1(wy84) mutants.

(A) Schematic diagram of the MIG-17 protein, corresponding conserved protein domains (colored) and genetic lesions for the alleles used in this study. (B–G) Confocal micrographs of the AIY presynaptic sites labeled with the synaptic vesicle marker GFP::RAB-3 (pseudo-colored green) in adult wild type (B), cima-1(wy84) (C), cima-1(wy84);mig-17(ola226) (D), cima-1(wy84);mig-17(shc8) (E), cima-1(wy84);mig-17(ola226) animals expressing a wild type copy of the mig-17 genomic DNA (Pmig-17::mig-17) (F), and cima-1(wy84);mig-17(ola226) animals expressing a copy of the mig-17 genomic DNA with a point mutation in the metalloprotease domain (Pmig-17::mig-17(E303A)) (G). Brackets indicate the AIY Zone 1 region, and asterisks indicate the Zone 2 region. The scale bar in (B) is 10 μm and applies to all images. (H) Quantification of the percentage of animals with ectopic synapses in the AIY Zone 1 region in the indicated genotypes. In the graph, the transgene rescue with wild type copy of the mig-17 genomic DNA control data is the same as in Figure 4H. The total number of animals (N) and the number of times scored (n1) are indicated in each bar for each genotype, as are, for the transgenic lines created, the number of transgenic lines (n2) examined (all using the convention N/n1/n2). Bars are pseudocolored by experiments, with controls in black, comparisons across mig-17 alleles in blue and rescue experiments in red. Statistical analyses are based on one-way ANOVA by Tukey’s multiple comparison test. Error bars represent SEM, N.S.: not significant, ****p<0.0001 compared to wild type (if on top of bar graph), unless brackets are used between two compared genotypes.

MIG-17 regulates basement membrane proteins to modulate synaptic allometry

To determine if MIG-17 remodels the basement membrane to modulate synaptic allometry, we examined the proteome through liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses in wild type and mig-17(ola226) mutant animals. Consistent with the known importance of MIG-17 in remodeling the basement membrane in other biological contexts (Kim and Nishiwaki, 2015), we observed significant and reproducible differences in the protein levels of basement membrane components for mig-17(ola226) mutants compared to wild type, including EMB-9/Collagen IV α1 chain, LET-2/Collagen IV α2 chain, OST-1/Sparc, UNC-52/Perlecan, NID-1/nidogen, EPI-1/laminin-α, LAM-1/laminin-β, and LAM-2/laminin-γ (Figure 7A and Supplementary file 1).

Figure 7 with 1 supplement see all
Download asset Open asset
MIG-17 modulates synaptic allometry through the regulation of the basement membrane.

(A) List of basement membrane components upregulated in the mass spectrometry analyses (see also Supplementary file 1), and alleles tested with cima-1 for their capacity to suppress the synaptic allometry phenotypes in adult worms. (B) Quantification of the percentage of animals with ectopic synapses in the Zone 1 region of AIY for the indicated the genotypes. Bars are pseudocolored by experiment, with black bars corresponding to controls, light pink bars corresponding to emb-9 alleles, and red bars corresponding to alleles for genes known to regulate emb-9, such as unc-52 and fbl-1. (C–F) Confocal micrographs of the pharynx (dashed line) of animals with a CRISPR-engineered MIG-17::mNeonGreen imaged at larva stage 1 (C), larva stage 3 (D), larva stage 4 (E) and 1 day-old adults (F) in wild-type animals. (G) Quantification of the average MIG-17::mNeonGreen intensity in the pharyngeal area (outlined with dashed lines in C-F) at the indicated developmental stages. (H–L) As (C–G), but imaging an integrated EMB-9::mCherry strain (a gift from David Sherwood) in wild type animals. (M–Q) As (H–K) but in adults of wild type (M); cima-1(wy84) (N); mig-17(ola226) (O); cima-1(wy84);mig-17(ola226) (P) and quantified in (Q). The statistics are based on one-way ANOVA by Tukey’s multiple comparison test. In the graphs, the total number of animals (N) and the number of times scored (n) are indicated in each bar for each genotype as N/n. Error bars represent SEM, N.S.: not significant, **p<0.01, ***p<0.001, ****p<0.0001 for indicated comparison. For all images, scale bars are 10 μm. The scale bar in (M) applies to (N–P).

EMB-9/Collagen IV α1 is a core component of the basement membrane regulated by ADAMTS proteins (Graham et al., 1997; Guo et al., 1991; Sibley et al., 1993) and plays important roles in post-embryonic neuromuscular junction morphology (Kurshan et al., 2014; Qin et al., 2014). We wondered whether the AIY presynaptic sites, which have a different relationship to BM than do NMJs, would have altered morphology in emb-9 mutant animals. Since EMB-9 null alleles are embryonic lethal (Guo et al., 1991; Gupta et al., 1997), we used neomorphic or hypomorphic missense alleles that disrupt NMJ morphology and are predicted to produce overabundant or disorganized collagen (Gotenstein et al., 2018; Gupta et al., 1997; Kubota et al., 2012; Kurshan et al., 2014; Qin et al., 2014). We did not observe detectable defects in the AIY presynaptic site distribution or morphology in emb-9(xd51) or emb-9(b189) mutants (Figure 7—figure supplement 1).

Interestingly, EMB-9/Collagen IV can also become overabundant or disorganized in ADAMTs mutants (Kim and Nishiwaki, 2015). We therefore hypothesized that the neomorphic or hypomorphic alleles of emb-9 could phenocopy mig-17 mutants and suppress the synaptic allometry defects for cima-1 mutants. Indeed, we observed that neomorphic and hypomorphic emb-9 alleles significantly suppressed the ectopic presynaptic sites in cima-1(wy84) mutant animals, although the penetrance of the suppression phenotype varied by the specific allele (Figure 7B). Therefore, while the emb-9 alleles do not affect the morphology of AIY presynaptic sites (as they do for NMJ synapses), they significantly suppress the synaptic allometry defects for cima-1 mutants.

We hypothesized that cima-1 mutants are suppressed both by mig-17 and the neomorphic and hypomorphic emb-9 alleles because in these mutants the material properties of the basement membrane are altered. This, in turn, would prevent the movement of glia during growth and suppress the ectopic contacts between glia and AIY seen for cima-1 mutants. If our hypothesis were correct, we would expect that other molecules known to modulate the levels or conformation of EMB-9 would also similarly affect synaptic allometry, as basement membrane properties would be altered. To test this, we imaged AIY presynaptic sites in alleles of unc-52/Perlecan and fbl-1/Fibulin, both of which can regulate the trafficking or function of EMB-9 (Kubota et al., 2004; Kubota et al., 2012; Morrissey et al., 2016; Qin et al., 2014). Consistent with our model, loss-of-function alleles of unc-52/Perlecan, which is known to functionally antagonize EMB-9/Collagen IV (Qin et al., 2014), significantly suppressed the ectopic presynaptic sites observed in cima-1(wy84) mutants (Figure 7—figure supplement 1 and Figure 7B). Similarly, the gain-of-function fbl-1(k201) allele (Kubota et al., 2004), which is predicted to cause an overabundance of EMB-9 (Kubota et al., 2012), also suppressed the ectopic presynaptic sites observed in cima-1(wy84) mutants (Figure 7—figure supplement 1 and Figure 7B). We also determined that the levels of suppression in cima-1(wy84);mig-17(ola226);fbl-1(k201) are similar to those seen in either cima-1(wy84);mig-17(ola226) or in cima-1(wy84);fbl-1(k201) mutants, consistent with mig-17 and fbl-1 genetically acting in the same pathway (Figure 7B).

Our findings indicate that while the different alleles of emb-9 and emb-9-regulators might have different effects on the conformation or levels of the EMB-9 protein in the basement membrane, they all suppress the ectopic presynaptic site phenotype in cima-1(wy84) mutants. Their shared ability to suppress cima-1(wy84) mutants suggests that lesions resulting in defects in the basement membrane might prevent the repositioning of glia that gives rise to the ectopic presynaptic sites in cima-1(wy84) mutants.

MIG-17 regulates EMB-9/Collagen IV α1 during post-embryonic growth

To better elucidate the relationship between MIG-17 and EMB-9 during growth, we examined MIG-17 and EMB-9 protein levels in vivo during post-embryonic development using an EMB-9::mCherry translational reporter (Ihara et al., 2011) and a MIG-17::mNeonGreen knock-in allele (via CRISPR-Cas9 strategies as described in Dickinson et al., 2013; Figure 6—figure supplement 1B). We observed that both MIG-17 and EMB-9 localize in the head-region to a pattern reminiscent of the extracellular matrix proximal to the pharynx bulb (Ihara et al., 2011). We also determined that the levels of MIG-17 and EMB-9 were regulated during post-embryonic growth. MIG-17 protein levels were detectable in larva stage one through larva stage 4, but became undetectable upon reaching the adult stage (Figure 7C–G, these results are consistent with previous in situ and western blot studies; Ihara and Nishiwaki, 2008). Conversely, EMB-9 protein levels increased as animals progress through the larval stages, achieving maximal expression in the adult stage (Figure 7H–L). Therefore, during post-embryonic growth, high protein levels of MIG-17 correlate with low protein levels of EMB-9.

The in vivo characterization of the protein levels of MIG-17 and EMB-9 are consistent with our proteomic results, and suggest that, directly or indirectly, MIG-17 regulates EMB-9 and basement membrane properties. Consistent with these findings, EMB-9::mCherry levels in mig-17(ola226) mutant animals were upregulated as compared to wild type (Figure 7M–Q). Interestingly, this increase in EMB-9 levels observed for mig-17(ola226) mutant was suppressed in cima-1(wy84);mig-17(ola226) double mutants, suggesting the existence of other cima-1-dependent mechanisms that modulate EMB-9 levels in the absence of MIG-17 (Figure 7P and Q). Importantly, our observations indicate that MIG-17 regulates EMB-9 and basement membrane properties to modulate synaptic allometry during post-embryonic growth.

Together, our findings support a model in which secreted metalloprotease MIG-17, whose levels are regulated during post-embryonic growth, dynamically regulates the muscle-derived basement membrane. Through regulation of the basement membrane, MIG-17 modulates cima-1-dependent epidermal-glial crosstalk to regulate glia position and morphology and sustain synaptic allometry during growth.

MIG-17 and EGL-15/FGFR promote ectopic presynaptic site formation in cima-1(wy84)

CIMA-1 modulates epidermal-glial cell adhesion via regulation of EGL-15/FGFR ectodomain which acts, not in its canonical signaling role, but as an extracellular adhesion factor (Bülow et al., 2004; Shao et al., 2013). Consistent with this model, CIMA-1 is required to regulate EGL-15(5A)/FGFR protein levels, and overexpression of the EGL-15(5A)/FGFR ectodomain in wild-type animals phenocopied cima-1 mutants (Shao et al., 2013). What is the relationship between MIG-17 and the glia-epidermis contacts modulated by CIMA-1 and EGL-15(5A)/FGFR?

We first examined if mig-17 mutants could enhance egl-15/FGFR suppression of cima-1. We determined that cima-1(wy84);egl-15(n484) double mutants, cima-1(wy84);mig-17(ola226) double mutants and cima-1(wy84);mig-17(ola226);egl-15(n484) triple mutants all suppressed the cima-1(wy84) phenotype of ectopic presynaptic sites in a similar manner (Figure 8A–D). We note that while the observed suppression was not a complete reversion to wild-type phenotypes, it is consistent with the degree of suppression observed for glia-ablated animals (Shao et al., 2013). Importantly, these findings indicate that alleles for mig-17 and egl-15 similarly suppress the cima-1 phenotype, and are incapable of enhancing each other’s effect on the suppression of cima-1, consistent with them acting in different tissues, but in similar genetic pathways to suppress cima-1 mutant defects in synaptic allometry.

Figure 8
Download asset Open asset
MIG-17 genetically interacts with EGL-15/Fibroblast Growth Factor Receptor to regulate synaptic allometry.

(A–C) Confocal micrographs of the AIY synaptic vesicle marker GFP::RAB-3 (green) in adult wild type (A), cima-1(wy84) (B), cima-1(wy84);egl-15(n484) (C). (D) Quantification of percentage of animals with ectopic synapses in the indicated genotypes. (E–G) Confocal micrographs of AIY synaptic vesicle marker GFP::RAB-3 (green) and VCSC glia (red) in adult wild type (E), wild-type animals overexpressing EGL-15(isoform 5A) in epidermal cells by using Pdpy-7::egl-15(5A) (F) and mig-17(ola226) overexpressing EGL-15(isoform 5A) in epidermal cells by using Pdpy-7::egl-15(5A) (G). (H–I) Quantification of percentage of animals with ectopic synapses (H) or ectopic glia (I) in the indicated genotypes. (J) Schematic model of the multi-tissue regulation of synaptic allometry in AIY, as in Figure 1I, but with the new findings on mig-17. In all images (A–C, E–G), brackets indicate the AIY Zone 1 region, asterisks mark the Zone 2 region. Scale bar in (A), 10 μm, applies to all images. In the graphs (D, H, I), the total number of animals (N), the number of times scored (n1) are indicated in each bar for each genotype, as are, for the transgenic lines created, the number of transgenic lines (n2) examined (all using the convention N/n1/n2). Statistical analyses are based on one-way ANOVA by Tukey’s multiple comparison test. Error bars represent SEM, N.S.: not significant, **p<0.01, ***p<0.001, ****p<0.0001 as compared to wild type (if on top of bar graph), unless brackets are used between two compared genotypes.

To further probe the relationship between EGL-15(5A)/FGFR and MIG-17, we examined synapses and glia in animals overexpressing EGL-15(5A)/FGFR. Overexpression of EGL-15(5A)/FGFR in epidermal cells promotes VCSC glia end-feet extension and ectopic presynaptic sites in AIY. This result phenocopies cima-1(wy84) mutants, and supports the idea that cima-1 acts antagonistically to the EGL-15/FGF Receptor (Figure 8F,H,I and Shao et al., 2013). Interestingly, we observed that mig-17(ola226) suppresses VCSC glia extension and the AIY ectopic presynaptic sites that arise during postembryonic growth in animals over-expressing EGL-15(5A)/FGFR (Figure 8G–I). This result is consistent with MIG-17 and EGL-15/FGFR acting in the same inter-tissue synaptic allometry pathway.

Together, our genetic findings indicate that EGL-15(5A)/FGFR and MIG-17 genetically interact to position glia and regulate synaptic allometry during growth (Figure 8J). The finding that mig-17(ola226) suppresses VCSC glia extension and ectopic synapses in animals over-expressing EGL-15(5A)/FGFR indicates that mig-17 is epistatic to egl-15. While we cannot exclude the possibility that EGL-15(5A)/FGFR is a substrate of MIG-17, their epistatic relationship suggests that mig-17 acts downstream (or in parallel) to modulate the role of egl-15 in positioning glia and regulating synaptic allometry (Figure 8J). Together with our other findings, we favor a model whereby MIG-17 modifies the basement membrane to modulate the effects of CIMA-1 and EGL-15 regulated epidermal-glial crosstalk on glia location and morphology during growth.

VCSC Glia bridge epidermal-derived growth signals with the muscle-secreted basement membrane to sustain synaptic allometry

How do these molecules, which are derived from non-neuronal tissues (muscle cells and epidermal cells) that do not contact the synapses act together to regulate synaptic allometry? To understand this, we examined electron micrographs and fluorescent microscopy images that show the anatomical relationship among synapses in AIY interneurons, VCSC glia, epidermal cells, basement membrane and muscles (Altun, 2019; White et al., 1986).

The AIY Zone 2 synaptic region lies in the ventral base of the nerve ring bundle and is in direct contact with the nerve ring-facing side of VCSC glia (Altun, 2019; White et al., 1986). No basement membrane is observed between VCSC glia and nerve ring neurons (Figure 9 and Figure 9—figure supplement 1). On the pseudocoelom-facing side, VCSC glia contact two distinct non-neuronal tissues: epidermal cells and muscle-derived basement membrane. VCSC are in direct contact with epidermal cells, which regulate glia morphology during growth through the expression of CIMA-1 and the EGL-15/FGF Receptor (Figure 9A, Figure 9—figure supplement 1D and Shao et al., 2013). No basement membrane is observable between VCSC glia and epidermal cells (Figure 9B–D, Figure 9—figure supplement 1D). However, we observed that at regions where glia are apposed to muscle cells, VCSC glia were decorated with basement membrane on the side facing the pseudocoelom cavity (Figure 9B–D, Figure 9—figure supplement 1D). Thus, VCSC glia have three surface regions: direct contact with neurons (on the nerve ring-facing side), direct contact with the epidermal cells (on the pseudocoelom-facing side), and contact with muscle-derived basement membrane (also on the pseudocoelom-facing side) (Figure 9D, Figure 9—figure supplement 1D).

Figure 9 with 1 supplement see all
Download asset Open asset
Glia maintain synaptic allometry by bridging epidermal-derived growth signals with the muscle-secreted basement membrane.

(A) Schematic of the head of C. elegans, as in Figure 1C, with indicated tissues pseudocolored. Box corresponds to cross sections examined in (B–D). (B) Segmented electron micrograph from a wild type animal (JSH236 from White et al., 1986). The EM corresponds to the Zone 2 region of AIY with muscles (pseudo-colored green), basement membrane (BM, pseudo-colored red), VCSC glia (pseudo-colored teal), epidermal cell (pseudo-colored beige) and the ventral bundle of the nerve ring (pseudo-colored pink, including AIY Zone two pseudo-colored dark pink). (C) Zoom-in of the dashed-boxed region in (B). The pseudo-coloring opacity is decreased as to show that the basement membrane is specifically observed between muscle and VCSC glia, but not between glia and epidermal cells or between glia and neurons. (D) A cartoon diagram depicting the cross-section of the C. elegans nerve ring as shown in (C) (modified from WormAtlas.org), and represented as a molecular and cellular model of our in vivo data regarding the role of non-neuronal cells in glia position and morphology to regulate synaptic allometry during growth. As illustrated in the cartoon and the EM image, body wall muscle (green), the nerve ring (pink) and glia (teal) are proximal to the epidermal cells (beige). The nerve ring bundle is surrounded by VCSC glia, which contact it directly. At the other side of the glia cell, it faces the pseudocoelum and interacts with muscle-derived basement membrane (red) and epidermal cells (beige).

Our data collectively indicate that secreted MIG-17 modulates the basement membrane. Regulation of the basement membrane by MIG-17 during post-embryonic growth acts in opposition to the CIMA-1-mediated epidermal-glial crosstalk. Therefore, muscles and epidermal cells interact with glia on the pseudocoelom-facing side and cooperate to regulate glia position (and morphology) during growth. Glia contact synapses on their nerve ring-facing side and sustain synaptic positions. Our data suggest that glia act as guideposts during growth, translating growth information from epidermal cells and muscles to guide synaptic allometry and preserve the embryonically-derived synaptic patterns during post-embryonic growth.

Discussion

We uncovered a muscle-epidermis-glia signaling axis, modulated by mig-17 and the basement membrane, which sustains synaptic allometry during growth. Suppressor forward genetic screens in the cima-1 mutant background identified mig-17, which encodes a secreted ADAMTS metalloprotease (Nishiwaki et al., 2000). We found that secreted mig-17 modulates basement membrane proteins. The basement membrane does not directly contact the affected synapses. Instead, muscle-derived basement membrane coats the pseudocoelum-facing side of glia, while glia contact synapses on their other cellular side. MIG-17 is regulated during growth and remodels the basement membrane to modulate glia morphology, which then modulates presynaptic positions during growth. Our findings underscore the critical role of non-neuronal cells in sustaining synaptic allometry in vivo.

Glia act as guideposts to regulate presynaptic positions during growth. We previously demonstrated that glia play critical roles, both during embryonic development and during post-embryonic growth, to sustain presynaptic positions in C. elegans. During embryonic development, VCSC glia secrete a chemotrophic factor (Netrin) to coordinate synaptic spatial specificity between AIY and its post-synaptic partner, called RIA (Colón-Ramos et al., 2007). Notably, postsynaptic RIA is not necessary for AIY to correctly establish the position of presynaptic specializations, underscoring the role of non-neuronal cells in presynaptic positioning, and coordinated synapse assembly, during development (Colón-Ramos et al., 2007). During post-embryonic growth, the same VCSC glia are required to sustain presynaptic positions but through distinct, Netrin-independent signaling pathways (Shao et al., 2013). Our current study demonstrates that sustaining synaptic allometry depends on the relative position of the glia end-feet with respect to the AIY neurite. By using genetic and in vivo cell biological manipulations, we could alter the position of both VCSC glia and AIY. Even when both cells were mispositioned in the animal, as long as their contact relationship was sustained, correct synaptic allometry was sustained (Figure 3E–H). Our findings are consistent with vertebrate and invertebrate studies supporting essential roles for glia in regulating synaptic assembly and function in vivo (Allen and Eroglu, 2017; Van Horn and Ruthazer, 2019). We extend these findings to highlight a role for glia in sustaining the embryonically established synaptic pattern during post-embryonic allometric growth.

Glia morphology and positions are actively maintained during growth. Growth in C. elegans relies on coordinated signals from epidermal cells and body wall muscles (Chisholm and Hardin, 2005). Epidermal cells express genes that regulate molting, body morphogenesis and animal size (Chisholm and Hsiao, 2012a; Chisholm and Xu, 2012b). Body wall muscle contractions regulate elongation during embryogenesis, and influence epidermal cytoskeletal remodeling via tension-sensing mechanisms (Chisholm and Hsiao, 2012a; Chisholm and Xu, 2012b; Williams and Waterston, 1994). While we do not yet understand how organisms sense growth, our findings uncovered a cooperative signaling pathway that emerges from these two growth-regulating cell types to position glia, which then drives synaptic positioning during allometry. Our genetic studies demonstrate that secreted MIG-17 is epistatic to epidermally derived CIMA-1 and EGL-15/FGFR. These results show a multi-tissue, non-neuronal pathway that converges to transduce growth information and position glia to regulate synaptic allometry. Thus, our findings uncover a non-cell autonomous, two-component system that cooperates to transduce growth information to the nervous system through glia.

During post-embryonic growth, ADAMTS protease MIG-17 regulates the basement membrane to modulate synaptic allometry. In Drosophila, the development of the peripheral nervous system and the maintenance of central nervous system architecture require homologous ADAMTS Stl and AdamT-A proteins (Lhamo and Ismat, 2015; Skeath et al., 2017). In general, ADAMTS metalloproteases function to degrade and remodel the extracellular matrix (Krishnaswamy et al., 2019). In humans, lesions in ADAMTS genes produce biomedically important defects, including short stature and neuronal developmental disorders, among other problems (Cheng et al., 2018; Howell et al., 2012; Miguel et al., 2005). Remodeling the extracellular matrix in C. elegans also contributes to gonad organogenesis and pharynx growth. These processes are partially mediated by the MIG-17 metalloprotease (Kim and Nishiwaki, 2015; Kubota et al., 2004; Kubota et al., 2008; Nishiwaki et al., 2000; Shibata et al., 2016).

Our proteomic, genetic and cell biological findings strongly suggest that the basement membrane is a dynamic structure that remodels, and that MIG-17 regulates synaptic allometry by modulating the basement membrane. Common among the genetic manipulations presented here—loss-of-function mig-17 and unc-52 alleles, gain of function fbl-1 alleles or hypomorphic and neomorphic emb-9 alleles—is a resulting disorganized basement membrane. All these alleles also suppress the ectopic synapses observed for cima-1 mutants. We hypothesize that these alleles all suppress cima-1 mutants because the material properties of the basement membrane prevent the movement of the glia during growth. This inability to reposition does not disrupt synaptic allometry as long as the glia and AIY relationship is preserved, as is the case in mig-17 and other basement membrane single mutants. But synaptic allometry defects occur when the relationship between glia and the AIY neurite is altered, as in the cima-1 mutants, in which epidermal-glia adhesion abnormally extends glia posteriorly. Therefore, MIG-17 and the basement membrane proteins act in opposition to CIMA-1 in positioning glia and regulating synaptic allometry during growth.

Our results demonstrate that modulating glia morphology and synaptic positions requires a muscle-epidermis-glia signaling axis, which utilizes MIG-17 dependent regulation of the extracellular matrix. We note that while basement membrane proteins can also regulate neuromuscular junction synapses (Ackley et al., 2003; Kurshan et al., 2014; Patton, 2003; Qin et al., 2014; Rogers and Nishimune, 2017), NMJs are in direct contact with the basement membrane. The neurons examined in this study, which are in the nerve ring, are not in direct contact with the basement membrane (White et al., 1986). Instead VCSC glia ensheath the nerve ring to form a physical barrier between the neuropil and adjacent tissues, including the pseudocoelom, the basement membrane and the epidermal cells (Shaham, 2015). At one side, VCSC glia contact neurons in the nerve ring, while at the other side they are either decorated by basement membrane or in direct contact with epidermal cells. Interactions among the VCSC glia, basement membrane and epidermal cells reflect the genetic relationships we uncovered in our forward genetic screens, as epidermal CIMA-1 and EGL-15/FGFR modulate glia morphology through epidermal-glial adhesion, and secreted MIG-17 modulate glia morphology through the muscle-derived extracellular matrix.

The muscle-epidermis-glia signaling axis described here is reminiscent of the neurovascular unit of the blood-brain barrier in Drosophila and vertebrates. In the vertebrate neurovascular unit, muscle-related pericyte cells interact with vascular endothelial cells and astrocytes through the basement membrane (Xu et al., 2019). Pericytes, endothelial cells and the basement membrane are not in direct contact with neurons. Instead, astrocytes mediate signaling between these non-neuronal cells and neurons, including coupling the developmental programs that coordinate vasculature development and neurodevelopment (Tam and Watts, 2010), and the functional programs that coordinate neuronal activity with blood flow (Allan, 2006; Koehler et al., 2009). We note that the extracellular matrix of the blood-brain barrier is molecularly similar to the basement membrane of C. elegans, and includes molecules we tested here, such as laminin, collagen IV and fibulin (Thomsen et al., 2017). While the role of these components in vertebrate synaptic allometry has not been examined, we speculate that the functional neurovascular unit may transduce information from the vasculature to sustain synaptic positions during allometric growth. Our findings therefore uncover a novel muscle-epidermis-glia signaling axis, which communicates in part through the remodeling of the basement membrane to sustains synaptic specificity during the organism’s allometric growth. We hypothesize that analogous structures in other organisms may represent conserved signaling axis that couple glia-mediated communication among non-neuronal cells and neurons to position synapses.

Materials and methods

Strains

All strains were grown at 22°C on NGM agar plates seeded with Escherichia coli OP50 (Brenner, 1974), except temperature sensitive strain emb-9(b189), grown at 16°C until L1 stage and then transferred to 22.5°C (Gupta et al., 1997). C. elegans N2 bristol was used as the wild-type strain.

The following alleles were utilized in this study:

  • LGII: unc-52(gk3), unc-52(e1421)

  • LGIII: emb-9(tk75), emb-9(xd51), emb-9(b189))

  • LGIV: cima-1(wy84), fbl-1(k201), dpy-4(e1166)

  • LGV: mig-17(ola226), mig-17(k113), mig-17(k174), mig-17(shc8), mig-17(shc19), nid-1(cg118), nid-1(cg119), lon-3(e2175)

  • LGX: let-2(k193), let-2(b246), egl-15(n484)

The following transgenic lines were used in this study: shcEx1126, shcEx1127 and shcEx1128[Pttx-3::syd-1::GFP;Pttx-3::rab-3::mCherry;Punc-122::RFP], shcEx1146 and shcEx1147[Pmig-17::mig-17 genomics;Phlh-17::mCherry], shcEx1129[Pmig-17::mig-17::SL2::GFP;Pdpy-4::mCherry], shcEx1130[Pmig-17::mig-17::SL2::GFP;Pmyo-3::mCherry], shcEx1131[Pmig-17::mig-17::SL2::GFP;Phlh-17::mCherry], shcEx1410[Pmig-17::mig-17::SL2::GFP;Prab-3::mCherry], shcEx845[Phlh-17::mCherry], shcEx1145[Pdpy-4::mCherry], shcEx1402[Pmyo-3::mCherry], shcEx1403[Prab-3::mCherry], shcEx1414 and shcEx1415 [Pmig-17::mig-17(E303A); Phlh-17::mCherry], shcEx1133, shcEx1134 and shcEx1135[Pmyo-3::mig-17;Phlh-17::mCherry], shcEx1676, shcEx1677 and shcEx1678[Plim-4::mig-17;Phlh-17::mCherry], shcEx1139 and shcEx1140[Phlh-17::mig-17;Phlh-17::mCherry], shcEx1142 and shcEx1143[Pdpy-7::mig-17;Punc-122::GFP], shcEx1675, shcEx1684 and shcEx1685 [Pttx-3::mig-17;Phlh-17::mCherry], qyIs46[unc119;emb-9::mCherry], shcEx776, shcEx777, shcEx778, shcEx780 and shcEx781[Phlh-17::mCherry;Pttx-3::GFP::rab-3], shcEx424, shcEx425, shcEx536, shcEx537 and shcEx538[Pdpy-7::egl-15(5A);Phlh-17::mCherry;Pttx-3::GFP:: rab-3], shcEx1252 and shcEx1253 [Pmig-17::mig-17(genomic);Phlh-17::mCherry], shcEx1682 and shcEx1683 [Prab-3::mig-17; Phlh-17::mCherry], shcEx1695 and shcEx1696[Pmyo-3::GFP], shcEx1697 and shcEx1698[Pttx-3::GFP], shcEx1699[Phlh-17::GFP].

Details on strains used in this study are listed in Supplementary file 2.

EMS screen and mutant identification

Request a detailed protocol

To identify cima-1 suppressors, animals that exhibited normal presynaptic distribution at the adult stage were isolated from a forward Ethyl Methane-Sulphonate (EMS) screen performed on the cima-1(wy84) mutants. The suppressor ola226 was isolated from this screen. The causative genetic lesion was identified through SNP mapping and whole genome sequencing (Minevich et al., 2012) to be a G to A point mutation in the first exon of mig-17, turning E19 into K in the protein. Fosmid WRM0616aB07, which includes the mig-17 gene, rescues the observed suppression of the AIY presynaptic distribution in cima-1(wy84); ola226.

Germline transformation

Request a detailed protocol

Transformations were carried out by microinjection of plasmid DNA into the gonad of adult hermaphrodites (Mello et al., 1991). Plasmids were injected at 5–20 ng/μl concentrations.

Plasmids

The following constructs were created by Gateway cloning (Invitrogen): Pmig-17::SL2::GFP; Pmig-17::mig-17(E303A)::GFP; Phlh-17::mig-17; Punc-14::mig-17; Pdpy-7::mig-17; Pmyo-3::mig-17. The mig-17 promoter is 1.7 kb sequence upstream from the start codon. The remaining constructs are listed in Supplementary file 3. Detailed cloning information is available upon request.

We constructed two Cas9-sgRNAs with pDD162 for each strain according to the method in Dickinson et al. (2015). The repair template of mig-17::mNeonGreen was modified from pDD268 and is illustrated in Figure 6—figure supplement 1B. Briefly, mNeonGreen was flanked by 1.2 kb genomic sequence upstream or downstream of the mig-17 stop codon. To prevent Cas9 from cutting the donor template, we also introduced one synonymous mutation in the protospacer adjacent motif (PAM). The repair template of mig-17(E303A) includes 1.2 kb upstream and 1.2 kb downstream of mig-17 genomic sequence, which flank the Glutamic acid at the 303 site. We mutated the Glutamic acid (GAA) to Alanine (GCA) and introduced eight synonymous mutations to prevent Cas9 from cutting the donor template (Figure 6—figure supplement 1A). mig-17(E303A) point mutation or mig-17::mNeonGreen knock-in animals were generated by microinjection of 50 ng/μl Cas9-sgRNA plasmids, 20 ng/μl repair template, and 5 ng/μl Pmyo-3::mCherry as a co-injection marker. The engineered strains were screened by PCR and verified by Sanger sequencing. We examined the glia morphology and gonad defect in mig-17::mNeonGreen knock-in animals, and observe that they behave as wild type, suggesting that MIG-17::neonGreen does not compromise MIG-17 function.

Protein extraction, digestion, and labeling

Request a detailed protocol

The samples were lysed in buffer (8 M guanidine hydrochloride, 100 mM TEAB) and sonicated. Samples were then centrifuged at 20,000 g for 30 min at 4°C, and the supernatant collected. Proteins were submitted to reduction by incubation with 10 mM DTT at 37°C for 45 min, followed by alkylation using 100 mM acrylamide for 1 hr at room temperature and digestion with Lys-C and trypsin using the FASP method (Wiśniewski et al., 2009). After stable isotope dimethyl labeling in 100 mM TEAB, peptides were mixed with light, intermediate and heavy (formaldehyde and NaBH3CN) isotopic reagents (1:1:1), respectively (Boersema et al., 2009). The peptide mixtures were desalted on a Poros R3 microcolumn according to the previous method (Huang et al., 2018).

Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

Request a detailed protocol

LC-ESI-MS/MS analyses were performed using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled with a nanoflow EASY-nLC 1000 system (Thermo Fisher Scientific, Odense, Denmark). A two-column system was adopted for proteomic analysis. The mobile phases were in Solvent A (0.1% formic acid in H2O) and Solvent B (0.1% formic acid in ACN). The derivatized peptides were eluted using the following gradients: 2–5% B in 2 min, 5–28% B in 98 min, 28–35% B in 5 min, 35–90% B in 2 min, 90% B for 13 min at a flow rate of 200 nl/min. Data-dependent analyses were used in MS analyses. The top 15 abundant ions in each MS scan were selected and fragmented in HCD mode.

Raw data was processed by Proteome Discover (Version 1.4, Thermo Fisher Scientific, Germany) and matched to the C. elegans database (20161228, 17,392 sequences) through the Mascot server (Version 2.3, Matrix Science, London, UK). Data was searched using the following parameters: 10 ppm mass tolerance for MS and 0.05 Da for MS/MS fragment ions; up to two missed cleavage sites were allowed; carbamidomethylation on cysteine, dimethyl labeling as fixed modifications; oxidation on methionine as variable modifications. The incorporated Target Decoy PSM Validator in Proteome Discoverer was used to validate the search results with only the hits with FDR ≤ 0.01. Three technical replicates were performed for the proteomic analyses.

Microscopy and image analyses

Request a detailed protocol

Animals were anaesthetized with 50 mM Muscimol (Tocris) on 2% agarose pads (Biowest, Lot No.: 111860), and examined with either with Perkin Elmer or Andor Dragonfly Spinning-Disk Confocal Microscope Systems. Image processing was performed by using Volocity, Image J, Adobe Photoshop CS6 or Imaris software (Andor).

Quantification

Request a detailed protocol

To quantify the percentage of animals with ectopic pre-synapses of AIY Zone one and posterior extension of glia, animals were synchronized by being selected at larva stage 4 (L4), and then examined 24 hr later using a Nikon Ni-U fluorescent microscope. Each dataset was collected from at least three biological replicates. At least 20 animals were scored for each group. For each germline transformation, multiple transgenic lines were examined. For synaptic allometric quantification, the ectopic synapses were defined as the presence of synaptic fluorescent markers the AIY Zone one region, an asynaptic area in wild type AIY neurons (Colón-Ramos et al., 2007; Shao et al., 2013). We also quantified the ratio of presynaptic length as the ratio of ventral length to total synaptic length (b/(a+b) in Figure 2F; Shao et al., 2013). The overlap of VCSC glia and ectopic synapses was defined as the VCSC glia and synaptic area of overlap at the Zone one and Zone two regions. The length of VCSC glial anterior process and ventral process (as shown in Figure 3A) were measured from confocal images taken in synchronized 1-day-old adults. The length of the pharynx and the body length were measured via DIC microscopy performed in synchronized 1-day-old adults.

The fluorescent intensity of MIG-17::mNeonGreen and EMB-9::mCherry in the pharyngeal region was normalized by the area with Image J from confocal images at the specified developmental stages. The mCherry clusters are likely intracellular accumulations of mCherry in the lysosome, as has been shown for other mCherry-tagged proteins. To minimize quantifying fluorescence from the intercellular EMB-9::mCherry clusters, we only quantified the mCherry in the second pharyngeal bulb region as shown in Figure 7.

Electron microscopy

Request a detailed protocol

L4 animals were prepared for EM by high pressure freezing and freeze substitution as described (Xuan et al., 2017). Serial sections of 40 nm thickness cut on a Ultracut 7 (Leica) and collected on formvar-covered, carbon-coated copper grids (EMS, FCF2010-Cu), and post-stained with 2.5% uranyl acetate and lead citrate. Images were acquired on a FEI Tecnai G2 Spirit BioTWIN. AIY Zone 2 was identified based on anatomical landmarks at the base of the ventral nerve bundle (White et al., 1986).

Statistical analysis

Request a detailed protocol

Specified statistical analyses were based on student’s t-test for comparisons between two groups or one-way ANOVA by Tukey’s multiple comparison test for three or more groups. All were analyzed using Prism 6.

Data availability

All data is presented in the figures or supplementary figures.

References

    1. Brenner S
    (1974)
    The genetics of Caenorhabditis elegans
    Genetics 77:71–94.
    1. Chisholm AD
    2. Hardin J
    (2005) Epidermal morphogenesis
    WormBook : The Online Review of C. Elegans Biology 1:1–22.
    https://doi.org/10.1895/wormbook.1.35.1
    1. Nishiwaki K
    (1999)
    Mutations affecting symmetrical migration of distal tip cells inCaenorhabditis elegans
    Genetics 152:985–997.
    1. White JG
    2. Southgate E
    3. Thomson JN
    4. Brenner S
    (1986) The structure of the nervous system of the nematode Caenorhabditis elegans
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 314:1–340.
    https://doi.org/10.1098/rstb.1986.0056

Decision letter

  1. Didier YR Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany
  2. Oliver Hobert
    Reviewing Editor; Howard Hughes Medical Institute, Columbia University, United States
  3. Peri Kurshan
    Reviewer; Albert Einstein College of Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "ADAMTS-family MIG-17 regulates synaptic allometry by modifying extracellular matrix and glia morphology during growth" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andrew D Chisholm (Reviewer #2); Peri Kurshan (Reviewer #3).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Your manuscript addresses the important question of how synapses are maintained after embryonic development and how they are scaled during postembryonic growth (a process the authors refer to as allometry, which raised concerns with one of the reviewers). Using the nematode C. elegans, the authors had identified in a previous study the putative solute carrier transporter cima-1 and the fibroblast growth factors as genes that function in the epidermis to shape glia, which are required to maintain the position of synapses of AIY interneurons. This current study extends these findings. The authors show that the cima-1 loss of function defects in AIY synapse maintenance can be partially suppressed by loss of the ADAMTS metalloprotease mig-17. Overall, the topic is timely and important as our knowledge of synaptic maintenance is limited. However, the reviewers raised a number of concerns. The reviewers all agreed that a journal like Development or Genetics would be a perfect home for this story, but that the extent of novelty and mechanistic insight did not rise to what is expected from an eLife paper.

1) The extensive amount of previous work on mig-17 in other cellular context somewhat diminished the novelty of this work. While mig-17 is placed in a novel cellular context, the reviewers were not entirely convinced that this novel context is actually quite properly framed (is this really synaptic allometry? See below) or whether some of the findings are not merely a quite simple reflection of glial overgrowth which then resulted in several secondary phenomena (as discussed more below).

2) The authors propose that the mig-17 ADAMTS enzyme negatively regulates the abundance of basement membrane proteins (such as collagen, which they show by both mass spec and fluorescence reporters). The interpretation is that this increase in BM proteins somehow suppresses defects caused by loss of the cima-1 solute carrier protein, which itself negatively regulates the fibroblast growth factor in an adjacent tissue. How do all these molecules identified by the authors function together? Do the authors envision that mig-17 is involved in the degradation of different types of basement membrane? Are the BM proteins direct or indirect targets? How would increased concentrations of BM proteins counteract the increased adhesion as a result of increased fibroblast growth factor coming from the epidermis that the authors propose in Shao et al.? Could mig-17 have an effect on the fibroblast growth factor? Do some of these factors physically interact? Some of these questions are obviously beyond the scope of this manuscript, but we would have liked to see somewhat more mechanistic insight into this important and interesting process.

3) The picture emerging from the author's analysis of genetic interactions is not entirely clear. The set of relationships between many different molecular players is very hard to keep straight, and the authors make sometimes contradictory statements about those relationships. For example, the authors state that the different molecules "act in the same pathway", which is confusing because they later explain (and their model figure shows) that there are two separate pathways (one from epidermal cells and one from muscle) that both converge on glial outgrowth. As another example, their explanation for how basement membrane components such as collagen (EMB-9) factor in to the pathway are confusing, perhaps owing to the fact that the alleles they use are not completely well-characterized. But a careful piecing together of all the data and genetic interactions suggests that an over-abundance (or extra stable version) of EMB-9 seems to suppress glial overgrowth (and therefore ectopic synapse formation). Their model figure uses arrow to show that relationships exist between MIG-17 and basement membrane components such as EMB-9 and glial cells, but a more accurate model would show that MIG-17 negatively regulates EMB-9, which in turn negatively regulates glial over-growth. Which is why either a loss-of-function mig-17 allele or a gain-of-function emb-9 allele, which both would lead to an increase in EMB-9 levels, suppress the cima-1 phenotype.

4) Extending on the points above: The mig-17 secreted protease likely functions from muscle to suppress the cima-1 mutant defects and requires enzymatic activity. Thus, different tissues (epidermis and muscle) are involved in maintaining glia shape, which in turn is necessary to maintain AIY synapses. The authors further show by mass spectrometry and fluorescent reporters that the abundance of basement membrane (BM) proteins is increased in mig-17 mutants. This thorough genetics study clearly establishes a role for mig-17 in the process of synapse maintenance, which is distinct from the role of mig-17 in shaping gonad development. Their data suggests that BM proteins such as collagens are involved in the process by which mig-17 functions to establish and maintain glial shape. Conceptually, however, the idea that mig-17 remodels the basement membrane is not novel (even if there are differences in molecular mechanism from e.g. gonad development). For example, mig-17 catalytic activity is required for function in other contexts (Nishiwaki et al., 2000, using the same missense mutation as used here). Other details (mig-17 effects on organ size; mig-17 reporter pattern, developmental regulation of mig-17 and emb-9) also cover similar ground to that reported by others (as properly acknowledged by the authors).

5) The authors frame their work in the context of “synaptic allometry” (maintenance of synaptic position during growth) yet it remains unclear if the ectopic synapses of cima-1 mutants result from disrupted allometry, or are a more specific result of the abnormal glial morphology. For example, most of the genetic manipulations appear to have little effect on synaptic allometry of “normal” (non-ectopic) synapses. Taken together, "allometry" may be an over-statement, as the phenotype can be boiled down to glial overgrowth (and resulting ectopic presynaptic puncta formation). The main novelty seems to be that both muscle and epidermal-derived signals impinge on glia morphology, but I'm not sure if that rises to the level of significance required.

6) There are also a number of additional concerns that seem minor in isolation, but together add up to the level of substantial concerns:

a) The statistical analyses used are not appropriate – the authors use t-tests for every individual comparison within a group, when they instead should use an ANOVA for multiple comparisons between the members of the group. Some of their statistical significance may erode upon this more stringent analysis.

b) The localization pattern of EMB-9::mCherry does not look like what you would expect from something that is supposed to be encasing muscles… what are all the clusters? Are they overexpression artifacts? mCherry aggregation artifacts? How was the quantification done and did it include the clusters? The colocalization between MIG-17::neonGreen and EMB-9::mCherry is not convincing.

c) They use Punc-14 promoter for ubiquitous neuronal expression is not standard protocol anymore. There is evidence for ubiquitous expression of this promoter.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "A muscle-epidermis-glia signaling axis sustains synaptic specificity during allometric growth in C. elegans" for consideration by eLife. Your article has been reviewed by two of the three peer reviewers that had seen an earlier version of the manuscript that was rejected, an additional new reviewer, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Peri Kurshan (Reviewer #1).

The two original reviewers have very much appreciated the revision of the manuscript and they agree (together with the new reviewer) that this manuscript now properly emphasizes an important conceptual advance by (a) framing the problem along the allometry concept, and by (b) demonstrating complex interactions between different tissues types that ensure allometry. However, one important experiment remains to be done (point #4 by reviewer #2) that we agreed would be necessary to strengthen an important, and somewhat understated (see reviewer #1) premise of the paper, namely that the effect of the ECM on synapse maintenance is indirect. Together with other points in the manuscript, we consider this to be an important novelty that would make the manuscript of interest to eLife. As per further discussion between the reviewers, this point should not only be addressed by examining the emb-9 marker, but also by a more comprehensive analysis of available EM sections.

All other comments by reviewer #2 can be editorially addressed.

Reviewer #1:

I find this version of the paper significantly more compelling.

The authors now emphasize that they have uncovered a signaling cascade mediating synapse position during growth that begins in the epidermis and muscle, goes through the glia, and ends at the neuron. I do think this is an interesting finding, and the paper now does a good job of laying out the novelty.

The question in my mind is how translatable this is to other systems. On the one hand, C. elegans as a model system has the advantage of enabling the authors to work out a complex genetic cascade such as this one within its in vivo context. But on the other hand, we already knew that glia are important for dictating where synapses form. In this case, the glia are getting signals from other structural cells (e.g. epidermis). But that is not going to be a widespread mechanism in the mammalian CNS… the authors would have been better served by focusing on the fact that the extracellular matrix is dictating glia position and thereby synapse position (rather than emphasizing the source of those signals as they do with the title of the paper)… at least that is something that could be directly relevant in other systems.

So in my opinion, what is most interesting is that ECM structure and composition can dictate synapse position not only directly, as has been shown before, but also indirectly by "repositioning" glia, as shown here. That's a slightly different spin than the one they've put on this.

The authors still have not really figured out the mechanisms by which this process occurs, and it is confusing that both putative lof and gof alleles of collagen seem to have the same effect on glial positioning.

Reviewer #2:

This manuscript by Fan and colleagues is a revised version of an earlier manuscript. It addresses the important question of how synapses are maintained in relation to other tissues after embryonic development and how they are scaled during postembryonic growth (a process the authors term allometry). Using the nematode C. elegans, the authors had previously identified the putative solute carrier transporter cima-1 and the fibroblast growth factors as genes that function in the epidermis to shape glia, which in turn are required to maintain the position of synapses of AIY interneurons. This current study extends these findings. Using a forward genetic screen, the authors identify a genetic suppressor of the cima-1 loss of function phenotype. This suppressor turns out to be mig-17, an ADAMTS protease, which is secreted to directly or indirectly remodel the basement membrane between the muscle and the glial cell. Through masspectrometry, the authors identify basement membrane proteins that are upregulated in mig-17 mutants. Removing some of the upregulated basement membrane proteins also suppresses the cima-1 loss of function phenotype suggesting that defective basement membranes are responsible for the suppression. Overall, the genetic results are clear and support the model the authors propose. On the downside, some of the genetic effects are possibly rather unspecific. For example, both gain and loss of function mutations of the collagen emb-9 suppress the cima-1 phenotype, suggesting that general basement membrane “malaise” is sufficient for the observed suppression. The authors acknowledge this, if not in these words. Along those lines, the genes that are mediating synaptic allometry as part of common genetic pathways in different tissues have, with some exceptions, little mechanistic connection, leaving many of the molecular details of this interesting phenomenon in the dark. That all being said, the finding that the glial cells (which mediate the allometry), are not only regulated by the adjacent epidermis, but also are influenced by a basement membrane that is secreted (or remodeled) by another adjacent tissue, namely muscle, is an important extension of the concept of how the AIY synapses are maintained. The technical and conceptual criticisms of the initial version have by and large been addressed and the writing is much improved, making the concepts much clearer.

Reviewer #3:

This solid and important manuscript reports on cellular and molecular bases of sustained synapse position in the face of organismal allometric growth. The authors identify a cooperative mechanism between non-neuronal cells (epidermis, BM-secreting muscle cells, and glia), whereby maintained glia morphology/position leads to the correct positioning of presynaptic specializations. Whereas the authors link mig-17 and BM components to this mechanism, no precise mechanistic/molecular understanding is provided, diminishing the significance of the study, especially in view of previously described work on mig-17 (which the authors cite), and the lab's previous work with cima-1 and egl-15. However, important conceptual novelty arises from the authors' identification of a multipartite system in which secreted molecules from distinct classes of non-neuronal cells coordinately impact glia position, thereby influencing synaptic development. Such multicellular coordination driving synaptic development may be at play in other species as well. The resolution enabled by C. elegans reveals an important mechanistic principle.

1) The use of the term "synapse allometry" may be an overstatement in view of the fact that only presynaptic specializations are affected/examined here. The position of these presynaptic structures appears hard wired, based solely on the region of contact between glia and presynaptic neurite, which is very interesting. But can these be considered bona fide synapses?

2) To further highlight the importance of non-neuronal cells in synapse positioning/development, the authors could/should underscore the fact that the post-synaptic partner (RIA) has no impact on presynaptic specialization assembly in the Discussion.

3) An allele of mig-17, ola226, was isolated as a suppressor mutation of cima-1 mutants, which otherwise display altered VCSC glia morphology and concomitant ectopic presynaptic specializations. Rescue assays for mig-17 function are logically carried out in double mutants cima-1;mig-17, testing whether transgenic animals are rescued back to cima-1 mutant phenotype, i.e. now displaying ectopic synapses. Since "rescue" in this situation is the manifestation of a defective phenotype, the interpretation provided would much strengthened if transgenic expression of mig-17 in the WT background showed no ectopic synapses.

4) Based on White's electron micrographs, the authors write that "No BM is present between the VCSC and the nerve ring neurons". While the image presented does appear to show this, BM on EM may not always be so clearly visible, especially if cells are densely apposed with each other. The authors could use confocal microscopy with fluorescent reporters for VCSC glia and EMB-9::mCherry, as well as for the AIY neuron and EMB-9::mCherry, to further validate this point. This is important as much of the novelty of this work relies on BM not directly impacting these synapses, and rather an indirect effect of BM on synapse position via glia.

5) The authors write that EMB-9::mCherry accumulations "are likely intracellular/lysosomal" and are therefore excluded from the analysis. While this may be plausible, colocalization with a lysosomal marker would enable a clearer interpretation.

6) Authors say that ola226 mutants have abnormal glia morphology and that AIY neurite and soma are anteriorly positioned. Anteriorly positioned with respect to what? Using the pharynx as a reference, as it appears from the schemtics presented, when the mutants under study affect pharynx size, may be problematic. How positions are evaluated should be described.

https://doi.org/10.7554/eLife.55890.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Your manuscript addresses the important question of how synapses are maintained after embryonic development and how they are scaled during postembryonic growth (a process the authors refer to as allometry, which raised concerns with one of the reviewers). Using the nematode C. elegans the authors had identified in a previous study the putative solute carrier transporter cima-1 and the fibroblast growth factors as genes that function in the epidermis to shape glia, which are required to maintain the position of synapses of AIY interneurons. This current study extends these findings. The authors show that the cima-1 loss of function defects in AIY synapse maintenance can be partially suppressed by loss of the ADAMTS metalloprotease mig-17. Overall, the topic is timely and important as our knowledge of synaptic maintenance is limited. However, the reviewers raised a number of concerns. The reviewers all agreed that a journal like Development or Genetics would be a perfect home for this story, but that the extent of novelty and mechanistic insight did not rise to what is expected from an eLife paper.

We received the constructive comments from the reviewers, and based on their thoughtful suggestions re-wrote the paper to better highlight the significance of the study. We also integrated all their other suggestions, including analyses of new alleles, statistical methods, etc. While the manuscript contains similar data as the original submission (with the suggested additions from the reviewers), we believe the reframing and rewriting improves it significantly to better underscore its main findings.

Briefly, we agree with the reviewers that the role of MIG-17 in remodeling the basement membrane is not a new finding. Upon rereading the critiques and the manuscript, we also agree that in our detailed genetic analyses we unintentionally placed too much emphasis on the molecules that emerged from our proteomic and genetic screens, drawing away from the important concepts that we learned. We thank the reviewers for pointing this out and helping us more clearly frame the value of this study.

The main finding here, which we now emphasize by re-writing the manuscript, is that a muscle-epidermis-glia signaling axis sustains synaptic positions during growth in C. elegans. The contribution of the paper is on the concept of synaptic allometry, and the role of non-neuronal cells, like muscles and the epidermal cells, in collaborating towards the maintenance of glia position, which in turn sustains synaptic positions during growth. I have presented this work in several forums in which it has been well received, particularly by the scientific community which work on glia and the role of non-neuronal cells in regulating nervous system architecture and function. The rigor with which we examine the contribution of specific tissues in positioning synapses in vivo is not possible in most other systems, and the important conceptual lessons which emerge from the study help inform examination of the process of synaptic allometry in other contexts.

1) The extensive amount of previous work on mig-17 in other cellular context somewhat diminished the enthusiasm of the novelty of this work. While mig-17 is placed in a novel cellular context, the reviewers were not entirely convinced that this novel context is actually quite properly framed (is this really synaptic allometry? See below) or whether some of the findings are not merely a quite simple reflection of glial overgrowth which then resulted in several secondary phenomena (as discussed more below).

We have rewritten the paper to better explain its conceptual contributions.

Briefly, the concept emerging from this study, beyond MIG-17, is that the circuit architecture established during embryogenesis is maintained during growth through interactions with non-neuronal cells, particularly muscles and epidermal cells. We find that muscles regulate this process through the modulation of the basement membrane via MIG-17. To be sure, the role of ADAMTS family protein GON-1 in synapse morphology has been described for NMJs. Yet, an important difference between those synapses and the ones studied here is that the basement membrane in NMJs is in direct contact, and likely directly signals, to NMJ synapses. In this study, the examined synapses in the nerve ring are not in direct contact with the basement membrane. So then how is it that the BM helps sustains synaptic positions during growth? We found the answer is by modifying, not directly the synapse, but glia position and morphology, which in turn positions the synapses during growth.

So, as the reviewers correctly point out, the synaptic phenotype emerges from the changing of glial morphology. We do not see this as a trivial finding. Based on our experience publishing in glia relationship with synapses, I can confidently state that there are very few studies linking glia to synaptic positions in vivo, and to my knowledge, no studies linking signaling from non-neuronal cells and the BM in positioning glia during growth to then regulate synaptic positions. Given that the relationship we examine here between BM:glia:synapses is also present in mammalian CNS systems (including the blood-brain barrier), and given that sustaining synaptic positions during growth (which we term synaptic allometry) is a conserved principle, we believe the concepts learned here might have important implications for our understanding of how synaptic positions are sustained during growth, and the role of non-neuronal cells, such as glia, muscles and epidermal cells, in forming a signaling axis that links growth information with the scaling of the synaptic pattern to preserve embryonically-derived circuit architecture.

2) The authors propose that the mig-17 ADAMTS enzyme negatively regulates the abundance of basement membrane proteins (such as collagen, which they show by both mass spec and fluorescence reporters). The interpretation is that this increase in BM proteins somehow suppresses defects caused by loss of the cima-1 solute carrier protein, which itself negatively regulates the fibroblast growth factor in an adjacent tissue. How do all these molecules identified by the authors function together? Do the authors envision that mig-17 is involved in the degradation of different types of basement membrane? Are the BM proteins direct or indirect targets? How would increased concentrations of BM proteins counteract the increased adhesion as a result of increased fibroblast growth factor coming from the epidermis that the authors propose in Shao et al.? Could mig-17 have an effect on the fibroblast growth factor? Do some of these factors physically interact? Some of these questions are obviously beyond the scope of this manuscript, but we would have liked to see somewhat more mechanistic insight into this important and interesting process.

We have rewritten these sections to address these points.

Briefly, our model is that epidermal cells establish an adhesion with glia through the FGF receptor. That adhesion is negatively regulated during growth by CIMA-1. Reduction of CIMA-1 will result in more adhesion and the posterior displacement of the glia. But the displacement of the glia requires modification of the basement membrane by MIG-17. When the basement membrane is not properly modified, like in BM mutants, or in mig-17 mutants, the glia can’t be posteriorly displaced and the morphological phenotypes (and ectopic synapses) are suppressed.

We explain this now much more clearly when discussing, for instance, the different alleles tested for emb-9, and for emb-9- regulating proteins. While these alleles are known to have different effects on the conformation or levels of the EMB-9 protein in the basement membrane, they all suppress the synaptic phenotype in cima-1(wy84) mutants. Their shared ability to suppress cima- 1(wy84) mutants suggests that lesions resulting in defects in the basement membrane prevent the repositioning of glia that gives rise to the ectopic synapses. Together with our genetic and proteomic findings, they support a model whereby MIG-17 regulates basement membrane proteins, such as EMB-9, to modulate glia position during growth, and synaptic allometry.

While we did not overexpress BM proteins, overexpression of MIG-17 results in glia extension and phenotypes like CIMA-1. The synaptic allometric defect of EGL-15A overexpression also depends on MIG-17. We cannot exclude a role of MIG-17 in regulating the FGF receptor (which we now mention in the paper), we found that MIG-17 is epistatic to the FGF receptor, suggesting that mig-17 acts downstream (or in parallel) to modulate the role of elg-15 in positioning glia and regulating synaptic allometry. We better discuss these findings in the Results and the Discussion.

3) The picture emerging from the author's analysis of genetic interactions is not entirely clear. The set of relationships between many different molecular players is very hard to keep straight, and the authors make sometimes contradictory statements about those relationships. For example, the authors state that the different molecules "act in the same pathway", which is confusing because they later explain (and their model figure shows) that there are two separate pathways (one from epidermal cells and one from muscle) that both converge on glial outgrowth. As another example, their explanation for how basement membrane components such as collagen (EMB-9) factor in to the pathway are confusing, perhaps owing to the fact that the alleles they use are not completely well-characterized. But a careful piecing together of all the data and genetic interactions suggests that an over-abundance (or extra stable version) of EMB-9 seems to suppress glial overgrowth (and therefore ectopic synapse formation). Their model figure uses arrow to show that relationships exist between MIG-17 and basement membrane components such as EMB-9 and glial cells, but a more accurate model would show that MIG-17 negatively regulates EMB-9, which in turn negatively regulates glial over-growth. Which is why either a loss-of-function mig-17 allele or a gain-of-function emb-9 allele, which both would lead to an increase in EMB-9 levels, suppress the cima-1 phenotype.

We thank the reviewers for this comment. While the genetic interactions are consistent with our model, we agree that the large number of double and triple mutants in the examination of our genotypes make it both hard to read and unclear. We addressed this in two ways:

1) We add schematics to underscore the pathways we found and the tissues in which they act. Briefly, when we (confusingly) referred to a single pathway, we meant it in a genetic sense – a genetic pathway culminating in synaptic allometry. We clarify this and other points raised by the reviewer sin the text and figures.

2) We re-wrote the text with professional editorial help to better explain the phenotypes in the context of the examined mutants.

4) Extending on the points above: The mig-17 secreted protease likely functions from muscle to suppress the cima-1 mutant defects and requires enzymatic activity. Thus, different tissues (epidermis and muscle) are involved in maintaining glia shape, which in turn is necessary to maintain AIY synapses. The authors further show by mass spectrometry and fluorescent reporters that the abundance of basement membrane (BM) proteins is increased in mig-17 mutants. This thorough genetics study clearly establishes a role for mig-17 in the process of synapse maintenance, which is distinct from the role of mig-17 in shaping gonad development. Their data suggests that BM proteins such as collagens are involved in the process by which mig-17 functions to establish and maintain glial shape. Conceptually, however, the idea that mig-17 remodels the basement membrane is not novel (even if there are differences in molecular mechanism from e.g. gonad development). For example, mig-17 catalytic activity is required for function in other contexts (Nishiwaki et al., 2000, using the same missense mutation as used here). Other details (mig-17 effects on organ size; mig-17 reporter pattern, developmental regulation of mig-17 and emb-9) also cover similar ground to that reported by others (as properly acknowledged by the authors).

We agree that, while we go into a rigorous dissection of the proteomic and genetic findings to establish the link between MIG-17 and the BM, that is not the main novelty of the study. The novelty is that through the reorganization of the BM, MIG-17 cooperates with signals from epidermal cells to maintain glia position, and in that way modulate synaptic positions during growth (as explained in more detail in point #1 above).

5) The authors frame their work in the context of “synaptic allometry” (maintenance of synaptic position during growth) yet it remains unclear if the ectopic synapses of cima-1 mutants result from disrupted allometry, or are a more specific result of the abnormal glial morphology. For example, most of the genetic manipulations appear to have little effect on synaptic allometry of “normal” (non-ectopic) synapses. Taken together, "allometry" may be an over-statement, as the phenotype can be boiled down to glial overgrowth (and resulting ectopic presynaptic puncta formation). The main novelty seems to be that both muscle and epidermal-derived signals impinge on glia morphology, but I'm not sure if that rises to the level of significance required.

This is an important point which we better addressed in the revised manuscript:

A) In this manuscript we introduce the term “synaptic allometry”, an important concept, in our opinion, for the community. As we define it here, synaptic allometry refers to sustaining the synaptic pattern during growth. This is different than maintaining the morphology/position/function/protein composition of one synapse. It is a nuanced, but important point. We would argue that sustaining the synaptic positions is what is important to sustain the synaptic pattern that underlies the connectivity. We now re-wrote the text to define clearly what we mean by synaptic allometry.

B) Extending on this point, we now show in the supplementary figures the relationship between the ectopic synapses in the cima-1 mutants and the postsynaptic partner RIA, demonstrating that the change in the presynaptic pattern due to glia morphology affects the relative positions of the components of the synapse, and the connectivity.

C) As the reviewers point out, the synaptic phenotype emerges from the changes in glial morphology. We do not see this as a trivial finding. There are no studies to our knowledge linking signalling from non-neuronal cells and the BM in positioning glia during growth to then regulate synaptic positions. The role of nonneuronal cells, in vivo, in sustaining synaptic positions is an important question which we address here. How glia morphology is maintained, and how this maintenance then impinges on the circuit architecture is essentially unknown and an important question in neuroscience. We find here that the glia mediate signals from epidermal cells and the BM regulated by muscles, to maintain synaptic positions during growth.

6) There are also a number of additional concerns that seem minor in isolation, but together add up to the level of substantial concerns:

a) The statistical analyses used are not appropriate – the authors use t-tests for every individual comparison within a group, when they instead should use an ANOVA for multiple comparisons between the members of the group. Some of their statistical significance may erode upon this more stringent analysis.

Thanks for the comments. We now use the ANOVA analysis for comparison among groups of three or more. None of the conclusions changed, but this is an important point and we are thankful for the correction.

b) The localization pattern of EMB-9::mCherry does not look like what you would expect from something that is supposed to be encasing muscles… what are all the clusters? Are they overexpression artifacts? mCherry aggregation artifacts? How was the quantification done and did it include the clusters? The colocalization between MIG-17::neonGreen and EMB-9::mCherry is not convincing.

The clusters are likely mCherry accumulations in lysosomes. While GFP gets quenched in acidic environments, mCherry does not (we carefully characterized this for a different protein and its relationship to the autophagy pathway in (Hill et al., 2019)). For this study, and to avoid quantifying these intracellular lysosomal aggregates, we carefully quantified the fluorescent intensity in the region of second pharyngeal bulb without scoring the fluorescence from the aggregates. This is now indicated both in the figures and the Materials and methods.

c) They use Punc-14 promoter for ubiquitous neuronal expression is not standard protocol anymore. There is evidence for ubiquitous expression of this promoter.

This is a good point. We redid these experiments with a number of additional promoters, and now demonstrate that, consistent with mig-17 being a secreted protein, expression from any cell-specific promoter rescues the phenotype (including the pan-neuronal rab-3 promoter, nerve ring specific lim-4 promoter and AIY specific ttx-3 promoter. These data are now included in Figure 5—figure supplement 1). Note that this findings are different from the negative result we obtained with the unc-14 promoter, and we revisit our interpretation based on these more rigorous experiments. While they do not change the overall conclusion, they extend our understanding of mig-17 as a secreted protein, and we present that accordingly.

[Editors’ note: what follows is the authors’ response to the second round of review.]

The two original reviewers have very much appreciated the revision of the manuscript and they agree (together with the new reviewer) that this manuscript now properly emphasizes an important conceptual advance by (a) framing the problem along the allometry concept, and by (b) demonstrating complex interactions between different tissues types that ensure allometry. However, one important experiment remains to be done (point #4 by reviewer #2) that we agreed would be necessary to strengthen an important, and somewhat understated (see reviewer #1) premise of the paper, namely that the effect of the ECM on synapse maintenance is indirect. Together with other points in the manuscript, we consider this to be an important novelty that would make the manuscript of interest to eLife. As per further discussion between the reviewers, this point should not only be addressed by examining the emb-9 marker, but also by a more comprehensive analysis of available EM sections.

All other comments by reviewer #2 can be editorially addressed.

Please find our revised manuscript “A muscle-epidermis-glia signaling axis sustains synaptic positions during growth in C. elegans”. We integrated most of the reviewer’s comments, as suggested. In particular, we have now added additional data demonstrating the relationship between the basement membrane and the nerve ring (new Figure 9—figure supplement 1). We do this by showing the relative position of EMB-9 marker with a body muscle, with glia and with AIY marker, and demonstrate by fluorescence microscopy that EMB-9 localizes primarily to muscles (pharynx and body wall muscles), near the glia, but separate from the neuron (even AIY, which is near the edge of the nerve ring). Notably, in the images one can observe a darker area where the nerve ring is located, indicative of reduced of EMB-9 labeling. We also performed electron microscopy in AIY. Although it is only a panel in the Figure 9—figure supplement 1, we are very proud of this achievement, as it is technically challenging to identify the AIY neuron in the context of the nerve ring. We carefully inspected new electron micrographs we generated, and, consistent with John White’s published electron micrographs, we observe that the basement membrane near the nerve ring is only visible in the region abutting the pseudocoelum. We confirmed our observations by communicating directly with EM expert David Hall (personal communication), who also reported to us that in the hundreds of electron micrographs he has reconstructed and inspected, he does not observe basement membrane in the nerve ring. Our findings are consistent with the observations made in other systems that the basement membrane is an organized extracellular matrix structure that is 30-70 nanometers thick, and not present in the fascicles of the CNS (Heikkinen et al., 2014; Krishnaswamy et al., 2019). We make the distinction in the figure legend, however, that while the structure of the basement membrane might not be present inside the nerve ring, neurons in the nerve ring could express basement membrane proteins.

https://doi.org/10.7554/eLife.55890.sa2

Article and author information

Author details

  1. Jiale Fan

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Conceptualization, Resources, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing
    Contributed equally with
    Tingting Ji and Kai Wang
    Competing interests
    No competing interests declared

  2. Tingting Ji

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Investigation
    Contributed equally with
    Jiale Fan and Kai Wang
    Competing interests
    No competing interests declared

  3. Kai Wang

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Investigation
    Contributed equally with
    Jiale Fan and Tingting Ji
    Competing interests
    No competing interests declared

  4. Jichang Huang

    State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  5. Mengqing Wang

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  6. Laura Manning

    Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    Contribution
    Investigation, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1597-0600

  7. Xiaohua Dong

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Conceptualization, Resources, Investigation, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared

  8. Yanjun Shi

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  9. Xumin Zhang

    State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China
    Contribution
    Conceptualization, Resources, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2810-6363

  10. Zhiyong Shao

    Department of Neurosurgery, the State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, the Institutes of Brain Science, and Zhongshan Hospital, Fudan University Shanghai, Shanghai, China
    Contribution
    Conceptualization, Resources, Investigation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    shaozy@fudan.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6475-7681

  11. Daniel A Colón-Ramos

    1. Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    2. Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico, San Juan, Puerto Rico
    Contribution
    Conceptualization, Resources, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    daniel.colon-ramos@yale.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0223-7717

Funding

National Natural Science Foundation of China (31471026)

  • Jiale Fan
  • Tingting Ji
  • Kai Wang
  • Mengqing Wang
  • Xiaohua Dong
  • Yanjun Shi
  • Zhiyong Shao

NIH Office of the Director (DP1NS111778)

  • Laura Manning
  • Daniel A Colón-Ramos

National Institutes of Health (R01NS076558)

  • Laura Manning
  • Daniel A Colón-Ramos

Howard Hughes Medical Institute (Faculty Scholar)

  • Daniel A Colón-Ramos

National Natural Science Foundation of China (31872762)

  • Jiale Fan
  • Tingting Ji
  • Mengqing Wang
  • Xiaohua Dong
  • Yanjun Shi
  • Zhiyong Shao

Shanghai Municipal Science and Technology Major Project (2018SHZDZX01)

  • Zhiyong Shao

National Natural Science Foundation of China (31870822)

  • Jichang Huang
  • Xumin Zhang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank ZF Altun and DH Hall from WormAtlas for help with schematic figures. We also thank Shiqing Cai, Yidong Shen, Kiyoji Nishiwaki, Mei Ding (Chinese Academy of Sciences), Yan Zou (Shanghai tech), David Sherwood (Duke University) and the Caenorhabditis Genetic Center (funded by NIH (P40 OD010440) for providing strains and plasmids. We thank members in Shao lab and the Colón-Ramos lab for insightful discussions on the work and advice on the project. We thank Mi Zhou for providing technical support on image acquisition. We thank the Yale CCMI Electron Microscopy Facility for use of their equipment. We thank the Research Center for Minority Institutions program, the Marine Biological Laboratories (MBL), and the Instituto de Neurobiología de la Universidad de Puerto Rico for providing meeting and brainstorming platforms. Research in the ZS lab was supported by the National Natural Science Foundation of China (31471026, 31872762), Shanghai Municipal Science and Technology Major Project (No. 2018SHZDZX01) and ZJLab. Research in the Zhang lab was supported by the National Natural Science Foundation of China (31870822). Research in the DAC-R lab was supported by NIH R01NS076558, DP1NS111778 and by an HHMI Scholar Award. We thank Life Science Editors for editing assistance.

Senior Editor

  1. Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Oliver Hobert, Howard Hughes Medical Institute, Columbia University, United States

Reviewer

  1. Peri Kurshan, Albert Einstein College of Medicine, United States

Publication history

  1. Received: February 10, 2020
  2. Accepted: April 5, 2020
  3. Accepted Manuscript published: April 7, 2020 (version 1)
  4. Version of Record published: April 17, 2020 (version 2)

Copyright

© 2020, Fan et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,556
    Page views
  • 317
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Jiale Fan
  2. Tingting Ji
  3. Kai Wang
  4. Jichang Huang
  5. Mengqing Wang
  6. Laura Manning
  7. Xiaohua Dong
  8. Yanjun Shi
  9. Xumin Zhang
  10. Zhiyong Shao
  11. Daniel A Colón-Ramos
(2020)
A muscle-epidermis-glia signaling axis sustains synaptic specificity during allometric growth in Caenorhabditis elegans
eLife 9:e55890.
https://doi.org/10.7554/eLife.55890

Categories and tags

Research organism

Further reading

    1. Cell Biology
    2. Neuroscience

    APOE expression and secretion are modulated by mitochondrial dysfunction

    Meghan E Wynne, Oluwaseun Ogunbona ... Victor Faundez
    Research Article Updated

    Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer’s disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

    1. Neuroscience

    The nematode worm C. elegans chooses between bacterial foods as if maximizing economic utility

    Abraham Katzen, Hui-Kuan Chung ... Shawn R Lockery
    Research Article Updated

    In value-based decision making, options are selected according to subjective values assigned by the individual to available goods and actions. Despite the importance of this faculty of the mind, the neural mechanisms of value assignments, and how choices are directed by them, remain obscure. To investigate this problem, we used a classic measure of utility maximization, the Generalized Axiom of Revealed Preference, to quantify internal consistency of food preferences in Caenorhabditis elegans, a nematode worm with a nervous system of only 302 neurons. Using a novel combination of microfluidics and electrophysiology, we found that C. elegans food choices fulfill the necessary and sufficient conditions for utility maximization, indicating that nematodes behave as if they maintain, and attempt to maximize, an underlying representation of subjective value. Food choices are well-fit by a utility function widely used to model human consumers. Moreover, as in many other animals, subjective values in C. elegans are learned, a process we find requires intact dopamine signaling. Differential responses of identified chemosensory neurons to foods with distinct growth potentials are amplified by prior consumption of these foods, suggesting that these neurons may be part of a value-assignment system. The demonstration of utility maximization in an organism with a very small nervous system sets a new lower bound on the computational requirements for utility maximization and offers the prospect of an essentially complete explanation of value-based decision making at single neuron resolution in this organism.

    1. Neuroscience

    Spatiotemporal neural dynamics of object recognition under uncertainty in humans

    Yuan-hao Wu, Ella Podvalny, Biyu J He
    Research Article Updated

    While there is a wealth of knowledge about core object recognition—our ability to recognize clear, high-contrast object images—how the brain accomplishes object recognition tasks under increased uncertainty remains poorly understood. We investigated the spatiotemporal neural dynamics underlying object recognition under increased uncertainty by combining MEG and 7 Tesla (7T) fMRI in humans during a threshold-level object recognition task. We observed an early, parallel rise of recognition-related signals across ventral visual and frontoparietal regions that preceded the emergence of category-related information. Recognition-related signals in ventral visual regions were best explained by a two-state representational format whereby brain activity bifurcated for recognized and unrecognized images. By contrast, recognition-related signals in frontoparietal regions exhibited a reduced representational space for recognized images, yet with sharper category information. These results provide a spatiotemporally resolved view of neural activity supporting object recognition under uncertainty, revealing a pattern distinct from that underlying core object recognition.