Our previous work showed that the cell adhesion molecule SAX-7 forms an elaborate pattern in Caenorhabditis elegans epidermal cells, which instructs PVD dendrite branching. However, the molecular mechanism forming the SAX-7 pattern in the epidermis is not fully understood. Here, we report that the dynein light intermediate chain DLI-1 and the fusogen EFF-1 are required in epidermal cells to pattern SAX-7. While previous reports suggest that these two molecules act cell-autonomously in the PVD, our results show that the disorganized PVD dendritic arbors in these mutants are due to the abnormal SAX-7 localization patterns in epidermal cells. Three lines of evidence support this notion. First, the epidermal SAX-7 pattern was severely affected in dli-1 and eff-1 mutants. Second, the abnormal SAX-7 pattern was predictive of the ectopic PVD dendrites. Third, expression of DLI-1 or EFF-1 in the epidermis rescued both the SAX-7 pattern and the disorganized PVD dendrite phenotypes, whereas expression of these molecules in the PVD did not. We also show that DLI-1 functions cell-autonomously in the PVD to promote distal branch formation. These results demonstrate the unexpected roles of DLI-1 and EFF-1 in the epidermis in the control of PVD dendrite morphogenesis.
Neuronal morphogenesis requires the coordinated actions of both extrinsic and intrinsic factors (Dong et al., 2015). Axons and dendrites are guided by diffusible or membrane-tethered molecular cues during development. These extracellular cues modify intracellular signaling mechanisms and cytoskeletal organization to pattern the growth of neurites and specify neuronal morphology (Jan and Jan, 2010).
The multimodal PVD sensory neurons in Caenorhabditis elegans have highly branched and ordered dendrites. Studies on PVD dendrites have provided insights into the molecular mechanisms of dendrite morphogenesis and the interaction between dendrites and their environment (Albeg et al., 2011; Liu and Shen, 2012; Liu et al., 2016; Oren-Suissa et al., 2010; Smith et al., 2010). Several intrinsic and extrinsic factors have been found to function in PVD dendrite morphogenesis.
SAX-7–L1CAM and MNR-1–menorin form a co-ligand complex on the epidermis to guide PVD dendrite development (Dong et al., 2013; Salzberg et al., 2013). The leucine-rich repeat protein DMA-1 acts as the cognate receptor on PVD dendrites for SAX-7 and MNR-1 (Dong et al., 2013; Liu and Shen, 2012; Salzberg et al., 2013). In addition, LECT-2, which is secreted by muscles, forms a complex with SAX-7, MNR-1 and DMA-1, and is essential to activate DMA-1 (Diaz-Balzac et al., 2016; Zou et al., 2016). A number of intracellular molecules are required for normal PVD morphogenesis. For example, the POU domain transcription factor UNC-86 and the LIM domain transcription factor MEC-3 are critical cell fate factors for PVD dendrite development (Smith et al., 2013).
Building a complex dendritic arbor also requires cytoskeleton- and molecular-motor-related proteins. For example, loss of the dynein regulator Bicaudal-D (bicd-1), causes ectopic branching in the proximal region of the PVD arbor and reduced branching at the distal arbor (Aguirre-Chen et al., 2011). RNAi of dnc-1 and dli-1, which encode components of the cytoplasmic dynein, show very similar phenotypes to that of the bicd-1 mutant (Aguirre-Chen et al., 2011). Genetic mosaic analyses show that bicd-1 acts primarily within the PVD neuron to regulate the patterning of its dendrite development. Selective expression of BICD-1 in the PVD neuron partially rescues the ectopic branching phenotype, suggestive of a cell-autonomous requirement of bicd-1 (Aguirre-Chen et al., 2011).
EFF-1, the essential fusogen for cell fusion in C. elegans, has also been reported to function in the PVD to sculpt dendritic arborization (Oren-Suissa et al., 2010). In the eff-1 mutant, PVD dendrites display a highly disorganized branching pattern with reduced sensitivity to strong mechanical stimuli (Oren-Suissa et al., 2010). eff-1 is expressed in the PVD, although this is likely to be at a low level. Furthermore, PVD-specific expression of EFF-1 partially rescues the dendritic arbor phenotype in the eff-1 mutants, suggesting that eff-1 functions cell autonomously in the PVD to control dendrite morphology (Oren-Suissa et al., 2010). EFF-1 is also expressed in many other cell types, including epidermal cells. In C. elegans, the hyp7 epidermal cell is a multinuclear syncytium resulting from several rounds of cell fusion. It has been shown that eff-1 acts as a fusogen in syncytium formation (Mohler et al., 2002).
In this study, we report the surprising findings that a dynein light intermediate chain protein, DLI-1, and the fusogen EFF-1 both have a non-autonomous function in PVD dendrite formation. Furthermore, we present evidence that both dli-1 and eff-1 are required in the epidermal cell for the correct pattern of SAX-7. Since epidermal SAX-7 plays an instructive role for PVD morphogenesis, DLI-1 and EFF-1 are indirectly involved in shaping the PVD arbors by controlling the subcellular localization pattern of SAX-7. By analysing the dli-1 mutant, we also confirm the previous findings that the dynein complex has a cell-autonomous function to promote distal PVD dendrite development.
The dli-1 mutant has over-branching of proximal dendrites and reduced distal branching
From forward genetic screening for a PVD dendrite morphology defect, we isolated a lethal allele, wy50053, which showed numerous, disorganized dendritic arbors near the cell body region and a reduced number of dendrites at the distal region. In wy50053 mutants, ectopic branches occurred most frequently at the region between primary and tertiary branches (Fig. 1C). To quantify this phenotype, we counted the branches that did not belong to the regular menorahs. More than 20 ectopic branches per 100 μm were found in the proximal dendrites of the wy50053 mutant whereas fewer than five were found in the wild type (WT) (Fig. 1B,C,J). In addition, the mutant showed a dramatically simplified arbor at the anterior distal region (Fig. 1F,G). Using snip-SNP mapping and whole-genome sequencing, we identified a mutation in dli-1, which encodes the dynein light intermediate chain. wy50053 caused a stop codon at position 198Q, likely producing a truncated protein. In addition to the PVD dendrite phenotypes, wy50053 exhibited similar phenotypes compared to a previously characterized strong loss-of-function allele, ku266, which contains an early stop codon at position 117W (Yoder and Han, 2001). Both alleles were sterile and caused a protruding vulva (Fig. S1D), suggesting wy50053 is a null or strong loss-of-function allele.
DLI-1 functions in both PVD and epidermis to control dendrite branching
To understand where DLI-1 functions to regulate PVD dendrites, we performed tissue-specific rescue experiments in the dli-1 mutant. Our previous study showed that two cell types are particularly important for PVD dendrite morphogenesis: the PVD neurons and the epidermal cell, on which the dendrites elaborate its branches (Dong et al., 2013; Liu and Shen, 2012). In addition, a previous study showed that the dynein regulator BICD-1 functions in PVD neurons to control PVD dendrite development (Aguirre-Chen et al., 2011). Surprisingly, expression of dli-1 in the PVD using the ser-2Prom3 promoter (Dong et al., 2013; Salzberg et al., 2014) did not rescue the disorganized arbors in the proximal dendritic region of dli-1 mutants, although it did rescue the reduced branches at the distal region. Furthermore, the rescued distal dendrites also showed a disorganized pattern similar to the proximal dendrites in this mutant (Fig. 1C,D,G,H,K). On the contrary, when we expressed dli-1 in epidermis using the Pdpy-7 promoter, which specifically drives expression in epidermis (Fig. S2), it fully rescued the numerous disorganized dendrites, but did not rescue the reduced branching phenotype in distal dendrites (Fig. 1C,E,G,I,J,K). The above data suggest that DLI-1 has two functions in PVD dendrite morphogenesis: a cell non-autonomous function in epidermis to generate the orderly patterns of PVD dendrites in the proximal region and a PVD autonomous function to promote distal dendrite branching.
Disrupted epidermal SAX-7 pattern in the dli-1 mutant
The cell non-autonomous function of dli-1 is surprising in light of the known functions of dynein in PVD. Next, we investigated the mechanism of dli-1 in epidermis. Previously, we have shown that the cell adhesion molecule SAX-7 (L1CAM) plays instructive roles in PVD morphogenesis. SAX-7 functions in the epidermis and is localized to orderly stripes both sublaterally and in the border of the muscle quadrant to guide the branching of PVD dendrites (Dong et al., 2013; Liang et al., 2015; Liu et al., 2016). Interestingly, the epidermal SAX-7 localization pattern was dramatically altered in the dli-1 mutants. In the wild type, SAX-7::GFP forms bright stripes at the lateral and sublateral lines (Fig. 2A). In dli-1 mutants, there are numerous ectopic patches of SAX-7 between lateral and sublateral lines (Fig. 2B). Coincidently, most of the ectopic PVD dendrites in the mutant localized on or at the rim of the SAX-7 patches (Fig. 2B,D). Importantly, Pdpy-7::dli-1 rescued both the SAX-7 patchy phenotype and the ectopic PVD dendrite phenotype (Fig. 2C,D). This result strongly suggests that DLI-1 is required to pattern the SAX-7 adhesion molecule in the epidermis, which in turn, patterns the PVD branches.
EFF-1 functions in epidermis to control dendrite branching
The numerous disorganized dendrites near the PVD cell body of the dli-1 mutant were similar to dendrites in the eff-1 mutant (Fig. 3A,B) (Oren-Suissa et al., 2010). It was previously reported that EFF-1 functions cell-autonomously in the PVD to support dendrite-to-dendrite fusion which impacts dendrite morphogenesis (Oren-Suissa et al., 2010). Surprisingly, we found that PVD-specific expression of EFF-1 (ser-2Prom3::eff-1 genome DNA) failed to rescue the PVD dendrite phenotypes in the eff-1 mutant (Fig. 3C,E). This is supported by four independent transgenic strains injected at different concentrations (Fig. S3). Since dli-1 functions in epidermis to regulate the PVD dendrite in the cell body region, and eff-1 has been reported to be important for epidermal syncytium formation by promoting cell–cell fusion (Mohler et al., 2002). We wondered if eff-1 also functions in the epidermis to regulate PVD dendrites. Oren-Suissa et al. (2010) reported that epidermis-specific expression of Pdpy-7::eff-1 (injected at 1 ng/µl) causes lethality. We observed similar lethality phenotypes when we injected Pdpy-7::eff-1 into eff-1 mutant at 5 ng/µl. However, from this injection, we did isolate one transgenic strain, which fully rescued the dendrite branching defect of the eff-1 mutant (Fig. 3D,E). It also rescued the Dpy phenotype of eff-1 mutants. To circumvent the lethality issue, we injected Pdpy-7::eff-1 at 1 ng/µl and obtained 5 transgenic lines; 3 out of these 5 strains fully rescued the PVD dendrite phenotype of the eff-1 mutant (Fig. S3).
The C isoform of eff-1 was found to be partially secreted, which interferes with the autonomy analysis by tissue-specific transgene expression. To test if the non-autonomous rescue of eff-1 was caused by the secreted isoform, we expressed Pdpy-7::eff-1c, and found that none of the three transgenic lines could rescue the PVD phenotype of the eff-1 mutant (Fig. S3), suggesting that this isoform is not essential in PVD dendrite development. Taken together, this evidence supports the idea that eff-1 functions mainly in epidermal cells to pattern the PVD dendrites.
eff-1 mutant has a disrupted epidermal SAX-7 pattern
Since dli-1 affects the PVD dendrite morphology by controlling the SAX-7 pattern in epidermal cells, we followed the same logic to test if eff-1 affects the distribution of SAX-7 in epidermal cells for PVD dendrite growth. In eff-1 mutants, the orderly stripes of SAX-7 were also largely absent and replaced by patch-like patterns. Interestingly, most of the disorganized ectopic PVD dendrites localized on or at the edges of the SAX-7 patches (Fig. 4A,B,E). Furthermore, expression of eff-1 in the epidermis (Pdpy-7::eff-1) fully rescued the disorganized SAX-7 pattern, whereas expression of EFF-1 in the PVD (Pser-2Prom3::eff-1) did not rescue the SAX-7 pattern (Fig. 4B–D). These data strongly suggest that eff-1 functions in a cell non-autonomous manner to regulate PVD dendrite development. Specifically, eff-1 is required in the epidermis to form the right SAX-7 localization pattern, which acts as a guide for PVD development.
The hyp7 cell is a multinuclear syncytium formed through rounds of cell fusion. EFF-1 is the essential fusogen required for the correct formation of the syncytium. We speculate that the abnormal SAX-7 pattern in the eff-1 mutant is probably due to cell fusion defects during epidermal development. The SAX-7-positive patches in the eff-1 mutant might correspond to the unfused epidermal precursor cells. To test this hypothesis, we marked the cell junctions using AJM-1::GFP to see if the disorganized ectopic branches were correlated with the unfused hypodermal cell. Consistent with our hypothesis, most of (>90%) the disorganized ectopic branches were localized to the unfused cells outlined by the AJM-1 marker, and most of the unfused cells contain ectopic branches in eff-1(hy21) mutant (Fig. S4).
The SAX-7–MNR-1–DMA-1 pathway is epistatic to eff-1
To further test if eff-1 functions by regulating the pattern of SAX-7, we examined genetic interactions between eff-1 and the PVD patterning receptor–ligand genes. If eff-1 controls the PVD dendrite through SAX-7 patterning, sax-7 should be epistatic to eff-1. Indeed, the eff-1(hy21); sax-7(nj48) double mutant resembled the sax-7 single mutant phenotype, in which tertiary and quaternary branches were nearly completely absent, and the secondary branches were disorganized (Fig. 5A–D,I,J). Our previous result showed that MNR-1 functions in the SAX-7 pathway, and both MNR-1 and SAX-7 are required to activate the DMA-1 receptor on the PVD dendrite. Similarly, the eff-1(hy21); mnr-1(wy758) double mutant also resembled the mnr-1 single mutant phenotype, in which tertiary and quaternary branches were nearly completely absent, and the secondary branches were disorganized (Fig. 5E,F,I,J). Similarly, the eff-1(hy21); dma-1(wy686) double mutant resembled dma-1 single mutant phenotype, in which tertiary and quaternary branches were nearly completely absent, and the number of secondary branches was decreased (Fig. 5G,H–J). These results suggest that the SAX-7–MNR-1–DMA-1 pathway is required for the ectopic PVD dendrite phenotype in the eff-1 mutant.
PVD dendrites are patterned by several extrinsic and intrinsic factors. The main extrinsic factors come from the epidermal cell adhesion molecule SAX-7 and MNR-1, as well as the secreted factor LECT-2 from muscles. SAX-7, MNR-1 and LECT-2 form a ligand–receptor complex with the transmembrane protein DMA-1, which serves as a receptor to guide the growth and branching of dendrites. SAX-7 forms an orderly striped pattern on the epidermal cell around the tertiary and quaternary region to guide PVD dendrite growth. It is not well understood which molecular mechanisms are required to form the pattern of SAX-7. Our tissue-specific rescue experiment indicates that the dynein complex is required in the epidermal cell to pattern SAX-7.
Dynein is the major minus-end motor along microtubule and functions in many cellular processes such as cell division and axonal transport. Dynein has also been reported to function cell-autonomously in organelle transport in dendrites (Zheng et al., 2008), delivering Golgi complexes to axons and dendrites (Jaarsma and Hoogenraad, 2015). These are consistent with our autonomous function of DLI-1 in PVD distal dendrite development. It is likely that the dynein complex is required to transport cargoes to anterior distal dendrites to support dendrite formation or maintenance there. In the anterior PVD dendrite, the microtubules are oriented with their minus ends pointing to the distal dendrite. This polarity dictates that dynein is the motor that is responsible for trafficking materials towards the distal arbors. Importantly, we also found an unexpected non-autonomous function of dynein in the epidermis to pattern the PVD dendrite. We found that DLI-1 also functions in epidermal cells to pattern the adhesion molecule SAX-7, which, in turn, guides formation of PVD dendrite branches. It is not clear yet how DLI-1 affects the SAX-7 localization. Using the nuclear marker Pdpy-7::GFP::SV40, we found that the dli-1 mutant has a reduced number of epidermal nuclei and enlarged nuclei (Fig. S1). This result suggests that a lack of dli-1 in the epidermis might cause pleiotropic defects, which indirectly affects localization of SAX-7.
Our data are most consistent with the model whereby the abnormal dendritic morphology in the eff-1 mutant is caused by the defective epidermal SAX-7 pattern. Based on the essential function of EFF-1 in cell–cell fusion, it is not surprising that there are unfused epidermal cells in the mutant, as shown by the abnormal pattern of AJM-1::GFP. The fact that the majority of the ectopic PVD dendrites localized to unfused epidermal cells further suggests that a defect in epidermal cell fusion is the likely cause for the abnormal SAX-7 patterning in the eff-1 mutant. These conclusions disagree with a previous paper (Oren-Suissa et al., 2010). In that paper, it was shown that PVD expression of eff-1 with the des-2 promoter partially rescues the eff-1 mutant, but expression of eff-1 in the epidermis did not rescue the phenotype. We found that Pdes-2 is expressed not only in PVD dendrites but also in muscle and other neurons (Fig. S5). It is conceivable that the partial rescue reported in the previous study might result from the potentially very weak expression of eff-1 in the epidermis. We used a more-specific promoter, ser-2Prom3, which is mainly expressed in two PVD and two OLL neurons (dim expression in PDE and another ventral cord neuron), and did not detect any rescuing activity.
In the previously mentioned paper, Oren-Suissa et al. (2010) and colleagues were not able to assess the effect of the epidermal expression of eff-1 because of lethality problems when Pdpy-7::eff-1 was injected at 1 ng/μl. Injection at a lower concentration (0.1 ng/μl) failed to exhibit any rescuing activity (Oren-Suissa et al., 2010). Consistent with their data, at 0.1 ng/μl, we found that only one of the four lines showed a partial rescue activity, suggesting that injection at this concentration might be too low to achieve significant expression of eff-1 in the epidermis. However, we were successful in obtaining transgenic lines at higher injection concentrations. One transgenic line at 5 ng/μl and 3 transgenic lines at 1 ng/μl completely rescued the eff-1 mutant, strongly suggesting that eff-1 functions in the epidermal cell in a dose-dependent manner to regulate the pattern of SAX-7 and PVD dendrite morphology.
Recently, Neumann et al. (2015) reported that eff-1 mediates regenerative axonal fusion in PLM through a cell autonomous manner. While it is likely that EFF-1 does function in neurons, our results showing that epidermal expression of EFF-1 rescued the mutant phenotype challenge the function of cell-autonomous dendrite fusion in sculpting PVD dendritic arbors.
MATERIALS AND METHODS
C. elegans strains
All the strains used come from the WT worm strain N2 (see Table S1). Hermaphrodite young adults were analyzed for all the data. C. elegans strains used in this study were cultured at 20°C on nematode growth medium (NGM) plates seeded with the Escherichia coli OP50 using standard methods (Brenner, 1974). The eff-1(hy21) worms were cultured at 15°C before injection and transferred to 25°C after injection for phenotype quantification. eff-1(hy21);jcIs1/wyIs581 were quantified at 20°C.
Constructs and transgenes
PCR and molecular cloning techniques were used to construct plasmids (see Table S2). Transgenic worms were generated by injection using standard methods (Mello and Fire, 1995). The Pdpy-7::EFF-1::eff-1_3′UTR plasmid was injected to the eff-1(hy21) mutant at the concentration of 0.1, 1 and 5 ng/µl. ser-2Prom3::EFF-1::eff-1_3′UTR was injected at 15 and 50 ng/µl. Podr-1::GFP was used as the co-injection marker. ser-2Prom3:: DLI-1::GFP and ser-2Prom3::myri-mCherry plasmids were injected to dli-1(wy50053)/unc-26(e205); dpy-4(e1166) mutant at concentrations of 30 ng/µl and 20 ng/µl, respectively. Pdpy-7::DLI-1::GFP, Pdpy-7::DLI-1 and ser-2Prom3::myri-mCherry plasmids were injected to dli-1(wy50053)/unc-26(e205) dpy-4(e1166) mutant at the concentration of 30 ng/µl and 20 ng/µl, respectively. Pdpy-7::SAX-7S::GFP and ser-2Prom3::myri-mCherry plasmids were injected to dli-1(wy50053)/ unc-26(e205); dpy-4(e1166) mutant at the concentration of 15 ng/µl and 25 ng/µl, respectively. Podr-1::GFP and Podr-1::RFP were used as the co-injection marker.
Young adult animals were mounted to a drop of M9 containing 1 mg/ml levamisole on a 3% agar pad. Fluorescent images were captured as described previously (Chai et al., 2012). Confocal images were recorded with Micro-Manager and processed using ImageJ.
All quantifications were carried out randomly. For Figs 1–4 and Figs S3,S4, to quantify the number of ectopic branches, the images of the cell body region were taken using spinning-disk confocal microscopy. The focal plane that contained the most ectopic branches was chosen in each worm. The ectopic branches were defined as the branches which grow from primary branch but were not perpendicular to it, the branches from secondary branches but failed to localize at sublateral site, and the retrograde branches from tertiary branches which showed an opposite direction to the quaternary branches. All of the free endings of these ectopic branches were quantified and finally normalized to 100 μm. To quantify the penetrance of patch dendrites, the total length of the ectopic branches and patch-related ectopic branches were measured with ImageJ in one field. For Fig. 5, the ectopic branches were counted from the whole animal with a Zeiss compound fluorescent microscope, and the ectopic branch was defined following above.
We are grateful to the Caenorhabditis Genetics Center and Dr G. S. Ou for strains. We thank X. T. Dong for some strains construction.
Conceptualization: X.-M.W., K.S.; Methodology: T.Z., X.L., X.-M.W., K.S.; Validation: T.Z., X.L., X.-M.W., K.S.; Formal analysis: T.Z., X.L., X.-M.W., K.S.; Investigation: T.Z., X.L., X.-M.W.; Resources: X.-M.W., K.S.; Data curation: T.Z., X.L., X.-M.W., K.S.; Writing - original draft: X.-M.W., K.S.; Writing - review & editing: X.-M.W., K.S.; Visualization: X.-M.W., K.S.; Supervision: X.-M.W., K.S.; Project administration: X.-M.W., K.S.
This work was supported by the National Basic Research Program of China (2013CB910103), grants from the National Natural Science Foundation of China (NSFC; 31571061, 31771138, 31428009) to X.-M.W. and K.S., the Howard Hughes Medical Institute and National Institute of Neurological Disorders and Stroke (NINDS; 1R01 NS082208) to K.S., and the CAS/SAFEA International Partnership Program for Creative Research Teams to K.S., and Youth Innovation Promotion Association CAS funding to X.-M.W. Deposited in PMC for release after 12 months.
The authors declare no competing or financial interests.