Abstract
Phenotypic plasticity is a critical component of an organism’s ability to thrive in an ever-changing environment. The free-living nematode, Caenorhabditis elegans, adapts to unfavorable environmental conditions by pausing reproductive development and entering a stress-resistant larval stage known as dauer. The transition into dauer is marked by vast morphological changes – including remodeling of epidermis, neurons and muscle. Though many of these dauer-specific traits have been described, the molecular basis of dauer-specific remodeling is still poorly understood. Here we show that the nidogen-domain containing protein DEX-1 functions downstream of the dauer decision to facilitate stage-specific tissue remodeling during dauer morphogenesis. DEX-1 was previously shown to regulate sensory dendrite formation during embryogenesis. We find that DEX-1 is also required for the proper remodeling of the stem cell-like epidermal seam cells and maintenance of seam cell quiescence during dauer. dex-1 mutant dauers lack distinct lateral cuticular alae during dauer and have increased sensitivity to sodium dodecyl sulfate (SDS). Furthermore, we find that DEX-1 mediated seam cell remodeling is required for proper dauer mobility. We show that DEX-1 acts cell autonomously in the seam cells during dauer and that dex-1 expression during dauer is regulated through DAF-16/FOXO-mediated derepression. Finally, we show that dex-1 interacts with a family of zona pellucida-domain encoding genes to regulate dauer-specific epidermal remodeling. Taken together, our data indicates that DEX-1 plays a central role in C. elegans epidermal remodeling during dauer.
Introduction
In order to survive changing environments, organisms modify their phenotype (i.e. phenotypic plasticity). Tissue remodeling is an important component of stress-induced phenotypic plasticity. For example, desert locusts are capable of altering their morphology between distinct ‘gregarious’ and ‘solitarious’ phases based on population density (Pener and Simpson 2009), and certain species of butterfly also change their body morphology and wing color based on seasonal cues (Windig 1994). While these large-scale displays of phenotypic plasticity are readily observed, the molecular basis of tissue remodeling in response to environmental inputs is often unclear.
C. elegans is a useful animal to investigate the molecular mechanisms that facilitate stress-induced remodeling. Under favorable growth conditions, C. elegans develops continuously through four larval stages (L1-L4) into a reproductive adult. However, under unfavorable environmental conditions, C. elegans larvae arrest their development at the second larval molt and enter the stress-resistant dauer stage (Cassada and Russell 1975; Golden and Riddle 1984). Dauers are specialized, non-feeding larvae capable of withstanding extended periods of adverse environmental conditions. Dauer-specific stress resistance is likely facilitated by several morphological changes that occur during dauer formation. For example, dauers display both structural and biochemical differences in their epidermis and cuticle compared with non-dauers (Cox et al. 1981; Blaxter 1993). Dauer formation also corresponds with a general radial shrinkage of the body and the formation of longitudinal cuticular ridges called alae (Fig. 1A).
Radial shrinkage and alae formation are regulated by a set of lateral hypodermal seam cells (Singh and Sulston 1978; Melendez et al. 2003). Seam cell function and remodeling are critical for proper dauer morphology and increased environmental resistance. The seam cells also have stem cell-like properties. During non-dauer development, the seam cells undergo asymmetrical divisions at larval molts to produce an anterior differentiated cell and a posterior seam cell. Alternatively, if the animal enters dauer diapause, the seam cells shrink and stop dividing.
Here, we characterize the role of DEX-1, a protein similar to mammalian tectorin and SNED1, in remodeling of the seam cells during dauer formation. DEX-1 comprises a transmembrane protein with two extracellular nidogen domains that is required for proper sensory neuron dendrite formation during embryogenesis (Heiman and Shaham 2009). We find that DEX-1 is also required during dauer formation for seam cell remodeling and resistance to environmental stressors. Furthermore, we find that dex-1 is upregulated in seam cells during dauer in a DAF-16 dependent manner. DEX-1 was previously shown to interact with a zona pellucida (ZP) domain containing protein to mediate dendrite extension. Our data suggest that DEX-1 interacts with additional ZP-domain proteins, to regulate seam cell remodeling. Finally, we implicate DEX-1 in regulating seam cell quiescence. Combined with previous data demonstrating a role for DEX-1 in sensory dendrite adhesion (Heiman and Shaham 2009), our data suggest that DEX-1 plays a role in modulating cell shape of several cell types throughout development.
Materials and Methods
Strains
All strains were grown under standard conditions unless otherwise noted (Brenner 1974). The wild-type Bristol N2 strain and the following mutant strains were used: CHB27 dex-1(ns42) III; SP1735 dyf-7(m537) X; CB1372 daf-7(e1372) III; DR129 daf-2(e1370) unc-32(e189) III; DR27 daf-16(m27) I; FX01126 cut-1(tm1126) IIl; RB1574 cut-6(ok1919) III; RB1629 cut-5(ok2005) X. dex-1(ns42) was given to us by Dr. Maxwell Heiman (Department of Genetics, Harvard University). cut-1(tm1126) was provided by the Mitani Consortium (Department of Physiology, Tokyo Women’s Medical University School of Medicine, Japan). All other mutant strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The following transgenic animals were also provided by the CGC: ST65 ncIs13[ajm-1::GFP]; JR667 wIs51 [SCMp::GFP+unc-119(+)]. The IL2 neurons were observed using PT2660 myIs13[klp-6p::GFP+pBx] III (Schroeder et al., 2013); PT2762 myIs14[klp-6p::GFP +pBx] V and JK2868 qIs56[lag-2p::GFP] (Blelloch et al.; Ouellet et al. 2008; Schroeder et al. 2013).
The following plasmids were the generous gift of Dr. Maxwell Heiman: pMH7 dex-1p::dex-1, pMH8 pha-4p::dex-1, pMH111 dex-1p(5.7kb)::gfp, pMH125 dex-1p(2.1kb)::gfp (Heiman and Shaham 2009). Novel plasmids were constructed using Gibson Assembly (NEB, E2611S). The seam cell-specific expression plasmid was built by replacing the dex-1 promoter from pMH7 with a 1.21kb cut-5 promoter region (for a complete list of primers used to construct plasmids, see Table S1). The hypodermal-specific dex-1 plasmid was constructed by replacing the dex-1 promoter in pMH7 with the dpy-7 promoter (Gilleard et al. 1997). The IRS sequence was deleted from pMH111 using the Q5 Site Directed Mutagenesis Kit (NEB, E05525).
Animals containing extrachromosomal arrays were generated using standard microinjection techniques (Mello et al. 1991) and genotypes confirmed using PCR analysis and observation of co-injection markers. Each animal was injected with 20μL of plasmid and 80μL of unc-122p::gfp as the co-injection marker.
Dauer Formation
Dauers were induced by one of two methods. For non-temperature sensitive strains, we used plates containing crude dauer pheromone extracted by previously established procedures (Vowels and Thomas 1992; Schroeder and Flatt 2014). For animals with mutations in daf-7(e1372) or daf-2(e1370), dauers were induced using the restrictive temperature of 25°C (Riddle et al. 1981).
Microscopy and Rescue Analysis
Animals were mounted onto 4% agarose pads and anesthetized with 1.0 mM or 0.1 mM levamisole for dauers and non-dauer or partial dauers, respectively. A Zeiss AxioImager microscope equipped with DIC and fluorescent optics was used to collect images. Images were analyzed using FIJI. For radial constriction experiments, measurement data was analyzed using a One-Way ANOVA with Bonferroni’s multiple comparisons test using Graphpad Prism 6 software. The seam cell area was measured for V2pap, V2ppp and V3pap and averaged to give one measurement per animal (Sulston and Horvitz 1977).
SDS Sensitivity Assays
Sodium dodecyl sulfate (SDS) dose-response assays were performed using 12-well culture dishes with wells containing M9 buffer and specified concentrations of SDS dissolved in water. Twenty dauers from each treatment were transferred into SDS. Dauer animals were exposed to SDS for 30 minutes and scored using established criteria (Liu et al. 2013) Briefly, animals were stimulated once with an eyelash and were scored as alive if movement was observed. Each concentration was tested in triplicate with each experiment containing a separate wild-type (N2) control. The LD50 and confidence interval of each concentration was calculated using probit analysis in Minitab 18.
Fluorescent Bead Feeding Assay
Feeding assays were carried out using established methods (Nika et al. 2016).Briefly, fluorescent beads (Sigma L3280) were added to a 10X concentrated OP50 E. coli overnight culture. Fresh NGM plates were then seeded with 65 µL of the bead/bacteria suspension and allowed to dry. Twenty dauer or non-dauer animals were added to the plate and incubated at 20°C for 40 minutes. Worms were then observed for the presence of fluorescent beads in the intestinal tract. Each experiment was performed twice.
Locomotion Assays
Animals were transferred to unseeded NGM plates and allowed to sit at room temperature for 10 minutes before being assayed. Animals were stimulated near the anus with an eyelash and the number of body bends was scored. Counting was stopped if the animal did not complete another body bend within 5 seconds of stopping, or if the animal reversed direction. Each animal was scored twice and then removed from the plate. Counts were averaged and then analyzed using a Kruskal-Wallis test with Dunn’s multiple comparisons test for dauers and the Mann-Whitney U test for adult animals using Graphpad Prism 6 software.
DEX-1 Expression Analysis
The fluorescent intensities of the V2pap, V2ppp and V3pap seam cells were measured using established methods (McCloy et al. 2014). Briefly, each cell was outlined and the area, integrated density and mean gray value were measured. Measurements were also taken for areas without fluorescence surrounding the cell. The total corrected cell fluorescence (TCCF = integrated density - (area of selected cell * mean fluorescence of background reading)) was then calculated for each cell. The intensities of the three cells from each worm were averaged such that each nematode comprised a single data point. The data were analyzed using One-Way ANOVA and Bonferroni’s multiple comparisons test. Ten animals were measured for each genotype.
Results
DEX-1 is required for proper dauer morphology and behavior
Wild-type C. elegans dauers have a distinctive morphology due to radial shrinkage that leads to a thin appearance compared with non-dauers (Figure 1A). We found that dex-1(ns42) mutants are defective in dauer radial shrinkage (Figure 1C). dex-1 mutant dauers are significantly wider in diameter when compared to wild-type dauers, leading to a ‘dumpy dauer’ phenotype (Figure 1, B and C). The defect in body size appears specific to the dauer stage, as non-dauer dex-1 mutants show no differences in body size compared with wild-type non-dauers (Figure S1). Radial shrinkage in dauers is correlated with the formation of longitudinal cuticular ridges on the lateral sides of the animal called the alae (Cassada and Russell 1975). The lateral alae of dex-1 mutant dauers are indistinct compared with wild-type dauers (Figures 1B and 2S). To confirm dex-1 as the causative mutation, we rescued the radial shrinkage phenotype of dex-1 mutants with dex-1 cDNA under the control of its endogenous promoter (Figure 1C). Together, these data suggest that dex-1 mutants form partial dauers with defects in epidermal remodeling.
In addition to radial shrinkage, dauers have several structural modifications compared with non-dauer animals that lead to increased resistance to environmental insults (Cassada and Russell 1975; Androwski et al. 2017). We characterized several dauer-specific phenotypes in dex-1 mutants. First, we exposed dauer animals to SDS. Wild-type dauers survive for hours in 1% SDS (Cassada and Russell 1975). We found that while dex-1 mutant dauers were sensitive to 1% SDS, they were able to survive significantly higher concentrations of SDS that wild-type non-dauer animals (Figure 1D) Similar to our radial shrinkage data, we could effectively rescue the SDS phenotype with a wild-type copy of dex-1 (Figure 1E). Second, we used a fluorescent bead assay that determines whether feeding occurs (Nika et al. 2016). Non-dauers will readily ingest beads and show fluorescence throughout the digestive system. Dauers suppress pharyngeal pumping and have a buccal plug that prevents entry of fluorescent beads. We found that while fluorescence was never observed in dex-1 mutant dauer intestines, we occasionally observed fluorescence in the buccal cavity (Figure S3). We never observed pharyngeal pumping in dex-1 dauers. These data suggest that while pharyngeal pumping is efficiently suppressed, dex-1 dauers have low-penetrance defects in buccal plug formation. Finally, we examined dex-1 dauers for the presence of dauer-specfic gene expression of lag-2p::gfp in the IL2 neurons during dauer (Ouellet et al. 2008). Similar to wild-type dauers, dex-1 dauers showed appropriate dauer-specific expression, indicating that they are indeed dauers with defects limited to epidermal remodeling (data not shown).
The decision to enter dauer is based on the ratio of population density to food availability (Golden and Riddle 1984). C. elegans constitutively secrete a pheromone mixture that is sensed by animals and, at high levels, triggers dauer formation (Golden and Riddle, 1982). Dauers can be picked from old culture plates (starved) or can be induced using purified dauer pheromone. We found no difference in the dauer phenotype between starved or pheromone-induced dex-1 mutant dauers. The C. elegans insulin/IGF-like and TGF-β signaling pathways function in parallel to regulate the dauer formation decision. Reduced insulin and TGF-β signaling induced by overcrowding and scarce food promotes dauer formation. Disruption of either the insulin-receptor homolog DAF-2 or the TGF-β homolog DAF-7 results in constitutive formation of dauers. Double mutants of daf-2 or daf-7 with dex-1 did not suppress the defects in dauer morphogenesis of dex-1, suggesting that dex-1 is acting downstream of the dauer decision pathway (data not shown).
DEX-1 functions in the stem cell-like seam cells to regulate dauer morphogenesis
dex-1 is expressed at high levels during embryogenesis to regulate sensory dendrite formation (Heiman and Shaham 2009). However, previous microarray data suggested that dex-1 is also upregulated during dauer (Liu et al. 2004). We generated transgenic animals expressing GFP driven by a 5.7kb 5’ dex-1 upstream promoter and observed bright fluorescence in the seam cells and glia socket cells of the anterior and posterior deirid neurons starting in the pre-dauer L2 (L2d) stage. Expression of dex-1p::gfp in the seam cells and deirid socket cells persists throughout dauer and is downregulated upon recovery from dauer (Figure 2, A and B). We also observed weak dex-1p::gfp expression in unidentified pharyngeal cells during all larval stages (Figure S4).
Dauer-specific radial shrinkage and subsequent lateral alae formation are facilitated by shrinkage of the seam cells (Singh and Sulston, 1978; Melendez et al, 2003). Using the ajm-1::gfp apical junction marker (Koppen et al., 2001), we found that dex-1 mutant dauer seam cells are larger and have jagged, rectangular edges unlike the smooth, elongated seam cells of wild type dauers (Figure 3). These data suggest that DEX-1 is required for seam cell remodeling. The seam cells also have stem cell-like properties. During non-dauer development, seam cells divide at larval molts to produce a seam cell daughter and a differentiated daughter cell (Sulston and Horvitz, 1977). During dauer, seam cells enter a quiescent state and only resume division following recovery from dauer. To determine if dex-1 is required for maintaining seam cell quiescence during dauer, we used a seam cell nuclei marker to examine the number of seam cell nuclei in wild-type and dex-1 backgrounds. We found that dex-1 mutant dauers have a slight, but statistically significant, greater number of seam cell nuclei compared with wild type dauers (dex-1 , WT , p = 0.0010, n = 40). This could suggest that DEX-1 plays a role in maintaining seam cell quiescence during dauer.
To determine where dex-1 acts to regulate seam cell remodeling, we expressed dex-1 cDNA under the control of cell-specific promoters. First, we expressed dex-1 in the seam cells using the cut-5 promoter. cut-5 was previously shown to be expressed specifically in the seam cells during L1 and dauer (Sapio et al. 2005). Using this seam cell-specific promoter, we partially rescued SDS sensitivity in dex-1 mutant dauers (Figure 1E). The seam cell-specific expression of dex-1 resulted in a mosaic rescue of the radial shrinkage phenotype (Figure S5). The rescued seam cells that underwent radial constriction also displayed intact lateral alae, while the non-rescued seam cells’ alae remained indistinct. Expression of dex-1 under a pharyngeal promoter failed to rescue the dex-1 seam cell phenotype suggesting a cell autonomous role for DEX-1. To verify the cell autonomous nature of dex-1, we sought to examine expression in a tissue with close contact to the seam cells. The seam cells are surrounded by a syncytial hypodermis. dex-1 expression in the surrounding hypodermis failed to rescue the dex-1 dauer-specific phenotype. Interestingly, expression of dex-1 in the hypodermis induced a dumpy phenotype in approximately 33% of non-dauer animals (Fig. S6). Together, these data indicate that dex-1 acts cell-autonomously to regulate seam cell remodeling during dauer.
DEX-1 acts in seam cells to regulate locomotion during dauer
Morphological changes during dauer are accompanied by changes in behavior. Wild-type dauer animals are general quiescent, but move rapidly when mechanically stimulated (Cassada and Russell 1975). Anecdotally, we noticed a higher percentage of quiescent dex-1 dauers than seen with wild-type dauers. To quantify this behavior, we developed a behavioral assay that quantifies movement following mechanical stimulation (see Materials and Methods). Although both dex-1 mutant and wild-type dauers initially respond to mechanical stimulation, the dex-1 mutant dauers have significantly reduced locomotion and display slightly uncoordinated body movements (Figure 1F and data not shown). This locomotion defect was dauer-specific, as non-dauer dex-1 animals moved at wild-type levels following mechanical stimulation (Figure S1).
Given that dex-1 was primarily expressed in the seam cells during dauer, we tested if seam cell-specific expression could rescue the behavioral phenotype. Surprisingly, seam cell-specific dex-1 expression completely rescued the dex-1 dauer locomotion defects (Figure 1F). In addition to seam cell remodeling, several neuron types remodel during dauer formation (Albert and Riddle 1983). For example, the IL2 and deirid sensory neurons remodel during dauer formation (Albert and Riddle 1983; Schroeder et al. 2013). The IL2s regulate dauer-specific behaviors (Lee et al. 2011; Schroeder et al. 2013), while the deirids respond to specific mechanical cues (Sawin et al. 2000). We, therefore, examined these neuron classes using fluorescent reporters; however, we observed no obvious difference in the neuronal structures between dex-1 and wild-type (data not shown).
dex-1 expression in dauers is regulated by DAF-16
To understand how dex-1 expression is regulated, we examined the 5’ upstream region of dex-1 for potential regulatory sites. Previous Chromatin Immunoprecipitation (ChIP-seq) data identified a putative DAF-16 binding site upstream of the dex-1 coding region (Celniker et al. 2009). DAF-16 is the sole C. elegans ortholog of the human Forkhead BoxO-type transcription factor and a major regulator of the dauer decision. To examine whether this region affects expression of dex-1, we first expressed GFP from a truncated 2.1kb dex-1 promoter. Unlike the 5.7kb dex-1 promoter fusion, the short dex-1 promoter drove GFP expression in the seam cells during all larval stages (Figure 2, C and D). This suggests there are elements within the long promoter that repress dex-1 expression during non-dauer stages. While mutations in daf-16 result in animals incapable of forming dauers, under conditions of high pheromone concentrations daf-16 mutants can enter into a partial dauer state (Vowels and Thomas 1992; Gottlieb and Ruvkun 1994). daf-16 partial dauers are identifiable by body morphology and the presence of indistinct lateral alae (Vowels and Thomas 1992). To determine if DAF-16 is regulating dex-1 expression during dauer, we first examined the expression of the 5.7 kb dex-1p::gfp reporter in daf-16 partial dauers. We found that the fluorescent intensity of dex-1p::gfp was significantly reduced in the daf-16 partial dauers compared to wild-type, suggesting that DAF-16 regulates dex-1 seam cell expression during dauer (Figure 4, B-D).
FOXO/DAF-16 binds to canonical DAF-16 binding elements and insulin response sequences (IRS) (Paradis and Ruvkun 1998; Obsil and Obsilova 2008). Within the ChIP-seq identified region (Celniker et al. 2009), we identified a putative insulin response sequence (IRS) binding site (Figure 4A). To determine whether the identified DAF-16 IRS site directly regulates dex-1 expression, we deleted the IRS sequence in the 5.7kb dex-1 promoter region used to drive GFP. We found that deleting the IRS sequence results in reduced GFP expression in the seam cells during dauer, similar to the levels observed in daf-16 partial dauers (Figure 4, B-E). Taken together, these results indicate that a repressor element lies within the 5.7kb promoter and that DAF-16 binding to the IRS acts to derepress dex-1 expression during dauer formation.
dex-1 interacts with genes encoding ZP-domain proteins
DEX-1 interacts with the ZP-domain protein DYF-7 to regulate primary dendrite extension during embryogenesis (Heiman and Shaham 2009). We, therefore, examined the dyf-7(m537) mutant for defects in dauer morphogenesis. Unlike dex-1 mutants, dyf-7 mutants are defective for dauer formation under typical dauer-inducing environmental conditions (Starich et al. 1995). We, therefore, examined dyf-7 mutants in dauer formation constitutive mutant backgrounds. Both daf-2; dyf-7 and daf-7; dyf-7 double mutants were normal for dauer-specific radial shrinkage, SDS resistance, and IL2 arborization (data not shown).
The cuticlin (CUT) proteins are a family of ZP-domain proteins including several that are required for proper dauer morphology. Disruption of cut-1, cut-5 and cut-6 result in dauers with incomplete radial shrinkage and defective alae formation (Sebastiano et al. 1991; Muriel et al. 2003; Sapio et al. 2005b). We asked whether these defects in CUT mutant larvae were due to seam cell remodeling. We found that, similar to dex-1, the seam cells of the CUT mutants were rectangular with jagged edges (Figure 5). Also similar to dex-1, we found that cut-1 and cut-5 dauers were more sensitive to SDS compared with wild type dauers (Table 1). Interestingly, while cut-6 mutant dauers were resistant to the standard 1% SDS treatment (Muriel et al. 2003), we found that the cut-6 mutant dauers were substantially more sensitive to SDS than wild-type dauers (Table 1). Taken together, these data indicate similar roles for DEX-1 and cuticlins during dauer remodeling.
We hypothesized that, similar to its interaction with DYF-7 during embryogenesis, DEX-1 may interact with the CUT proteins during dauer. We, therefore, examined double mutants of dex-1 with cut-1, cut-5, and cut-6. The dex-1; cut-1 double mutant did not enhance SDS sensitivity beyond the dex-1 single mutant, suggesting that they may act in the same pathway to regulate dauer remodeling. The dex-1; cut-5 double mutant was synthetically lethal during embryogenesis or early L1. This is similar to the dex-1; dyf-7 double mutant (Heiman and Shaham 2009), suggesting that in addition to roles in dauer remodeling, cut-5 and dex-1 have redundant roles during embryogenesis. However, unlike the dex-1; dyf-7 double mutant (Heiman and Shaham 2009), the dex-1; cut-5 synthetic lethality did not display temperature sensitivity (data not shown). Interestingly, the dex-1cut-6 double mutant was intermediate in SDS sensitivity between the dex-1 and cut-6 single mutants (Table 1).
We further tested the cut mutant phenotypes by generating double mutants between each of the cut mutants (Table 1). The cut-1;cut-5 double mutant showed a significant reduction of SDS resistance compared to single mutants alone, suggesting that they may be acting in parallel pathways. The cut-1;cut-6 double mutants retained the cut-6 SDS sensitivity phenotype, suggesting that cut-6 is epistatic to cut-1. The cut-6;cut-5 dauers showed a drastic increase in sensitivity to SDS compared to the single mutants, indicating that these genes may also play roles in parallel pathways during dauer remodeling. Interestingly, the cut-6; cut-5 double mutant showed a severe dumpy phenotype in all developmental stages. These results confirm previous work demonstrating a broad role for ZP-domain proteins in development (Heiman and Shaham 2009; Gill et al. 2016).
Discussion
The dauer stage of C. elegans is an excellent example of a polyphenism, where distinct phenotypes are produced by the same genotype via environmental regulation (Simpson et al. 2011). Compared to the decision to enter dauer, little is known about the molecular mechanisms controlling remodeling of dauer morphology. DEX-1 was previously characterized as a component of embryonic neuronal development (Heiman and Shaham 2009). Our data shows that DEX-1 also functions during dauer-specific remodeling of the stem cell-like seam cells. We demonstrate a cell-autonomous role for DEX-1 in the regulation of seam cell remodeling during dauer morphogenesis.
We found that dex-1 is required for the shrinkage of the seam cells and formation of lateral alae during dauer. Furthermore, we found that dex-1 dauers have significantly more seam cells than wild-type dauers, suggesting ectopic divisions during dauer diapause. In mammalian cell lines, stem cell shape regulates differentiation (McBeath et al. 2004; Kilian et al. 2010). For example, mesenchymal stem cells will differentiate into adipocytes or osteoblasts depending on whether the cell is round or flat, respectively (McBeath et al. 2004). We speculate that the shrinkage observed during dauer may be important for maintenance of seam cell quiescence.
We also found the dex-1 mutant dauers have defects in locomotion when mechanically stimulated. We originally assumed that this could be due to the lack of lateral alae; however, our seam-cell specific rescue of dex-1 resulted in a mosaic pattern of alae formation while having complete rescue of behavior. We, therefore, speculate that dex-1 mutant dauers may have defects in other neuron classes that result in locomotion defects during dauer. Previous RNAi data shows that knockdown of dex-1 results in low penetrance defects in motor neuron commissure formation (Schmitz et al. 2007). Dauers exhibit changes in somatic muscle structure (Dixon et al. 2008). It will be interesting to determine if dex-1 mutants have dauer-specific defects in neuromuscular junctions.
Dissection of the genetic pathways regulating the decision to enter dauer has revealed insights into TGF-β, insulin and hormone signaling. The FOXO transcription factor, DAF-16, is a well-known regulator of the dauer formation decision by acting downstream of the insulin/IGF-1 receptor DAF-2. Dauer-inducing environmental conditions lead to a translocation of DAF-16 to the nucleus where it activates dauer formation pathways (Lee et al. 2001; Fielenbach and Antebi 2008). We found that dex-1 expression during dauer is regulated by DAF-16. Based on our results we propose that DEX-1 is normally repressed during non-dauer post-embryonic stages and DAF-16 serves to derepress dex-1 expression via an upstream insulin response sequence. The dex-1p::gfp fluorescence was not completely eliminated in the daf-16 partial dauers and the IRS-deletion dauers suggesting that both dex-1 repressors and other regulatory elements remain to be identified.
Together with previous work (Heiman and Shaham 2009), our data suggests that DEX-1 may interact with ZP-domain proteins. While the cut mutants are all deletion alleles that disrupt the ZP-domains and, therefore, likely functional nulls, the sole dex-1(ns42) allele is a nonsense mutation that disrupts a region resembling mammalian zonadhesin (Heiman and Shaham 2009). It is possible that dex-1 is a non-null and, therefore, genetic interaction experiments should be interpreted with caution. It has been proposed that biochemical compaction of the cuticlins in the extracellular space between the seam and the hypodermis causes radial constriction, and thus forms the lateral alae via a ‘cuticlin tether’ (Sapio et al. 2005). We add to this by proposing that DEX-1 may function as a molecular binding hub for multimerized cuticlins. Our data indicate that DEX-1 acts in a cell autonomous manner within the seam cells. DEX-1 includes a transmembrane domain and, therefore, may function as a transmembrane anchor while simultaneously binding the multimerized cuticlins in the extracellular space (Figure 6). Alternatively, DEX-1 could act as a secreted factor with restricted localization to the apical border of the seam cell. During embryogenesis, DEX-1 is secreted and localized to the dendritic tips (Heiman and Shaham, 2009). In both models, DEX-1 may serve to couple physical interactions between the remodeled cuticular extracellular matrix and seam cell shape (Figure 6). Failure of these tissues to properly compact and thicken due to a loss of DEX-1 could lead to an overall weakening of the cuticle and thus result in SDS sensitivity observed in dex-1 mutant dauers. Interestingly, DEX-1 is similar to the human extracellular matrix protein SNED1 (Sushi, nidogen, and EGF-like Domain 1) that promotes invasiveness during breast cancer metastasis (Naba et al. 2014), suggestive of a mechanical role in tissue remodeling. Previous research demonstrated a role for autophagy dauer-specific seam cell remodeling (Melendez et al. 2003). It will be interesting to determine if dauer-specific changes to autophagy are influenced through DEX-1 mediated mechanical forces.
Acknowledgments
We thank Max Heiman for reagents and advice, and Scott Robinson at the Beckman Institute for assistance with electron microscopy. This work was supported by the National Institutes of Health (grant R01GM111566 to NES).