Epidermal Collagen Reduction Drives Selective Aspects of Aging in Sensory Neurons

ABSTRACT

Despite advances in understanding molecular and cellular changes in the aging nervous system, the upstream drivers of these changes remain poorly defined. Here, we investigate the roles of non‐neural tissues in neuronal aging, using the cutaneous PVD polymodal sensory neuron in Caenorhabditis elegans as a model. We demonstrate that during normal aging, PVD neurons progressively develop excessive dendritic branching, functionally correlated with age‐related proprioceptive deficits. Our study reveals that decreased collagen expression, a common age‐related phenomenon across species, triggers this process. Specifically, loss‐of‐function in dpy‐5 or col‐120, genes encoding cuticular collagens secreted to the epidermal apical surface, induces early‐onset excessive dendritic branching and proprioceptive deficits. Adulthood‐specific overexpression of dpy‐5 or col‐120 mitigates excessive branching in aged animals without extending lifespan, highlighting their specific roles in promoting neuronal health span. Notably, collagen reduction specifically drives excessive branching in select sensory neuron subclasses but does not contribute to PVD dendritic beading, another aging‐associated neurodegenerative phenotype associated with distinct mechanosensitive dysfunction. Lastly, we identify that rig‐3, an immunoglobulin superfamily member expressed in interneurons, acts upstream of collagen genes to maintain PVD dendritic homeostasis during aging, with collagen's regulatory role requiring daf‐16/FOXO. These findings reveal that age‐related collagen reduction cues neuronal aging independently of collagen's traditional structural support function, possibly involving bi‐directional communication processes between neurons and non‐neuronal cells. Our study also offers new insights into understanding selective neuron vulnerability in aging, emphasizing the importance of multi‐tissue strategies to address the complexities of neuronal aging.

Reduced collagen on the epidermal apical surface drives selective aging in sensory neurons of Caenorhabditis elegans , leading to excessive dendritic branching and related functional deficits. An immunoglobulin superfamily protein in interneurons acts upstream of collagen genes to maintain dendritic homeostasis during aging, with a downstream requirement of DAF‐16/FOXO. These findings suggest that collagen reduction cues neuronal aging independently of structural support, possibly through bi‐directional communication between neurons and non‐neuronal cells.

1. Introduction

Age represents a significant risk factor for many neurological disorders, including peripheral neuropathies. While several factors such as diabetes, autoimmune diseases, physical injury, and vitamin imbalances contribute to neuropathies, aging not only increases susceptibility to these conditions but also raises the prevalence of idiopathic neuropathies (Hicks et al. 2021). Currently, our limited ability to treat neuropathies in elderly populations largely stems from gaps in understanding the mechanisms driving the aging of the nervous system. Signaling from non‐neural tissues may be a promising avenue to address this gap.

Non‐neural tissues, such as the skin, play essential roles in the development of the peripheral nervous system. A study shows that neurotrophic factors in the skin guide sensory innervation, with overexpression of neurotrophin‐4 in mouse epidermal basal cells leading to excessive sensory endings in the dermal footpad (Krimm et al. 2006). Similarly in C. elegans , the morphogenesis of the PVD neuron, a cutaneous sensory neuron with an elaborate dendritic arbor, relies on SAX‐7, an epidermally‐expressed cell‐adhesion molecule, along with other extracellular proteins, that precisely guide dendritic arborization (Dong et al. 2013; Salzberg et al. 2013). Epidermal ensheathment is a process where epidermal cells wrap around growing neurites of sensory neurons to regulate branching morphogenesis, which is also highly dependent on two‐way signaling between the neuron and the skin; blocking sheath formation in Drosophila and zebrafish destabilizes dendrite branches and reduces nociceptive function (Jiang et al. 2019). However, whether non‐neural tissues also actively contribute to the aging of the peripheral nervous system remains less understood.

As neurons age, they undergo structural deterioration, leading to synapse loss and impaired signal transmission, and ultimately behavioral abnormalities. Neuritic beading, also known as focal swelling or blebbing, is an evolutionarily conserved aging‐associated structural change indicative of neurodegeneration (Takeuchi et al. 2005). These neuritic beads, often consisting of broken‐down cytoskeletal and motor protein components, disrupt organelle transport, such as mitochondrial trafficking, leading to bioenergetic dysfunction, and eventual neuronal death (Takeuchi et al. 2005). Our previous research in C. elegans has shown that age‐related upregulation of antimicrobial peptides (AMPs) in the skin triggers dendritic beading in PVD neurons, accompanied by mechanosensory decline, demonstrating a significant role for skin‐derived molecular cues in sensory neuron aging (E et al. 2018).

Increased aberrant neurite branching is another age‐related structural change, documented in the peripheral nervous systems of elderly human patients with sensory neuropathies (Lauria et al. 1999). While it has been speculated that the increased branching may compensate for other aging‐associated structural and functional losses, this counterintuitive phenomenon is not well understood. Interestingly, C. elegans PVD neurons also exhibit a pronounced increase in dendritic branching in advanced age, as noted in our prior work (E et al. 2018) and in a separate study by Kravtsov, Oren‐Suissa, and Podbilewicz (2017), though the functional implications and precise underlying mechanisms remain unknown. It is noteworthy that the aging‐associated excessive branching, unlike the dendritic beading, does not appear to be regulated by skin AMPs (E et al. 2018), suggesting the involvement of alternative mechanisms.

In this study, we provide an in‐depth characterization of aging‐associated excessive dendritic branching in PVD neurons, demonstrating its functional relevance to age‐related decline in proprioception. Using PVD neuron as a model, we further discover that mutations or downregulation of two cuticular collagen genes, dpy‐5 and col‐120, induce early‐onset excessive dendritic branching and proprioceptive deficits, suggesting that age‐dependent reduction of skin collagens plays a causative role in sensory neuron aging. Moreover, overexpression of these collagen genes mitigates the severity of excessive branching in aged animals. Our results also indicate that the regulatory role of skin collagens is specific to branching integrity during aging and is limited to certain types of cutaneous sensory neurons. Additionally, we identify RIG‐3, an immunoglobulin superfamily protein expressed in interneurons, as an upstream partner in skin collagen's regulation of PVD dendritic integrity, with downstream involvement of DAF‐16/FOXO. These findings reveal a novel role for collagens in actively regulating sensory neuron aging, expanding our understanding of the upstream molecular mechanisms that drive nervous system aging.

2. Results

2.1. Aging Triggers Progressive Development of Excessive Higher‐Order Dendritic Branching in PVD Sensory Neurons

To investigate how skin tissues impact sensory neuron aging, we used the PVD neuron in C. elegans as a model. PVD is a cutaneous mechanosensory neuron, involved in harsh touch, cold sensation, and proprioception, extending dendrites across the body (Chatzigeorgiou et al. 2010; Tao et al. 2019). Each animal possesses two PVD neurons, PVDR on the right and PVDL on the left, with dendritic branches extending dorsally and ventrally. Each PVD has a single axon that synapses ventrally with AVA and PVC interneurons (Figure 1a) (Smith et al. 2010; White et al. 1986). From the PVD soma, two primary (1°) dendritic branches extend anteriorly and posteriorly. Secondary (2°) dendrites emerge orthogonally from 1° branches at regular intervals, forming nonoverlapping “menorah‐like” repeating units mainly composed of tertiary (3°) and quaternary (4°) dendrites (Figure 1b) (Smith et al. 2010), with higher‐order branches (5° and above) rarely observed in young, healthy animals. Our previous studies have shown that aging in PVD neurons often involves the progressive development of bead‐like structures along the dendrites (Figure S1a) (E et al. 2018), resembling morphological changes observed in human patients with peripheral neuropathies (Faerman et al. 1982). Majority of the beading structures in PVD dendrites indicate a disrupted microtubule network and are associated with age‐related deficits in the harsh touch sensing function of PVD neurons (Figure S1b) (E et al. 2018).

FIGURE 1.

Aging triggers the progressive development of excessive higher‐order dendritic branching in PVD neurons. (a) Schematic diagram of PVD neuron in control (CT) animals [F49H12.4::GFP(wdIs51)]. Black boxes indicate regions imaged. (b) A “menorah” structure with orders of dendritic branching indicated. (c) Representative images of anterior (Zone 1) and posterior (Zone 2) sections of PVD neuron at Day 1 (D1) and D9 of adulthood in CT animals. Blue and red boxes indicate zoomed in sections for Zone 1 and 2 respectively. Yellow and blue arrowheads indicate PVD soma and axon respectively. White and red arrowheads indicate 5^o and 6^o dendritic branches respectively. Scale bar = 20 μm. Mild autofluorescence of gut granules, a normal physiological feature in C. elegans , appears as small punctate structures in the background. (d, e) Quantification of 5° (d) and 6° (e) dendrites during aging in CT animals (D1: n = 70, D3: n = 50, D6: n = 98, D9: n = 100). (f, g) 5° (f) and 6° (g) dendrites in CT animals cultured at different temperatures (D6 15°C: n = 36, D6 20°C: n = 39, D6 25°C: n = 41, D9 15°C, 20°C, 25°C: n = 20). (h) 5° dendrites in CT and daf‐16(mu86) mutant animals (CT D3: n = 51, CT D7: n = 70, daf‐16 D3: n = 47, daf‐16 D7: n = 46). Experiments performed without FUDR. (i–p) Number of 5° dendrites normalized to number of 4° dendrites or menorahs. PVD soma was used as a reference point to determine anterior and posterior sections. (i) D3: n = 21, D7: n = 19. (j) D3: n = 22, D7: n = 39. (k) D3: n = 21, D7: n = 19. (l) D3: n = 22, D7: n = 39. (m–p) D3: n = 43, D7: n = 58. Experiments performed without FUDR. (q) Representative time‐lapse images of actin dynamics in higher‐order dendritic branches during an 8‐min session at 2‐min intervals in D1 and D9 CT animals [ser‐2(3)p::GFP::UtrCH + ser‐2(3)p::mCherry‐PH(lxyEx51)]. mCherry::PH was used to visualize the membrane structures of PVD dendrites. Yellow arrows indicate 2° and 3° dendrites. White and red arrowheads indicate 5° and 6° dendrites, with asterisks indicating dendrites where a dynamic actin remodeling event occurs. Scale bar = 10 μm. (r, s) Changes in the length of higher‐order dendrites relative to their initial lengths were tracked using time‐lapse imaging data obtained from D1 (r) and D9 (s) CT animals as in (q). Measurements were taken for 5° and 6° dendrites branching from 4° to 5° dendrites, respectively, with each line representing a single higher‐order dendrite. n (animals imaged) = 3/group. (t) Absolute change in branch length over 8 min in D1 and D9 CT animals (measurements from (r to s)), with each point representing a higher‐order dendrite. n = 3/group. *p < 0.05, ***p < 0.001, ****p < 0.0001.

In this study, we focused on and conducted a thorough characterization of another distinctive aging phenotype of PVD neurons—excessive dendritic branching, previously noted in literature (E et al. 2018; Kravtsov, Oren‐Suissa, and Podbilewicz 2017) yet lacking clarification regarding its functional implications and underlying mechanisms. Our analysis revealed a progressive and substantial increase in the number of 5° and 6° dendritic branches from Day 1 (D1) to D9 of adulthood in control (CT) animals (Figure 1c–e and S1c–f), while the number of 4° dendrites or PVD menorahs remained mostly unchanged (Table S1). This phenotype was observed consistently regardless of FUDR use for synchronizing age (see Section 4) (Table S1). Notably, no age‐related ectopic branching was detected in the axons. To confirm that this branching is an inherent consequence of aging, we first utilized the environment temperature‐lifespan correlation in C. elegans , where higher temperatures accelerate aging, while lower temperatures decelerate it (Klass 1977). Our findings revealed a temperature‐dependent variation in the severity and progression of PVD dendritic branching (Figure 1f,g). We further assessed branching in lifespan‐affecting mutants, focusing on DAF‐2/insulin‐like signaling and mitochondrial pathway, given their well‐established roles in aging (Ishii et al. 1998; Lee and Lee 2022). In short‐lived daf‐16(mu86) mutants, excessive branching appeared earlier and progressed rapidly with age; mev‐1(kn1) mutants with similarly short lifespans showed the same trend (Figures 1h and S1g–i). Notably, long‐lived daf‐2(e1370) mutants displayed excessive branching similar to CT animals at D9 (Figure S1j), consistent with previous findings by Kravtsov, Oren‐Suissa, and Podbilewicz (2017).

Additionally, the increase in higher‐order (≥ 5°) dendritic branching persisted after accounting for the slight age‐related increase in body length (Figure S1m,n), indicating it is not merely growth‐related. The extent and progression of branching were comparable between PVDL and PVDR throughout aging (Table S1). While slight variations were noted between the dorsal and ventral sides, the overall increase in branching remained consistent on both sides (Table S1). This pattern persisted after normalizing for the number of PVD “menorah” structures or 4° branches (Figure 1i–l, Table S1). Using the PVD soma position as a reference point, a similar increase was observed in both anterior and posterior sections (Figure 1m–p, Table S1), Together, these data suggest that excessive branching in PVD higher‐order dendrites is an intrinsic feature of aging.

2.2. Cytoskeletal Composition of Higher‐Order Dendrites in Aging PVD Neurons

During development, stabilized microtubules are often found in large primary dendrites, while actin is abundant in small higher‐order dendrites or dendritic spines, supporting anatomical plasticity (Kaech et al. 2001). To examine the cytoskeletal composition of the aging‐associated higher‐order dendritic branches in PVD neurons, we used transgenic strains expressing ser‐2(3)p::EMTB::GFP and ser‐2(3)p::GFP::UtrCH to visualize polymerized microtubules and actin filaments, respectively, in PVD neurons (E et al. 2018). We found that microtubules were absent in > 3° branches in both aged and young adult CT animals (Figure S2a). Time‐lapse imaging analysis of young adult CTs revealed abundant F‐actin in 4° dendrites and at the leading edges of occasional higher‐order branches, with the excessive 5°/6° branches in aged CTs primarily consisting of F‐actin as well (Figure 1q and S2b). Further quantitative analysis indicated dynamic remodeling of F‐actin‐rich higher‐order branches in both young and aged CTs, with frequent outgrowths and retractions (Figure 1r,s). Notably, during the same observation period, ≥ 5° branches in aged animals tended to gain length, while those in young adults appeared more tightly regulated from further growth (Figure 1t). This regulation is likely independent of pruning or phagocytosis, as no fluorescent neuronal debris was observed near remodeled regions. These findings suggest that a localized mechanism restricting dendritic growth may be lost with aging.

2.3. Aging‐Associated Excessive Dendritic Branching in PVD Neurons Correlates With Proprioceptive Deficits

To explore the functional implications of the excessive branching in aging PVD neurons, we first examined its relevance to noxious harsh touch sensation, a process relying on proper PVD synaptic connections with downstream command interneurons for initiating escape movements (Chatzigeorgiou et al. 2010). To correlate neuronal morphological changes with functional outcomes, we performed harsh touch assays with individual animals and subsequently analyzed PVD branching in the same animals. Interestingly, age‐related decline in harsh touch response in CTs (E et al. 2018) showed no correlation with excessive branching (Figure S3a).

As a polymodal neuron, PVD senses multiple stimuli. The integrity of its dendritic menorah structures is essential for the sensory aspect of proprioceptive regulation, independent of its axon's synaptic function (Tao et al. 2019). Proprioception, the processing of sensorimotor input for posture control and movement, can be assessed in C. elegans by measuring track patterns left by individual worms during sinusoidal movements on bacterial lawns (Figure 2a). Mutations in dma‐1 or related genes disrupting PVD menorah structures (characterized by absence of ≥ 2° branches) reduce track wavelength and amplitude (Tao et al. 2019). We therefore hypothesized that excessive branching in aging PVD neurons could manifest as proprioceptive deficits.

FIGURE 2.

Aging‐associated excessive dendritic branching in PVD neurons correlates with proprioceptive deficits. (a) Representative images of movement tracks of CT animals at D3 and D9. Scale bar = 500 μm. (b, c) Mean amplitude (b) and wavelength (c) measured from 100 tracks per animal in CT animals. Normalized for animal body length (D3: n = 42, D7: n = 36). (d, e) Variability in amplitude (d) and wavelength (e) measured via coefficient of variability (CV) from 100 tracks per animal in CT animals (D3: n = 42, D7: n = 36). (f–i) Correlation between proprioception measurements and number of PVD 5° dendritic branches. Each data point represents an individual animal. ****p < 0.0001. All experiments in this figure performed without FUDR.

Our data showed that both the mean amplitude and mean wavelength of sinusoidal movements were significantly reduced in aged CT animals, compared to young (Figure 2b,c). Additionally, aged CT animals displayed increased intra‐individual variability in both amplitude and wavelength (Figure 2d,e). These age‐related changes resemble the gait pattern changes observed in elderly humans, such as decreased stride length/step width and greater variability, which are strongly linked to a decline in proprioception (Callisaya et al. 2010; Wiesmeier, Dalin, and Maurer 2015). A within‐individual correlation analysis further showed that the extent of “higher‐order” branches, particularly dorsal branches, was negatively correlated with mean amplitude and wavelength, while the severity of excessive branching positively correlated with variability in track amplitude and wavelength (Figure 2f–i, Table S2). These findings reveal a novel link between aging‐associated excessive branching of PVD dendrites and proprioceptive deficits in aging.

Interestingly, there appears to be an absence of concurrence of PVD dendritic beading and excessive branching within individual animals; rather, a negative correlation seems evident, with animals showing pronounced beading less likely to exhibit severe branching, and vice versa (Figure S3b,c). Combined with the distinct behavioral manifestations of beading and excessive branching (E et al. 2018) (Figures 2, S1b and S3a), these observations suggest that aging neurons undergo a variety of morphological changes, each linked to specific functional outcomes, potentially as a compensatory mechanism to preserve certain functions, especially in polymodal neurons.

2.4. Loss‐Of‐Function Mutations in Epidermal Collagen Genes Induce Early‐Onset Excessive PVD Dendritic Branching and Proprioceptive Deficits

We next investigated the molecular mechanisms underlying aging‐associated excessive branching. Previously, we found that aging‐associated upregulation of a skin AMP contributes to PVD dendritic beading during aging, indicating that age‐related skin changes can influence PVD neuron aging (E et al. 2018). However, AMP upregulation does not account for the excessive branching phenotype (E et al. 2018), prompting us to explore whether other molecular changes in the aging skin might contribute. A prime candidate is age‐related decline in skin collagen gene expression, a phenomenon well‐documented across species, including humans. Among the downregulated collagen genes in aging C. elegans (Figure S4a, GSE176088) (Teuscher et al. 2024), we examined several, including col‐120, col‐129, col‐141, dpy‐5, dpy‐10 and rol‐6, which have been previously studied for their roles in morphogenesis and other contexts. We found that disruption of col‐120, col‐141, dpy‐5 and dpy‐10 via RNA interference (RNAi) or mutation resulted in early‐onset excessive branching in PVD dendrites by D3, while the others had no effects (Figures 3a–i and S4b–i), suggesting specific skin collagens may play a role in neuronal aging. In this study, we focused on col‐120 and dpy‐5, as neither impacts skin integrity or AMP gene expression, unlike col‐141 and dpy‐10 (Sandhu et al. 2021; Zhu et al. 2023), which could confound PVD aging analysis (E et al. 2018). Furthermore, the expression of dpy‐5 and col‐120 are not temporally clustered, and their proteins localize to distinct areas within the epidermal apical surface (Teuscher et al. 2024; Thacker, Sheps, and Rose 2006), suggesting that they likely do not interact with each other functionally.

FIGURE 3.

Loss of epidermal collagen leads to early onset of aging‐associated excessive branching and proprioceptive deficit. (a) Domain structures of COL‐120 and DPY‐5, with key features annotated. Adapted from CeCoIDB (Teuscher et al. 2019). (b, c) Representative images of excessive PVD dendritic branching in dpy‐5(e907) mutant (b) and col‐120(sy1526) mutant (c) animals at D3. White and red arrowheads indicate 5° and 6° dendrites respectively. Scale bar = 20 μm. (d) Quantification of 5° dendrites in CT (n = 50), dpy‐5 mutant (n = 50), and endogenous promoter‐driven rescue in dpy‐5 mutant (n = 49). (e) 5° dendrites in CT, dpy‐5 mutant, and epidermis‐specific rescue in dpy‐5 mutant (driven by dpy‐7p; line 1) (n = 60 for all groups). (f) 5° dendrites in CT (n = 40), col‐120 mutant (n = 38), and endogenous promoter‐driven rescue in col‐120 mutant (n = 38). (g) 5° dendrites in CT (n = 82), col‐120 mutant (n = 100), and epidermis‐specific rescue in col‐120 mutant (driven by dpy‐7p and col‐19p; line 1) (n = 55). (h) 5° dendrites in empty vector (EV, control treatment) (n = 70) and dpy‐5 RNAi treated animals (n = 88; RNAi clone 1). (i) 5° dendrites in EV (n = 70) and col‐120 RNAi treated animals (n = 70; RNAi clone 1). (j) Representative images of movement tracks of D3 CT, D9 CT and D3 col‐120(sy1526) animals. Scale bar = 500 μm. (k, l) Mean amplitude (k) and wavelength (l) measured from 100 tracks per animal for D3 CT (n = 28), D9 CT (n = 51), and D3 col‐120 mutant (n = 25). Normalized for animal body length. ns—not significant, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

In C. elegans , the skin is primarily composed of a simple epithelium that secretes a collagenous cuticle to the apical side as a surface coat, creating an additional protective barrier (Chisholm and Hsiao 2012). DPY‐5 and COL‐120 are classified as cuticular collagens (Teuscher et al. 2019; Thacker, Sheps, and Rose 2006), with a domain structure similar to mammalian membrane‐associated or fibril‐associated collagens with interrupted triple‐helices, distinguishing them from basement membrane collagens (Figure 3a). Importantly, DPY‐5 and COL‐120 protein levels both decrease with age in wild‐type animals (Teuscher et al. 2024; Thacker, Sheps, and Rose 2006). Although mutations in either dpy‐5 or col‐120 do not affect skin integrity (Sandhu et al. 2021; Van de Walle et al. 2019), these mutants have reduced lifespans and altered body sizes, with dpy‐5(e907) being shorter and wider and col‐120(sy1526) slightly elongated (Figure S4k–m). To account for body size differences, we normalized PVD dendritic branching to individual body length, confirming that early‐onset excessive branching persisted in both col‐120(sy1526) and dpy‐5(e907) at D3 (Figure S1k–n).

We further performed rescue experiments by expressing wild‐type dpy‐5 or col‐120 cDNA under their respective endogenous promoters or an epidermis‐specific promoter. Both approaches successfully rescued the excessive branching phenotype in each mutant (Figure 3d–g, additional rescue lines in Figure S5). PVD neurons complete dendritic arborization around the late L4 stage (last/4th larval stage) (Smith et al. 2010). At L4, dpy‐5 and col‐120 mutants showed a slight increase in dendritic branching compared to CT animals, though not reaching the extent observed at D3 (Figure S5i–j). To confirm that excessive branching in dpy‐5 or col‐120 mutants is genuinely aging‐associated, we performed adulthood‐specific systemic RNAi knockdowns of these genes, initiating at late L4 and phenotyping PVD dendrites 3 days into adulthood (D3). With over 180 cuticular collagen genes in C. elegans (Wormbase), multiple predicted paralogs exist for both col‐120 and dpy‐5. To ensure specificity, our RNAi clones targeted unique coding regions without homologous sequences in other collagen genes (see Section 4). RNAi effectiveness was validated by RT‐qPCR (Figure S4j). CT animals fed on E. coli HT115 (DE3) bacteria with empty vector L4440 (EV, the control treatment for RNAi experiments) exhibited aging‐associated excessive branching in PVD similar to those fed on OP50 (Figure S5k–l). Our adulthood‐specific RNAi approach was sufficient to induce excessive higher‐order branches by D3 (Figures 3h,i and S5m–s).

Importantly, we found that D3 col‐120(sy1526) mutants exhibited a significant decrease in both mean wavelength and amplitude of sinusoidal movement, closely resembling CT animals at D9, though without increased variability in these parameters (Figure 3j–l and S5t–u). Collectively, these findings demonstrate that epidermal collagens, DPY‐5 and COL‐120, play a critical role in maintaining PVD dendritic branching integrity and PVD‐navigated proprioceptive function during aging.

2.5. Adulthood‐Specific Overexpression of Epidermal Collagen Genes Delays Onset of Excessive PVD Dendritic Branching

Using the col‐19 promoter (Thein et al. 2003) we overexpressed col‐120 or dpy‐5 in the epidermis, specifically during adulthood, and found that it mitigated excessive branching in PVD when examined at D9 (Figure 4a,b and S6a–d). Moreover, dpy‐5 overexpression appeared to shift proprioceptive function parameters in D9 animals toward those of younger animals, though this effect did not reach statistical significance (Figure S6f–i). It is important to note that age‐related declines in other tissues, such as muscles and motor neurons, may complicate assessing the sensory aspect of proprioception at this stage of life. Interestingly, neither dpy‐5 nor col‐120 epidermis‐specific overexpression extended lifespan (Figures 4c,d and S6e). These findings suggest that the beneficial effects of epidermal collagen overexpression on aging appears to primarily target neuronal health, rather than promoting lifespan extension.

FIGURE 4.

Adulthood‐specific overexpression of collagen genes mitigates aging‐associated excessive dendritic branching in PVD neurons. (a) Quantification of 5° dendrites in CT (n = 46) and dpy‐5 epidermis‐specific overexpression driven by col‐19p (n = 50). (b) 5° dendrites in CT (n = 53) and col‐120 epidermis‐specific overexpression driven by col‐19p (n = 54; line 2). (c, d) Lifespan comparison between CT (n = 100) and dpy‐5 epidermis‐specific overexpression driven by col‐19p (n = 100) (c), as well as CT (n = 100) and col‐120 epidermis‐specific overexpression driven by col‐19p (n = 100; line 1) (d). No statistical significance compared to CT. Additional replicates in Figure S6. Experiments performed without FUDR. ***p < 0.001, ****p < 0.0001.

2.6. DPY‐5 and COL‐120 Are Not Involved in Aging‐Associated PVD Dendritic Beading

To investigate whether skin collagen genes regulate the overall aging of PVD neurons, we examined the effects of dpy‐5 or col‐120 mutation on the previously documented aging‐associated dendritic beading phenotype (Figure S1a) (E et al. 2018). Notably, the extent of PVD dendritic beading in D3 dpy‐5(e907) or col‐120(sy1526) mutant animals was comparable to that observed in age‐matched CT (Figure 5a). Coupled with our observations of a potential inverse relationship between aging‐associated dendritic beading and excessive dendritic branching in older CT animals (Figure S3b,c), these results suggest that dpy‐5 and col‐120 regulate PVD dendritic structure via mechanisms separate from those leading to the dendritic beading degenerative phenotype, such as the skin AMP NLP‐29 (E et al. 2018). This is also supported by previous research showing that mutations in dpy‐5 and col‐120 do not impact nlp‐29 expression (Zhu et al. 2023). Collectively, our findings support the notion that distinct non‐neural molecular mechanisms may be underlying different dimensions of neuronal aging, even within an individual neuron.

FIGURE 5.

Selective impact of epidermal collagens on the aging process of sensory neurons. (a) Quantification of PVD dendritic beading in control (CT, n = 29), dpy‐5(e907) (n = 26), and col‐120(sy1526) (n = 29). Scored based on severity of beading phenotype. (b) Schematic diagram of ALM and PLM neuron, with a representative image of the ALM neuron in CT [mec‐4p::GFP(zdIs5)] animals at D3 and D11. Scale bar = 10 μm. White and yellow arrowheads indicate the ectopic branching from ALM anterior neurite and the ALM soma respectively. (c, d) Ectopic branches in ALM (c) and PLM neuron (d) in CT (n = 78) and dpy‐5(e907) (n = 131). Number of ectopic branches observed per animal is indicated. (e, f) Ectopic branches in ALM (e) and PLM neuron (f) in CT (n = 67) and 120(sy1526) (n = 81). ns—not significant, *p < 0.05, ****p < 0.0001.

2.7. ALM Sensory Neurons Exhibit Ectopic Branching in Response to Loss‐Of‐Function Mutation in dpy‐5 or col‐120, but PLM Neurons Do Not

To determine whether the regulatory role of skin collagens in neuron structural integrity is specific to certain neuron populations, we examined two additional sub‐types of cutaneous sensory neurons in C. elegans : ALM and PLM touch receptor neurons, which are crucial for the perception of gentle touch (Corsi, Wightman, and Chalfie 2015). Intriguingly, both dpy‐5(e907) and col‐120(sy1526) animals exhibited an increase in ectopic, aberrant branching from ALM anterior neurites at D3 when compared to CT animals, while PLM neurons showed no morphological abnormalities (Figure 5b–f). This discrepancy indicates that the regulatory role of examined collagens in neuritic branching may not extend uniformly across all cutaneous sensory neurons, suggesting that certain neuron sub‐types may possess unique features rendering them particularly vulnerable to the effects of skin collagen loss during aging.

2.8. RIG‐3/IgSF in Interneurons Acts Upstream of Epidermal Collagens to Regulate Excessive PVD Dendritic Branching in Aging

Next, we explored the downstream mechanisms by which collagen genes regulate PVD dendritic integrity during aging. In C. elegans , the polarized structure of epidermal cells segregates apically secreted cuticular collagens from basement membrane collagens anatomically and functionally (Chisholm and Xu 2012), making direct interaction between COL‐120 or DPY‐5 and sensory neurons (located on the basal side of the epidermis) unlikely. Thus, the age‐related decline in these collagens may contribute to neuronal aging through indirect mechanisms, potentially involving cell signaling rather than direct structural support. To test this hypothesis, we first examined several neuronal cell surface receptors using adulthood‐specific RNAi. We found that knockdown of ina‐1, pat‐2 or rig‐3 led to early‐onset excessive dendritic branching in PVD (Figure 6a, Figure S7a–c). While ina‐1 and pat‐2 (both α‐integrins) are widely expressed, rig‐3, encoding an immunoglobulin superfamily (IgSF) protein, is expressed selectively in a small set of neurons but notably absent in PVD (Babu et al. 2011; Taylor et al. 2021). This selective expression pattern makes rig‐3 a particularly intriguing candidate for a non‐cell‐autonomous role in regulating PVD morphology. Analysis of rig‐3(ok2156) mutants confirmed our RNAi findings of early‐onset excessive dendritic branching but showed that rig‐3 mutation does not significantly impact PVD morphogenesis during development (Figures 6b and S7d–f).

FIGURE 6.

RIG‐3/IgSF acts as upstream of epidermal collagens to regulate PVD dendritic integrity in aging. (a) Quantification of 5° dendrites in empty vector (EV) and rig‐3 RNAi treated CT animals at D3 (n = 84 for both groups). (b) 5° dendrites in CT (n = 91) (ser‐2(3)p::GFP(lxyEx77), see Section 4) and rig‐3(ok2156) mutant (n = 90). (c) 5° dendrites in CT (n = 37), rig‐3(ok2156) (n = 40), and pan‐neuronal rescue (driven by unc‐33p) in rig‐3(ok2156) (n = 37). (d) 5° dendrites in CT (n = 41), rig‐3(ok2156) (n = 46), and interneuron rescue (driven by nmr‐1p) in rig‐3(ok2156) (n = 37). (e) 5° dendrites in dpy‐5(e907) mutant animals treated with EV (n = 84) or rig‐3 RNAi (n = 85), as well as in col‐120(sy1526) mutant animals treated with EV (n = 88) or rig‐3 RNAi (n = 86). (f) 5° dendrites in CT (n = 76), rig‐3 pan‐neuronal overexpression driven by unc‐33p (n = 57), rig‐3 overexpression in dpy‐5(e907) mutant background (n = 10), and rig‐3 overexpression in col‐120(sy1526) mutant background (n = 37). (g) 5° dendrites in CT (ser‐2(3)p::GFP(lxyEx77)) (n = 30), dpy‐5 epidermis‐specific overexpression driven by col‐19p (n = 22), and dpy‐5 overexpression in rig‐3(ok2156) mutant background (n = 18). (h) 5° dendrites in CT (n = 10), col‐120 epidermis‐specific overexpression driven by col‐19p (n = 19; line 1), and col‐120 overexpression (line 1) in rig‐3(ok2156) mutant background (n = 20). ns—not significant, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

We then conducted a rescue experiment using a pan‐neuronal promoter to drive wild‐type rig‐3 expression in rig‐3(ok2156), fully restoring normal PVD dendritic branching (Figure 6c and S7g). Pan‐neuronal overexpression of rig‐3 also significantly suppressed excessive branching in aged animals (Figure 6f), similar to the effects of col‐120 or dpy‐5 overexpression (Figure 4). Since rig‐3 is not detected in PVD, we sought to identify the specific neuron(s) through which it acts. rig‐3 is highly expressed in interneurons, especially AVA, where it regulates locomotion by modulating neuropeptide release, indirectly affecting sensory neurons connected to AVA (Bhardwaj, Pandey, and Babu 2020). Notably, PVD axon also synapses with AVA (White et al. 1986), leading us to hypothesize that RIG‐3 may regulate PVD dendritic integrity from AVA. Using the nmr‐1 promoter, which is active in fewer than 10 neuron subsets—primarily interneurons including AVA, AVD, AVE and PVC, all of which also endogenously express rig‐3 (Figure S7i) (Brockie et al. 2001; Taylor et al. 2021), we conducted a rescue experiment in rig‐3(ok2156). This fully rescued the excessive branching phenotype, suggesting rig‐3 non‐cell‐autonomously regulates PVD dendritic branching from within this small set of interneurons (Figure 6d and S7h).

To determine if rig‐3 mediates the effects of skin collagens on PVD branching, we performed adulthood RNAi of rig‐3 in col‐120 or dpy‐5 mutants. We found that it did not further enhance excessive branching in collagen mutants, suggesting rig‐3 functions in the same genetic pathway as dpy‐5 and col‐120 in regulating PVD dendritic integrity during aging (Figure 6e and S7j). Further epistasis analysis surprisingly revealed that rig‐3 overexpression's beneficial effect on branching in aged animals was completely blocked by col‐120 or dpy‐5 mutations (Figure 6f and Figure S7k). Conversely, col‐120 or dpy‐5 overexpression's beneficial effects remained despite introduction of the rig‐3 mutation (Figures 6g,h and S7l,m). Contrary to our expectations, these data suggest that neuronal RIG‐3 functions upstream of collagens in maintaining PVD dendritic health over the course of aging.

2.9. Beneficial Effect of COL‐120 on PVD Dendritic Integrity Requires DAF‐16/FOXO

To further explore downstream pathways of skin collagen genes, we analyzed transcriptomic data (GSE19310) comparing wild‐type animals and dpy‐10(e128) mutants, another cuticular collagen mutation associated with early‐onset excessive PVD branching (Figure S4h,i). KEGG pathway analysis of differentially expressed genes in dpy‐10(e128) revealed significant downregulation in several signaling pathways, including FOXO signaling (Figure S7n). We sought to examine potential interactions between collagen genes and daf‐16/FOXO, focusing on col‐120, given its stronger effect in suppressing excessive branching when overexpressed (Figure 4). We found that daf‐16(mu86) mutation completely suppressed the beneficial effect of epidermis‐specific col‐120 overexpression (Figure S7o,p). Since col‐120 overexpression was driven by the col‐19 promoter specifically in adulthood and previous studies show that col‐19 transcription is unaffected by daf‐2 or daf‐16 (Ewald et al. 2015; Uno et al. 2013), the branching suppression observed with daf‐16 mutation is unlikely due to reduced expression of col‐19p::col‐120. Additionally, the suppression effect cannot be simply attributed to a potential lifespan reduction caused by daf‐16 mutation. This interpretation is supported by the observation that D7 daf‐16(mu86) animals with col‐120 overexpression displayed branching levels more comparable to age‐matched CTs (Figure S7o,p), in contrast to the significant difference observed between D7 CTs and D7 daf‐16(mu86) single mutants (Figures 1h and S1g). Together, these results suggest that daf‐16/FOXO may act downstream of col‐120 in regulating PVD dendritic branching during aging.

3. Discussion

Our study demonstrates that age‐related reduction in epidermal collagen drives both structural and functional changes in sensory neuron aging process. Interestingly, the impacts exhibit selectivity, manifested at two levels: firstly, collagen reduction contributes to excessive branching but not neuritic beading in aging neurons, pointing to a phenotype‐specific effect; secondly, their role is confined to regulating branching integrity in specific sub‐types of cutaneous sensory neurons, highlighting cell‐type‐targeted neuron‐epidermal interactions. Unexpectedly, we identify RIG‐3/IgSF in interneurons as an upstream partner of collagens, coordinating neuronal homeostasis during aging. Taken together, our findings suggest that the regulatory role of skin collagens is mediated through intricate, bi‐directional communication processes between neurons and epidermal cells, emphasizing the need for a paradigm shift toward exploring multi‐tissue strategies to comprehensively address the complexities of neuronal aging.

Although age‐related declines in collagen production in various tissues have been well‐documented, their impacts on the nervous system during aging remain underexplored. One study reported that aged mice lacking COL6A1, a ubiquitously expressed collagen VI gene, exhibited more severe deficits in motor coordination and spatial memory compared to age‐matched WT animals (Cescon et al. 2016). Our current study extends this area by demonstrating that non‐basement membrane collagens, particularly those from non‐neural tissues, play a distinctive regulatory role in maintaining neuronal structure and function during aging. Notably, the beneficial effects of overexpressing these collagen genes appears to primarily target neuronal health rather than promoting longevity. Interestingly, previous studies have reported a slight lifespan extension with col‐120 overexpression driven by its native promoter (Ewald et al. 2015; Teuscher et al. 2024), including when overexpression was induced post‐developmentally via CRISPR activation (Goyala and Ewald 2023). These discrepancies with our findings may stem from differences in experimental design, such as the promoters and 3′UTR sequences used to construct transgenic strains, or the bacterial diets provided. For example, while we used standard food E. coli OP50 in our experiments, Goyala et al. used E. coli HT115 to deliver sgRNA targeting the col‐120 promoter, which also served as the animals' food source. Given that C. elegans lifespan is known to vary with different bacterial diets (Stuhr and Curran 2020), these factors could contribute to the differing results. The distinction could also indicate the complexity of collagen's role in aging and the importance of considering tissue‐specific and systemic factors when evaluating its effects.

While our data suggest that DAF‐16/FOXO mediates COL‐120's effects on PVD dendritic health, questions remain about whether DAF‐16 acts within epidermal cells, whether COL‐120 engages additional signaling pathways that interact with DAF‐16 elsewhere, and which specific DAF‐16 target genes are involved. Importantly, changes in key collagen genes can have broad effects, such as altering extracellular matrix (ECM) composition as seen with col‐120 (Teuscher et al. 2024), or influencing signaling pathways like Wnt and TGF‐β with dpy‐10 (Figure S7n). In fact, such extensive downstream effects may explain why rescuing the PVD excessive branching phenotype in col‐120 or dpy‐5 mutants was challenging, even with endogenous promoters, as compensatory responses induced by collagen mutations could interfere with attempts to fully restore normal function.

An unexpected finding from our study is the role of RIG‐3/IgSF as an upstream partner of skin collagens in modulating PVD dendritic branching. Our data suggest that RIG‐3 acts from interneurons, likely adjacent to PVD, yet the beneficial effects of rig‐3 overexpression are blocked by the loss of skin collagens. This observation implies a signaling loop between neurons and epidermal cells, rather than a simple neuron–neuron interaction, in RIG‐3's regulation of neuronal aging. RIG‐3 shares homology with mammalian neural cell adhesion molecule 1 (NCAM1), also an IgSF protein (Babu et al. 2011). Studies in a rat sciatic nerve injury model have shown that NCAM1 on axonal membrane is essential for axonal fasciculation during nerve regeneration, requiring interaction between extracellular collagen VI and the fibronectin type III (FNIII) domain of NCAM1 (Sun et al. 2022). While RIG‐3 also contains a FNIII domain (Babu et al. 2011), the segregation of cuticular collagens to the epidermal apical surface makes direct interaction with COL‐120 or DPY‐5 unlikely. Notably, RIG‐3 can function as either a cell surface or secreted protein (Babu et al. 2011); future studies may clarify whether its secretion is required for its role in regulating PVD branching and assess its potential to act over long distances.

Excessive branching in aging peripheral neurons is a conserved phenomenon. For example, elderly humans with sensory neuropathy exhibit increased branching of epidermal nerve fibers in the proximal thigh compared to healthy controls (Lauria et al. 1999). Our time‐lapse imaging revealed that excessive high‐order dendritic branches in PVD neurons are enriched with F‐actin but lack microtubules, indicating high structural plasticity, much like dendritic spines, known to affect neuronal signaling and synaptic plasticity. Therefore, it is tempting to speculate that increased branching observed in aging reflects a compensatory plasticity for future neurodegeneration at more advanced ages. Mechanotransduction may play a role in this phenomenon. As C. elegans age, their cuticle becomes stiffer, while the ECM weakens, reducing resistance to mechanical stress. Studies have shown that col‐120 expression can be upregulated in response to mild mechanical compression, and loss of col‐120 decreases cuticle mechanical resistance, particularly in long‐lived daf‐2 mutants, suggesting a feedback loop between ECM homeostasis and mechanotransduction (Rahimi, Sohrabi, and Murphy 2022; Teuscher et al. 2024). Mechanosensitive sodium channels, such as MEC‐10, UNC‐8, DEL‐1, and DEGT‐1, are localized to PVD dendritic branches and are essential for proprioceptive and mechanonociceptive signaling (Tao et al. 2019). Age‐related collagen loss could compromise ECM stability, allowing cumulative mechanical stress to alter channel sensitivity over time and, in turn, influence dendritic branching through modified sensory feedback to mechanical cues. This effect may ultimately contribute to compensatory branching, inadvertently engaging sensorimotor networks beyond their usual functional scope.

During nervous system development, transmembrane and extracellular proteins are crucial for shaping neuronal structure and guiding migration. Proteins such as SAX‐7/L1CAM, MNR‐1, and DMA‐1 (transmembrane LRR protein) have been extensively studied for their roles in PVD dendritic morphogenesis; however, deficiencies in these proteins primarily affect 1°‐ 4° dendritic branches and do not contribute to excessive higher‐order branching (Dong et al. 2013; Salzberg et al. 2013). Additionally, we did not observe a clear link between abnormal dendrite overlapping, often due to self‐avoidance defects (Kravtsov, Oren‐Suissa, and Podbilewicz 2017; Oren‐Suissa et al. 2010), and excessive branching, either during aging or in collagen mutants. These observations suggest that the molecular mechanisms responsible for neuron morphogenesis in development may be distinct from those driving neuron structural deterioration in aging.

Our findings reveal that polymodal sensory neurons can undergo multiple, distinct morphological changes during aging, each corresponding to specific functional declines. In PVD neurons, simultaneous excessive dendritic branching and dendritic beading are rare, suggesting that animals may avoid experiencing major impariments in both harsh touch function and proprioception concurrently. While it is unclear whether polymodal neurons prioritize preserving one function over another as they age, the current study combined with our previous research (E et al. 2018) highlight that within multifunctional neurons, certain structures and functions may be selectively vulnerable to aging, likely driven by distinct non‐neural signals.

At a broader level, neurons across different anatomical regions also exhibit varying susceptibility or resistance to aging, leading to differences in the onset timing and progression of neurodegenerative processes. This selective vulnerability is commonly observed in both normal aging and age‐related neurodegenerative diseases, and evident across species. For instance, ALM, PLM, and PVD neurons in C. elegans age at varying rates and respond differently to the same skin‐derived aging cue, which can be mediated by cell surface proteins that are differentially expressed in different neurons (E et al. 2018). Given that each neuron class/sub‐type has a unique gene expression profile, certain molecular signatures may determine their responsiveness to non‐neural or extracellular aging cues, contributing to selective vulnerability.

4. Materials and Methods

4.1. Caenorhabditis elegans Strains and Maintenance

C. elegans strains were maintained at 20°C, or as otherwise indicated, on Nematode Growth Medium (NGM) plates seeded with E. coli OP50. Only hermaphrodites were used for data generating experiments. For age‐synchronized progeny, plates with gravid hermaphrodites were bleached to eliminate microbes, and animals were transferred to NGM plates containing 100 μM 5′fluorodeoxyuridine (FUDR) (VWR, 76345–984) at the mid‐ or late‐L4 stage to prevent egg hatching. Day 1 of adulthood (D1) was defined as 24 h post‐L4. Given the potential confounding effects of FUDR on aging (Feldman, Kosolapov, and Ben‐Zvi 2014; Van Raamsdonk and Hekimi 2011), key experiments were repeated without FUDR, moving animals onto fresh plates daily until D5, every 2 days thereafter. FUDR‐free experiments are noted in figure legends. See Table S3 for strain information.

4.2. DNA Constructs and Generation of Transgenes

DNA expression constructs were generated using Gateway cloning technology (Thermo Fisher), with details in Table S4. Primers were acquired from Millipore and Thermo Fisher (Table S5). Transgenic animals were generated by microinjection, with plasmid DNAs used at 10–100 ng/μL, co‐injection marker ttx‐3p::RFP or ttx‐3p::GFP at 50 ng/μL (Tables S3 and S4). At least two independent transgenic lines were analyzed per construct. Promoters used include—dpy‐5p (−854 bp from start codon), col‐120p (−348 bp), nmr‐1p (−1104 bp), dpy‐7p (−304 bp), col‐19p (−649 bp), and unc‐33p (−1974 bp).

4.3. Fluorescent Microscopy

Neurons were visualized using fluorescent reporters and phenotyped on a Zeiss Axio Imager M2 using 0.5%–1% M9 dilution of 1‐phenoxy‐2‐propanol (Fisher) for immobilization. Confocal images were taken on a Leica SP8 with Z‐stack (0.5‐1 μm/slice) at 63X magnification, using polystyrene beads (Polysciences, 00876–15) for immobilization.

4.4. Quantification of Neuron Morphological Defects

wdIs51(F49H12.4::GFP) (Smith et al. 2010) was used to visualize PVD neurons. Excessive dendritic branching was quantified by counting 5° and 6° branches, identified within “menorah” structures (Figure 1b). 5° and 6° branches for each animal were counted for either PVDL or PVDR for either the dorsal or ventral side, depending on which was clearly visible during the imaging session. PVD dendritic beading phenotype was assessed by counting bead‐ or bubble‐like structures along the dendrites of either PVDL or PVDR across the entire dendritic tree. Animals with ≥ 10 beads across ≥ 5 menorahs were considered positive for degeneration (E et al. 2018). Severity scoring: in Figure 5a, animals with 0–10 beading structures was scored as normal, 10–20 as mild, 20–50 as moderate, and > 50 as severe; in Figure S1b, a finer scale was used: score 1 for 0–10 structures, 2 for 10–20, 3 for 20–30, 4 for 30–50, 5 for 50–100, 6 for 100–200, and 7 for over 200. ser‐2(3)p::mCherry‐PH was used to visualize PVD dendritic membrane structures with respect to F‐actin or microtubules. For rig‐3 mutants, ser‐2(3)p::GFP (lxyEx77) was constructed to visualize PVD neurons due to challenges in crossing rig‐3(ok2156)X with wdIs51X. ser‐2(3)p::GFP(lxyEx77) animals had larger body size than wdIs51, likely due to genetic background differences, which may account for the branching number variation at D1 between the two reporter strains.

zdIs5(mec‐4p::GFP) was used to visualize ALM and PLM neurons, and the number of ectopic branches sprouting from their main neurites were counted.

For each experimental group, a minimum of three replicates of 30 animals, or indicated otherwise, were quantified alongside a control group, with experimenters blinded to sample identity to minimize bias.

4.5. PVD Function Analysis: Harsh Touch Response

The measurement of the harsh touch response was conducted using a mec‐4(u253) mutant background with non‐functional touch receptor neurons but intact PVD neuron functionality, as previously described (E et al. 2018), with modifications to the scoring system. A score of 1 was given if the animal moved over one body length, 0.5 if it moved between one‐third and one body length, and 0 if it moved less than one‐third or not at all. Each animal underwent 10 trials with 10–15 min intervals. Cumulative scores ranged from 0 (severely impaired response) to 10 (optimal response).

4.6. PVD Function Analysis: Proprioception

Proprioceptive sensing specific to PVD neurons was assessed using a locomotion assay as described previously (Tao et al. 2019; Tavernarakis et al. 1997) with some modifications. On the day of assay, animals were transferred to fresh NGM plates seeded with OP50, one animal per plate, and allowed to roam freely for 2–3 h at 20°C, until sufficient tracks were made in the bacterial lawn. The plate was then imaged using a digital camera attached to a Leica S9i stereomicroscope at 1X magnification. Three independent replicates of 10 animals each were assayed for each group. A total of 100 randomly selected wavelengths (distance between two adjacent peaks) and amplitudes (distance between peak and wavelength, perpendicular to wavelength) were measured using Fiji. All measurements were used in cases where animals had fewer than 100 measurements, with a minimum of 40 measurements per animal. All measurements were normalized to the body lengths of individual animals and units were converted to millimeters utilizing a reference slide. All animals subjected to proprioception assay were also examined under the Zeiss to quantify PVD dendritic branching.

4.7. RT‐qPCR

To quantify the change in expression level of collagens in aging, N2 WT animals at D1 and D7 were collected, washed with M9 solution for three 30‐min washes, and total RNA was isolated with TRI reagent (Millipore Sigma).

To test RNAi efficacy, 10‐worm lysis method (Ly, Reid, and Snell 2015) was used to prepare lysates. Briefly, 10 worms were cleaned in H₂O and placed in 1 μL of lysis buffer (0.25 mM EDTA, 5 mM Tris pH 8., 0.5% Triton X‐100, 0.5% Tween‐20, 1 mg/mL proteinase K). Samples were lysed in a thermal cycler at 65°C for 15 min, 85°C for 1 min, and stored at −80°C for at least 12 h. An additional 1.5 μL of lysis buffer was then added, and the cycle was repeated. Samples were treated with dsDNAse (Thermo Fisher; 0.5 μL each of 10X buffer and dsDNAse, 1.5 μL ultrapure H₂O) and incubated at 37°C for 15 min, 55°C for 5 min, and 65°C for 1 min, followed by reverse transcription.

Reverse transcription was performed with iScript Reverse Transcription Supermix for RT‐qPCR (BioRad). RT‐qPCR was performed using either TaqMan Gene Expression Assays (ThermoFisher, Ce02462726_m1, Ce02417736_s1, Ce02457574_g1) and iTaq Universal Probes Supermix (Bio‐Rad) or using designed primers (Table S5) and iTaq Universal SYBR Green (Bio‐Rad). ama‐1 and cdc‐42 were used as housekeeping controls. Primer efficiency was verified utilizing standard curve method. Bio Rad CFX96 Touch Deep Well Real‐time PCR Detection System was used to perform the qPCR and relative mRNA levels were calculated using comparative ΔΔCT method.

4.8. Lifespan Assay

L4 animals were transferred to assay plates and scored daily. An animal was considered dead when it no longer responded to gentle prodding with a platinum wire and displayed no pharyngeal pumping. Lifespan was calculated from the time animals were first placed on the assay plates until they were scored as dead. Animals that died due to protruding/bursting vulva, internal hatching, as well as animals that crawled off the agar were censored. For lifespan assays examining collagen overexpression strains, the PVD::GFP background strain (wdIs51) was used as the control.

4.9. Time‐Lapse Imaging

Imaging of PVD branches was done on the Leica SP8 using 2% agarose pads with polystyrene beads for immobilization. Control animals [ser2(3)p::mCherry‐PH; ser2(3)p::GFP::UtrCH(lxyEx51)] were imaged at D1 and D9. Each session lasted 8 min with 2‐min intervals, and images were Z‐stack projections captured with a 63X objective in both red and green channels. Branch dynamics were quantified in Fiji by measuring the length of individual higher‐order branches at each time‐point, and actin dynamics were assessed by observing GFP‐labeled actin movement.

4.10. Adulthood‐Specific RNAi

Feeding method was used to perform RNAi experiments (Conte Jr. et al. 2015). FUDR at a concentration of 100 μM was externally added to RNAi plates following growth of the HT115 bacterial lawn. The RNAi clones were obtained from the Vidal RNAi library (Horizon Discovery, Table S4) or amplified from cDNA using PCR. The section of cDNA included for each RNAi clone are dpy‐5::L4440 (1) (PELZ88: 210 bp section downstream of dpy‐5 start codon) and col‐120::L4440 (1) (PELZ45: 256 bp section downstream of col‐120 start codon). These regions were selected to ensure RNAi specificity, confirmed by BLAST search to ensure no homology with other collagen genes. Additional RNAi clones were also included: dpy‐5::L4440 (2) (PELZ48: 554 bp section 301 bp downstream of dpy‐5 start codon) and col‐120::L4440 (2) (PELZ46: 605 bp section 337 bp downstream of col‐120 start codon). All RNAi clones were sequenced to verify their identities. Empty L4440 plasmid was used as a negative control. TJ356 [zIs356 (daf‐16p::daf‐16a/b::GFP)] animals with empty‐vector or daf‐2 RNAi treatment were used as a positive control. Successful RNAi condition was indicated by nuclear translocation of DAF‐16::GFP in daf‐2 RNAi‐treated animals, while cytoplasmic localization indicated unsuccessful treatment.

4.11. Gene Expression Datasets and Pathway Analysis

Gene expression datasets (GSE176088, GSE19310) were obtained from the NCBI Gene Expression Omnibus. Data were analyzed using GEO2R to identify differentially expressed genes (DEGs), with criteria of log₂‐fold change ≥ 1.5 and a p < 0.05. KEGG enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (Sherman et al. 2022), applying an FDR < 0.01 for statistical significance. The top 10 significant KEGG pathways were visualized in a bubble graph, with the rich factor representing the proportion of DEGs within each pathway term.

4.12. Statistical Analysis

Data processing, statistical analyses, and graph plotting were performed with GraphPad Prism software. Normality was assessed using the D'Agostino & Pearson test. For comparisons between experimental and control groups, unpaired t‐test for normally distributed data, and the Mann–Whitney test for non‐normally distributed data were used. When comparing three or more groups, either one‐way ANOVA or the Kruskal‐Wallis test, followed by post hoc analysis. Pearson's correlation test was applied for correlation analyses. Log‐Rank test was used to analyze lifespan data. Bar graphs display the mean ± SEM for normally distributed data, and the median with interquartile range for non‐normally distributed data.

Author Contributions

Lezi E conceived the project. Lezi E and Meera M. Krishna designed the experiments; Meera M. Krishna, Lezi E, Swapnil G. Waghmare, Ariel L. Franitza, and Emily C. Maccoux performed the experiments and collected the data. Meera M. Krishna, Swapnil G. Waghmare and Lezi E analyzed and interpreted the data. Meera M. Krishna and Lezi E drafted the manuscript. Meera M. Krishna, Swapnil G. Waghmare, Ariel L. Franitza, Emily C. Maccoux and Lezi E edited and proofread the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.