Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Mar 31;217(3):iyaa047.
doi: 10.1093/genetics/iyaa047.

Specific collagens maintain the cuticle permeability barrier in Caenorhabditis elegans

Anjali Sandhu  1 , Divakar Badal  2 , Riya Sheokand  1 , Shalini Tyagi  1 , Varsha Singh  1   2   3
Affiliations

Specific collagens maintain the cuticle permeability barrier in Caenorhabditis elegans

Anjali Sandhu et al. Genetics. .

Abstract

Collagen-enriched cuticle forms the outermost layer of skin in nematode Caenorhabditis elegans. The nematode's genome encodes 177 collagens, but little is known about their role in maintaining the structure or barrier function of the cuticle. In this study, we found six permeability determining (PD) collagens. Loss of any of these PD collagens-DPY-2, DPY-3, DPY-7, DPY-8, DPY-9, and DPY-10-led to enhanced susceptibility of nematodes to paraquat (PQ) and antihelminthic drugs- levamisole and ivermectin. Upon exposure to PQ, PD collagen mutants accumulated more PQ and incurred more damage and death despite the robust activation of antioxidant machinery. We find that BLMP-1, a zinc finger transcription factor, maintains the barrier function of the cuticle by regulating the expression of PD collagens. We show that the permeability barrier maintained by PD collagens acts in parallel to FOXO transcription factor DAF-16 to enhance survival of insulin-like receptor mutant, daf-2. In all, this study shows that PD collagens regulate cuticle permeability by maintaining the structure of C. elegans cuticle and thus provide protection against exogenous toxins.

Keywords: BLMP-1; Caenorhabditis elegans; collagens; cuticle; dumpy; hypodermis; oxidative stress; paraquat; permeability; survival.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Four collagens encoded in C. elegans genome maintain cuticle barrier function. (A) Pictorial representation of Hoechst staining in C. elegans. Hoechst 33258 staining-based permeability assay of (B) bus-8(e2698) and (C) EV, (D) dpy-7, (E) dpy-8, (F) dpy-9, (G) dpy-10, (H) dpy-4, and (I) dpy-5 RNAi animals. Scale bar, 50 µm. n = 3; N ≥ 15.
Figure 2
Figure 2
PD collagens maintain the ultra-structure of C. elegans cuticle. Scanning electron micrograph of (A) WT, (B) dpy-4(e1166), (C) dpy-5(e61), (D) dpy-7(e88), (E) dpy-8(e1281), (F) dpy-9(e12), and (G) dpy-10(e128) animals (50,000× magnification). Annuli, A, furrows, F, anterior, Ant, and posterior, Post., of the worms are indicated. Scale bar, 1 µm.
Figure 3
Figure 3
PD collagens positively regulate survival of C. elegans on exogenous toxins. Kaplan–Meier survival curves of (A) WT, dpy-7(e88) and dpy-8(e1281) animals on 20 mM paraquat (PQ), (B) WT, dpy-9(e12) and dpy-10(e128) animals on 20 mM PQ, (C) dpy-9(e12) animals with RNAi of EV, dpy-7, dpy-8 and dpy-10 on 20 mM PQ, (D) dpy-10(e128) animals with RNAi of EV, dpy-7, dpy-8 and dpy-9 on 20 mM PQ. n = 3; N ≥ 50 for panels A–D. (E) qRT-PCR analysis of PD collagens in WT animals exposed to 20 mM PQ for 8 h. (F) qRT-PCR analysis of collagen processing enzymes in WT animals exposed to 20 mM PQ for 8 and 24 h. (G) Hoechst 33258 staining in WT animals with EV and pdi-2 RNAi. n = 2; N ≥ 15. Kaplan–Meier survival curves of WT animals with (H) EV and pdi-2 RNAi animals on 20 mM PQ. n = 3; N ≥ 50. (I) Hoechst 33258 staining in WT animals with EV and bli-4 RNAi. n = 2; N = 15. Kaplan–Meier survival curves of WT animals with (J) EV and bli-4 RNAi animals on 20 mM PQ. n = 3; N ≥ 40. Percent paralyzed of WT, dpy-4(e1166), dpy-5(e61), dpy-7(e88), dpy-8(e1281), dpy-9(e12), and dpy-10(e128) animals upon (K) exposure to 125 µM levamisole and, (L) 50 µM Ivermectin (IVM). n = 3; N ≥ 40 for panels K and L. Error bars indicate SEM. n = 3; N ≥ 50. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, NS—not significant, for Student’s t-test and Mantel–Cox test for survival curves. P-value for survival curves are indicated next to genotypes. For TD50 values in survival assays, see Supplementary Table S2.
Figure 4
Figure 4
PD collagen mutants show enhanced accumulation of PQ and increased damage during PQ exposure. Kaplan–Meier survival curves of (A) EV and dpy-9 RNAi animals treated with or without 5 mM NAC, on 20 mM PQ, (B) of EV and dpy-10 RNAi animals treated with or without 5 mM NAC, on 20 mM PQ. n = 3; N ≥ 50 for panels A and B. (C) Pharyngeal pumping rate in WT, dpy-9 (e12), and dpy-10 (e128) animals upon exposure to 20 mM PQ for 0, 6, and 12 h. n = 3; N ≥ 10. (D) LC-MS/MS analysis of PQ accumulation in EV, dpy-9, and dpy-10 RNAi animals upon exposure to 20 mM PQ for 12 h. (E) Quantification of PQ accumulation per animal. Error bars indicate SEM. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; NS—not significant, P ≥ 0.05, significance based on Student’s t-test and Mantel–Cox test for survival curves. P-value for survival curves are indicated next to genotypes. For TD50 values in survival assays, see Supplementary Table S2.
Figure 5
Figure 5
dpy-9 and dpy-10 mutants display robust oxidative stress response. (A) Comparison of inducibility of detoxification genes in WT, dpy-9 (e12), and dpy-10 (e128) animals upon exposure to 20 mM PQ compared to untreated animals for 6 animals upon exposure to e SEM. Significance is provided for comparisons between inducibility observed in mutants over WT. (B) gst-4p::GFP induction in EV and dpy-10 RNAi animals upon exposure to 20 mM PQ for 6 h. Arrow heads indicate pharynx and intestine junction. In addition, see Table 1 for inducibility of genes due to PQ exposure. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; NS—not significant, significance based on Student’s t-test.
Figure 6
Figure 6
Transcription factor BLMP-1 regulates cuticle permeability and survival of worms on PQ. Hoechst 33258 permeability assay in (A) EV and (B) blmp-1 RNAi in WT animals. Scale bar, 50 µm. n = 3; N ≥ 15. Kaplan–Meier survival curves of (C) EV control and blmp-1 RNAi animals against 20 mM PQ. n = 3; N ≥ 50. qRT-PCR analysis of transcripts for collagens and collagen processing enzymes upon (D) blmp-1 RNAi in WT animals compared to EV control. Error bars indicate SEM. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; NS—not significant, P ≥ 0.05, significance based on Student’s t-test and Mantel–Cox test for survival curves. P-value for survival curves are indicated next to genotypes. For TD50 values in survival assays, see Supplementary Table S2.
Figure 7
Figure 7
DPY-10 does not affect DAF-16 dependent antioxidants expression in daf-2 mutant. Hoechst 33258 staining-based permeability assay in (A) EV, (B) dpy-7, (C) dpy-8 and (D) dpy-10 RNAi in daf-2 (e1370) animals. Scale bar, 50 µm. n = 3; N ≥ 15. Kaplan–Meier survival curves of (E) EV and dpy-7 RNAi, (F) EV and dpy-8 RNAi, (G) EV and dpy-10 RNAi treated WT and daf-2 (e1370) animals against 20 mM PQ. n = 3; N ≥ 50 for panels E–G. Scanning electron micrographs of (H) EV, (I) dpy-10 and (J) daf-16 RNAi in daf-2 (e1370) animals and, (K) daf-16 RNAi in WT animals at 50,000× magnification. Kaplan–Meier survival curves of EV, dpy-10 and daf-16 RNAi in daf-2 (e1370) animals and, EV RNAi in WT animals against (L) 20 mM paraquat and (M) heat stress at 32°C. n = 3; N ≥ 50 for panels L and M. Hoechst 33258 stained nuclei in (N) daf-16 RNAi in daf-2 (e1370) animals and (O) daf-16 RNAi in WT animals. Scale bar, 50 µm. n = 3; N ≥ 15 for panels N and O. qRT-PCR analysis of detoxification genes gpx-6, gst-10, sod-3, ctl-2, ctl-3, and mtl-1 at (P) basal level in EV, dpy-10, and daf-16 RNAi in daf-2 (e1370) animals and (Q) upon 20 mM PQ exposure in daf-2 animals. (R) Inducibility of detoxification genes in daf-2 (e1370) animals with EV, dpy-10, and daf-16 RNAi, exposed to PQ for 6 h over untreated controls. Error bars indicate SEM. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; NS—not significant, P ≥ 0.05, significance based on Student’s t-test and Mantel–Cox test for survival curves. P-value for survival curves are indicated next to genotypes. Stars in gray color repesent comparision with daf-2; EV. For TD50 values in survival assays, see Supplementary Table S2. (S) Proposed model for enhanced susceptibility of PD collagen defective animals to exogenous toxins.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. An JH, Blackwell TK.. 2003. SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev. 17:1882–1893. - PMC - PubMed
    1. Atchison WD, Geary TG, Manning B, VandeWaa EA, Thompson DP.. 1992. Comparative neuromuscular blocking actions of levamisole and pyrantel-type anthelmintics on rat and gastrointestinal nematode somatic muscle. Toxicol Appl Pharmacol. 112:133–143. - PubMed
    1. Beck CD, Rankin CH.. 1995. Heat shock disrupts long-term memory consolidation in Caenorhabditis elegans. Learn Mem. 2:161–177. - PubMed
    1. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O.. 2012. Oxidative stress and antioxidant defense. World Allergy Organ J. 5:9–19. - PMC - PubMed
    1. Boguniewicz M, Leung DYM.. 2011. Atopic dermatitis: a disease of altered skin barrier and immune dysregulation. Immunol Rev. 242:233–246. - PMC - PubMed

Publication types