TY  - GEN
T1  - LIR-1 and PDTF-1 regulate the permeability barrier function of the C. elegans cuticle
AU  - Sandhu, Anjali
AU  - Sheokand, Riya
AU  - Singh, Varsha
DO  - 10.17912/micropub.biology.000434
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000434/
AB  - C. elegans skin consists of two layers- cuticle and hypodermis. The cuticle is the outer most layer that is predominately composed of cross-linked collagens. It acts as a physical barrier against exogenous toxins and helps in locomotion (Page and Johnstone 2007; Altun and Hall 2009; Sandhu et al. 2021). Cuticle is secreted by the underlying hypodermis, a single cell layer. In a recent study, we uncovered the role of six collagens- DPY-2, -3, -7, -8, -9 and -10 in maintenance of permeability barrier function of the cuticle to a nucleic acid dye Hoechst 33258. Lack of any of these collagens or transcription factor BLMP-1 led to enhanced Hoechst staining and susceptibility to exogenous toxins (Bus et al. 1976; Kass et al. 1980; Bus and Gibson 1984; Atchison et al. 1992; Sandhu et al. 2021). To see if there were additional regulators of cuticle permeability, we performed an RNAi screen for 286 transcription factors known to be expressed in the hypodermis (Kaletsky et al. 2018) using Hoechst 33258 staining assay (Extended Data, Table 1). Animals treated with empty vector RNAi control remained impermeable to Hoechst stain. As expected, we found that blmp-1 RNAi caused staining (Extended Data, Table 1; Sandhu et al. 2020). In addition, we found that two transcription factors, lir-1 and T26A8.4, also caused staining (Fig. 1A). We name T26A8.4 permeability-determining transcription factor PDTF-1. Importantly, RNAi of either lir-1 or pdtf-1 in WT animals enhanced their susceptibility towards exogenous toxins such as paraquat (PQ), levamisole, and ivermectin (IVM) (Fig. 1, B-D). This could result from their ability to regulate collagen expression or through other mechanism(s). To test the former, we examined the effect of lir-1 and pdtf-1 RNAi on COL-19::GFP expression, reporter for adult collagen COL-19 (Thein et al. 2003) expressed in circumferential ridges called annuli in C. elegans cuticle. Furrows delineate the annuli which pattern the entire C. elegans cuticle. COL-19::GFP showed regular expression in annuli separated by parallel furrows in wild type animals as expected, whereas its expression was disrupted proximal to alae in both lir-1 and pdtf-1 RNAi animals (marked in Fig. 1G). To test if permeability determining transcription factors regulate PD collagen expression, we examined level of transcripts for collagens and collagen processing enzymes in lir-1 and pdtf-1 RNAi animals by qRT-PCR (Fig. 1, E-F). We found that expression of transcripts for PD collagens as well as DPY-4 and DPY-5 collagens was reduced in lir-1 RNAi animals. pdtf-1 RNAi only altered the expression of transcripts for DPY-5 collagen and PDI-2, a collagen processing enzyme. This is consistent with our previous study where we showed that pdi-2 knockdown also causes permeability defects (Sandhu et al. 2021). In all, our analysis of transcription factors in the hypodermis indicates that LIR-1 and PDTF-1 are transcription regulators of cuticle permeability barrier function, collagen expression, and protection of C. elegans from exogenous toxins.
PY  - 2021
JO  - microPublication Biology
ER  -