A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans

Abstract

Whole-mount in situ hybridization (WISH) is an outstanding method to decipher the spatiotemporal expression patterns of microRNAs (miRNAs) and provides important clues for elucidating their functions. The first WISH method for miRNA detection was developed in zebrafish. Although this method was quickly adapted for other vertebrates and fruit flies, WISH analysis has not been successfully used to detect miRNAs in Caenorhabditis elegans. Here, we show a novel WISH method for miRNA detection in C. elegans. Using this method, mir-1 miRNA was detected in the body-wall muscle where the expression and roles of mir-1 miRNA have been previously elucidated. Application of the method to let-7 family miRNAs, let-7, mir-48, mir-84, and mir-241, revealed their distinct but partially overlapping expression patterns, indicating that miRNAs sharing a short common sequence were distinguishably detected. In pash-1 mutants that were depleted of mature miRNAs, signals of mir-48 miRNA were greatly reduced, suggesting that mature miRNAs were detected by the method. These results demonstrate the validity of WISH to detect mature miRNAs in C. elegans.

Somatic and Germline MicroRNAs Form Distinct Silencing Complexes to Regulate Their Target mRNAs Differently

“…We selected germline-and soma-enriched miRNAs, miR-35 and miR-241, respectively, that have similar expression levels in young adult animals, allowing us to purify similar amounts of miRNA complexes (Figure 2A). A comparative analysis between wild-type and glp-4(bn2) germline-less animals grown at restrictive temperature confirmed the previously reported germline enrichment of miR-35 (Alvarez-Saavedra and Horvitz, 2010; Wu et al, 2010;McEwen et al, 2016) and the somatic enrichment of miR-241 (Andachi and Kohara, 2016) (Figure 2A). We performed mass spectrometry analyses to compare proteins that copurified with either miR-35 or miR-241 miRISCs recovered from wild-type young adult lysates ( Figure 2B).…”

An updatedC. elegansnuclear body muscle transcriptome for studies in muscle formation and function

“…Let-7 and the closely related miR-241 are also present. These miRNAs temporally regulate larval development and have been previously identified in the pharyngeal and vulval muscles (Martinez et al 2008; Andachi and Kohara 2016). Among the predicted target genes, we identified several genes in the DAF insulin pathway that control muscle health and aging (Mallick et al 2020; Basu et al 2021).…”

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging

“…mir-1 is a highly conserved microRNA expressed predominantly in C. elegans muscle tissues including pharyngeal muscle, body wall muscle, and sex muscles ( Andachi and Kohara, 2016 ; Martinez et al, 2008 ). While microRNAs are known as important developmental regulators ( Ivey and Srivastava, 2015 ), miR-1 expression also persists throughout adulthood, declining by half from day 1 to day 14 of adulthood ( Figure 1—figure supplement 1A , data from Zhou et al, 2019 ).…”