Establishment of a tissue-specific RNAi system in C. elegans

Abstract

In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal-and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues.

Fig. 1

Strategy of tissue-specific RNAi using the rde-1 mutant A: Yellow colour shows RNAi resistant cells. Red colour shows RNAi sensitive cells. Expression of rde-1 in tissue A should result in RNAi sensitivity in tissue A, but not tissue B. B: Plasmids used in this study. The lin-26 promoter (3.7 kb 5′ sequence) was placed upstream of NLS-GFP or rde-1, resulting in hypodermis-specific NLS-GFP or rde-1 expression. The myo-3 promoter (2.4 kb 5′ sequence) was fused to NLS-GFP, resulting in a muscle-specific NLS-GFP expressing plasmid (pPD93.97). The hlh-1 promoter (3.0 kb 5′ sequence) was fused to rde-1, resulting in a muscle-specific rde-1 expressing plasmid.

Fig. 2

Estimation of tissue specific RNAi system using GFP markers “Strain” shows the worm strain used for each assay with its properties. Purple bar represents percentage of GFP positive embryos after RNAi for GFP. Blue bar represents percentage of GFP positive embryos after no RNAi. Double sided T-bar shows standard deviation.

Fig. 3

Estimation of the efficacy of the tissue specific RNAi system using embryonic lethal genes “Strain” shows the worm strain used for each assay with its properties. “RNAi for” shows which gene was used in the assay. Black bar represents percentage of embryos that showed lethality.

Fig. 4

Tissue-specific depletion of AJM-1 via ajm-1(RNAi) in wild type and hypodermis-rescued rde-1 embryos. a–c, MH27 (a monoclonal antibody specific to AJM-1) staining of wild type, ajm-1(RNAi); rde-1 [plin-26::rde-1] and ajm-1(RNAi) embryos. Hypodermal MH27 signal is shown in red (a projection of three confocal sections of the surface of the embryo), and pharyngeal and intestinal signal is shown in green (a projection of two central confocal sections). In the wild type, MH27 staining is bright and even in all epithelia (a). ajm-1(RNAi) in the strain rescued for rde-1 in the hypodermis (b) results in marked reduction of MH27 staining in the hypodermis, but no effect in the pharynx and intestine. ajm-1(RNAi) in a wild-type embryo causes almost complete loss of MH27 signal from all embryonic epithelia (c). d–f, Nomarski phenotypes of wild type, ajm-1(RNAi); rde-1 [plin-26::rde-1] and ajm-1(RNAi) embryos. A wild type embryo is shown at the 3-fold stage (d). ajm-1(RNAi) causes a nearly identical 2-fold arrest phenotype in both the strain rescued for rde-1 in hypodermis (e) and in the wild type (f). However, in the case of the strain rescued for rde-1 in hypodermis, embryonic lethality is only observed in 50% of the embryos, while 100% lethality is observed in a wild-type background. Scale bar represents 10 μm. Arrowheads indicate vacuoles characteristic of the ajm-1 loss of function phenotype.

Fig. 5

Muscle-specific feeding RNAi for the unc-98 gene in adult worms All three panels show polarized light microscope images of progeny from worms fed with bacteria expressing dsRNA for unc-98. (a) Wild type strain; (b) the rde-1 mutant strain; (c) muscle-specific RNAi strain. Birefringent needles, which are characteristic of unc-98 mutants, are observed in (a) and (c). Scale bar represents 20 μm.