acn-1, a C. elegans homologue of ACE, genetically interacts with the let-7 microRNA and other heterochronic genes

Abstract

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors - lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.

Keywords: APP; acn-1; developmental timing; heterochronic genes; let-7; lin-41.

Figure 1.

Knockdown of acn-1 partially suppresses let-7 lethality. This graph shows the result of an RNAi screen for let-7 suppressors. RNAi constructs targeting C. elegans homologues of the genes listed in the table in this figure were screened for suppression of the bursting-vulva phenotype of let-7(n2853) at the L4/adult transition. Except when noted, let-7(n2853) animals were placed on RNAi bacteria at L1. Animals marked with L2* were fed with empty vector RNAi clones for the first 16 hours before being moved to plates with the indicated RNAi constructs. Animals marked with L2** were fed with the OP50 strain of E. coli for 16 hours before being moved. Empty L4440 vector was used as a negative control; daf-12 RNAi was used as a positive control.

Figure 2.

Loss of acn-1 enhanced the apl-1(RNAi) phenotype and affected apl-1 expression. (A) RNAi-mediated knockdown of acn-1 and apl-1 enhanced the suppression of the let-7 bursting-vulva phenotype. This effect was dose-dependent. A gradient of the quantity of RNAi was achieved by mixing the indicated percentage of tested RNAi construct with the corresponding amount of empty vector RNAi (experimental details in Materials and Methods). We observed a significant enhancement of the suppression phenotype in animals fed with a double RNAi construct (dbRNAi) targeting both acn-1 and apl-1 sequences. 10–19% of animals fed with dbRNAi stalled at the L1 or L2 stage and were excluded from the calculation of the percentage of animals exhibiting the bursting-vulva phenotype. The L1 and L2 labels designate the developmental stage at which animals were placed on RNAi bacteria. (B) Expression of the apl-1::gfp::unc-54 3′UTR construct in hypodermal seam cells at different developmental stages in nematodes fed empty vector (L4440) or acn-1 RNAi. Shown is the percentage of animals with seam cell GFP expression.

Figure 3.

lin-41 influences acn-1’s oscillating expression pattern. (A and B) qRT-PCR of acn-1 mRNA from wild-type (N2) nematodes shows oscillating expression. These time points cover the period between early L2 and early L3 (the L2/L3 transition takes place between 18 and 20 hrs post-L1.) (A) hbl-1(mg285) mutation led to a slight rightward phase shift in the oscillation of acn-1 expression but no significant differences in any oscillation parameters, while (B) lin-41(ma104) mutation significantly dampened the amplitude of acn-1’s oscillations.

Figure 4.

A proposed model of the relationships between acn-1 and the heterochronic genes let-7, hbl-1, and lin-41.