To investigate the roles of miRNAs in animal survival during starvation-induced L1 diapause, we impaired the overall miRISC function with loss-of-function (lf) mutants of ain-1 (ku322, ku425, and tm3681) and ain-2(tm2432) and examined their L1 starvation survival rate (Materials and Methods). The strong suppression of the mir-71(lf) defect by hbl-1(RNAi), and the relatively weak effect of miR-71 on hbl-1 expression, are consistent with the idea that miR-71 exerts its role by modulating activities of multiple genes related to hbl-1 function in developmental timing. In contrast, the nuclear-localized GFP expression under the control of the 3′UTR of age-1(Fig. 3 C and D) or unc-31 (Fig. 3 E and F) was strongly repressed in the control worms, but prominently derepressed in mir-71(lf) mutant worms. If the 3′UTR of age-1 or unc-31 is repressed by miR-71, the GFP expression will be repressed in tissues where miR-71 is expressed in wild-type worms, but derepressed in the same tissues of mir-71(lf) worms. (A) The mir-71(n4115, lf) mutant displayed severe reduction in L1 starvation survival rate, and the reduced survival rate of mir-71(lf) was suppressed by a reduction-of-function allele of age-1(hx546). (C) The reduced L1 starvation survival rate of ain-1(lf) mutants was significantly suppressed by a null allele of unc-31.
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Elegans Genetic Center (reference 257) and an N2 strain from the laboratory stock, respectively. Wild-type strains A and B are an N2 strain recently obtained from the C. (A) Survival rate curves of wild-type and mutant strains, as indicated. This is consistent with the previous reports that AIN-1 and AIN-2 are functional homologs with overlapping biochemical roles (16, 17). The roles of InsRs have also been implicated in arresting the cell cycle in germ cells and a portion of somatic cells during L1 diapause (2, 4). Contributed new reagents/analytic tools; X.Z., R.Z., and M.H.
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When late, first larval stage (L1) worms sense unfavorable conditions, they enter an alternative and long-lived larval stage called dauer larvae (or dauer diapause). The nematode Caenorhabditis elegans responds to starvation by entering developmental arrest at multiple stages of its life cycle (1). Extreme climate events such as droughts and heatwaves are intensifying under climate change, yet their combined effects on plant recovery remain unclear. These pages contain all relevant country-specific information, including the recovery and resilience plans, the Commission’s assessment of the plans as well as information on payments requested by the Member States and funds paid out by the Commission.
- To identify individual miRNAs that play prominent roles in L1 diapause, we screened 72 available mutant strains of individual miRNAs and miRNA families (87 miRNAs in total) using the L1 starvation assay.
- Numerous animal species across multiple phyla enter developmental arrest for long-term survival in unfavorable environments and resume development upon stress removal.
- 1A because the ain-1 mutations reduce, but do not eliminate, miRISC functions.
- This will be followed by an ‘ex post evaluation’ in 2028, once the measures included in the recovery plans are fully implemented.
- Starting from its 2022 cycle, the European Semester process was adapted to take into account the creation of the Recovery and Resilience Facility and the implementation of the recovery and resilience plans.
- Biomass recovery was similar across growth strategies, suggesting that growth-related differences play a minimal role in short-term recovery; however, early regrowth was characterised by contrasting trait shifts.
- Among short-lived miRNA mutants, a mir-71 deletion mutant, mir-71(n4115) (referred to as mir-71(lf) hereafter), displayed a severe reduction in L1 starvation survival rate (Table S1 and Fig. 2A).
Here we show that compromising overall microRNA (miRNA) functions or mutating certain individual miRNAs impairs the long-term survival of nematodes during starvation-induced L1 diapause. Third-party accounts will also be restored if third-party backup was enabled on the old device. If you become locked out of those services and don’t have a backup of your accounts in Duo Mobile, you’ll need to contact the support team for that application (or perform the account recovery process for each of those third-party applications). However, whether an account can be restored depends upon Duo Restore being enabled by the administrator in the Duo Admin Panel or whether you’ve set a recovery password for reconnecting third-party accounts.
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- The computation-based prediction that age-1 and pdk-1 are potential targets of miR-71 was also reported in a recent study focusing on miRNA functions in aging where the mRNA level of pdk-1 was shown to be up-regulated in mir-71 worms (14).
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- (A) The mir-71(n4115, lf) mutant displayed severe reduction in L1 starvation survival rate, and the reduced survival rate of mir-71(lf) was suppressed by a reduction-of-function allele of age-1(hx546).
- To investigate the roles of miRNAs in animal survival during starvation-induced L1 diapause, we impaired the overall miRISC function with loss-of-function (lf) mutants of ain-1 (ku322, ku425, and tm3681) and ain-2(tm2432) and examined their L1 starvation survival rate (Materials and Methods).
- We further found that this survival rate reduction of ain-1 mutants was overcome by ectopic expression of the AIN-2 protein in the intestine but not in the muscle (Fig. 1A and Fig. S1A).
- These results indicate that miR-71 is not essential for arresting seam cell or M-cell divisions during L1 diapause, suggesting that miR-71 function is distinct from DAF-16 function.
We recommend that incorporating trait-based recovery dynamics is essential for predicting ecosystem stability under compound climate extremes. Biomass recovery was similar across growth strategies, suggesting that growth-related differences play a minimal role in short-term recovery; however, early regrowth was characterised by contrasting trait shifts. Solidago canadensis exhibited high tolerance to heat and drought, with early biomass and trait recovery, indicating potential for dominance under climate extremes. Biomass fully recovered within one month in both growth strategies, but leaf traits showed transient shifts, over-recovery in SLA and under-recovery in LDMC, likely reflecting production of new leaf tissues.
MiR-71 regulates vulval cell division during recovery of starved L1 worms. These results indicate that miR-71 is not essential for arresting seam cell or M-cell divisions during L1 diapause, suggesting that miR-71 function is distinct from DAF-16 function. DAF-16 (the FOXO homolog in C. elegans) has been shown to play an important role in cell cycle arrest and developmental progression partly by promoting cki-1 expression in some somatic cells during L1 arrest (2).
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The numbers on each image indicate how many worms of the examined ones displayed the indicated phenotype. (Right panels) The gonad of the same animals in the Left panels to indicate the similar developmental stage. (A) Differential interference contrast (DIC) images showing L4 worms recovered from 4-d–starved L1 worms.
Support figures
We further examined worms recovering from 4 d of L1 starvation and found that around 90% of the mir-71(lf) mutants displayed retarded vulval precursor cell (VPC) division, compared with less than 5% in wild type (Fig. 4A). We found that the 3′UTRs of several genes of the InsR pathway, including unc-31, age-1, pdk-1, akt-2, and sgk-1, contain predicted miR-71 targeting sites (as predicted by TargetScan and mirWIP). (H and I) Fluorescence images (H) and statistical data (I) showing that the M cell diveded in fed animals but remained undivided in 4-, 7-, or 11-d–starved L1 wild-type and mir-71(lf) worms. (E) Fluorescence and DIC images showing that the unc-31 3′UTR reporter was repressed in mir-71(+)worms (2/2 transgenic lines) but not in mir-71(lf) worms (4/4 transgenic lines). We found that the poor survival rate of daf-16(mu86)(lf) was further decreased by mir-71(lf) (Fig. 2C), consistent with the notion that a portion of miR-71 activities regulate genes that act in parallel to UNC-31–mediated InsR/PI3K signaling for long-term survival during L1 diapause. Mutating miR-71 drastically reduces the survival rate of animals in L1 diapause, and the effect can be suppressed by mutations of insulin receptor pathway genes age-1 and unc-31.
(E) DIC images showing that hbl-1(RNAi) caused precocious VPC divisions in late L2/early L3 in both wild-type and mir-71(lf) worms recovered from 4 d of L1 starvation. Note that the daf-16(lf) worms recovering from 3 d of L1 starvation displayed a ∼12-h delay in overall development and that the mir-71(lf); daf-16(lf) double mutants displayed an ∼24-h delay. (C) Bar graph showing that the delayed VPC timing defects of mir-71(lf) worms was suppressed by an unc-31(lf) mutation and partially suppressed by an age-1(rf) mutation. In worms that recovered from 4 d of L1 starvation, we also found that a significant portion of the mir-71(lf) mutants displayed egg-laying defects and overproliferating or precociously reflexed gonads.
(D) Fractions of worms that carry 3′UTR reporter transgene and show no GFP expression GFP(−), weak GFP expression GFP(+/−), and comparable GFP expression to mCherry GFP(+). We found that the mRNA level of UNC-31 was up-regulated by about 20% in mir-71(lf) (Fig. 3A). These results suggest that a significant portion of the miR-71 activities in L1 diapause survival may be devoted to regulating the activities of UNC-31–mediated InsR/PI3K signaling and that the rest of miR-71 activity may regulate UNC-31–independent pathways. We next examined the relationship between miR-71 and UNC-31, which functions upstream of AGE-1 during L1 diapause by regulating calcium-regulated dense-core vesicle fusion and the release of an insulin-like ligand (3). We identified 10 miRNA mutants that showed reduced survival rates with a stringent standard, as well as a few miRNA mutants with slightly increased survival rates (Table S1, Fig. 1D, and Fig. S1B).
S1A indicated a dominant role of intestinal miRNAs in regulating L1 starvation survival. We used a dual-color 3′UTR reporter system (18) to test the computational, prediction-based hypothesis that the 3′UTRs of age-1 and unc-31 are directly regulated by miR-71 (Fig. 3B and Materials and Methods). Among these potential miRNA targets, the predicted miR-71–targeting sites in the 3′UTRs of age-1 and unc-31 are conserved between C.
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This result is consistent with the observation that miR-71 is specifically required for the starvation-induced stress response (Fig. S5). For example, we observed a robust retarded mutant phenotype in the vulval lineage but did not see obvious defects in seam cell differentiation or alae formation. It seems plausible that miRNAs that control developmental timing are also involved in regulating the metabolic rate through repressing the InsR pathway activity.
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To test the hypothesis that these developmental timing genes mediate the regulatory role of miR-71 in larval development during recovery from starvation-induced L1 diapause, we examined whether knocking down HBL-1 function can suppress the retarded VPC timing defect of mir-71(lf). Reduction-of-function mutation (rf) in the age-1/PI3 kinase gene, age-1(hx546), made worms long-lived in the L1 starvation assay and was able to suppress the reduced L1 survival rate of mir-71(lf); the rate of the double mutants was comparable to that of wild type (Fig. 2A). Our genetic analysis indicated that for both L1 diapause survival and developmental recovery functions, miR-71 regulates expressions of genes in both the insulin receptor-dependent and -independent pathways.
The two ain-1 loss-of-function alleles displayed significant reductions in L1 starvation survival rate. We further found that this survival rate reduction of ain-1 mutants was overcome by ectopic expression of the AIN-2 protein in the intestine but not in the muscle (Fig. 1A and Fig. S1A). We found that ain-1 but not ain-2 mutants displayed a significant reduction in L1 starvation survival rate compared with that of wild type (Fig. 1 A and D). Furthermore, a recent study suggests that the expression of certain miRNAs is differentially regulated by starvation-induced dauer diapause (15). Consistent with these ideas, several recent lines of evidence suggest that miRNA let-7 and the heterochronic genes lin-42 and hbl-1 are required to regulate the starvation-induced dauer diapause (10–12) and that a number of miRNAs including lin-4 and mir-71 are involved in regulating life span (13, 14).
