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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If you haven’t enabled third-party account restore in Duo Mobile then app backups to Google account backup (Android) or iCloud (iOS) accounts DO NOT contain any private key or other sensitive data. Duo Mobile’s restore functionality lets you back up Duo-protected accounts and third-party OTP accounts (such as Google or Facebook) for recovery to the same device or to a new device. We speculate that the expression of heterochronic genes controlling the L2/L3 programs, including that of hbl-1 and lin-42, are increased during L1 diapause to arrest the developmental progression, and miR-71 is probably required to suppress these “excess” signals during the recovery phase (Fig. S5). Furthermore, the observed derepression of individual genes by mir-71(lf) seemed too weak to account for the phenotype, consistent with the idea that a prominent phenotype of an miRNA mutation is caused by the collective effect of changing expression in many genes, an important property of miRNA-mediated gene regulation. (F) Fluorescence and DIC images showing that an hbl-1 3′UTR reporter was repressed in mir-71(+) worms and slightly derepressed in mir-71(lf) mutants.
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- 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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- However, miR-71 does not appear to regulate all postembryonic development during L1 diapause recovery.
- To understand how miR-71 affects VPC division, we searched its predicted targets for potential genes involved in regulating developmental timing.
Previous studies showed that the release of postdocking calcium-regulated dense-core vesicles, the insulin receptor (InsR) pathway, the AMPK pathway, and protein revery play login chaperones are required for the long-term survival of starved L1 worms (2–4). Unlike dauer diapause, L1 diapause is not accompanied by life cycle changes and has not been shown to require certain signaling pathways that control the formation of dauer diapause such as TGF-β signaling (daf-1, daf-7) and nuclear hormone receptor (daf-12) (2, 3). The coordinated entrance into developmental arrest, long-term survival, and the reinitiation of development upon food availability are important biological processes to investigate. Different organisms have developed versatile growth arrest strategies to overcome starvation-induced metabolic and developmental problems. The presented results indicate that interactions between multiple miRNAs and likely a large number of their mRNA targets in multiple pathways regulate the response to starvation-induced L1 diapause.
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The reporter construct, the control plasmid, and a transformation marker plasmid were coinjected into worms to generate the extrachromosomal arrays for analysis. Elegans and Caenorhabditis briggsae, leading us to focus further analyses on these two genes. We further examined the functional relationship between miR-71 and DAF-16, a FOXO transcription factor acting critically and negatively downstream of AGE-1/PI3K in the InsR pathway. Because the InsR pathway was previously shown to play a prominent role in L1 diapause (2, 3), we examined genetic interactions between miR-71 and different components of the InsR pathway.
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(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.
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Consistent with the observation described above, the 4-d–starved mir-71(lf) mutants recovering on the RNAi control plates displayed the highly penetrant retarded defect in VPC division. If this were true, the starved mir-71(lf); daf-16(lf) double-mutant worms should show a slow growth phenotype similar to that of daf-16(lf) worms, but no specific VPC timing defect. (H) Fluorescence and DIC images showing that a lin-42 3′UTR reporter was repressed in mir-71(+) worms (2/2 transgenic lines) and prominently derepressed in mir-71(−) worms (2/2 transgenic lines). We thus asked whether miR-71 was required for the reinitiation of developmental programs during the recovery phase after L1 starvation. These results suggest that miR-71 regulates the expression of unc-31 and age-1 through their 3′UTRs. Note that there are extra GFP-positive cells (red arrows) in mir-71(lf) mutants.
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- This is consistent with hbl-1 being one of the downstream targets of miR-71, although this modest effect alone is not expected to account for the vulval developmental phenotype in mir-71 mutant.
Whereas the vulva of wild-type worms developed into the pyramidal stage (81 of 82 worms), the P6.p of mir-71(n4115, lf) mutant worms divided only once (83 of 89 worms). 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). (C) Fluorescence and differential interference contrast (DIC) images showing that the age-1 3′UTR reporter was repressed in mir-71(+) worms (3/4 transgenic lines) but not in mir-71(lf) worms (4/4 transgenic lines). The transcript level of unc-31 was increased in mir-71(lf) worms, compared with that of wild-type controls that were normalized to the value of 1. MiR-71 represses the expression of age-1 and unc-31 through the actions on their 3′UTR, but miR-71 is not required for arresting M cell division during L1 diapause.
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.
These results compelled us to examine specific interactions between individual miRNAs and their targets to gain mechanistic insights. This result suggests that miR-71 likely functions upstream of, or in parallel to, HBL-1 in regulating VPC timing. Moreover, the expression of hbl-1 is repressed by let-7 family miRNAs at L3 during normal development, and the hyperactivity of hbl-1 caused by failure of miRNA regulation leads to retarded development (26).
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(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).
Although the complete removal of miRNA functions causes embryonic lethality or infertility in worms, a partial disruption of overall miRNA functions by mutating either ain-1 or ain-2 provides an effective way to investigate miRNA functions (16, 17). However, we found that the reporter transgene with the lin-42 3′UTR was significantly repressed in wild-type worms, but derepressed in the mir-71(lf) worms (Fig. 4 H and I). This is consistent with hbl-1 being one of the downstream targets of miR-71, although this modest effect alone is not expected to account for the vulval developmental phenotype in mir-71 mutant. In starved L1 worms, we detected only a slight increase in the mRNA level of hbl-1 in mir-71 mutants compared with that in wild type (∼10%), which may not be biologically significant. In contrast, the mir-71(lf) mutant worms recovering on hbl-1(RNAi) displayed precocious VPC divisions similar to that seen in wild type (Fig. 4E).
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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.