The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3' UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions.     

Molecular mechanisms of developmental timing in C. elegans and Drosophila.

Characterization of the heterochronic genes has provided a strong foundation for understanding the molecular mechanisms of developmental timing in C. elegans. In apparent contrast, studies of developmental timing in Drosophila have demonstrated a central role for gene cascades triggered by the steroid hormone ecdysone. In this review, I survey the molecular mechanisms of developmental timing in C. elegans and Drosophila and outline how common regulatory pathways are beginning to emerge.     

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

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

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.     

Alterations in ribosome biogenesis cause specific defects in C. elegans hermaphrodite gonadogenesis

Ribosome biogenesis is a cell-essential process that influences cell growth, proliferation, and differentiation. How ribosome biogenesis impacts development, however, is poorly understood. Here, we establish a link between ribosome biogenesis and gonadogenesis in Caenorhabditis elegans that affects germline proliferation and patterning. Previously, we determined that pro-1(+)activity is required in the soma--specifically, the sheath/spermatheca sublineage--to promote normal proliferation and prevent germline tumor formation. Here, we report that PRO-1, like its yeast ortholog IPI3, influences rRNA processing. pro-1 tumors are suppressed by mutations in ncl-1 or lin-35/Rb, both of which elevate pre-rRNA levels. Thus, in this context, lin-35/Rb acts as a soma-autonomous germline tumor promoter. We further report the characterization of two additional genes identified for their germline tumor phenotype, pro-2 and pro-3, and find that they, too, encode orthologs of proteins involved in ribosome biogenesis in yeast (NOC2 and SDA1, respectively). Finally, we demonstrate that depletion of additional C. elegans orthologs of yeast ribosome biogenesis factors display phenotypes similar to depletion of progenes. We conclude that the C. elegans distal sheath is particularly sensitive to alterations in ribosome biogenesis and that ribosome biogenesis defects in one tissue can non-autonomously influence proliferation in an adjacent tissue.     

Deficiencies in C20 polyunsaturated fatty acids cause behavioral and developmental defects in Caenorhabditis elegans fat-3 mutants

Arachidonic acid and other long-chain polyunsaturated fatty acids (PUFAs) are important structural components of membranes and are implicated in diverse signaling pathways. The Delta6 desaturation of linoleic and linolenic acids is the rate-limiting step in the synthesis of these molecules. C. elegans fat-3 mutants lack Delta6 desaturase activity and fail to produce C20 PUFAs. We examined these mutants and found that development and behavior were affected as a consequence of C20 PUFA deficiency. While fat-3 mutants are viable, they grow slowly, display considerably less spontaneous movement, have an altered body shape, and produce fewer progeny than do wild type. In addition, the timing of an ultradian rhythm, the defecation cycle, is lengthened compared to wild type. Since all these defects can be ameliorated by supplementing the nematode diet with gamma-linolenic acid or C20 PUFAs of either the n6 or the n3 series, we can establish a causal link between fatty acid deficiency and phenotype. Similar epidermal tissue defects and slow growth are hallmarks of human fatty acid deficiency.     

let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein

Genetic analysis previously suggested that the let-60 gene controls the switch between vulval and hypodermal cell fates during C. elegans vulval induction. We have cloned the let-60 gene, and shown that it encodes a gene product identical in 84% of its first 164 amino acids to ras gene products from other vertebrate and invertebrate species. This conservation suggests that the let-60 product contains all the biochemical functions of ras proteins. Extrachromosomal arrays of let-60 ras DNA cause cell-type misspecification (extra vulval fates) phenotypically opposite to that caused by let-60 ras loss-of-function mutations (no vulval fates), and suppress the vulvaless phenotype of mutations in two other genes necessary for vulval induction. Thus, the level and pattern of let-60 ras expression may be under strict regulation; increase in let-60 ras activity bypasses or reduces the need for upstream genes in the vulval induction pathway.     

Pheromones and Nutritional Signals Regulate the Developmental Reliance on let-7 Family MicroRNAs in C. elegans

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Control of cell cycle timing during C. elegans embryogenesis

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P₃ (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.     

The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans.

The lin-4 gene encodes a small RNA that is required to translationally repress lin-14 toward the end of the first larval stage of Caenorhabditis elegans development. To determine if the timing of LIN-14 protein down-regulation depends on the temporal profile of lin-4 RNA level, we analyzed the stage-specificity of lin-4 RNA expression during wild-type development and examined the phenotypes of transgenic worms that overexpress lin-4 RNA during the first larval stage. We found that lin-4 RNA first becomes detectable at approximately 12 h of wild-type larval development and rapidly accumulates to nearly maximum levels by 16 h. This profile of lin-4 RNA accumulation corresponded to the timing of LIN-14 protein down-regulation. Transgenic strains that express elevated levels of lin-4 RNA prior to 12 h of development display reduced levels of LIN-14 protein and precocious phenotypes consistent with abnormally early loss of lin-14 activity. These results indicate that the temporal profile of lin-4 RNA accumulation specifies the timing of LIN-14 down-regulation and thereby controls the timing of postembryonic developmental events.     

The P-type ATPase CATP-1 is a novel regulator of C. elegans developmental timing that acts independently of its predicted pump function

During postembryonic stages, metazoans synchronize the development of a large number of cells, tissues and organs by mechanisms that remain largely unknown. In Caenorhabditis elegans larvae, an invariant cell lineage is tightly coordinated with four successive molts, thus defining a genetically tractable system to analyze the mechanisms underlying developmental synchronization. Illegitimate activation of nicotinic acetylcholine receptors (nAChRs) by the nicotinic agonist dimethylphenylpiperazinium (DMPP) during the second larval stage (L2) of C. elegans causes a lethal heterochronic phenotype. DMPP exposure delays cell division and differentiation without affecting the molt cycle, hence resulting in deadly exposure of a defective cuticle to the surrounding environment. In a screen for DMPP-resistant mutants, we identified catp-1 as a gene coding for a predicted cation-transporting P-type ATPase expressed in the epidermis. Larval development was specifically slowed down at the L2 stage in catp-1 mutants compared with wild-type animals and was not further delayed after exposure to DMPP. We demonstrate that CATP-1 interacts with the insulin/IGF and Ras-MAPK pathways to control several postembryonic developmental events. Interestingly, these developmental functions can be fulfilled independently of the predicted cation-transporter activity of CATP-1, as pump-dead engineered variants of CATP-1 can rescue most catp-1-mutant defects. These results obtained in vivo provide further evidence for the recently proposed pump-independent scaffolding functions of P-type ATPases in the modulation of intracellular signaling.