The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation

lin-4 encodes a small RNA that is complementary to sequences in the 3' untranslated region (UTR) of lin-14 mRNA and that acts to developmentally repress the accumulation of LIN-14 protein. This repression is essential for the proper timing of numerous events of Caenorhabditis elegans larval development. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results indicate that the rate of synthesis of lin-14 mRNA, its state of polyadenylation, its abundance in the cytoplasmic fraction, and its polysomal sedimentation profile do not change in response to the accumulation of lin-4 RNA. Our results indicate that association of lin-4 RNA with the 3' UTR of lin-14 mRNA permits normal biogenesis of lin-14 mRNA, and normal translational initiation, but inhibits step(s) thereafter, such as translational elongation and/or the release of stable LIN-14 protein.     

Isoform-specific mutations in the Caenorhabditis elegans heterochronic gene lin-14 affect stage-specific patterning

The Caenorhabditis elegans heterochronic gene lin-14 specifies the temporal sequence of postembryonic developmental events. lin-14, which encodes differentially spliced LIN-14A and LIN-14B1/B2 protein isoforms, acts at distinct times during the first larval stage to specify first and second larval stage-specific cell lineages. Proposed models for the molecular basis of these two lin-14 gene activities have included the production of functionally distinct isoforms and the generation of a temporal gradient of LIN-14 protein. We report here that loss of the LIN-14B1/B2 isoforms alone affects one of the two lin-14 temporal patterning functions, the specification of second larval stage lineages. A temporal expression difference between LIN-14A and LIN-14B1/B2 is not responsible for the stage-specific phenotype: protein levels of all LIN-14 isoforms are high in early first larval stage animals and decrease during the first larval stage. However, LIN-14A can partially substitute for LIN-14B1/B2 when expressed at a higher-than-normal level in the late L1 stage. These data indicate that LIN-14B1/B2 isoforms do not provide a distinct function of the lin-14 locus in developmental timing but rather may contribute to an overall level of LIN-14 protein that is the critical determinant of temporal cell fate.     

Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in Caenorhabditis elegans

The timing of postembryonic developmental programs in Caenorhabditis elegans is regulated by a set of so-called heterochronic genes, including lin-28 that specifies second larval programs. lin-66 mutations described herein cause delays in vulval and seam cell differentiation, indicating a role for lin-66 in timing regulation. A mutation in daf-12/nuclear receptor or alg-1/argonaute dramatically enhances the retarded phenotypes of the lin-66 mutants, and these phenotypes are suppressed by a lin-28 null allele. We further show that the LIN-28 protein level is upregulated in the lin-66 mutants and that this regulation is mediated by the 3'UTR of lin-28. We have also identified a potential daf-12-response element within lin-28 3'UTR and show that two microRNA (miRNA) (lin-4 and let-7)-binding sites mediate redundant inhibitory activities that are likely lin-66-independent. Quantitative PCR data suggest that the lin-28 mRNA level is affected by lin-14 and miRNA regulation, but not by daf-12 and lin-66 regulation. These results suggest that lin-28 expression is regulated by multiple independent mechanisms including LIN-14-mediated upregulation of mRNA level, miRNAs-mediated RNA degradation, LIN-66-mediated translational inhibition and DAF-12-involved translation promotion.     

LIN-14 inhibition of LIN-12 contributes to precision and timing of C. elegans vulval fate patterning

Studies of C. elegans vulval development have illuminated mechanisms underlying cell fate specification and elucidated intercellular signaling pathways [1]. The vulval precursor cells (VPCs) are spatially patterned during the L3 stage by the EGFR-Ras-MAPK-mediated inductive signal and the LIN-12/Notch-mediated lateral signal. The pattern is both precise and robust [2] because of crosstalk between these pathways [3]. Signaling is also regulated temporally, because constitutive activation of the spatial patterning pathways does not alter the timing of VPC fate specification [4, 5]. The heterochronic genes, including the microRNA lin-4 and its target lin-14, constitute a temporal control mechanism used in different contexts [6-8]. We find that lin-4 specifically controls the activity of LIN-12/Notch through lin-14, but not other known targets, and that persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction. In the L2 stage, there is sufficient lin-14 activity to inhibit constitutive lin-12. Our results suggest that lin-4 and lin-14 contribute to spatial patterning through temporal gating of LIN-12. We propose that in the L2 stage, lin-14 sets a high threshold for LIN-12 activation to help prevent premature activation of LIN-12 by ligands expressed in other cells in the vicinity, thereby contributing to the precision and robustness of VPC fate patterning.     

The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. We identified a new heterochronic gene, lin-46, from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. Our analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. We also show that lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. Our findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development.     

lin-28 controls the succession of cell fate choices via two distinct activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.     

Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites

The heterochronic gene lin-28 is a regulator of developmental timing in the nematode Caenorhabditis elegans. It must be expressed in the first larval stage and downregulated by the second stage for normal development. This downregulation is mediated in part by lin-4, a 21-nt microRNA. If downregulation fails due to a mutation in a short sequence in the lin-28 3' UTR that is complementary to lin-4, then a variety of somatic cell lineages fail to progress normally in development. Here, we report that Lin-28 homologues exist in diverse animals, including Drosophila, Xenopus, mouse, and human. These homologues are characterized by the LIN-28 protein's unusual pairing of RNA-binding motifs: a cold shock domain (CSD) and a pair of retroviral-type CCHC zinc knuckles. Conservation of LIN-28 proteins shows them to be distinct from the other conserved family of CSD-containing proteins of animals, the Y-box proteins. Importantly, the LIN-28 proteins of Drosophila, Xenopus, and mouse each appear to be expressed and downregulated during development, consistent with a conserved role for this regulator of developmental timing. In addition, the extremely long 3' UTRs of mouse and human Lin-28 genes show extensive regions of sequence identity that contain sites complementary to the mammalian homologues of C. elegans lin-4 and let-7 microRNAs, suggesting that microRNA regulation is a conserved feature of the Lin-28 gene in diverse animals.     

LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts

Background

Biological timing mechanisms that integrate cyclical and successive processes are not well understood. C. elegans molting cycles involve rhythmic cellular and animal behaviors linked to the periodic reconstruction of cuticles. Molts are coordinated with successive transitions in the temporal fates of epidermal blast cells, which are programmed by genes in the heterochronic regulatory network. It was known that juveniles molt at regular 8–10 hr intervals, but the anticipated pacemaker had not been characterized.

Results

We find that inactivation of the heterochronic gene lin-42a, which is related to the core circadian clock gene PERIOD (PER), results in arrhythmic molts and continuously abnormal epidermal stem cell dynamics. The oscillatory expression of lin-42a in the epidermis peaks during the molts. Further, forced expression of lin-42a leads to anachronistic larval molts and lethargy in adults.

Conclusions

Our results suggest that rising and falling levels of LIN-42A allow the start and completion, respectively, of larval molts. We propose that LIN-42A and affiliated factors regulate molting cycles in much the same way that PER-based oscillators drive rhythmic behaviors and metabolic processes in mature mammals. Further, the combination of reiterative and stage-specific functions of LIN-42 may coordinate periodic molts with successive development of the epidermis.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

Novel heterochronic functions of the Caenorhabditis elegans period-related protein LIN-42

LIN-42, the Caenorhabditis elegans homolog of the Period (Per) family of circadian rhythm proteins, functions as a member of the heterochronic pathway, regulating temporal cell identities. We demonstrate that lin-42 acts broadly, timing developmental events in the gonad, vulva, and sex myoblasts, in addition to its well-established role in timing terminal differentiation of the hypodermis. In the vulva, sex myoblasts, and hypodermis, lin-42 activity prevents stage-specific cell division patterns from occurring too early. This general function of timing stage-appropriate cell division patterns is shared by the majority of heterochronic genes; their mutation temporally alters stage-specific division patterns. In contrast, lin-42 function in timing gonad morphogenesis is unique among the known heterochronic genes: inactivation of lin-42 causes the elongating gonad arms to reflex too early, a phenotype which implicates lin-42 in temporal regulation of cell migration. Three additional isoforms of lin-42 are identified that expand our view of the lin-42 locus and significantly extend the homology between LIN-42 and other PER family members. We show that, similar to PER proteins, LIN-42 has a dynamic expression pattern; its levels oscillate relative to the molts during postembryonic development. Transformation rescue studies indicate lin-42 is bipartite with respect to function. Intriguingly, the hallmark PAS domain is dispensable for LIN-42 function in transgenic animals.     

A Network of Genes Antagonistic to the LIN-35 Retinoblastoma Protein of Caenorhabditis elegans

The Caenorhabditis elegans pRb ortholog, LIN-35, functions in a wide range of cellular and developmental processes. This includes a role of LIN-35 in nutrient utilization by the intestine, which it carries out redundantly with SLR-2, a zinc-finger protein. This and other redundant functions of LIN-35 were identified in genetic screens for mutations that display synthetic phenotypes in conjunction with loss of lin-35. To explore the intestinal role of LIN-35, we conducted a genome-wide RNA-interference-feeding screen for suppressors of lin-35; slr-2 early larval arrest. Of the 26 suppressors identified, 17 fall into three functional classes: (1) ribosome biogenesis genes, (2) mitochondrial prohibitins, and (3) chromatin regulators. Further characterization indicates that different categories of suppressors act through distinct molecular mechanisms. We also tested lin-35; slr-2 suppressors, as well as suppressors of the synthetic multivulval phenotype, to determine the spectrum of lin-35-synthetic phenotypes that could be suppressed following inhibition of these genes. We identified 19 genes, most of which are evolutionarily conserved, that can suppress multiple unrelated lin-35-synthetic phenotypes. Our study reveals a network of genes broadly antagonistic to LIN-35 as well as genes specific to the role of LIN-35 in intestinal and vulval development. Suppressors of multiple lin-35 phenotypes may be candidate targets for anticancer therapies. Moreover, screening for suppressors of phenotypically distinct synthetic interactions, which share a common altered gene, may prove to be a novel and effective approach for identifying genes whose activities are most directly relevant to the core functions of the shared gene.     

Regulatory mutations of mir-48, a C. elegans let-7 family MicroRNA, cause developmental timing defects

The C. elegans heterochronic genes program stage-specific temporal identities in multiple tissues during larval development. These genes include the first two miRNA-encoding genes discovered, lin-4 and let-7. We show that lin-58 alleles, identified as lin-4 suppressors, define another miRNA that controls developmental time. These alleles are unique in that they contain point mutations in a gene regulatory element of mir-48, a let-7 family member. mir-48 is expressed prematurely in lin-58 mutants, whereas expression of mir-241, another let-7 family member residing immediately upstream of mir-48, appears to be unaffected. A mir-48 transgene bearing a lin-58 point mutation causes strong precocious phenotypes in the hypodermis and vulva when expressed from multicopy arrays. mir-48::gfp fusions reveal expression in these tissues, and inclusion of a lin-58 mutation causes precocious and enhanced gfp expression. These results suggest that lin-58 alleles disrupt a repressor binding site that restricts the time of miR-48 action in wild-type animals.     

Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation

The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.