Ann E. Rougvie

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3′ untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3′ untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA–RNA interactions with their 3′ untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.

The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged

Protein-DNA cross-linking of cultured Drosophila cells has shown that, in vivo, prior to the induction of heat shock, there is approximately one molecule of RNA polymerase II associated with the promoter region of the major heat shock gene, hsp70. Here, we show that this promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain of approximately 25 nucleotides in length, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction. The detection of a post-initiation RNA polymerase complex on the promoter region of the inactive gene suggests that there is a transcriptional control mechanism that acts at a step early in transcript elongation.

Chromosome cohesion is regulated by a clock gene paralogue TIM-1.

Faithful transmission of the genome requires that a protein complex called cohesin establishes and maintains the regulated linkage between replicated chromosomes before their segregation. Here we report the unforeseen participation of Caenorhabditis elegans TIM-1, a paralogue of the Drosophila clock protein TIMELESS, in the regulation of chromosome cohesion. Our biochemical experiments defined the C. elegans cohesin complex and revealed its physical association with TIM-1. Functional relevance of the interaction was demonstrated by aberrant mitotic chromosome behaviour, embryonic lethality and defective meiotic chromosome cohesion caused by the disruption of either TIM-1 or cohesin. TIM-1 depletion prevented the assembly of non-SMC (structural maintenance of chromosome) cohesin subunits onto meiotic chromosomes; however, unexpectedly, a partial cohesin complex composed of SMC components still loaded. Further disruption of cohesin activity in meiosis by the simultaneous depletion of TIM-1 and an SMC subunit decreased homologous chromosome pairing before synapsis, revealing a new role for cohesin in metazoans. On the basis of comparisons between TIMELESS homologues in worms, flies and mice, we propose that chromosome cohesion, rather than circadian clock regulation, is the ancient and conserved function for TIMELESS-like proteins.

Control of developmental timing in animals.

The molecular mechanisms that time development are now being deciphered in various organisms, particularly in Caenorhabditis elegans. Key recent findings indicate that certain C. elegans timekeeping genes are conserved across phyla, and their developmental expression patterns indicate that a timing function might also be conserved. Small regulatory RNAs have crucial roles in the timing mechanism, and the cellular machinery required for production of these RNAs intersects with that used to process double-stranded RNAs during RNA interference.

Intrinsic and extrinsic regulators of developmental timing: from miRNAs to nutritional cues.

A fundamental challenge in biology is to understand the reproducibility of developmental programs between individuals of the same metazoan species. This developmental precision reflects the meticulous integration of temporal control mechanisms with those that specify other aspects of pattern formation, such as spatial and sexual information. The cues that guide these developmental events are largely intrinsic to the organism but can also include extrinsic inputs, such as nutrition or temperature. This review discusses the well-characterized developmental timing mechanism that patterns the C. elegans epidermis. Components of this pathway are conserved, and their links to developmental time control in other species are considered, including the temporal patterning of the fly nervous system. Particular attention is given to the roles of miRNAs in developmental timing and to the emerging mechanisms that link developmental programs to nutritional cues.

C. elegans AMPKs promote survival and arrest germline development during nutrient stress

Summary Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic &agr; subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.

miRNAs give worms the time of their lives: small RNAs and Temporal Control in Caenorhabditis elegans

Alteration in the timing of particular developmental events can lead to major morphological changes that have profound effects on the life history of an organism. Insights into developmental timing mechanisms have been revealed in the model organism Caenorhabditis elegans, in which a regulatory network of heterochronic genes times events during larval development, ensuring that stage‐specific programs occur in the appropriate sequence and on schedule. Developmental timing studies in C. elegans led to the landmark discovery of miRNAs and continue to enhance our understanding of the regulation and activity of these small regulatory molecules. Current views of the heterochronic gene pathway are summarized here, with a focus on the ways in which miRNAs contribute to temporal control and how miRNAs themselves are regulated. Finally, the conservation of heterochronic genes and their functions in timing, as well as their related roles in stem cells and cancer, are highlighted. Developmental Dynamics 239:1477–1489, 2010.

The C. elegans developmental timing protein LIN-42 regulates diapause in response to environmental cues

Environmental conditions can have a major impact on developmental progression in animals. For example, when C. elegans larvae encounter harsh conditions they can reversibly halt the passage of developmental time by forming a long-lived dauer larva at the end of the second larval stage. Here, we show that the period homolog lin-42, known to control developmental time, also acts as a component of a switch that mediates dauer entry. Loss of lin-42 function renders animals hypersensitive to dauer formation under stressful conditions, whereas misexpression of lin-42 in the pre-dauer stage inhibits dauer formation, indicating that lin-42 acts as a negative regulator of this life history decision. These phenotypes place LIN-42 in opposition to the ligand-free form of the nuclear receptor DAF-12, which indirectly senses environmental conditions and helps to integrate external cues into developmental decisions. Mutations that impair DAF-12 ligand binding are exquisitely sensitive to the absence of lin-42, whereas overexpression of LIN-42 can suppress the dauer constitutive phenotype of a ligand-insensitive daf-12 mutant, suggesting that LIN-42 and DAF-12 are intimate partners in controlling the decision to become a dauer larva. The functional outputs of Period family proteins and nuclear receptors also converge in other organisms, suggesting that the relationship between lin-42 and daf-12 represents an ancient genetic framework for responding to environmental stimuli.

Chapter 14 Protein—DNA Cross-Linking as a Means to Determine the Distribution of Proteins on DNA in Vivo

Publisher Summary The protein-DNA cross-linking can be used to determine the distribution of proteins on DNA in vivo . The temporal and spatial distributions of particular proteins on specific DNA sequences provide key insights into the regulation and mechanics of transcription and chromosome replication and basic information on chromatin structure. The chapter describes a method for directly mapping the distribution of a protein on DNA in living cells by ultraviolet (UV)-induced cross-linking. UV irradiation of cells cross-links protein to DNA at the points of contact. The protein–DNA adducts are purified from cells, and then the protein of interest is immunoprecipitated with an appropriate antibody. The distribution of the protein on DNA in the cell is revealed by identifying specific restriction fragments that coprecipitate with the protein via their UV-induced covalent attachment to the protein. The UV-cross-linking method provides a unique opportunity to evaluate mechanistic questions in vivo . The method has been used to demonstrate that Drosophila topoisomerase I is recruited during heat shock to activated transcription units. It has also provided a direct measure of the relative in vivo density of RNA polymerase II on a variety of Drosophila genes.

Caenorhabditis elegans period homolog lin-42 regulates the timing of heterochronic miRNA expression

Significance Caenorhabditis elegans studies have contributed greatly to our understanding of the temporal regulation of development. These studies led to the discovery of microRNAs (miRNAs) and revealed that a succession of stage-specifically expressed miRNAs act as switches to guide life-stage transitions in cell fates. These miRNAs are conserved to humans and cause disease states when misexpressed, highlighting the importance of their temporal regulation. Much is known about the biogenesis and function of miRNAs, but how these processes are temporally sculpted is not well understood. This study reports that LIN-42, the C. elegans homolog of the circadian rhythm protein Period, regulates timing of miRNA expression by modulating transcription. These findings further our understanding of the functions of developmental timekeepers and regulation of miRNAs. MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally via the 3′ UTR of target mRNAs and were first identified in the Caenorhabditis elegans heterochronic pathway. miRNAs have since been found in many organisms and have broad functions, including control of differentiation and pluripotency in humans. lin-4 and let-7–family miRNAs regulate developmental timing in C. elegans, and their proper temporal expression ensures cell lineage patterns are correctly timed and sequentially executed. Although much is known about miRNA biogenesis, less is understood about how miRNA expression is timed and regulated. lin-42, the worm homolog of the circadian rhythm gene period of flies and mammals, is another core component of the heterochronic gene pathway. lin-42 mutants have a precocious phenotype, in which later-stage programs are executed too early, but the placement of lin-42 in the timing pathway is unclear. Here, we demonstrate that lin-42 negatively regulates heterochronic miRNA transcription. let-7 and the related miRNA miR-48 accumulate precociously in lin-42 mutants. This defect reflects transcriptional misregulation because enhanced expression of both primary miRNA transcripts (pri-miRNAs) and a let-7 promoter::gfp fusion are observed. The pri-miRNA levels oscillate during larval development, in a pattern reminiscent of lin-42 expression. Importantly, we show that lin-42 is not required for this cycling; instead, peak amplitude is increased. Genetic analyses further confirm that lin-42 acts through let-7 family miRNAs. Taken together, these data show that a key function of lin-42 in developmental timing is to dampen pri-miRNAs levels, preventing their premature expression as mature miRNAs.