A transient apical extracellular matrix relays cytoskeletal patterns to shape permanent acellular ridges on the surface of adult C. elegans.

Epithelial cells secrete apical extracellular matrices to form protruding structures such as denticles, ridges, scales, or teeth. The mechanisms that shape these structures remain poorly understood. Here, we show how the actin cytoskeleton and a provisional matrix work together to sculpt acellular longitudinal alae ridges in the cuticle of adult C. elegans. Transient assembly of longitudinal actomyosin filaments in the underlying lateral epidermis accompanies deposition of the provisional matrix at the earliest stages of alae formation. Actin is required to pattern the provisional matrix into longitudinal bands that are initially offset from the pattern of longitudinal actin filaments. These bands appear ultrastructurally as alternating regions of adhesion and separation within laminated provisional matrix layers. The provisional matrix is required to establish these demarcated zones of adhesion and separation, which ultimately give rise to alae ridges and their intervening valleys, respectively. Provisional matrix proteins shape the alae ridges and valleys but are not present within the final structure. We propose a morphogenetic mechanism wherein cortical actin patterns are relayed to the laminated provisional matrix to set up distinct zones of matrix layer separation and accretion that shape a permanent and acellular matrix structure.

Feedback between a retinoid-related nuclear receptor and the let-7 microRNAs controls the pace and number of molting cycles in C. elegans.

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. Caenorhabditis elegans undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns and accelerated in let-7(-) mutants, but timed similarly in let-7(-) nhr-23(-) double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7-binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also coregulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.

A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans.

Biological tubes must develop and maintain their proper diameter to transport materials efficiently. These tubes are molded and protected in part by apical extracellular matrices (aECMs) that line their lumens. Despite their importance, aECMs are difficult to image in vivo and therefore poorly understood. The Caenorhabditis elegans vulva has been a paradigm for understanding many aspects of organogenesis. Here we describe the vulva luminal matrix, which contains chondroitin proteoglycans, Zona Pellucida (ZP) domain proteins, and other glycoproteins and lipid transporters related to those in mammals. Confocal and transmission electron microscopy revealed, with unprecedented detail, a complex and dynamic aECM. Different matrix factors assemble on the apical surfaces of each vulva cell type, with clear distinctions seen between Ras-dependent (1°) and Notch-dependent (2°) cell types. Genetic perturbations suggest that chondroitin and other aECM factors together generate a structured scaffold that both expands and constricts lumen shape.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Delayed accumulation of intestinal coliform bacteria enhances life span and stress resistance in Caenorhabditis elegans fed respiratory deficient E. coli.

BACKGROUND: Studies with the nematode model Caenorhabditis elegans have identified conserved biochemical pathways that act to modulate life span. Life span can also be influenced by the composition of the intestinal microbiome, and C. elegans life span can be dramatically influenced by its diet of Escherichia coli. Although C. elegans is typically fed the standard OP50 strain of E. coli, nematodes fed E. coli strains rendered respiratory deficient, either due to a lack coenzyme Q or the absence of ATP synthase, show significant life span extension. Here we explore the mechanisms accounting for the enhanced nematode life span in response to these diets. RESULTS: The intestinal load of E. coli was monitored by determination of worm-associated colony forming units (cfu/worm or coliform counts) as a function of age. The presence of GFP-expressing E. coli in the worm intestine was also monitored by fluorescence microscopy. Worms fed the standard OP50 E. coli strain have high cfu and GFP-labeled bacteria in their guts at the L4 larval stage, and show saturated coliform counts by day five of adulthood. In contrast, nematodes fed diets of respiratory deficient E. coli lacking coenzyme Q lived significantly longer and failed to accumulate bacteria within the lumen at early ages. Animals fed bacteria deficient in complex V showed intermediate coliform numbers and were not quite as long-lived. The results indicate that respiratory deficient Q-less E. coli are effectively degraded in the early adult worm, either at the pharynx or within the intestine, and do not accumulate in the intestinal tract until day ten of adulthood. CONCLUSIONS: The findings of this study suggest that the nematodes fed the respiratory deficient E. coli diet live longer because the delay in bacterial colonization of the gut subjects the worms to less stress compared to worms fed the OP50 E. coli diet. This work suggests that bacterial respiration can act as a virulence factor, influencing the ability of bacteria to colonize and subsequently harm the animal host. Respiratory deficient bacteria may pose a useful model for probing probiotic relationships within the gut microbiome in higher organisms.

MORC family ATPases required for heterochromatin condensation and gene silencing.

Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause derepression of DNA-methylated genes and TEs but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, which are predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes.

Toward a unified model of developmental timing: A "molting" approach.

Animal development requires temporal coordination between recurrent processes and sequential events, but the underlying timing mechanisms are not yet understood. The molting cycle of C. elegans provides an ideal system to study this basic problem. We recently characterized LIN-42, which is related to the circadian clock protein PERIOD, as a key component of the developmental timer underlying rhythmic molting cycles. In this context, LIN-42 coordinates epithelial stem cell dynamics with progression of the molting cycle. Repeated actions of LIN-42 may enable the reprogramming of seam cell temporal fates, while stage-specific actions of LIN-42 and other heterochronic genes select fates appropriate for upcoming, rather than passing, life stages. Here, we discuss the possible configuration of the molting timer, which may include interconnected positive and negative regulatory loops among lin-42, conserved nuclear hormone receptors such as NHR-23 and -25, and the let-7 family of microRNAs. Physiological and environmental conditions may modulate the activities of particular components of this molting timer. Finding that LIN-42 regulates both a sleep-like behavioral state and epidermal stem cell dynamics further supports the model of functional conservation between LIN-42 and mammalian PERIOD proteins. The molting timer may therefore represent a primitive form of a central biological clock and provide a general paradigm for the integration of rhythmic and developmental processes.

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.

Regulation of the C. elegans molt by pqn-47.

C. elegans molts at the end of each of its four larval stages but this cycle ceases at the reproductive adult stage. We have identified a regulator of molting, pqn-47. Null mutations in pqn-47 cause a developmental arrest at the first larval molt, showing that this gene activity is required to transit the molt. Mutants with weak alleles of pqn-47 complete the larval molts but fail to exit the molting cycle at the adult stage. These phenotypes suggest that pqn-47 executes key aspects of the molting program including the cessation of molting cycles. The pqn-47 gene encodes a protein that is highly conserved in animal phylogeny but probably misannotated in genome sequences due to much less significant homology to a yeast transcription factor. A PQN-47::GFP fusion gene is expressed in many neurons, vulval precursor cells, the distal tip cell (DTC), intestine, and the lateral hypodermal seam cells but not in the main body hypodermal syncytium (hyp7) that underlies, synthesizes, and releases most of the collagenous cuticle. A functional PQN-47::GFP fusion protein localizes to the cytoplasm rather than the nucleus at all developmental stages, including the periods preceding and during ecdysis when genetic analysis suggests that pqn-47 functions. The cytoplasmic localization of PQN-47::GFP partially overlaps with the endoplasmic reticulum, suggesting that PQN-47 is involved in the extensive secretion of cuticle components or hormones that occurs during molts. The mammalian and insect homologues of pqn-47 may serve similar roles in regulated secretion.

MLT-10 defines a family of DUF644 and proline-rich repeat proteins involved in the molting cycle of Caenorhabditis elegans.

The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X(2)-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10.

The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25.

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. Here, we show that the paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family.

Functional genomic analysis of C. elegans molting.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development.