Larval Arrest Research Articles

Loss of the Na+/K+ cation pump CATP-1 suppresses nekl-associated molting defects.

The conserved Caenorhabditis elegans protein kinases NEKL-2 and NEKL-3 regulate membrane trafficking and are required for larval molting. Through a forward genetic screen we identified a mutation in catp-1 as a suppressor of molting defects in synthetically lethal nekl-2; nekl-3 double mutants. catp-1 encodes a membrane-associated P4-type ATPase involved in Na+-K+ exchange. A previous study found that wild-type worms exposed to the nicotinic agonist dimethylphenylpiperazinium (DMPP) exhibited larval arrest and molting-associated defects, which were suppressed by inhibition of catp-1. By testing a spectrum catp-1 alleles, we found that resistance to DMPP toxicity and the suppression of nekl defects did not strongly correlate, suggesting key differences in the mechanism of catp-1-mediated suppression. Through whole genome sequencing of additional nekl-2; nekl-3 suppressor strains, we identified two additional coding-altering mutations in catp-1. However, neither mutation, when introduced into nekl-2; nekl-3 mutants using CRISPR, was sufficient to elicit robust suppression of molting defects, suggesting the involvement of other loci. Endogenously tagged CATP-1 was primarily expressed in epidermal cells within punctate structures located near the apical plasma membrane, consistent with a role in regulating cellular processes within the epidermis. Based on previous studies, we tested the hypothesis that catp-1 inhibition induces entry into the pre-dauer L2d stage, potentially accounting for the ability of catp-1 mutants to suppress nekl molting defects. However, we found no evidence that loss of catp-1 leads to entry into L2d. As such, loss of catp-1 may suppress nekl-associated and DMPP-induced defects by altering electrochemical gradients within membrane-bound compartments.

Soul is a master control gene governing the development of the Drosophila prothoracic gland

The prothoracic gland (PG) is a major insect endocrine organ. It is the principal source of insect steroid hormones, and critical for key developmental events such as the molts, the establishment of critical weight (CW), pupation, and sexual maturation. However, little is known about the developmental processes that regulate PG morphology. In this study, we identified soul, which encodes a PG-specific basic helix-loop-helix (bHLH) transcription factor. We demonstrate that Tap, also a bHLH protein, dimerizes with Soul. Both are expressed in the developing PG. Interfering with either soul or tap function caused strikingly similar phenotypes, resulting in small and fragmented PGs, the abolishment of steroid hormone-producing gene expression, larval arrest, and a failure to undergo metamorphosis. Furthermore, both soul and tap showed expression peaks just prior to the CW checkpoint. Disrupting soul- or tap-function before, but not after, the CW checkpoint caused larval arrest, and perturbed highly similar gene cohorts, which were enriched for regulators and components of the steroid hormone biosynthesis pathway. Intriguingly, a chitin-based cuticle gene, Cpr49Ah, and a POU domain transcription factor gene, pdm3, are direct target genes of the Soul/Tap complex, and disruption of either phenocopied key aspects of soul/tap loss-of-function phenotypes. Taken together, our findings demonstrate that the Soul/Tap heterodimer resides at the top of a complex gene hierarchy that drives PG development, CW establishment, and steroid hormone production.

SPP-5 affects larval arrest via insulin signaling pathway in Caenorhabditis elegans.

Diapause is an endocrine-mediated metabolic and growth arrest state in response to unfavorable external environments. The nematode Caenorhabditis elegans can enter diapause/arrest during embryonic, larval, or adult stages when subjected to detrimental external environments.Larval stage 1 (L1) arrest happens when animals hatch without food. Previous work has shown that the insulin pathway plays a prominent role in regulating L1 arrest. However, the downstream signal molecular mechanisms and biomarkers are still missing. In this study, we showed that SaPosin-like Protein family member SPP-5 is significantly upregulated during L1 arrest, suggesting that it could act as an L1 arrest biomarker. Using RNA interference we demonstrated that spp-5 knockdown accelerated larval development, while the overexpression resulted in L1 arrest. Consistently,SPP-5 level was significantly up-regulated in the L1 arrest daf-2(e1370) mutants, and spp-5(RNAi) suppressed the daf-2(e1370) induced L1 arrest. These results suggest that SPP-5 can serve as an L1 arrest biomarker and promote the arrest probably via the insulin signaling pathway.

Role of CYP311A1 in wing development of Drosophila melanogaster.

Lipid homeostasis is crucial for growth and development of organisms. Several cytochrome P450 monooxygenases (CYPs) are involved in lipid metabolism. The function of Cyp311a1 in the anterior midgut as a regulator of phosphatidylethanolamine (PE) metabolism in Drosophila melanogaster has been demonstrated, as depletion of Cyp311a1 caused larval growth arrest that was partially rescued by supplying PE. In this study, we investigated the role of CYP311A1 in wing morphogenesis in Drosophila. Using the GAL4-UAS system, Cyp311a1 was selectively knocked down in the wing disc. A deformed wing phenotype was observed in flies with reduced Cyp311a1 transcripts. BODIPY and oil red O staining revealed a reduction of neutral lipids in the wing disc after the depletion of Cyp311a1. In addition, we observed an enhanced sensitivity to Eosin Y penetration in the wings of Cyp311a1 knocked-down flies. Moreover, the reduction of CYP311A1 function in developing wings does not affect cell proliferation and apoptosis, but entails disordered Phalloidin or Cadherin distribution, suggesting an abnormal cell morphology and cell cortex structure in wing epithelial cells. Taken together, our results suggest that Cyp311a1 is needed for wing morphogenesis by participating in lipid assembly and cell homeostasis.

Domain 3a mutation of VPS33A suppresses larval arrest phenotype in the loss of VPS45 in Caenorhabditis elegans.

The Sec1/Munc18 (SM) protein VPS45 is a key regulator of SNARE-mediated membrane fusion in endosomal trafficking, but its precise role remains unknown. To understand the function of VPS45 in vivo , we performed a genetic suppressor screen in Caenorhabditis elegans . We found that the temperature-sensitive lethality caused by the loss of VPS-45 can be suppressed by a mutation in another SM protein, VPS33A. The VPS33A M376I mutation is located in domain 3a, which is predicted to be essential for SNARE complex assembly. These results highlight the functional importance of domain 3a in endosomal SM proteins and its role in specific membrane fusion.

Exploring the Impact of Onobrychis cornuta and Veratrum lobelianum Extracts on C. elegans: Implications for MAPK Modulation, Germline Development, and Antitumor Properties.

In an era of increasing interest in the potential health benefits of medicinal foods, the need to assess their safety and potential toxicity remains a critical concern. While these natural remedies have garnered substantial attention for their therapeutic potential, a comprehensive understanding of their effects on living organisms is essential. We examined 316 herbal extracts to determine their potential nematocidal attributes in Caenorhabditis elegans. Approximately 16% of these extracts exhibited the capacity to induce diminished survival rates and larval arrest, establishing a correlation between larval arrest and overall worm viability. Certain extracts led to an unexpected increase in male nematodes, accompanied by a discernible reduction in DAPI-stained bivalent structures and perturbed meiotic advancement, thereby disrupting the conventional developmental processes. Notably, Onobrychis cornuta and Veratrum lobelianum extracts activated a DNA damage checkpoint response via the ATM/ATR and CHK-1 pathways, thus hindering germline development. Our LC-MS analysis revealed jervine in V. lobelianum and nine antitumor compounds in O. cornuta. Interestingly, linoleic acid replicated phenotypes induced by O. cornuta exposure, including an increased level of pCHK-1 foci, apoptosis, and the MAPK pathway. Mutants in the MAPK pathway mitigated the decline in worm survival, underscoring its importance in promoting worm viability. This study reveals complex interactions between herbal extracts and C. elegans processes, shedding light on potential antitumor effects and mechanisms. The findings provide insights into the complex landscape of herbal medicine's impact on a model organism, offering implications for broader applications.

A spontaneous TIR1 loss-of-function allele in C. elegans.

The auxin-inducible degron (AID) system is a widely-used system for conditional protein depletion. During the course of an experiment, we depleted the nuclear hormone receptor transcription factor NHR-23 to study molting, and we recovered a spontaneous suppressor allele that bypassed the L1 larval arrest caused by NHR-23 depletion. These mutants also failed to deplete a BFP::AID reporter in the strain background, suggesting a broader defect in the AID system. These animals carried an in-frame 18 base pair insertion that produced a 6 amino acid repeat in TIR1. The larval arrest in these animals could be restored by expressing a wild-type TIR1 transgene from an extrachromosomal array. Sister siblings that lost this array developed normally on auxin. Together, these experiments indicate that the TIR1 mutation was causing the loss of developmental arrest in the nhr-23::AID strain. This result highlights the importance of setting up a robust secondary screen to detect such mutants if performing forward genetic screens in conjunction with the AID system.

Cannabinoids activate the insulin pathway to modulate mobilization of cholesterol in C. elegans.

The nematode Caenorhabditis elegans requires exogenous cholesterol to survive and its depletion leads to early developmental arrest. Thus, tight regulation of cholesterol storage and distribution within the organism is critical. Previously, we demonstrated that the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) plays a key role in C. elegans since it modulates sterol mobilization. However, the mechanism remains unknown. Here we show that mutations in the ocr-2 and osm-9 genes, coding for transient receptors potential V (TRPV) ion channels, dramatically reduce the effect of 2-AG in cholesterol mobilization. Through genetic analysis in combination with the rescue of larval arrest induced by sterol starvation, we found that the insulin/IGF-1signaling (IIS) pathway and UNC-31/CAPS, a calcium-activated regulator of neural dense-core vesicles release, are essential for 2-AG-mediated stimulation of cholesterol mobilization. These findings indicate that 2-AG-dependent cholesterol trafficking requires the release of insulin peptides and signaling through the DAF-2 insulin receptor. These results suggest that 2-AG acts as an endogenous modulator of TRPV signal transduction to control intracellular sterol trafficking through modulation of the IGF-1 signaling pathway.

Histone deacetylase HDA-4-mediated epigenetic regulation in space-flown C. elegans

Epigenetic changes during long-term spaceflight are beginning to be studied by NASA’s twin astronauts and other model organisms. Here, we evaluate the epigenetic regulation of gene expression in space-flown C. elegans by comparing wild type and histone deacetylase (hda)-4 mutants. Expression levels of 39 genes were consistently upregulated in all four generations of adult hda-4 mutants grown under microgravity compared with artificial Earth-like gravity (1G). In contrast, in the wild type, microgravity-induced upregulation of these genes occurred a little. Among these genes, 11 contain the domain of unknown function 19 (DUF-19) and are located in a cluster on chromosome V. When compared with the 1G condition, histone H3 trimethylation at lysine 27 (H3K27me3) increased under microgravity in the DUF-19 containing genes T20D4.12 to 4.10 locus in wild-type adults. On the other hand, this increase was also observed in the hda-4 mutant, but the level was significantly reduced. The body length of wild-type adults decreased slightly but significantly when grown under microgravity. This decrease was even more pronounced with the hda-4 mutant. In ground-based experiments, one of the T20D4.11 overexpressing strains significantly reduced body length and also caused larval growth retardation and arrest. These results indicate that under microgravity, C. elegans activates histone deacetylase HDA-4 to suppress overregulation of several genes, including the DUF-19 family. In other words, the expression of certain genes, including negative regulators of growth and development, is epigenetically fine-tuned to adapt to the space microgravity.

The role of Desmodium intortum, Brachiaria sp. and Phaseolus vulgaris in the management of fall armyworm Spodoptera frugiperda (J. E. Smith) in maize cropping systems in Africa

BACKGROUNDThe fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) is a serious pest of maize. Farming systems such as push‐pull or maize‐legume intercropping have been reported to reduce FAW infestations significantly. However, the exact mechanisms involved in FAW management have not been practically elucidated. We therefore assessed larval host preference, feeding and survival rate when exposed to four host plants commonly used in push‐pull and legume intercropping. We also compared adult moths' oviposition preference between maize and other grasses used as trap crops in push‐pull.RESULTSThe larval orientation and settlement study showed that maize was the most preferred host plant followed by bean, desmodium and Brachiaria brizantha cv Mulato II. The larval arrest and dispersal experiment showed that mean number of larvae was significantly higher on maize than on Desmodium or B. brizantha cv Mulato II. However, no significant differences were found between maize and bean after 24 h. Maize was the most consumed plant, followed by bean, desmodium and finally brachiaria. The mean percentage of survival to the pupation stage was significantly higher on maize. The study on FAW oviposition preference showed no significant differences in egg deposited between maize and other grasses. However, B. brizantha cv Xaraes, which received more eggs than maize, could be a promising alternative to B. brizantha cv Mulato II for the control of FAW.CONCLUSIONThe study provides a better understanding of the mechanisms involved in the control of fall armyworm under the push‐pull and maize legume intercropping. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis

An insect’s capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period – results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

Effect of Larval Food Availability on Adult Aedes Aegypti (Diptera: Culicidae) Fitness and Susceptibility to Zika Infection.

Aedes (Stegomyia) aegypti (Linnaeus, 1762) is a mosquito species of significant medical importance. The use of this vector in research studies usually requires a large number of mosquitoes as well as rearing and maintenance in a laboratory-controlled environment. However, laboratory conditions may be different from field environments, presenting stressful challenges such as low food concentration, especially during larval stages, which may, in turn, impair vector biology. Therefore, we tested herein if larval food availability (0.004, 0.009, 0.020, and 0.070% diets) would affect overall adult insect fitness. We observed slower development in mosquitoes fed a 0.004% diet 15 d post-eclosion (DPE) and shorter mean time in mosquitoes fed a 0.020% diet (7 DPE). Larval diet and adult mosquito weight were positively correlated, and heavier females fed higher larval diets exhibited greater blood feeding capacity and oviposition. In addition, larval diet concentrations led to median adult lifespan variations (male/female in days-0.004%: 30 ± 1.41, 45 ± 1.3; 0.009%: 31.5 ± 1.33, 41 ± 1.43; 0.020%: 26 ± 1.18, 41 ± 1.45; 0.070%: 29 ± 1.07, 44 ± 1.34), reduced tolerance to deltamethrin (1 mg/m2) and changes in detoxification enzyme activities. Moreover, in the larval 0.070% diet, females presented higher Zika susceptibility (plaque-forming unit [PFU]: 1.218 × 106) compared with other diets (0.004%: 1.31 × 105; 0.009%: 2.0 × 105; 0.020%: 1.25 × 105 PFU). Altogether, our study demonstrates that larval diet restriction results not only in larval developmental arrest but also in adult fitness impairment, which must be considered in future assessments.

Defining Caenorhabditis elegans as a model system to investigate lipoic acid metabolism

Lipoic acid (LA) is a sulfur-containing cofactor that covalently binds to a variety of cognate enzymes that are essential for redox reactions in all three domains of life. Inherited mutations in the enzymes that make LA, namely lipoyl synthase, octanoyltransferase, and amidotransferase, result in devastating human metabolic disorders. Unfortunately, because many aspects of this essential pathway are still obscure, available treatments only serve to alleviate symptoms. We envisioned that the development of an organismal model system might provide new opportunities to interrogate LA biochemistry, biology, and physiology. Here we report our investigations on three Caenorhabditis elegans orthologous proteins involved in this post-translational modification. We established that M01F1.3 is a lipoyl synthase, ZC410.7 an octanoyltransferase, and C45G3.3 an amidotransferase. Worms subjected to RNAi against M01F1.3 and ZC410.7 manifest larval arrest in the second generation. The arrest was not rescued by LA supplementation, indicating that endogenous synthesis of LA is essential for C. elegans development. Expression of the enzymes M01F1.3, ZC410.7, and C45G3.3 completely rescue bacterial or yeast mutants affected in different steps of the lipoylation pathway, indicating functional overlap. Thus, we demonstrate that, similarly to humans, C. elegans is able to synthesize LA de novo via a lipoyl-relay pathway, and suggest that this nematode could be a valuable model to dissect the role of protein mislipoylation and to develop new therapies.

Inhibition of acetyl-CoA carboxylase by spirotetramat causes growth arrest and lipid depletion in nematodes

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-mobile systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. However, in nematodes the mode of action of SPT and its effect on their development have not been studied so far. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids and fatty acid composition. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by SPT, a well-known systemic insecticide for sucking pest control.

Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint.

The final body size of any given individual underlies both genetic and environmental constraints. Both mammals and insects use target of rapamycin (TOR) and insulin signaling pathways to coordinate growth with nutrition. In holometabolous insects, the growth period is terminated through a cascade of peptide and steroid hormones that end larval feeding behavior and trigger metamorphosis, a nonfeeding stage during which the larval body plan is remodeled to produce an adult. This irreversible decision, termed the critical weight (CW) checkpoint, ensures that larvae have acquired sufficient nutrients to complete and survive development to adulthood. How insects assess body size via the CW checkpoint is still poorly understood on the molecular level. We show here that the Drosophila transcription factor Snail plays a key role in this process. Before and during the CW checkpoint, snail is highly expressed in the larval prothoracic gland (PG), an endocrine tissue undergoing endoreplication and primarily dedicated to the production of the steroid hormone ecdysone. We observed two Snail peaks in the PG, one before and one after the molt from the second to the third instar. Remarkably, these Snail peaks coincide with two peaks of PG cells entering S phase and a slowing of DNA synthesis between the peaks. Interestingly, the second Snail peak occurs at the exit of the CW checkpoint. Snail levels then decline continuously, and endoreplication becomes nonsynchronized in the PG after the CW checkpoint. This suggests that the synchronization of PG cells into S phase via Snail represents the mechanistic link used to terminate the CW checkpoint. Indeed, PG-specific loss of snail function prior to the CW checkpoint causes larval arrest due to a cessation of endoreplication in PG cells, whereas impairing snail after the CW checkpoint no longer affected endoreplication and further development. During the CW window, starvation or loss of TOR signaling disrupted the formation of Snail peaks and endocycle synchronization, whereas later starvation had no effect on snail expression. Taken together, our data demonstrate that insects use the TOR pathway to assess nutrient status during larval development to regulate Snail in ecdysone-producing cells as an effector protein to coordinate endoreplication and CW attainment.

Nutrient Sensing and Response Drive Developmental Progression in Caenorhabditis elegans.

In response to nutrient limitation, many animals, including Caenorhabditis elegans, slow or arrest their development. This process requires mechanisms that sense essential nutrients and induce appropriate responses. When faced with nutrient limitation, C. elegans can induce both short and long-term survival strategies, including larval arrest, decreased developmental rate, and dauer formation. To select the most advantageous strategy, information from many different sensors must be integrated into signaling pathways, including target of rapamycin (TOR) and insulin, that regulate developmental progression. Here, how nutrient information is sensed and integrated into developmental decisions that determine developmental rate and progression in C. elegans is reviewed.

The Enigmatic Canal-Associated Neurons Regulate Caenorhabditis elegans Larval Development Through a cAMP Signaling Pathway.

Caenorhabditis elegans larval development requires the function of the two Canal-Associated Neurons (CANs): killing the CANs by laser microsurgery or disrupting their development by mutating the gene ceh-10 results in early larval arrest. How these cells promote larval development, however, remains a mystery. In screens for mutations that bypass CAN function, we identified the gene kin-29, which encodes a member of the Salt-Inducible Kinase (SIK) family and a component of a conserved pathway that regulates various C. elegans phenotypes. Like kin-29 loss, gain-of-function mutations in genes that may act upstream of kin-29 or growth in cyclic-AMP analogs bypassed ceh-10 larval arrest, suggesting that a conserved adenylyl cyclase/PKA pathway inhibits KIN-29 to promote larval development, and that loss of CAN function results in dysregulation of KIN-29 and larval arrest. The adenylyl cyclase ACY-2 mediates CAN-dependent larval development: acy-2 mutant larvae arrested development with a similar phenotype to ceh-10 mutants, and the arrest phenotype was suppressed by mutations in kin-29. ACY-2 is expressed predominantly in the CANs, and we provide evidence that the acy-2 functions in the CANs to promote larval development. By contrast, cell-specific expression experiments suggest that kin-29 acts in both the hypodermis and neurons, but not in the CANs. Based on our findings, we propose two models for how ACY-2 activity in the CANs regulates KIN-29 in target cells.

MANF deletion abrogates early larval Caenorhabditis elegans stress response to tunicamycin and Pseudomonas aeruginosa

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is the only human neurotrophic factor with an evolutionarily-conserved C. elegans homolog, Y54G2A.23 or manf-1. MANF is a small, soluble, endoplasmic-reticulum (ER)-resident protein that is secreted upon ER stress and promotes survival of target cells such as neurons. However, the role of MANF in ER stress and its mechanism of cellular protection are not clear and the function of MANF in C. elegans is only beginning to emerge. In this study, we show that depletion of C. elegans manf-1 causes a slight decrease in lifespan and brood size; furthermore, combined depletion of manf-1 and the IRE-1/XBP-1 ER stress/UPR pathway resulted in sterile animals that did not produce viable progeny. We demonstrate upregulation of markers of ER stress in L1 larval nematodes, as measured by hsp-3 and hsp-4 transcription, upon depletion of manf-1 by RNAi or mutation; however, there was no difference in tunicamycin-induced expression of hsp-3 and hsp-4 between wild-type and MANF-deficient worms. Surprisingly, larval growth arrest observed in wild-type nematodes reared on tunicamycin is completely prevented in the manf-1 (tm3603) mutant. Transcriptional microarray analysis revealed that manf-1 mutant L1 larvae exhibit a novel modulation of innate immunity genes in response to tunicamycin. The hypothesis that manf-1 negatively regulates the innate immunity pathway is supported by our finding that the development of manf-1 mutant larvae compared to wild-type larvae is not inhibited by growth on P. aeruginosa. Together, our data represent the first characterization of C. elegans MANF as a key modulator of organismal ER stress and immunity.

Repression of Germline Genes in Caenorhabditis elegans Somatic Tissues by H3K9 Dimethylation of Their Promoters.

Repression of germline-promoting genes in somatic cells is critical for somatic development and function. To study how germline genes are repressed in somatic tissues, we analyzed key histone modifications in three Caenorhabditis elegans synMuv B mutants, lin-15B, lin-35, and lin-37—all of which display ectopic expression of germline genes in the soma. LIN-35 and LIN-37 are members of the conserved DREAM complex. LIN-15B has been proposed to work with the DREAM complex but has not been shown biochemically to be a member of the complex. We found that, in wild-type worms, synMuv B target genes and germline genes are enriched for the repressive histone modification dimethylation of histone H3 on lysine 9 (H3K9me2) at their promoters. Genes with H3K9me2 promoter localization are evenly distributed across the autosomes, not biased toward autosomal arms, as are the broad H3K9me2 domains. Both synMuv B targets and germline genes display a dramatic reduction of H3K9me2 promoter localization in lin-15B mutants, but much weaker reduction in lin-35 and lin-37 mutants. This difference between lin-15B and DREAM complex mutants likely represents a difference in molecular function for these synMuv B proteins. In support of the pivotal role of H3K9me2 in regulation of germline genes by LIN-15B, global loss of H3K9me2 but not H3K9me3 results in phenotypes similar to synMuv B mutants, high-temperature larval arrest, and ectopic expression of germline genes in the soma. We propose that LIN-15B-driven enrichment of H3K9me2 at promoters of germline genes contributes to repression of those genes in somatic tissues.

The intestinal intermediate filament network responds to and protects against microbial insults and toxins.

The enrichment of intermediate filaments in the apical cytoplasm of intestinal cells is evolutionarily conserved, forming a sheath that is anchored to apical junctions and positioned below the microvillar brush border, which suggests a protective intracellular barrier function. To test this, we used Caenorhabditiselegans, the intestinal cells of which are endowed with a particularly dense intermediate filament-rich layer that is referred to as the endotube. We found alterations in endotube structure and intermediate filament expression upon infection with nematicidal B.thuringiensis or treatment with its major pore-forming toxin crystal protein Cry5B. Endotube impairment due to defined genetic mutations of intermediate filaments and their regulators results in increased Cry5B sensitivity as evidenced by elevated larval arrest, prolonged time of larval development and reduced survival. Phenotype severity reflects the extent of endotube alterations and correlates with reduced rescue upon toxin removal. The results provide in vivo evidence for a major protective role of a properly configured intermediate filament network as an intracellular barrier in intestinal cells. This notion is further supported by increased sensitivity of endotube mutants to oxidative and osmotic stress.