Seam Cells Research Articles

Clock gene homologs lin-42 and kin-20 regulate circadian rhythms in C. elegans

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

Neuroprotective effect of erythropoietin on anesthesia-induced neurotoxicity through the modulation of autophagy in Caenorhabditis elegans.

The anti-oxidative, anti-inflammatory, and anti-apoptotic effects of erythropoietin may provide neuroprotective effects. Erythropoietin also modulates autophagy signaling that may play a role in anesthesia-induced neurotoxicity (AIN). Herein, we investigated whether AIN can be attenuated by the neuroprotective effect of erythropoietin in the Caenorhabditis elegans (C. elegans). Synchronized worms were divided into the control, Iso, EPO, and EPO-Iso groups. The chemotaxis index (CI) was evaluated when they reached the young adult stage. The lgg-1::GFP-positive puncta per seam cell were used to determine the autophagic events. The erythropoietin-mediated pathway of autophagy was determined by measuring the genetic expression level of let-363, bec-1, atg-7, atg-5, and lgg-3. Increased lgg-1::GFP puncta were observed in the Iso, EPO, and EPO-Iso groups. In the Iso group, only the let-363 level decreased significantly as compared to that in the control group (P = 0.009). bec-1 (P < 0.001), atg-5 (P = 0.012), and lgg-3 (P < 0.001) were expressed significantly more in the EPO-Iso group than in the Iso groups. Repeated isoflurane exposure during development decreased the CI. Erythropoietin could restore the decreased CI by isoflurane significantly in the EPO-Iso group. Erythropoietin showed neuroprotective effects against AIN and modulated the autophagic pathway in C. elegans. This experimental evidence of erythropoietin-related neuroprotection against AIN may be correlated with the induced autophagic degradation process that was sufficient for handling enhanced autophagy induction in erythropoietin-treated worms.

Analysis of Haemonchus embryos at single cell resolution identifies two eukaryotic elongation factors as intervention target candidates

Advances in single cell technologies are allowing investigations of a wide range of biological processes and pathways in animals, such as the multicellular model organism Caenorhabditis elegans – a free-living nematode. However, there has been limited application of such technology to related parasitic nematodes which cause major diseases of humans and animals worldwide. With no vaccines against the vast majority of parasitic nematodes and treatment failures due to drug resistance or inefficacy, new intervention targets are urgently needed, preferably informed by a deep understanding of these nematodes’ cellular and molecular biology – which is presently lacking for most worms. Here, we created the first single cell atlas for an early developmental stage of Haemonchus contortus – a highly pathogenic, C. elegans-related parasitic nematode. We obtained and curated RNA sequence (snRNA-seq) data from single nuclei from embryonating eggs of H. contortus (150,000 droplets), and selected high-quality transcriptomic data for > 14,000 single nuclei for analysis, and identified 19 distinct clusters of cells. Guided by comparative analyses with C. elegans, we were able to reproducibly assign seven cell clusters to body wall muscle, hypodermis, neuronal, intestinal or seam cells, and identified eight genes that were transcribed in all cell clusters/types, three of which were inferred to be essential in H. contortus. Two of these genes (i.e. Hc-eef-1A and Hc-eef1G), coding for eukaryotic elongation factors (called Hc-eEF1A and Hc-eEF1G), were also demonstrated to be transcribed and expressed in all key developmental stages of H. contortus. Together with these findings, sequence- and structure-based comparative analyses indicated the potential of Hc-eEF1A and/or Hc-eEF1G as intervention targets within the protein biosynthesis machinery of H. contortus. Future work will focus on single cell studies of all key developmental stages and tissues of H. contortus, and on evaluating the suitability of the two elongation factor proteins as drug targets in H. contortus and related nematodes, with a view to finding new nematocidal drug candidates.

Dynein directs prophase centrosome migration to control the stem cell division axis in the developing Caenorhabditis elegans epidermis.

The microtubule motor dynein is critical for the assembly and positioning of mitotic spindles. In Caenorhabditis elegans, these dynein functions have been extensively studied in the early embryo but remain poorly explored in other developmental contexts. Here, we use a hypomorphic dynein mutant to investigate the motor's contribution to asymmetric stem cell-like divisions in the larval epidermis. Live imaging of seam cell divisions that precede formation of the seam syncytium shows that mutant cells properly assemble but frequently misorient their spindle. Misoriented divisions misplace daughter cells from the seam cell row, generate anucleate compartments due to aberrant cytokinesis, and disrupt asymmetric cell fate inheritance. Consequently, the seam becomes disorganized and populated with extra cells that have lost seam identity, leading to fatal epidermal rupture. We show that dynein orients the spindle through the cortical GOA-1Gα-LIN-5NuMA pathway by directing the migration of prophase centrosomes along the anterior-posterior axis. Spindle misorientation in the dynein mutant can be partially rescued by elongating cells, implying that dynein-dependent force generation and cell shape jointly promote correct asymmetric division of epithelial stem cells.

Exploring the mechanisms of host-specificity of a hyperparasitic bacterium (Pasteuria spp.) with potential to control tropical root-knot nematodes (Meloidogyne spp.): insights from Caenorhabditis elegans.

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950's the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells.

Acquired stress resilience through bacteria-to-nematode interdomain horizontal gene transfer.

Natural selection drives the acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisitions in immunity, metabolic, and reproduction function via interdomain HGT (iHGT) from bacteria. Here, we report that the nematode gene rml-3 has been acquired by iHGT from bacteria and that it enables exoskeleton resilience and protection against environmental toxins in Caenorhabditis elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most similar to bacterial enzymes that biosynthesize L-rhamnose, a cell-wall polysaccharide component. C. elegans rml-3 is highly expressed during larval development and upregulated in developing seam cells upon heat stress and during the stress-resistant dauer stage. rml-3 deficiency impairs cuticle integrity, barrier functions, and nematode stress resilience, phenotypes that can be rescued by exogenous L-rhamnose. We propose that interdomain HGT of an ancient bacterial rml-3 homolog has enabled L-rhamnose biosynthesis in nematodes, facilitating cuticle integrity and organismal resilience to environmental stressors during evolution. These findings highlight a remarkable contribution of iHGT on metazoan evolution conferred by the domestication of a bacterial gene.

Lipid kinase PIP5K1A regulates let-7 microRNA biogenesis through interacting with nuclear export protein XPO5.

MicroRNAs (miRNAs) are small non-coding RNAs first discovered in Caenorhabditis elegans. The let-7 miRNA is highly conserved in sequence, biogenesis and function from C. elegans to humans. During miRNA biogenesis, XPO5-mediated nuclear export of pre-miRNAs is a rate-limiting step and, therefore, might be critical for the quantitative control of miRNA levels, yet little is known about how this is regulated. Here we show a novel role for lipid kinase PPK-1/PIP5K1A (phosphatidylinositol-4-phosphate 5-kinase) in regulating miRNA levels. We found that C. elegans PPK-1 functions inthe lin-28/let-7 heterochronic pathway, which regulates the strict developmental timing of seam cells. In C. elegans and human cells, PPK-1/PIP5K1A regulates let-7 miRNA levels. We investigated the mechanism further in human cells and show that PIP5K1A interacts with nuclear export protein XPO5 in the nucleus to regulate mature miRNA levels by blocking the binding of XPO5 to pre-let-7 miRNA. Furthermore, we demonstrate that this role for PIP5K1A is kinase-independent. Our study uncovers the novel finding of a direct connection between PIP5K1A and miRNA biogenesis. Given that miRNAs are implicated in multiple diseases, including cancer, this new finding might lead to a novel therapeutic opportunity.

The C. elegans gene gvd-1 promotes late larval development and germ cell proliferation.

Limiting maternal resources necessitate deferring the development of adult-specific structures, notably the reproductive structures, to the postembryonic phase. These structures form postembryonically from blast cells generated during embryogenesis. A close coordination of developmental timing and pattern among the various postembryonic cell lineages is essential to form a functional adult. Here, we show that the C. elegans gene gvd-1 is essential for the development of several structures that form during the late larval stages. In gvd-1 mutant animals, blast cells that normally divide during the late larval stages (L3 and L4) fail to divide. In addition, germ cell proliferation is also severely reduced in these animals. Expression patterns of relevant reporter transgenes revealed a delay in G1/S transition in the vulval precursor cell P6.p and cytokinesis failure in seam cells in gvd-1 larvae. Our analyses of GVD-1::GFP transgenes indicate that GVD-1 is expressed in both soma and germ line, and functions in both. Sequence comparisons revealed that the sequence of gvd-1 is conserved only among nematodes, which does not support a broadly conserved housekeeping function for gvd-1. Instead, our results indicate a crucial role for gvd-1 that is specific to the larval development of nematodes.

Physical exercise attenuates age-related muscle atrophy and exhibits anti-ageing effects via the adiponectin receptor 1 signalling.

Although the adiponectin signalling exerts exercise-mimicking effects, whether this pathway contributes to the anti-ageing benefits of physical exercise has not been established yet. Swim exercise training and wheel running were used to measure lifespan in the nematode Caenorhabditis elegans and skeletal muscle quality in mice, respectively. Muscle weight, muscle fibre cross-sectional area (CSA) and myonuclei number were used to evaluate muscle mass. RNA sequencing (RNA-Seq) analysis of skeletal muscle in exercised mice was used to study the underlying mechanisms. Western blot and immunofluorescence were performed to explore autophagy- and senescence-related markers. The C.elegans adiponectin receptor PAQR-1/AdipoR1, but not PAQR-2/AdipoR2, was activated (3.55-fold and 3.48-fold increases in p-AMPK on Days 1 and 6, respectively, P<0.001), which was involved in lifespan extension in exercised worms. Exercise training increased skeletal muscle mass index (1.29-fold, P<0.01), muscle weight (1.75-fold, P<0.001), myonuclei number (1.33-fold, P<0.05), muscle fibre CSA (1.39-fold, P<0.05) and capillary abundance (2.19-fold, P<0.001 for capillary density; 1.58-fold, P<0.01 for capillary number) in aged mice. Physical exercise reduced protein (2.94-fold, P<0.001) and mRNA levels (1.70-fold, P<0.001) of p16INK4a , a marker for cellular senescence, in skeletal muscle of aged mice. These beneficial effects of exercise on skeletal muscle of mice were dependent on AdipoR1. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differentially expressed genes in skeletal muscle between exercised mice with and without AdipoR1 knockdown by RNA-Seq analysis revealed that several KEGG pathways, such as 'AMPK signalling pathway' (P<0.001), 'FOXO signalling pathway' (P<0.001) and 'autophagy' (P<0.001) were overrepresented. Knockdown of FoxO3a inhibited exercise-mediated beneficial effects on skeletal muscle quality of mice by inhibiting autophagy/mitophagy (3.81-fold reduction in LC3-II protein, P<0.001; 1.53-fold reduction in BNIP3 protein, P<0.05). Knockdown of daf-16, the FoxO homologue in C.elegans, reduced autophagy (2.77-fold and 2.06-fold reduction in GFP::LGG-1 puncta in seam cells and the intestine, respectively, P<0.05) and blocked lifespan extension by exercise in worms. Our findings provide insights into how the AdipoR1 pathway has an impact on the anti-ageing benefits of exercise and implicate that activation of the AdipoR1 signalling may represent a potential therapeutic strategy for reducing age-related loss of skeletal muscle.

Daf-42 is an evolutionarily young gene essential for dauer development in Caenorhabditis elegans.

Under adverse environmental conditions, nematodes arrest into dauer, an alternative developmental stage for diapause. Dauer endures unfavorable environments and interacts with host animals to access favorable environments, thus playing a critical role in survival. Here, we report that in Caenorhabditis elegans, daf-42 is essential for development into the dauer stage, as the null mutant of daf-42 exhibited a "no viable dauer" phenotype in which no viable dauers were obtained in any dauer-inducing conditions. Long-term time lapse microscopy of synchronized larvae revealed that daf-42 is involved in developmental changes from the pre-dauer L2d stage to the dauer stage. daf-42 encodes large, disordered proteins of various sizes that are expressed in and secreted from the seam cells within a narrow time window shortly before the molt into dauer stage. Transcriptome analysis showed that the transcription of genes involved in larval physiology and dauer metabolism is highly affected by the daf-42 mutation. Contrary to the notion that essential genes that control the life and death of an organism may be well conserved across diverse species, daf-42 is an evolutionarily young gene conserved only in the Caenorhabditis genus. Our study shows that dauer formation is a vital process that is controlled not only by conserved genes but also by newly emerged genes, providing important insights into evolutionary mechanisms.

Anthelmintic-Like Activity and Ultrastructure Changes Produced by Two Polyphenolic Combinations against Cooperia punctata Adult Worms and Infective Larvae.

Cooperia punctata is one of the most prevalent gastrointestinal nematodes affecting cattle under grazing conditions, and the increasing reports of anthelmintic resistance forces researchers to look for novel control measures. Previous reports have proposed the use of polyphenolic compound (PC) combinations (Coumarin:Quercetin (CuQ) and Caffeic-acid:Rutin (CaR)) against free-living stages (L3) of C. punctata. The objective of this study was to assess the in vitro motility inhibition of C. punctata adult worms and infective larvae using the Larval Motility Inhibition Assay (LMIA) and Adult Motility Inhibition Assay (AMIA), and to assess the structural and ultrastructural changes induced by both treatments using Scanning and Transmission Electron Microscopy. For the LMIA, infective larvae were incubated for 3 h in 0.8 mg mL-1 and 0.84 mg mL-1 of CuQ and CaR, respectively. For AMIA, six concentrations and five incubation periods (2, 4, 6, 12 and 24 h) were assessed using each PC combination. Cooperia punctata motility was calculated as a percentage and corrected using control motility percentages. A multiple comparisons Brown-Forsythe and Welch ANOVA test was used to compare larval motility; and to fit the dose-response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism® V.9.2.0. Although larval motility was barely affected by both treatments (p > 0.05), adult worm motility was inhibited 100% and 86.9% after 24 h incubation with CuQ and CaR, respectively (p < 0.05). The best fit EC50 for adult worm motility inhibition were 0.073 ± 0.071 mg mL-1 and 0.051 ± 0.164 mg mL-1 for CuQ and CaR, respectively. Main structural and ultrastructural lesions observed in both biological stages were: (i) L3 sheath-cuticle complex disruption, (ii) collagen fibers degradation; (iii) hypodermic detachment, (iv) seam cell apoptosis and (v) mitochondrial swelling. The alterations observed suggest that the PC combinations interfere with the anatomy and physiology of the locomotive apparatus of the nematodes.

NHR-23 activity is necessary for C. elegans developmental progression and apical extracellular matrix structure and function.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

Isolation and molecular identification of nematode surface mutants with resistance to bacterial pathogens.

Numerous mutants of the nematode Caenorhabditis elegans with surface abnormalities have been isolated by utilizing their resistance to a variety of bacterial pathogens (Microbacterium nematophilum, Yersinia pseudotuberculosis, and 2 Leucobacter strains), all of which are able to cause disease or death when worms are grown on bacterial lawns containing these pathogens. Previous work led to the identification of 9 srf or bus genes; here, we report molecular identification and characterization of a further 10 surface-affecting genes. Three of these were found to encode factors implicated in glycosylation (srf-2, bus-5, and bus-22), like several of those previously reported; srf-2 belongs to the GT92 family of putative galactosyltransferases, and bus-5 is homologous to human dTDP-D-glucose 4,6-dehydratase, which is implicated in Catel-Manzke syndrome. Other genes encoded proteins with sequence similarity to phosphatidylinositol phosphatases (bus-6), Patched-related receptors (ptr-15/bus-13), steroid dehydrogenases (dhs-5/bus-21), or glypiation factors (bus-24). Three genes appeared to be nematode-specific (srf-5, bus-10, and bus-28). Many mutants exhibited cuticle fragility as revealed by bleach and detergent sensitivity; this fragility was correlated with increased drug sensitivity, as well as with abnormal skiddy locomotion. Most of the genes examined were found to be expressed in epidermal seam cells, which appear to be important for synthesizing nematode surface coat. The results reveal the genetic and biochemical complexity of this critical surface layer, and provide new tools for its analysis.

An nhr-23::mScarlet::3xMyc knock-in allele for studying spermatogenesis and molting.

C. elegans NHR-23 is a nuclear hormone receptor transcription factor involved in molting, apical extracellular matrix structure, and spermatogenesis. To determine NHR-23 expression dynamics, we previously tagged the endogenous nhr-23 locus with a GFP::AID*::3xFLAG tag. To allow co-localization of NHR-23 with green fluorescent protein-tagged factors of interest, we generated an equivalent strain carrying an mScarlet::3xMyc tag to produce a C-terminal fusion. Similar to the GFP::AID*::3xFLAG knock-in, NHR-23 ::mScarlet::3xMyc was expressed in seam and hypodermal cells, vulval precursor cells, and the spermatogenic germline. We also observed a diffuse NHR-23::mScarlet expression pattern in spermatids and residual bodies after NHR-23 ceased to localize on chromatin. Further examination of this novel localization may provide insight into NHR-23 regulation of spermatogenesis.

An Exact Hypergraph Matching algorithm for posture identification in embryonic C. elegans.

The nematode Caenorhabditis elegans (C. elegans) is a model organism used frequently in developmental biology and neurobiology [White, (1986), Sulston, (1983), Chisholm, (2016) and Rapti, (2020)]. The C. elegans embryo can be used for cell tracking studies to understand how cell movement drives the development of specific embryonic tissues. Analyses in late-stage development are complicated by bouts of rapid twitching motions which invalidate traditional cell tracking approaches. However, the embryo possesses a small set of cells which may be identified, thereby defining the coiled embryo's posture [Christensen, 2015]. The posture serves as a frame of reference, facilitating cell tracking even in the presence of twitching. Posture identification is nevertheless challenging due to the complete repositioning of the embryo between sampled images. Current approaches to posture identification rely on time-consuming manual efforts by trained users which limits the efficiency of subsequent cell tracking. Here, we cast posture identification as a point-set matching task in which coordinates of seam cell nuclei are identified to jointly recover the posture. Most point-set matching methods comprise coherent point transformations that use low order objective functions [Zhou, (2016) and Zhang, (2019)]. Hypergraphs, an extension of traditional graphs, allow more intricate modeling of relationships between objects, yet existing hypergraphical point-set matching methods are limited to heuristic algorithms which do not easily scale to handle higher degree hypergraphs [Duchenne, (2010), Chertok, (2010) and Lee, (2011)]. Our algorithm, Exact Hypergraph Matching (EHGM), adapts the classical branch-and-bound paradigm to dynamically identify a globally optimal correspondence between point-sets under an arbitrarily intricate hypergraphical model. EHGM with hypergraphical models inspired by C. elegans embryo shape identified posture more accurately (56%) than established point-set matching methods (27%), correctly identifying twice as many sampled postures as a leading graphical approach. Posterior region seeding empowered EHGM to correctly identify 78% of postures while reducing runtime, demonstrating the efficacy of the method on a cutting-edge problem in developmental biology.

Hemicentin-mediated type IV collagen assembly strengthens juxtaposed basement membrane linkage.

Basement membrane (BM) matrices surround and separate most tissues. However, through poorly understood mechanisms, BMs of adjacent tissue can also stably link to support organ structure and function. Using endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging, we identified extracellular matrix proteins mediating a BM linkage (B-LINK) between the uterine utse and epidermal seam cell BMs in Caenorhabditis elegans that supports the uterus during egg-laying. We found that hemicentin is secreted by the utse and promotes fibulin-1 assembly to jointly initiate the B-LINK. During egg-laying, however, both proteins' levels decline and are not required for B-LINK maintenance. Instead, we discovered that hemicentin recruits ADAMTS9/20, which facilitates the assembly of high levels of type IV collagen that sustains the B-LINK during the mechanically active egg-laying period. This work reveals mechanisms underlying BM-BM linkage maturation and identifies a crucial function for hemicentin and fibulin-1 in initiating attachment and type IV collagen in strengthening this specialized form of tissue linkage.

Cabin1 domain-containing gene picd-1 interacts with pry-1/Axin to regulate multiple processes in Caenorhabditis elegans

The Axin family of scaffolding proteins control diverse processes, such as facilitating the interactions between cellular components and providing specificity to signaling pathways. While several Axin family members have been discovered in metazoans and shown to play crucial roles, their mechanism of action are not well understood. The Caenorhabditis elegans Axin homolog, pry-1, is a powerful tool for identifying interacting genes and downstream effectors that function in a conserved manner to regulate Axin-mediated signaling. Our lab and others have established pry-1’s essential role in developmental processes that affect the reproductive system, seam cells, and a posterior P lineage cell, P11.p. Additionally, pry-1 is crucial for lipid metabolism, stress responses, and aging. In this study, we expanded on our previous work on pry-1 by reporting a novel interacting gene named picd-1 (pry-1-interacting and Cabin1 domain-containing). PICD-1 protein shares sequence conservation with CABIN1, a component of the HUCA complex. Our findings have revealed that PICD-1 is involved in several pry-1-mediated processes, including stress response and lifespan maintenance. picd-1’s expression overlapped with that of pry-1 in multiple tissues throughout the lifespan. Furthermore, PRY-1 and PICD-1 inhibited CREB-regulated transcriptional coactivator homolog CRTC-1, which promotes longevity in a calcineurin-dependent manner. Overall, our study has demonstrated that picd-1 is necessary for mediating pry-1 function and provides the basis to investigate whether Cabin-1 domain-containing protein plays a similar role in Axin signaling in other systems.

Daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71.

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells.

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.

A Maternal-Effect Toxin Affects Epithelial Differentiation and Tissue Mechanics in Caenorhabditis elegans.

Selfish genetic elements that act as post-segregation distorters cause lethality in non-carrier individuals after fertilization. Two post-segregation distorters have been previously identified in Caenorhabditis elegans, the peel-1/zeel-1 and the sup-35/pha-1 elements. These elements seem to act as modification-rescue systems, also called toxin/antidote pairs. Here we show that the maternal-effect toxin/zygotic antidote pair sup-35/pha-1 is required for proper expression of apical junction (AJ) components in epithelia and that sup-35 toxicity increases when pathways that establish and maintain basal epithelial characteristics, die-1, elt-1, lin-26, and vab-10, are compromised. We demonstrate that pha-1(e2123) embryos, which lack the antidote, are defective in epidermal morphogenesis and frequently fail to elongate. Moreover, seam cells are frequently misshaped and mispositioned and cell bond tension is reduced in pha-1(e2123) embryos, suggesting altered tissue material properties in the epidermis. Several aspects of this phenotype can also be induced in wild-type embryos by exerting mechanical stress through uniaxial loading. Seam cell shape, tissue mechanics, and elongation can be restored in pha-1(e2123) embryos if expression of the AJ molecule DLG-1/Discs large is reduced. Thus, our experiments suggest that maternal-effect toxicity disrupts proper development of the epidermis which involves distinct transcriptional regulators and AJ components.