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High-quality DNA extraction from organoids is an important step in molecular genetics research. Here, we show that a lysis buffer from the field of Caenorhabditis elegans research, called Single Worm Lysis Buffer (SWLB), is a low-cost, yet reliable method for DNA extraction from mammalian organoids. SWLB is superior in terms of price, storage, hands-on time and sustainability compared to current standardized DNA extraction protocols, while equally effective. This work indicates that it is useful to compare methods from different model systems, such as mammalian organoids and invertebrate nematodes, to find useful alternatives for research methodologies., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that controls neuroblast migration in Caenorhabditis elegans as a tractable system to study a robust, cell-intrinsic timing mechanism in vivo. Single-molecule mRNA quantification showed that the expression of the receptor increases non-linearly, a dynamic that is predicted to enhance timing precision over an unregulated, linear increase in timekeeper abundance. We show that this upregulation depends on transcriptional activation, providing in vivo evidence for a model in which the timing of receptor expression is regulated through an accumulating activator that triggers expression when a specific threshold is reached. This timing mechanism acts across a cell division that occurs in the neuroblast lineage and is influenced by the asymmetry of the division. Finally, we show that positive feedback of receptor expression through the canonical Wnt pathway enhances temporal precision. We conclude that robust cell-intrinsic timing can be achieved by combining regulation and feedback of the timekeeper gene., Competing Interests: ES, SG, CD, EF, MF, AM, HK No competing interests declared, (© 2023, Schild et al.)

Polyploid cells contain more than 2 copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either 1 nucleus (mononucleation) or 2 or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of polyploidy is largely unknown. Here, we assess the function of multinucleation in Caenorhabditis elegans intestinal cells through specific inhibition of binucleation without altering genome ploidy. Through single-worm RNA sequencing, we find that binucleation is important for tissue-specific gene expression, most prominently for genes that show a rapid up-regulation at the transition from larval development to adulthood. Regulated genes include vitellogenins, which encode yolk proteins that facilitate nutrient transport to the germline. We find that reduced expression of vitellogenins in mononucleated intestinal cells leads to progeny with developmental delays and reduced fitness. Together, our results show that binucleation facilitates rapid up-regulation of intestine-specific gene expression during development, independently of genome ploidy, underscoring the importance of spatial genome organization for polyploid cell function., Competing Interests: The authors have declared that no competing interests exist.

RNA tomography or tomo-seq combines mRNA sequencing and cryo-sectioning to spatially resolve gene expression. We have adapted this method for the nematode Caenorhabditis elegans to generate anteroposterior gene expression maps at near-cellular resolution. Here, we provide a detailed overview of the method and present two approaches: one that includes RNA isolation for maximum sensitivity and one that is suitable for partial automatization and is therefore less time-consuming. For complete details on the use and execution of this protocol, please refer to Ebbing et al. (2018)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Members of the Wnt family of secreted glycoproteins regulate cell migration through distinct canonical and noncanonical signaling pathways. Studies of vertebrate development and disease have shown that these pathways can have opposing effects on cell migration, but the mechanism of this functional interplay is not known. In the nematode Caenorhabditis elegans , a switch from noncanonical to canonical Wnt signaling terminates the long-range migration of the QR neuroblast descendants, providing a tractable system to study this mechanism in vivo. Here, we show that noncanonical Wnt signaling acts through PIX-1/RhoGEF, while canonical signaling directly activates the Slt-Robo pathway component EVA-1/EVA1C and the Rho GTPase-activating protein RGA-9b/ARHGAP, which are required for migration inhibition. Our results support a model in which cross-talk between noncanonical and canonical Wnt signaling occurs through antagonistic regulation of the Rho GTPases that drive cell migration., Competing Interests: The authors declare no competing interest.

Divergence of gene function and expression during development can give rise to phenotypic differences at the level of cells, tissues, organs, and ultimately whole organisms. To gain insights into the evolution of gene expression and novel genes at spatial resolution, we compared the spatially resolved transcriptomes of two distantly related nematodes, Caenorhabditis elegans and Pristionchus pacificus, that diverged 60-90 Ma. The spatial transcriptomes of adult worms show little evidence for strong conservation at the level of single genes. Instead, regional expression is largely driven by recent duplication and emergence of novel genes. Estimation of gene ages across anatomical structures revealed an enrichment of novel genes in sperm-related regions. This provides first evidence in nematodes for the "out of testis" hypothesis that has been previously postulated based on studies in Drosophila and mammals. "Out of testis" genes represent a mix of products of pervasive transcription as well as fast evolving members of ancient gene families. Strikingly, numerous novel genes have known functions during meiosis in Caenorhabditis elegans indicating that even universal processes such as meiosis may be targets of rapid evolution. Our study highlights the importance of novel genes in generating phenotypic diversity and explicitly characterizes gene origination in sperm-related regions. Furthermore, it proposes new functions for previously uncharacterized genes and establishes the spatial transcriptome of Pristionchus pacificus as a catalog for future studies on the evolution of gene expression and function., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Cellular behaviors such as migration, division, and differentiation rely on precise timing, and yet the molecular events that govern these behaviors are highly stochastic. We investigate regulatory strategies that decrease the timing noise of molecular events. Autoregulatory feedback increases noise. Yet we find that in the presence of regulation by a second species, autoregulatory feedback decreases noise. To explain this finding, we develop a method to calculate the optimal regulation function that minimizes the timing noise. The method reveals that the combination of feedback and regulation minimizes noise by maximizing the number of molecular events that must happen in sequence before a threshold is crossed. We compute the optimal timing precision for all two-node networks with regulation and feedback, derive a generic lower bound on timing noise, and discuss our results in the context of neuroblast migration during Caenorhabditis elegans development.

Single-cell isolation and transcriptomic analysis of a specific cell type or tissue offers the possibility of studying cell function and heterogeneity in time-dependent processes with remarkable resolution. The reduced tissue complexity and highly stereotyped development of Caenorhabditis elegans , combined with an extensive genetic toolbox and the ease of growing large tightly synchronized populations makes it an exceptional model organism for the application of such approaches. However, the difficulty to dissociate and isolate single cells from larval stages has been a major constraint to this kind of studies. Here, we describe an improved protocol for dissociation and preparation of single cell suspensions from developmentally synchronized populations of C. elegans L1 larvae. Our protocol has been empirically optimized to allow efficient FACS-based purification of large number of single cells from rare cell types, for subsequent extraction and sequencing of their mRNA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Directional migration of neurons and neuronal precursor cells is a central process in nervous system development. In the nematode Caenorhabditis elegans , the two Q neuroblasts polarize and migrate in opposite directions along the anteroposterior body axis. Several key regulators of Q cell polarization have been identified, including MIG-21, DPY-19/DPY19L1, the netrin receptor UNC-40/DCC, the Fat-like cadherin CDH-4 and CDH-3/Fat, which we describe in this study. How these different transmembrane proteins act together to direct Q neuroblast polarization and migration is still largely unknown. Here, we demonstrate that MIG-21 and DPY-19, CDH-3 and CDH-4, and UNC-40 define three distinct pathways that have partially redundant roles in protrusion formation, but also separate functions in regulating protrusion direction. Moreover, we show that the MIG-21, DPY-19 and Fat-like cadherin pathways control the localization and clustering of UNC-40 at the leading edge of the polarizing Q neuroblast, and that this is independent of the UNC-40 ligands UNC-6/netrin and MADD-4. Our results provide insight into a novel mechanism for ligand-independent localization of UNC-40 that directs the activity of UNC-40 along the anteroposterior axis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Appropriate Wnt morphogen secretion is required to control animal development and homeostasis. Although correct Wnt globular structure is essential for secretion, proteins that directly mediate Wnt folding and maturation remain uncharacterized. Here, we report that protein disulfide isomerase-1 (PDI-1), a protein-folding catalyst and chaperone, controls secretion of the Caenorhabditis elegans Wnt ortholog EGL-20. We find that PDI-1 function is required to correctly form an anteroposterior EGL-20/Wnt gradient during embryonic development. Furthermore, PDI-1 performs this role in EGL-20/Wnt-producing epidermal cells to cell-non-autonomously control EGL-20/Wnt-dependent neuronal migration. Using pharmacological inhibition, we further show that PDI function is required in human cells for Wnt3a secretion, revealing a conserved role for disulfide isomerases. Together, these results demonstrate a critical role for PDIs within Wnt-producing cells to control long-range developmental events that are dependent on Wnt secretion., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

To advance our understanding of the genetic programs that drive cell and tissue specialization, it is necessary to obtain a comprehensive overview of gene expression patterns. Here, we have used spatial transcriptomics to generate high-resolution, anteroposterior gene expression maps of C. elegans males and hermaphrodites. To explore these maps, we have developed computational methods for discovering region- and tissue-specific genes. We have found extensive sex-specific gene expression differences in the germline and sperm and discovered genes that are specifically expressed in the male reproductive tract. These include a group of uncharacterized genes that encode small secreted proteins that are required for male fertility. We conclude that spatial gene expression maps provide a powerful resource for identifying tissue-specific gene functions in C. elegans. Importantly, we found that expression maps from different animals can be precisely aligned, enabling transcriptome-wide comparisons of gene expression patterns., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Van Gogh-like (Vangl) and Prickle (Pk) are core components of the non-canonical Wnt planar cell polarity pathway that controls epithelial polarity and cell migration. Studies in vertebrate model systems have suggested that Vangl and Pk may also inhibit signaling through the canonical Wnt/β-catenin pathway, but the functional significance of this potential cross-talk is unclear. In the nematode C. elegans, the Q neuroblasts and their descendants migrate in opposite directions along the anteroposterior body axis. The direction of these migrations is specified by Wnt signaling, with activation of canonical Wnt signaling driving posterior migration, and non-canonical Wnt signaling anterior migration. Here, we show that the Vangl ortholog VANG-1 influences the Wnt signaling response of the Q neuroblasts by negatively regulating canonical Wnt signaling. This inhibitory activity depends on a carboxy-terminal PDZ binding motif in VANG-1 and the Dishevelled ortholog MIG-5, but is independent of the Pk ortholog PRKL-1. Moreover, using Vangl1 and Vangl2 double mutant cells, we show that a similar mechanism acts in mammalian cells. We conclude that cross-talk between VANG-1/Vangl and the canonical Wnt pathway is an evolutionarily conserved mechanism that ensures robust specification of Wnt signaling responses., Competing Interests: The authors have declared that no competing interests exist.

Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.

Important cellular processes such as migration, differentiation, and development often rely on precise timing. Yet, the molecular machinery that regulates timing is inherently noisy. How do cells achieve precise timing with noisy components? We investigate this question using a first-passage-time approach, for an event triggered by a molecule that crosses an abundance threshold and that is regulated by either an accumulating activator or a diminishing repressor. We find that either activation or repression outperforms an unregulated strategy. The optimal regulation corresponds to a nonlinear increase in the amount of the target molecule over time, arises from a tradeoff between minimizing the timing noise of the regulator and that of the target molecule itself, and is robust to additional effects such as bursts and cell division. Our results are in quantitative agreement with the nonlinear increase and low noise of mig-1 gene expression in migrating neuroblast cells during Caenorhabditis elegans development. These findings suggest that dynamic regulation may be a simple and powerful strategy for precise cellular timing., Competing Interests: The authors have declared that no competing interests exist.

Invertebrate and vertebrate nervous systems generate different types of dopaminergic neurons in distinct parts of the brain. We have taken a genetic approach to understand how the four functionally related, but lineally unrelated, classes of dopaminergic neurons of the nematode Caenorhabditis elegans , located in distinct parts of its nervous system, are specified. We have identified several genes involved in the generation of a specific dopaminergic neuron type that is generated from the so-called postdeirid lineage, called PDE. Apart from classic proneural genes and components of the mediator complex, we identified a novel, previously uncharacterized zinc finger transcription factor, ztf-6 Loss of ztf-6 has distinct effects in different dopamine neuron-producing neuronal lineages. In the postdeirid lineage, ztf-6 is required for proper cell division patterns and the proper distribution of a critical cell fate determinant, the POP-1/TCF-like transcription factor., (Copyright © 2018 Doitsidou et al.)

Efficient sorting and transportation of integral membrane proteins, such as ion channels, nutrient transporters, signalling receptors, cell-cell and cell-matrix adhesion molecules is essential for the function of cellular organelles and hence organism development and physiology. Retromer is a master controller of integral membrane protein sorting and transport through one of the major sorting station within eukaryotic cells, the endosomal network. Subtle de-regulation of retromer is an emerging theme in the pathoetiology of Parkinson's disease. Here we summarise recent advances in defining the neuroprotective role of retromer and how its de-regulation may contribute to Parkinson's disease by interfering with: lysosomal health and protein degradation, association with accessory proteins including the WASH complex and mitochondrial health., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During development, cell migration plays a central role in the formation of tissues and organs. Understanding the molecular mechanisms that drive and control these migrations is a key challenge in developmental biology that will provide important insights into disease processes, including cancer cell metastasis. In this article, we discuss the Caenorhabditis elegans Q neuroblasts and their descendants as a tool to study cell migration at single-cell resolution in vivo. The highly stereotypical migration of these cells provides a powerful system to study the dynamic cytoskeletal processes that drive migration as well as the evolutionarily conserved signaling pathways (including different Wnt signaling cascades) that guide the cells along their specific trajectories. Here, we provide an overview of what is currently known about Q neuroblast migration and highlight the live-cell imaging, genome editing, and quantitative gene expression techniques that have been developed to study this process., (© 2016 Wiley Periodicals, Inc.)

The biogenesis of ribosomes and their coordination of protein translation consume an enormous amount of cellular energy. As such, it has been established that the inhibition of either process can extend eukaryotic lifespan. Here, we used next-generation sequencing to compare ribosome-associated RNAs from normal strains of Caenorhabditis elegans to those carrying the life-extending daf-2 mutation. We found a long noncoding RNA (lncRNA), transcribed telomeric sequence 1 (tts-1), on ribosomes of the daf-2 mutant. Depleting tts-1 in daf-2 mutants increases ribosome levels and significantly shortens their extended lifespan. We find tts-1 is also required for the longer lifespan of the mitochondrial clk-1 mutants but not the feeding-defective eat-2 mutants. In line with this, the clk-1 mutants express more tts-1 and fewer ribosomes than the eat-2 mutants. Our results suggest that the expression of tts-1 functions in different longevity pathways to reduce ribosome levels in a way that promotes life extension., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Members of the Wnt family of secreted signaling proteins are key regulators of cell migration and axon guidance. In the nematode C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism. Importantly, the transition from the first to the second phase, which is the main determinant of the final position of the QR descendants along the anteroposterior body axis, is mediated through a cell-autonomous process in which the time-dependent expression of a Wnt receptor turns on the canonical Wnt/β-catenin signaling response that is required to terminate long-range anterior migration. Our results show that, in addition to direct guidance of cell migration by Wnt morphogenic gradients, cell migration can also be controlled indirectly through cell-intrinsic modulation of Wnt signaling responses.

During the first stage of larval development, the Q neuroblasts and their descendants migrate to well-defined positions along the anteroposterior body axis, where they differentiate into sensory neurons and interneurons. The two Q neuroblasts are initially present at similar positions on the left and right lateral side, but this symmetry is broken when the Q neuroblast on the left side (QL) polarizes towards the posterior and the Q neuroblast on the right side (QR) towards the anterior. This left-right asymmetry is maintained when the descendants of the two Q neuroblasts migrate to their final positions in the posterior and anterior. The mechanisms that establish this asymmetry and control the migration of the Q descendants along the anteroposterior axis are surprisingly complex and include interplay between Wnt signaling pathways, homeotic genes, and the basic cell migration and polarity machinery. Here, we will give an overview of what is currently known about the mechanisms that mediate and control the development and migration of the Q neuroblasts and their descendants.

Wnt signaling plays a central role in development, adult tissue homeostasis, and cancer. Several steps in the canonical Wnt/β-catenin signaling cascade are regulated by ubiquitylation, a protein modification that influences the stability, subcellular localization, or interactions of target proteins. To identify regulators of the Wnt/β-catenin pathway, we performed an RNA interference screen in Caenorhabditis elegans and identified the HECT domain-containing ubiquitin ligase EEL-1 as an inhibitor of Wnt signaling. In human embryonic kidney 293T cells, knockdown of the EEL-1 homolog Huwe1 enhanced the activity of a Wnt reporter in cells stimulated with Wnt3a or in cells that overexpressed casein kinase 1 (CK1) or a constitutively active mutant of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6). However, knockdown of Huwe1 had no effect on reporter gene expression in cells expressing constitutively active β-catenin, suggesting that Huwe1 inhibited Wnt signaling upstream of β-catenin and downstream of CK1 and LRP6. Huwe1 bound to and ubiquitylated the cytoplasmic Wnt pathway component Dishevelled (Dvl) in a Wnt3a- and CK1ε-dependent manner. Mass spectrometric analysis showed that Huwe1 promoted K63-linked, but not K48-linked, polyubiquitination of Dvl. Instead of targeting Dvl for degradation, ubiquitylation of the DIX domain of Dvl by Huwe1 inhibited Dvl multimerization, which is necessary for its function. Our findings indicate that Huwe1 is part of an evolutionarily conserved negative feedback loop in the Wnt/β-catenin pathway.

Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF) and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG) domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. Here, we demonstrate that POP-1 (the C. elegans TCF) also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/ß-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation.

Secretion of Wnt proteins is mediated by the Wnt sorting receptor Wls, which transports Wnt from the Golgi to the cell surface for release. To maintain efficient Wnt secretion, Wls is recycled back to the trans-Golgi network (TGN) through a retromer dependent endosome to TGN retrieval pathway. It has recently been shown that this is mediated by an alternative retromer pathway in which the sorting nexin SNX3 interacts with the cargo-selective subcomplex of the retromer to sort Wls into a retrieval pathway that is morphologically distinct from the classical SNX-BAR dependent retromer pathway. Here, we investigated how sorting of Wls between the two different retromer pathways is specified. We found that when the function of the cargo-selective subcomplex of the retromer is partially disrupted, Wnt secretion can be restored by interfering with the maturation of late endosomes to lysosomes. This leads to an accumulation of Wls in late endosomes and facilitates the retrieval of Wls through a SNX-BAR dependent retromer pathway. Our results are consistent with a model in which spatial separation of the SNX3 and SNX-BAR retromer complexes along the endosomal maturation pathway as well as cargo-specific mechanisms contribute to the selective retrieval of Wls through the SNX3 retromer pathway., (© 2013.)

Wnt proteins are secreted lipid-modified glycoproteins that control many aspects of development in organisms ranging from sponges to vertebrates. Wnt proteins are also important regulators of C. elegans development, with functions in processes as diverse as cell fate specification, asymmetric cell division, cell migration and synapse formation. In this review, we will give an overview of what we currently know about the signaling mechanisms that mediate these different functions of Wnt.

Variability in gene expression contributes to phenotypic heterogeneity even in isogenic populations. Here, we used the stereotyped, Wnt signaling-dependent development of the Caenorhabditis elegans Q neuroblast to probe endogenous mechanisms that control gene expression variability. We found that the key Hox gene that orients Q neuroblast migration exhibits increased gene expression variability in mutants in which Wnt pathway activity has been perturbed. Distinct features of the gene expression distributions prompted us on a systematic search for regulatory interactions, revealing a network of interlocked positive and negative feedback loops. Interestingly, positive feedback appeared to cooperate with negative feedback to reduce variability while keeping the Hox gene expression at elevated levels. A minimal model correctly predicts the increased gene expression variability across mutants. Our results highlight the influence of gene network architecture on expression variability and implicate feedback regulation as an effective mechanism to ensure developmental robustness., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35 (fl) allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35 (∆/∆) organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35 (∆/∆) organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.

Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3-isoprostanes. F3-isoprostanes are prostaglandin-like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev-1 and clk-1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro-oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress-resistant and long-lived daf-2 insulin/IGF-1 receptor mutant involved distinct daf-16-dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS., (© 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-β-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt-β-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.

Forkhead box O (FOXO; DAF-16 in worms) transcription factors, which are of vital importance in cell-cycle control, stress resistance, tumor suppression, and organismal lifespan, are largely regulated through nucleo-cytoplasmic shuttling. Insulin signaling keeps FOXO/DAF-16 cytoplasmic, and hence transcriptionally inactive. Conversely, as in loss of insulin signaling, reactive oxygen species (ROS) can activate FOXO/DAF-16 through nuclear accumulation. How ROS regulate the nuclear translocation of FOXO/DAF-16 is largely unknown. Cysteine oxidation can stabilize protein-protein interactions through the formation of disulfide-bridges when cells encounter ROS. Using a proteome-wide screen that identifies ROS-induced mixed disulfide-dependent complexes, we discovered several interaction partners of FOXO4, one of which is the nuclear import receptor transportin-1. We show that disulfide formation with transportin-1 is required for nuclear localization and the activation of FOXO4/DAF-16 induced by ROS, but not by the loss of insulin signaling. This molecular mechanism for nuclear shuttling is conserved in C. elegans and directly connects redox signaling to the longevity protein FOXO/DAF-16., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Establishment of cell polarity is crucial for many biological processes including cell migration and asymmetric cell division. The establishment of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified and maintained by intracellular signaling networks usually using positive feedback regulation. Generally, these two processes are intertwined and it is challenging to determine which proteins contribute to the sensing or amplification process, particularly in multicellular organisms. Here, we integrated phenomenological modeling with quantitative single-cell measurements to separate the sensing and amplification components of Wnt ligands and receptors during establishment of polarity of the Caenorhabditis elegans P cells. By systematically exploring how P-cell polarity is altered in Wnt ligand and receptor mutants, we inferred that ligands predominantly affect the sensing process, whereas receptors are needed for both sensing and amplification. This integrated approach is generally applicable to other systems and will facilitate decoupling of the different layers of signal sensing and amplification.

Coordinated movement depends on the creation of synapses between specific neurons in the motor circuit. In C. elegans, this important decision is regulated by the UNC-4 homeodomain protein. unc-4 mutants are unable to execute backward locomotion because VA motor neurons are mis-wired with inputs normally reserved for their VB sisters. We have proposed that UNC-4 functions in VAs to block expression of VB genes. This model is substantiated by the finding that ectopic expression of the VB gene ceh-12 (encoding a homolog of the homeodomain protein HB9) in unc-4 mutants results in the mis-wiring of posterior VA motor neurons with VB-like connections. Here, we show that VA expression of CEH-12 depends on a nearby source of the Wnt protein EGL-20. Our results indicate that UNC-4 prevents VAs from responding to a local EGL-20 cue by disabling a canonical Wnt signaling cascade involving the Frizzled receptors MIG-1 and MOM-5. CEH-12 expression in VA motor neurons is also opposed by a separate pathway that includes the Wnt ligand LIN-44. This work has revealed a transcriptional mechanism for modulating the sensitivity of specific neurons to diffusible Wnt ligands and thereby defines distinct patterns of synaptic connectivity. The existence of comparable Wnt gradients in the vertebrate spinal cord could reflect similar roles for Wnt signaling in vertebrate motor circuit assembly.

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Although several Wnt signal transduction mechanisms have been described in detail, it is still largely unknown how cells are specified to adopt such different Wnt signaling responses. Here, we have used the stereotypic migration of the C. elegans Q neuroblasts as a model to study how two initially equivalent cells are instructed to activate either β-catenin dependent or independent Wnt signaling pathways to control the migration of their descendants along the anteroposterior axis. We find that the specification of this difference in Wnt signaling response is dependent on the thrombospondin repeat containing protein MIG-21, which acts together with the netrin receptor UNC-40/DCC to control an initial left-right asymmetric polarization of the Q neuroblasts. Furthermore, we show that the direction of this polarization determines the threshold for Wnt/β-catenin signaling, with posterior polarization sensitizing for activation of this pathway. We conclude that MIG-21 and UNC-40 control the asymmetry in Wnt signaling response by restricting posterior polarization to one of the two Q neuroblasts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The Wnt family of signalling proteins has essential functions in development and adult tissue homoeostasis throughout the animal kingdom. Although signalling cascades triggered by Wnt proteins have been extensively studied, much remains to be learned about how Wnts are produced and secreted. Over the past few years, it has become clear that the secretion of Wnt proteins requires a specialized trafficking pathway. As this pathway has been discussed in two recent reviews (Lorenowicz & Korswagen 2009, Port & Basler 2010), we will focus our discussion on the key questions that need to be addressed to gain a more complete understanding of the mechanism and regulation of this essential secretion pathway., (© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.)

Sorting nexins are a large family of evolutionarily conserved phosphoinositide-binding proteins that have fundamental roles in orchestrating cargo sorting through the membranous maze that is the endosomal network. One ancient group of complexes that contain sorting nexins is the retromer. Here we discuss how retromer complexes regulate endosomal sorting, and describe how this is generating exciting new insight into the central role played by endosomal sorting in development and homeostasis of normal tissues.

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.

The migration of neuroblasts along the anteroposterior body axis of C. elegans is controlled by multiple Wnts that act partially redundantly to guide cells to their precisely defined final destinations. How positional information is specified by this system is, however, still largely unknown. Here, we used a novel fluorescent in situ hybridization methods to generate a quantitative spatiotemporal expression map of the C. elegans Wnt genes. We found that the five Wnt genes are expressed in a series of partially overlapping domains along the anteroposterior axis, with a predominant expression in the posterior half of the body. Furthermore, we show that a secreted Frizzled-related protein is expressed at the anterior end of the body axis, where it inhibits Wnt signaling to control neuroblast migration. Our findings reveal that a system of regionalized Wnt gene expression and anterior Wnt inhibition guides the highly stereotypic migration of neuroblasts in C. elegans. Opposing expression of Wnts and Wnt inhibitors has been observed in basal metazoans and in the vertebrate neurectoderm. Our results in C. elegans support the notion that a system of posterior Wnt signaling and anterior Wnt inhibition is an evolutionarily conserved principle of primary body axis specification.

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1-MLL5. The activity of SET1 and MLL proteins relies on protein-protein interactions within large multisubunit complexes that include three core components: RbBP5, Ash2L, and WDR5. It remains unclear how the composition and specificity of these complexes varies between cell types and during development. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RbBP5, Ash2L, and WDR5. Here we show that SET-2 is responsible for the majority of bulk H3K4 methylation at all developmental stages. However, SET-2 and absent, small, or homeotic discs 2 (ASH-2) are differentially required for tri- and dimethylation of H3K4 (H3K4me3 and -me2) in embryos and adult germ cells. In embryos, whereas efficient H3K4me3 requires both SET-2 and ASH-2, H3K4me2 relies mostly on ASH-2. In adult germ cells by contrast, SET-2 serves a major role whereas ASH-2 is dispensable for H3K4me3 and most H3K4me2. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function in the maintenance of a functional germ line. This study demonstrates that individual subunits of SET1-related complexes can show tissue specificity and developmental regulation and establishes C. elegans as a model to study SET1-related complexes in a multicellular organism.

Studies using chemical inhibitors have suggested that p38 MAP kinase is a key regulator of Wnt/β-catenin signaling. In this issue, Verkaar et al. (2011) show that cross-reactivity of p38 inhibitors with casein kinase Iδ/ɛ is responsible for Wnt/β-catenin pathway inhibition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.

Background: MicroRNAs (miRNA) are an abundant and ubiquitous class of small RNAs that play prominent roles in gene regulation. A significant fraction of miRNA genes reside in the introns of the host genes in the same orientation and are thought to be co-processed from the host gene mRNAs and thus depend on the host gene promoter for their expression. However, several lines of evidence for independent expression of intronic miRNAs exist in the literature but the extent of this independence remains unclear., Results: We performed a systematic analysis of genomic regions surrounding intronic miRNAs in the nematode Caenorhabditis elegans and found that, in many cases, there are extended intronic sequences immediately upstream of the miRNAs that are well-conserved between the nematodes. We have generated transcriptional green fluorescent protein reporter fusions in transgenic C. elegans lines and demonstrated that, in all seven investigated cases, the conserved sequences show promoter properties and produce specific expression patterns that are different from the host gene expression patterns. The observed expression patterns are corroborated by the published small RNA sequencing data., Conclusions: Our analysis reveals that the number of intronic miRNAs that do not rely on their host genes for expression is substantially higher than previously appreciated. At least one-third of the same-strand intronic miRNAs in C. elegans posses their own promoters and, thus, could be transcribed independently from their host genes. These findings provide a new insight into the regulation of miRNA genes and will be useful for the analysis of interactions between miRNAs and their host genes.

Mutational activation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs in a wide variety of tumors, whereas activating Wnt pathway mutants are predominantly found in colon cancer. Because GSK3 is a key component of both pathways, it is widely assumed that active PI3K signaling feeds positively into the Wnt pathway by protein kinase B (PKB)-mediatefd inhibition of GSK3. In addition, PKB has been proposed to modulate the canonical Wnt signaling through direct stabilization and nuclear localization of beta-catenin. Here, we show that compartmentalization by Axin of GSK3 prohibits cross-talk between the PI3K and Wnt pathways and that Wnt-mediated transcriptional activity is not modulated by activation of the PI3K/PKB pathway.

Wnt proteins are members of a highly conserved family of signalling molecules that play a central role in development and disease. During the past years, the different signalling pathways that are triggered by Wnt proteins have been studied in detail, but it is still largely unknown how a functional Wnt protein is produced and secreted. The recent finding that Wnt proteins are post-translationally modified and the discovery of the Wnt binding protein Wntless and its trafficking by the retromer complex show that Wnt secretion is a complex and highly regulated process. In this review, we will give an overview of the Wnt maturation and secretion pathway and discuss how this process may influence the spreading and signalling activity of Wnt.

Early endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150(glued) component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila. To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.

Disabled proteins are a conserved family of monomeric adaptor proteins that in mammals are implicated in the endocytosis of lipoprotein receptors. Previous studies have shown that the sole Caenorhabditis elegans Disabled homologue, DAB-1, is involved in the lipoprotein receptor-mediated secretion of a fibroblast growth factor. We show here that DAB-1 is essential for the uptake of yolk protein by developing oocytes, and for the localisation of the yolk receptor RME-2. The localisation of DAB-1 in oocytes is itself dependent upon clathrin and AP2, consistent with DAB-1 acting as a clathrin-associated sorting protein during yolk protein endocytosis. DAB-1 is also required for the endocytosis of molecules from the pseudocoelomic fluid by the macrophage-like coelomocytes, and is broadly expressed in epithelial tissues, consistent with a general role in receptor-mediated endocytosis. We also show that dab-1 mutations are synthetic lethal in combination with loss-of-function mutations affecting the AP-1 and AP-3 complexes, suggesting that the reduced fluid and membrane uptake exhibited by dab-1 mutants sensitises them to defects in other trafficking pathways.

Migrating neuronal cells are directed to their final positions by an array of guidance cues. It has been shown that guidance molecules such as UNC-6/Netrin and SLT-1/Slit play a major role in controlling cell and axon migrations along the dorsal-ventral body axis. Much less is known, however, about the mechanisms that mediate migration along the anterior-posterior (AP) body axis. Recent research in Caenorhabditis elegans has uncovered an important role of the Wnt family of signalling molecules in controlling AP-directed neuronal cell migration and polarity. A common theme that emerges from these studies is that multiple Wnt proteins function in parallel as instructive cues or permissive signals to control neuronal patterning along this major body axis.