Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis.

During Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

A DNA replication-independent function of pre-replication complex genes during cell invasion in C. elegans.

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

Tissue-specific inhibition of protein sumoylation uncovers diverse SUMO functions during C. elegans vulval development.

The sumoylation (SUMO) pathway is involved in a variety of processes during C. elegans development, such as gonadal and vulval fate specification, cell cycle progression and maintenance of chromosome structure. The ubiquitous expression and pleiotropic effects have made it difficult to dissect the tissue-specific functions of the SUMO pathway and identify its target proteins. To overcome these challenges, we have established tools to block protein sumoylation and degrade sumoylated target proteins in a tissue-specific and temporally controlled manner. We employed the auxin-inducible protein degradation system (AID) to down-regulate the SUMO E3 ligase GEI-17 or the SUMO ortholog SMO-1, either in the vulval precursor cells (VPCs) or in the gonadal anchor cell (AC). Our results indicate that the SUMO pathway acts in multiple tissues to control different aspects of vulval development, such as AC positioning, basement membrane (BM) breaching, VPC fate specification and morphogenesis. Inhibition of protein sumoylation in the VPCs resulted in abnormal toroid formation and ectopic cell fusions during vulval morphogenesis. In particular, sumoylation of the ETS transcription factor LIN-1 at K169 is necessary for the proper contraction of the ventral vulA toroids. Thus, the SUMO pathway plays several distinct roles throughout vulval development.

Microfluidic-based imaging of complete Caenorhabditis elegans larval development.

Several microfluidic-based methods for Caenorhabditis elegans imaging have recently been introduced. Existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method that allows parallel live-imaging across multiple larval stages, while maintaining worm orientation and identity over time. This is achieved through an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition only. In this way, excellent quality images can be acquired with minimal impact on worm viability or developmental timing. The capabilities of the devices are demonstrated by observing the hypodermal seam and P-cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate feasibility of on-chip RNAi by perturbing basement membrane breaching during anchor cell invasion.

Polarized epidermal growth factor secretion ensures robust vulval cell fate specification in Caenorhabditis elegans.

The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC.

The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion.

Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.

ß-Integrin de-phosphorylation by the Density-Enhanced Phosphatase DEP-1 attenuates EGFR signaling in C. elegans.

Density-Enhanced Phosphatase-1 (DEP-1) de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover, dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling. Using a substrate-trapping DEP-1 mutant in a proteomics approach, we have identified the C. elegans ß-integrin subunit PAT-3 as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin recruitment. The non-phosphorylatable ß-integrin mutant pat-3(Y792F) partially suppresses the hyperactive EGFR signaling phenotype caused by loss of dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the ß-integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the αß-integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. We propose that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through αß-integrin activation.

Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA.

Human cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling.

The C. elegans hox gene lin-39 controls cell cycle progression during vulval development.

Cell fate specification during organogenesis is usually followed by a phase of cell proliferation to produce the required number of differentiated cells. The Caenorhabditis elegans vulva is an excellent model to study how cell fate specification and cell proliferation are coordinated. The six vulval precursor cells (VPCs) are born at the first larval stage, but they arrest in the G1 phase of the cell cycle until the beginning of the third larval stage, when their fates are specified and the three proximal VPCs proliferate to generate 22 vulval cells. An epidermal growth factor (EGF) signal from the gonadal anchor cell combined with lateral DELTA/NOTCH signaling between the VPCs determine the primary (1°) and secondary (2°) fates, respectively. The hox gene lin-39 plays a key role in integrating these spatial patterning signals and in maintaining the VPCs as polarized epithelial cells. Using a fusion-defective eff-1(lf) mutation to keep the VPCs polarized, we find that VPCs lacking lin-39 can neither activate lateral NOTCH signaling nor proliferate. LIN-39 promotes cell cycle progression through two distinct mechanisms. First, LIN-39 maintains the VPCs competent to proliferate by inducing cdk-4 cdk and cye-1 cyclinE expression via a non-canonical HOX binding motif. Second, LIN-39 activates in the adjacent VPCs the NOTCH signaling pathway, which promotes VPC proliferation independently of LIN-39. The hox gene lin-39 is therefore a central node in a regulatory network coordinating VPC differentiation and proliferation.

An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling.

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.

Emergent stem cell homeostasis in the C. elegans germline is revealed by hybrid modeling.

The establishment of homeostasis among cell growth, differentiation, and apoptosis is of key importance for organogenesis. Stem cells respond to temporally and spatially regulated signals by switching from mitotic proliferation to asymmetric cell division and differentiation. Executable computer models of signaling pathways can accurately reproduce a wide range of biological phenomena by reducing detailed chemical kinetics to a discrete, finite form. Moreover, coordinated cell movements and physical cell-cell interactions are required for the formation of three-dimensional structures that are the building blocks of organs. To capture all these aspects, we have developed a hybrid executable/physical model describing stem cell proliferation, differentiation, and homeostasis in the Caenorhabditis elegans germline. Using this hybrid model, we are able to track cell lineages and dynamic cell movements during germ cell differentiation. We further show how apoptosis regulates germ cell homeostasis in the gonad, and propose a role for intercellular pressure in developmental control. Finally, we use the model to demonstrate how an executable model can be developed from the hybrid system, identifying a mechanism that ensures invariance in fate patterns in the presence of instability.

WormQTL--public archive and analysis web portal for natural variation data in Caenorhabditis spp.

Here, we present WormQTL (http://www.wormqtl.org), an easily accessible database enabling search, comparative analysis and meta-analysis of all data on variation in Caenorhabditis spp. Over the past decade, Caenorhabditis elegans has become instrumental for molecular quantitative genetics and the systems biology of natural variation. These efforts have resulted in a valuable amount of phenotypic, high-throughput molecular and genotypic data across different developmental worm stages and environments in hundreds of C. elegans strains. WormQTL provides a workbench of analysis tools for genotype-phenotype linkage and association mapping based on but not limited to R/qtl (http://www.rqtl.org). All data can be uploaded and downloaded using simple delimited text or Excel formats and are accessible via a public web user interface for biologists and R statistic and web service interfaces for bioinformaticians, based on open source MOLGENIS and xQTL workbench software. WormQTL welcomes data submissions from other worm researchers.

PTEN negatively regulates MAPK signaling during Caenorhabditis elegans vulval development.

Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP(3) lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways.

The Caenorhabditis elegans homolog of the Opitz syndrome gene, madd-2/Mid1, regulates anchor cell invasion during vulval development.

Mutations in the human Mid1 gene cause Opitz G/BBB syndrome, which is characterized by various midline closure defects. The Caenorhabditis elegans homolog of Mid1, madd-2, positively regulates signaling by the unc-40 Netrin receptor during the extension of muscle arms to the midline and in axon guidance and branching. During uterine development, a specialized cell called anchor cell (AC) breaches the basal laminae separating the uterus from the epidermis and invades the underlying vulval tissue. AC invasion is guided by an UNC-6 Netrin signal from the ventral nerve cord and an unknown guidance signal from the vulval cells. Using genetic epistasis analysis, we show that madd-2 regulates AC invasion downstream of or in parallel with the Netrin signaling pathway. Measurements of AC shape, polarity and dynamics indicate that MADD-2 prevents the formation of ectopic AC protrusions in the absence of guidance signals. We propose that MADD-2 represses the intrinsic invasive capacity of the AC, while the Netrin and vulval guidance cues locally overcome this inhibitory activity of MADD-2 to guide the AC ventrally into the vulval tissue. Therefore, developmental cell invasion depends on a precise balance between pro- and anti-invasive factors.

LIN-39 and the EGFR/RAS/MAPK pathway regulate C. elegans vulval morphogenesis via the VAB-23 zinc finger protein.

Morphogenesis represents a phase of development during which cell fates are executed. The conserved hox genes are key cell fate determinants during metazoan development, but their role in controlling organ morphogenesis is less understood. Here, we show that the C. elegans hox gene lin-39 regulates epidermal morphogenesis via its novel target, the essential zinc finger protein VAB-23. During the development of the vulva, the egg-laying organ of the hermaphrodite, the EGFR/RAS/MAPK signaling pathway activates, together with LIN-39 HOX, the expression of VAB-23 in the primary cell lineage to control the formation of the seven vulval toroids. VAB-23 regulates the formation of homotypic contacts between contralateral pairs of cells with the same sub-fates at the vulval midline by inducing smp-1 (semaphorin) transcription. In addition, VAB-23 prevents ectopic vulval cell fusions by negatively regulating expression of the fusogen eff-1. Thus, LIN-39 and the EGFR/RAS/MAPK signaling pathway, which specify cell fates earlier during vulval induction, continue to act during the subsequent phase of cell fate execution by regulating various aspects of epidermal morphogenesis. Vulval cell fate specification and execution are, therefore, tightly coupled processes.

Cell-cycle regulation of NOTCH signaling during C. elegans vulval development.

C. elegans vulval development is one of the best-characterized systems to study cell fate specification during organogenesis. The detailed knowledge of the signaling pathways determining vulval precursor cell (VPC) fates permitted us to create a computational model based on the antagonistic interactions between the epidermal growth factor receptor (EGFR)/RAS/MAPK and the NOTCH pathways that specify the primary and secondary fates, respectively. A key notion of our model is called bounded asynchrony, which predicts that a limited degree of asynchrony in the progression of the VPCs is necessary to break their equivalence. While searching for a molecular mechanism underlying bounded asynchrony, we discovered that the termination of NOTCH signaling is tightly linked to cell-cycle progression. When single VPCs were arrested in the G1 phase, intracellular NOTCH failed to be degraded, resulting in a mixed primary/secondary cell fate. Moreover, the G1 cyclins CYD-1 and CYE-1 stabilize NOTCH, while the G2 cyclin CYB-3 promotes NOTCH degradation. Our findings reveal a synchronization mechanism that coordinates NOTCH signaling with cell-cycle progression and thus permits the formation of a stable cell fate pattern.

Prolonging somatic cell proliferation through constitutive hox gene expression in C. elegans.

hox genes encode a conserved family of homeodomain transcription factors that are essential to determine the identity of body segments during embryogenesis and maintain adult somatic stem cells competent to regenerate organs. In contrast to higher organisms, somatic cells in C. elegans irreversibly exit the cell cycle after completing their cell lineage and the adult soma cannot regenerate. Here, we show that hox gene expression levels in C. elegans determine the temporal competence of somatic cells to proliferate. Down-regulation of the central hox gene lin-39 in dividing vulval cells results in their premature cell cycle exit, whereas constitutive lin-39 expression causes precocious Pn.p cell and sex myoblast divisions and prolongs the proliferative phase of the vulval cells past their normal point of arrest. Furthermore, ectopic expression of hox genes in the quiescent anchor cell re-activates the cell cycle and induces proliferation until young adulthood. Thus, constitutive expression of a single hox transcription factor is sufficient to prolong somatic cell proliferation beyond the restriction imposed by the cell lineage. The down-regulation of hox gene expression in most somatic cells at the end of larval development may be one cause for the absence of cell proliferation in adult C. elegans.

The C. elegans MAGI-1 protein is a novel component of cell junctions that is required for junctional compartmentalization.

Cell junctions are essential to maintain polarity and tissue integrity. Epithelial cell junctions are composed of distinct sub-compartments that together ensure the strong adhesion between neighboring cells. In Caenorhabditis elegans epithelia, the apical junction (CeAJ) forms a single electron-dense structure, but at the molecular level it is composed of two sub-compartments that function redundantly and localize independently as two distinct but adjacent circumferential rings on the lateral plasma membrane. While investigating the role of the multi PDZ-domain containing protein MAGI-1 during C. elegans epidermal morphogenesis, we found that MAGI-1 localizes apical to both the Cadherin/Catenin (CCC) and AJM-1/DLG-1 (DAC) containing sub-domains. Removal of MAGI-1 function causes a loss of junctional compartmentalization along the lateral membrane and reduces the overall robustness of cell-cell adhesion mediated by either type of cell junctions. Our results suggest that MAGI-1 functions as an "organizer" that ensures the correct segregation of different cell adhesion complexes into distinct domains along the lateral plasma membrane. Thus, the formation of stable junctions requires the proper distribution of the CCC and DAC adhesion protein complexes along the lateral plasma membrane.

A dynamic physical model of cell migration, differentiation and apoptosis in Caenorhabditis elegans.

The germ line of the nematode C. elegans provides a paradigm to study essential developmental concepts like stem cell differentiation and apoptosis. Here, we have created a computational model encompassing these developmental landmarks and the resulting movement of germ cells along the gonadal tube. We have used a technique based on molecular dynamics (MD) to model the physical movement of cells solely based on the force that arises from dividing cells. This novel way of using MD to drive the model enables calibration of simulation and experimental time. Based on this calibration, the analysis of our model shows that it is in accordance with experimental observations. In addition, the model provides insights into kinetics of molecular pathways within individual cells as well as into physical aspects like the cell density along the germ line and in local neighbourhoods of individual germ cells. In the future, the presented model can be used to test hypotheses about diverse aspects of development like stem cell division or programmed cell death. An iterative process of evolving this model and experimental testing in the model system C. elegans will provide new insights into key developmental aspects.