Michael F Portereiko et al. (2001) International C. elegans Meeting "Pharynx tubulogenesis during C. elegans development"

Michael F Portereiko, Susan E Mango

[International C. elegans Meeting, 2001]

During C. elegans embryogenesis, the pharyngeal primordium develops from a ball of cells into a linear tube connected anteriorly to the buccal cavity and posteriorly to the midgut. Using GFP reporters localized to discrete subcellular regions, we have shown that pharyngeal tubulogenesis occurs in three stages: i) lengthening of the nascent pharyngeal lumen by reorientation of apicobasal polarity of anterior pharyngeal cells (Reorientation), ii) formation of an epithelium by the buccal cavity cells, which mechanically couples the buccal cavity to the pharynx and anterior epidermis (Epithelialization), and iii) a concomitant movement of the pharynx anteriorly and the epidermis of the mouth posteriorly to bring the pharynx, buccal cavity and mouth into close apposition (Contraction) (1). We call this three-step process pharyngeal extension. We have undertaken two approaches to identify loci required for pharyngeal extension. First, we have used RNA interference to determine the role, if any, of candidate genes previously shown to be expressed in the pharynx. Second, we are undertaking a mutagenesis screen to identify mutants that generate pun (pharynx unattached) phenotypes but are otherwise largely normal. From 2000 haploid genomes, we have recovered seven Pun mutants. Our current goals are to characterize the phenotypes of the mutants and to continue screening. 1. MF Portereiko and SE Mango, Morphogenesis of the C. elegans Pharynx, Dev. Biol, in press.

Michael F Portereiko et al. (2002) West Coast Worm Meeting "Identifying genetic loci that control early pharynx morphogenesis"

Susan E Mango, Michael F Portereiko

[West Coast Worm Meeting, 2002]

During C. elegans embryogenesis, the pharyngeal primordium develops from a ball of cells into a linear tube connected to the buccal cavity anteriorly and the midgut posteriorly. We call this process pharyngeal extension. We have shown that the connection of the pharynx to the buccal cavity occurs in three stages1 . Prior to extension, the pharyngeal cells are shaped like a wedge, with the tip of the wedge, which faces the interior of the primordium, defining the apical surface. I) During the first stage (Reorientation), the anterior pharyngeal cells reorient their apicobasal polarity to align it along the dorsoventral axis of the embryo. This behavior lengthens the nascent pharyngeal lumen towards the anterior of the embryo. II) In the second stage (Epithelialization), the arcade cells form an epithelium that is continuous with the pharynx posteriorly and the hypodermis anteriorly. Epithelialization mechanically couples the digestive tract to the surrounding hypodermis. III) During the third stage (Contraction), the pharynx moves anteriorly and the epidermis of the mouth posteriorly. These movements suggest that cells of the epidermis, buccal cavity and pharynx contract their apical surfaces, similar to a purse-string. To identify the molecules that regulate pharyngeal extension, we have performed a genetic screen for mutants that arrest with a pharynx unattached (Pun) phenotype at the L1 stage. We recovered 14 mutants from an EMS mutagenesis of 3000 haploid genomes. Pun animals could reflect either a failure to connect the pharynx with the buccal cavity or a 'snap-back' in which the attachment between the pharynx and the buccal cavity was not strong enough to withstand the forces of embryonic elongation. We used time-lapse videomicroscopy to distinguish between these possibilities and found that only 1/14 mutants belonged to the snap-back category. Of the remaining alleles, four are defective for Stage I Reorientation. The anterior cells appear to be positioned correctly within the pharyngeal primordium but fail to reorient their apicobasal polarity. Eight alleles are defective for Stage II. The anterior pharyngeal cells reorient their polarity but fail to form an epithelium with the arcade cells. For one allele, cells within the primordium appear disorganized, suggesting the extension defects may be a secondary effect. Importantly, the pharynx is well differentiated in all of the mutants, suggesting that the Pun phenotype does not reflect a general defect in pharyngeal development. We have assigned 9/13 alleles to linkage groups by snip-SNP mapping and have defined at least six genes.

Von Stetina, Stephen E. et al. (2009) International Worm Meeting "Finding Regulators of Cadherin-Independent Epithelialization."

Mango, Susan E., Von Stetina, Stephen E.

[International Worm Meeting, 2009]

How are polarized epithelia established and maintained? This question is of critical importance, as the loss of epithelial polarity is associated with metastasis(1). There are many well-studied protein complexes that lie in specific membrane compartments with roles integral to the epithelial cell. The E-cadherin-containing adherens junction serves to link neighboring epithelial cells together while the more basal tight junction functions to separate the apical and basolateral surfaces. For some cells, E-cadherin is the major initiator of cell polarity and epithelium formation via cell-cell adhesion(2). However, recent studies have discovered E-cadherin independent polarity pathways(3-6). C. elegans offers a powerful system to study this cadherin-independent mechanism, as E-cadherin is dispensible for the initiation of epithelial polarity in nematodes(4). We study cadherin-independent epithelium formation during pharynx development. Nine pharyngeal arcade cells undergo a mesenchymal-to-epithelial transition to link the pharynx to the outer epidermis(7). Ablation of the arcade cells results in a Pharynx unattached (Pun) phenotype, in which the pharynx fails to connect to the epidermis(7). Pun animals die as they are unable to eat. Our lab has undertaken a genetic screen for Pun mutants that fail to form the arcade cell epithelium (Portereiko and Mango, unpublished). This screen revealed that loss of the central-spindlin component ZEN-4/MKLP1 induces a Pun phenotype because the arcade cells fail to polarize(8). We are currently studying where and when ZEN-4 is needed for arcade cell polarization. We have also undertaken a structure/function analysis of this mitotic kinesin in order to elucidate its role in epithelialization. In addition, we are in the process of cloning several mutants that were isolated in the Pun mutagenesis screen. (1). J. M. Lee, S. Dedhar, R. Kalluri, E. W. Thompson, J Cell Biol 172, 973 (Mar 27, 2006). (2). L. N. Nejsum, W. J. Nelson, J Cell Biol 178, 323 (Jul 16, 2007). (3). A. F. Baas et al., Cell 116, 457 (Feb 6, 2004). (4). M. Costa et al., J Cell Biol 141, 297 (Apr 6, 1998). (5). T. J. Harris, M. Peifer, J Cell Biol 167, 135 (Oct 11, 2004). (6). W. B. Raich, C. Agbunag, J. Hardin, Curr Biol 9, 1139 (Oct 21, 1999). (7). M. F. Portereiko, S. E. Mango, Dev Biol 233, 482 (May 15, 2001). (8). M. F. Portereiko, J. Saam, S. E. Mango, Curr Biol 14, 932 (Jun 8, 2004).

Wei-meng Woo et al. (2004) West Coast Worm Meeting "The extracellular matrix protein F-spondin is essential for muscle attachment and epidermal elongation"

Chris Suh, Yishi Jin, Andrew D Chisholm, Mei Ding, Christie Emigh, Wei-Meng Woo, Jian Cao

[West Coast Worm Meeting, 2004]

In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to vab-13 and ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.

Bonnie Kaas et al. (2008) C.elegans Neuronal Development Meeting "Regulation of Levels of UNC-13 at Synapses"

Rebecca Lawson, Rebecca Kohn, Thomas Limouze, Ryan Wennell, Bonnie Kaas

[C.elegans Neuronal Development Meeting, 2008]

Release of neurotransmitters from neurons is highly regulated. Several proteins play roles in this process, including UNC-13, and decreased release of neurotransmitters in unc-13 mutants results in paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. elegans knockout consortium isolated a strain with a large deletion in the coding region of the gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, developmental rate, and body bends per minute. Aldicarb assays were used to determine whether a deletion in the gene coding for the F-box protein alters the response to inhibitors of acetylcholinesterase. We found that the deletion resulted in some changes in developmental rate and in aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding for the F-box protein and in unc-13.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Atsushi Yamanaka et al. (2001) International C. elegans Meeting "Biochemical and functional analysis of SKP1 family in Caenorhabditis elegans"

Yasumi Ohshima, Kei-ichi Nakayama, Masayoshi Yada, Atsushi Yamanaka, Hiroyuki Imaki

[International C. elegans Meeting, 2001]

The ubiuquitin-proteasome pathway is a key mechanism for substrate-specific degradation to control the abundance of a number of proteins. SCF complex, one of ubiquitin-protein ligases (E3s), regulates cell cycle progression, signal transduction, and many other biological systems. The SCF complex consists of invariable components, such as Skp1, Cul-1 and Rbx1, and variable components called F-box proteins that bind to Skp1 through the F-box motif. F-box proteins are substrate-specific adaptor subunits that recruit substrates to the SCF complex. Surprisingly, we found that the genome of Caenorhabditis elegans ( C. elegans ) contains at least 20 Skp1-like sequenses, whereas one or a few Skp1 is present in humans. Therefore, we studied C. elegans Skp1-like proteins (CeSkp1) that are likely to be variable components of SCF complex in addition to F-box proteins. At least, seven CeSkp1s were associated with C. elegans Cul-1 (CeCul-1) in yeast two-hybrid system as well as co-immunoprecipitation assay in mammalian cells, and these expression patterns were different in C. elegans . By RNA interference (RNAi), two of these CeSkp1s showed embyonic lethality and four showed the phenotype of slow growth. There were differences among CeSkp1s in ability to interact with F-box proteins. These results suggest that CeSkp1s, like F-box proteins, act as variable components of SCF complex in C. elegans .

Soto, Martha et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Arp2/3-based Regulation of Junctional Dynamics During Morphogenetic Cell Movements and Polarization."

Bernadskaya, Yelena, Patel, Falshruti, Soto, Martha

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Morphogenesis requires dynamic interaction between the adherens junctions and the actin cytoskeleton. This interaction is mediated by ?-catenin, which was proposed to be a stable bridge between the junctions and F-actin at the apical regions of polarized epithelial cells. However, ?-catenin cannot bind to F-actin and its adherens junction partner, ?-catenin, simultaneously, suggesting the bridge is dynamic and complex. ?-catenin was thought to inhibit Arp2/3-based branched actin at the junctions for normal maturation of adherens junctions. Our research suggests a new positive role for Arp2/3-dependent branched actin in junctional maturation and during embryonic cell movements of C. elegans . In this study we investigate how progressive WAVE/Scar and Arp2/3-depen dent accumulation of ?-catenin at the adherens junctions contributes to the accumulation of F-actin at apical regions of epithelial cells. We find that removing Arp2/3-dependent actin nucleation disrupts junctional maturation and results in altered levels of adherens junctional proteins in epithelial tissues. In particular, there is a drop in both ?-catenin and F-actin levels at the apical intestine, and altered organization of the apical intestine. Subcellular fractionation reveals that loss of Arp2/3-dependent actin nucleation reduces the amount of ?-catenin in the membrane-associated pool. These data demonstrate an essential role for Arp2/3 to dynamically remodel F-actin to support adherens junctions and polarized F-actin during cell migration and tissue morphogenesis.

Ge Gao et al. (2004) Mid-west Worm Meeting "Loss of pan-1 causes a Peter Pan-like phenotype in C. elegans"

Ge Gao, Karen Bennett

[Mid-west Worm Meeting, 2004]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans . PAN-1, a P-granule associated novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), the larvae arrest between the L1 and L2 stages and can survive eight days at 20 0 C. A pan-1(gk142) deletion strain exhibits the same 'forever-young' phenotype. mRNA analysis and protein expression show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to separate potential germline and somatic functions of PAN-1. If PAN-1 belongs to the family of F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

De Fruyt, Nathan et al. (2021) International Worm Meeting "Neuropeptidergic modulation of C. elegans learning behavior"

Van Damme, Sara, Schoofs, Liliane, Watteyne, Jan, De Fruyt, Nathan, Beets, Isabel, Fadda, Melissa

[International Worm Meeting, 2021]

Neuropeptides are an evolutionarily conserved group of neuromodulators that regulate a wide range of adaptive behaviors, such as learning. Yet, unravelling the molecular and circuit mechanisms underlying this neuropeptidergic modulation is challenging due to the diversity of neuropeptide signaling pathways, and their 'wireless' extrasynaptic mode of action. Using reverse pharmacology, we have constructed a molecular map of the C. elegans neuropeptide-receptor network. Phylogenetic reconstruction of the evolutionary history of nematode neuropeptide systems across bilaterian animals revealed several nematode-specific diversifications of neuropeptide signaling in addition to evolutionarily ancient neuropeptide pathways. One of these ancient, conserved pathways is a neuropeptide Y/F (NPY/F)-like signaling system that is an important regulator of learning behavior both in Proto- and Deuterostomia. We found that NPY/F-like FLP-34 neuropeptides are required in serotonergic neurons for aversive olfactory associative learning, which is functionally similar to the role of NPY in vertebrate learning as well as to the role of NPF in invertebrate learning. NPY/F-like neuropeptides are released from serotonergic neurons and signal through the G protein-coupled receptor NPR-11 in the excitatory AIA interneurons to facilitate olfactory aversive learning. In addition, signaling through NPY/F-like receptor NPR-11 also affects learning in salt gustatory plasticity, a gustatory associative learning paradigm. NPY/F-like signaling is not the only neuropeptidergic signaling system affecting learning behavior; we discovered additional neuropeptides that appear to be important to the learning process as well, including peptides that are expressed in non-neuronal cells. Our current research focuses on unravelling the functions of such non-neuronal neuropeptide messengers in learning and other types of behavioral plasticity.