Chen ZP et al. (1998) West Coast Worm Meeting "THE abi-1 GENE, A GENE REQUIRED FOR THE VULVAL MORPHOGENESIS, ENCODES A NUCLEAR HORMONE RECEPTOR"

Han M, Chen ZP

[West Coast Worm Meeting, 1998]

Using an efficient TS screen for mutants that have morphogenetic defects in the vulva, we have identified many novel genes, one of them being the abi-1 (abnormal invagination of the vulva) gene. The abi-1 (ku217) allele is a TS and recessive mutation, which produces defects in the morphology of the vulva, as well as in the male mating structures, the gonadal migration, viability, and fertility. Dosage analysis suggests that the ku217 allele is a loss-of-function mutation. From non-complementation screens, we have isolated several possible alleles of the abi-1 gene, all of them having more severe phenotypes than the ku217 allele. We have cloned the abi-1 gene using the DNA-mediated microinjection transformation. The predicted gene product, which is 472 a.a. long, is a homolog of members of the nuclear hormone receptor superfamily. Sequencing of the ku217 mutant shows a missense mutation in the N-terminus of the protein (L32F). The abi-1::GFP reporter gene is expressed in the 12 Pn.p cells and the hypodermal cells. The expression level increases in the P5.p, P6.p, and P7.p after the first round of division. Epistasis test shows that the abi-1(ku217) mutation, which by itself is slightly under-induced, can enhance the induction in the let-60/ras (gf) mutant animals. The mutant phenotype in the vulval morphogenesis exists in the let-60 (gf); abi-1 and lin-1(lf);abi-1 double mutant animals.

Cohen, J.D. et al. (2017) International Worm Meeting "A zona pellucida protein and a scavenger receptor play opposing roles in shaping a narrow tube."

Cohen, J.D., Sundaram, M.V., Forman-Rubinsky, R.

[International Worm Meeting, 2017]

A lipid-, sugar-, and glycoprotein-rich apical extracellular matrix (aECM) shapes and protects the narrowest tubes in the body. For example, lung surfactant helps narrow airways remain open, and a glycocalyx ("sweet husk") lines capillaries in the vascular system. Damage to the aECM may contribute to diseases of tube integrity such as chronic obstructive pulmonary disease and microvascular disease. Nevertheless, the roles that specific aECM components play in matrix organization and tube protection remain poorly understood. To study aECM in narrow tube maintenance, our lab uses the C. elegans excretory system, a simple conduit composed of three tandem, unicellular tubes. In particular, we have focused on understanding the maintenance of the excretory system's middle tube, the duct cell, as it elongates during embryogenesis. Genetic screening has identified apically secreted proteins that are required to maintain the duct lumen, including LET-653, which contains a Zona Pellucida (ZP) domain. ZP proteins are highly abundant in many aECMs, and ZP protein dysfunction is associated with human diseases, including chronic kidney disease and the microvascular disease hereditary hemorrhagic telangiecstasia (HHT), but how ZP proteins protect tube integrity is still unclear. ZP domains consist of two parts, ZP-N and ZP-C. In vitro studies suggest that ZP-N domains can polymerize to form fibrils, while ZP-C domains regulate ZP-N polymerization and/or bind specific partners to alter signaling. However, we found that the LET-653 ZP-C domain is sufficient to rescue let-653 mutants and to form a stable (though transient) layer in the aECM; we are now testing if it can polymerize on its own and are seeking to identify its potential partners. We sought to identify LET-653 interactors by screening for suppressors. Surprisingly, we found that loss of SCAV-2, a putative scavenger receptor, bypasses the requirements for LET-653 and several other aECM components. Scavenger receptors can transport lipids and the enzymes that process them; thus, scavenger receptors may regulate the amounts or types of lipids reaching the aECM. let-653 mutants show increased DiI staining of aECM lipids, suggesting that aberrant lipid accumulation may underlie let-653 phenotypes. Continuing work will identify the lipid classes that may interact with LET-653 in the aECM, and describe how SCAV-2 may regulate these lipids to alter the aECM.

Williams, Claire R. et al. (2013) International Worm Meeting "DYF-7 prevents rupture of a sensory epithelium made of neurons and glia."

Williams, Claire R., Heiman, Maxwell G.

[International Worm Meeting, 2013]

Cells come in a variety of shapes, all finely tuned to perform specific functions. To understand how these essential shapes arise, we have turned to neurons with highly stereotyped morphologies, those of the amphid sense organ in Caenorhabditis elegans. The amphid contains twelve neurons that each extend a single, unbranched dendrite to the nose tip, and two glia which ensheathe the distal endings of these dendrites; together, these cells, which make apical junctions with one another, form a sensory epithelium. We previously showed that amphid dendrites grow by a process termed retrograde extension, in which the dendrite tip remains stationary at the nose as the neuron cell body migrates away, stretching out the dendrite behind it. This process is dependent on DYF-7, a zona pellucida (ZP) domain protein, as without it the dendrite tip breaks away from the nose, resulting in a short dendrite phenotype. We tagged DYF-7 with superfolderGFP and found that its extracellular ZP domain is secreted and forms a cap structure surrounding the dendrite tip. When expressed ectopically in other regions of the embryo, DYF-7 re-localizes to dendrite tips, but also marks the luminal surfaces of the digestive tract, a classic epithelium. Immunostaining these embryos with antibodies against AJM-1 showed that DYF-7 localizes in the apical extracellular matrix adjacent to epithelial apical junctions, including junctions between amphid dendrites and their ensheathing glia. Precociously expressed DYF-7 does not become localized until after apical junctions have been established. In contrast to our previous model of DYF-7 acting as an anchor that holds sensory dendrites at the nose, we now propose that DYF-7 forms filaments across the apical surface of a sensory epithelium composed of neurons and glia, and prevents rupture of this epithelium in response to mechanical forces that result from cell migration. Further, since the primary domain of DYF-7 is a ZP domain, and ZP domains are found at the surfaces of nearly all epithelia in nearly all animals, we speculate that an ancestral function of ZP domain proteins may be to reinforce epithelia by forming a similar meshwork of filaments over their apical surfaces.

Shun-wa Tsang et al. (2005) International Worm Meeting "Identification of new components in ray morphogenesis of C. elegans"

Shun-wa Tsang, King-lau Chow, Yiu-man Lam, Horace Hok-yeung Lee

[International Worm Meeting, 2005]

Eight genes required for normal ray morphogenesis have been identified and characterized. These genes can be classified into three groups, modifying enzymes, collagens and novel membrane bound proteins. MAB-7 and RAM-5 belong to the last group. Both of them were proposed to be involved in the communication process between structural cells and hypodermis during ray morphogenesis. Their interacting partners and mechanistic interaction for cell-cell communication, however, remain to be identified and characterized. In order to uncover these missing components in ray morphogenesis, EMS screen, yeast two-hybrid screen and RNAi screen were initiated. From the genetic screen, a new ram gene, ram-6, was isolated. Its identity is to be determined by complementation rescue and subsequent characterization. In our yeast two-hybrid screen, the extracellular region of MAB-7 and RAM-5; cytoplasmic domain of RAM-5 were chosen as baits to screen for their potential interacting partners. Up to this point, over 2x105 clones were screened from the worm cDNA library. One of a number of positive clones screened out by MAB-7 bait was predicted to encode a large extracellular protein. The function of this extracellular protein is unclear. To further verify the relationships between these genetic components in and ray morphogenesis, RNAi knock down and expression of these genes were examined. The detail will be discussed. In addition, RAM-5 protein with an essential ZP domain located at its N-terminus has been implicated to interact with other ZP proteins in vitro. If such domain in RAM-5 shares similar function, RAM-5 should interact with ZP domain containing proteins in worm during ray morphogenesis. In C. elegans, there are 40 ZP domain containing proteins. These 40 genes will be systematically inactivated by RNAi and their corresponding phenotype related to ray morphogenesis will be examined. (The study is supported by Research Grants Council, Hong Kong.)

Heiman, Maxwell G. et al. (2009) International Worm Meeting "Different sensory dendrites use distinct machineries to attain their lengths."

Shaham, Shai, Heiman, Maxwell G.

[International Worm Meeting, 2009]

Each embryo encodes a map, with cell shape and cell contacts following set patterns. In C. elegans, these patterns are essentially invariant, with a given cell forming the same shape and contacts in every individual. To understand how this developmental map is encoded and read, we have taken a forward genetic approach, isolating mutants in which the map is perturbed. We focused first on the amphids, a well-characterized pair of sense organs in the head of the animal. Each amphid consists of 12 neurons, which extend unbranched sensory dendrites to the tip of the nose, and two glial cells, the sheath and the socket, which also extend processes to the nose where they surround dendritic endings of the neurons. Using time-lapse imaging to observe the formation of single dendrites, we found that these cells acquire their shapes by an unusual mechanism: the neurons first form contacts with the presumptive nose, and then the cell body migrates away, dragging out the dendrite behind it. This mechanism suggests a developmental map based on the formation of specific cell contacts, for example between dendrites and the nose tip. Indeed, we isolated mutants in which a "map failure" occurs, with the amphid failing to attain its proper length, and these mutants affect a pair of secreted extracellular matrix proteins, DYF-7 and DEX-1, required to anchor dendritic tips at the nose during cell migration. DYF-7 is a zona pellucida (ZP) domain protein expressed in most sensory neurons in the developing embryo; DEX-1 is a nidogen- and zonadhesin-containing protein expressed widely in the developing head and tail. Yet, the effects of these mutants are limited to the amphids and a similar pair of tail sense organs, the phasmids. The presence of 40 predicted ZP-encoding C. elegans genes, and several zonadhesin-like genes, suggests the possibility that part of the developmental map is encoded by combinatorial matchmaking between DYF-7- and DEX-1-like proteins. To test this hypothesis, we have isolated mutants in which sensory dendrites of the URX or CEP neurons fail to attach to the nose. Although URX and dorsal CEP neurons are lineal sisters, the mutants show that these cells attach to the nose using machineries distinct from each other and from that of the amphid. While not yet cloned, these mutants might affect additional ZP or ZP-interacting proteins. Indeed, the presence of ZP proteins in sense organs is highly conserved across species and sensory modalities. The involvement of distinct ZP proteins would support the model of a developmental map partly based on cells forming contacts with specific extracellular attachment sites.

Horace S.W. TSANG et al. (2007) International Worm Meeting "noah-2: new component in ray morphogenesis of C. elegans."

Horace S.W. TSANG, Agnes K.Y. HUI, King L. Chow

[International Worm Meeting, 2007]

Organ morphogenesis is an important process during development. We use male tail sensory ray as a model and screen for genes that required for this process. From previous forward genetics screens, we identified two novel transmembrane proteins, MAB-7 and RAM-5. Domain analysis of both MAB-7 and RAM-5 proteins showed that their extracellular domains are crucial for their function, therefore the extracellular domains were used as bait in yeast two hybrid screens. A number of potential candidates were identified. The results will be discussed. In addition, a genome-wide screen for interacting partner, targeted RNAi gene knock down was performed to identify potential components required for ray morphogenesis. Since RAM-5 contains a ZP domain curial for protein function and in vitro studies show that ZP proteins interact with each other, we evaluated the biological function of 40 ZP domain-containing proteins in C. elegans ray development. From the screens, we identified noah-2 (NOmpA Homolog, a Drosophila nompA counterpart) shows larval lethal phenotype and Ram phenotype. The gene is expressed in hypodermis from embryo to adult, which explains its requirement in larval development. To confirm the requirement of noah-2 in ray morphogenesis, dominant negative NOAH-2 protein expressing transgenic animals were generated. They display Ram phenotype revealing all features similar to other ram mutants. Furthermore, to test whether noah-2 works together with ram-5 in controlling ray morphogenesis, noah-2 was knocked down in ram-5(bx81) mutant. An enhanced Ram phenotype was observed, suggesting that they are acting synergistically in ray morphogenesis. The implication of this result in the context of ram genes interactions will be presented. (The research is funded by Research Grants Council, Hong Kong.).

Aldis Krizus et al. (2006) Development & Evolution Meeting "The Polyspermy Barrier Requires Eggshell Chitin, which Depends on SPE-11 for Synthesis/Extrusion"

James Dennis, Wendy Johnston, Aldis Krizus

[Development & Evolution Meeting, 2006]

One of the earliest post-fertilization events is the elaboration of a barrier to polyspermy. This barrier prevents penetration by multiple sperm, which would lead to aneuploidy and lethality. In mammals, oocytes are surrounded by the zona pelucida (ZP). Sperm contact with ZP3 initiates the acrosomal reaction, activating the single sperm, and allowing it to penetrate the ZP and fuse with the oocyte. This triggers a Ca2+ oscillation, stimulating the release of oocyte cortical granules and resulting in inactivation of ZP3 and prevention of further sperm penetration. Ca2+ oscillation also contributes to the development of a second, oocyte plasma membrane, barrier, although the biochemical nature of this block is unclear. C. elegans oocytes do not have a ZP and C. elegans sperm do not undergo the acrosomal reaction. However, C. elegans fertilization results in a Ca2+ wave, and C. elegans has a robust, albeit unidentified, polyspermy barrier. In addition to generating a polyspermy barrier, fertilization also results in elaboration of the eggshell, which contains the GlcNAc homopolymer, chitin ([(GlcNAcβ1,4GlcNAc)n]). The physical properties of chitin (tough, insoluble polymer) and its almost immediate synthesis following fertilization suggested to us that chitin might be part of the polyspermy barrier. Eggshell chitin is synthesized by CHS-1, and the substrate, UDP-GlcNAc, is provided by GNA-2. To test the hypothesis that eggshell chitin is required to prevent polyspermy, we quantified the number of sperm nuclei in embryos of AZ212 (unc-119(ed3);ruIS32[unc-119(+) pie-1::GFP::H2B]III), XA781(gna-2(qa705)I; unc-119(ed3);ruIS32[unc-119(+)pie-1::GFP::H2B]III) or AZ212 fed chs-1 RNAi, using histone H2B::GFP to mark chromatin. Polyspermy incidence was 0% in AZ212, 23% in XA781 and 60% in AZ212 fed chs-1 RNAi. Deficiency of the paternally provided protein, SPE-11 results in osmotic sensitivity, failed polar body extrusion and a 12 sister chromatid phenotype that resembles gna-2(qa705) and chs-1 RNAi. Staining of spe-11(hc90) embryos with the chitin-binding reagent showed that eggshell chitin was either absent or very reduced. These results support the conclusion that chitin is essential for the polyspermy barrier, and that the sperm-provided molecule, SPE-11, is required for chitin synthesis/extrusion.

Flatt, K. et al. (2017) International Worm Meeting "The role of DEX-1 in dauer formation and cuticle morphology."

Unal, C., Beshers, C., Flatt, K., Schroeder, N.

[International Worm Meeting, 2017]

The C. elegans dauer larvae undergo extensive tissue remodeling events that allow them to survive during times of unfavorable environmental conditions. These remodeling events include neuronal arborization and changes to the cuticle. To better understand these remodeling events we are currently investigating the role of the extracellular environment in dauer formation and morphogenesis. It has previously been shown that a set of sensory head neurons, the inner labial 2 (IL2) neurons, arborize in a dauer specific manner. To further investigate this phenotype, we looked at the extracellular matrix (ECM) protein, DEX-1, that is required during embryogenesis for proper dendrite morphology and functions via interactions with zona pellucida (ZP) domain-containing ECM proteins. We observed that dex-1 mutant dauers exhibit wild-type IL2 arborization, but are sensitive to SDS treatments and have defects in radial constriction and lateral alae formation. A 5kb dex-1p::gfp promoter fusion is expressed in the seam cells, a set of 16 stem cell-like hypodermal cells, exclusively during dauer. Interestingly, a truncated version of this dex-1 promoter is expressed in the seam cells during all larval stages. During dauer, the seam cells undergo autophagy-mediated shrinkage that facilitates radial shrinkage and alae formation. We also observed that dex-1 mutant dauers are less responsive to mechanical stimulation compared with wild-type dauers. Closer examination of the dex-1 expression pattern revealed dauer-specific expression in the glial cells of the mechanosensory deirid neurons. Reporter analysis with an ajm-1::gfp and preliminary electron microscopy data indicate that dex-1 dauers have defects in seam cell shrinkage. The seam cell mediated radial shrinkage and subsequent alae formation are facilitated by a family ZP domain-containing ECM proteins called cuticulins (CUT). Disruption of CUT-1, CUT-5 and CUT-6 causes similar dauer morphology phenotypes as dex-1 mutants, and their ZP domains make them good candidates for DEX-1 interactions. Indeed, our preliminary data suggests possible genetic interactions between dex-1, cut-1 and cut-5. Our results indicate that DEX-1 plays an important role in ECM remodeling during dauer formation.

Poggioli, Corey et al. (2013) International Worm Meeting "LET-653, a secreted ZP-domain and mucin-related protein, functions in the excretory duct/pore, and not the excretory canal cell."

Sundaram, Meera V., Bickard, Kevin, Poggioli, Corey

[International Worm Meeting, 2013]

Polarized epithelia secrete a specialized extracellular matrix (ECM) that strongly influences their cell morphology and tissue integrity. For example, the basal ECM includes collagens and laminins that interact with cell surface receptors such as integrins to influence cytoskeletal organization and junction strength. The apical ECM frequently contains glycoproteins such as mucins or zona pellucida (ZP)-domain proteins, as well as (in ecdysozoa) cuticle components such as collagen or chitin. There is increasing evidence that the apical ECM also can modulate cytoskeletal organization and junction strength, although in general this role is not well understood. Mutants lacking the C. elegans ZP-domain and mucin-like protein LET-653 arrest as "rod-like" L1 larvae with severe defects in morphology of the excretory canal cell (Buechner et al, 1999; Jones and Baillie, 1995), leading to a hypothesis that LET-653 might be part of the canal cell apical ECM. Here we show instead that let-653 functions in two other tubular components of the excretory system, the duct and pore cells, which connect the excretory canal cell to the outside environment to allow for fluid waste excretion. let-653 reporters are expressed in the excretory duct and other cuticle-lined epithelia, and let-653 transgene expression in the duct or pore, but not in the canal cell or body muscle, is sufficient to rescue let-653 lethality. The most prominent early phenotype of let-653 mutants is a swelling of the duct lumen. We propose that LET-653 is a component of the duct/pore apical ECM, and that canal cell defects in let-653 mutants are a secondary consequence of luminal defects in the duct/pore.

Frederick J Tan et al. (2004) West Coast Worm Meeting "Functions of the Outer Mitochondrial Membrane Protein CED-9"

R Blake Hill, Andrew Z Fire, Frederick J Tan

[West Coast Worm Meeting, 2004]

CED-9 (CEll Death abnormal) negatively regulates the programmed cell death pathway in C. elegans . This membrane associated pro-survival protein prevents the activation of the CED-3 caspase. Loss of function ced-9 animals arrest at early embryogenesis, presumably dying from ectopic cell deaths. Conversely, gain of function animals appear healthy, albeit with extra cells (Hengartner, Ellis and Horvitz, 1992). These effects have been ascribed to the ability of CED-9 to sequester the CED-4/CED-3 caspase complex at the mitochondria (Chen, et al. , 2000). We hope to understand the structural features of CED-9 that participate in its function. To this end, we are assaying the activity of various CED-9 mutants and derivatives for ability to prevent cell death and respond to appropriate modulating signals. A question that we consider to be central is whether the C-terminal transmembrane domain of CED-9 serves only as a targeting sequence to the mitochondrion, or plays a more active role. We have demonstrated that this domain is essential for mitochondrial localization and plays some role in the function of CED-9. Distinction between localization and other structural and functional roles is in progress. Hengartner MO, Ellis RE and HR Horvitz. 1992. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356 , 494-499. Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y, and HR Horvitz. 2000. Translocation of C. elegans CED-4 to Nuclear Membranes During Programmed Cell Death. Science 287 , 1485-1489.