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.

Chen ZP et al. (2000) Bioessays "Building a protein interaction map: research in the post-genome era."

Chen ZP, Han M

[Bioessays, 2000]

With the extensive amount of information generated by genome-wide sequencing, the entire set of gene products in an organism can now be predicted. The challenge of understanding the function of each gene in the genome has led to the development of many large-scale and high-throughput experimental techniques. Recently, two papers, Walhout et al.(1) and Uetz et al.,(2) have described studies that add a new functional dimension to research conducted on a genome-wide scale. These two groups have utilized the yeast two-hybrid system to identify interactions among the entire complement of proteins encoded by the Caenorhabditis elegans and the Saccharomyces cerevisiae genomes, respectively. Using a set of 29 genes that have been previously characterized, Walhout et al. demonstrated the feasibility and efficiency of this technique by building an interaction matrix among a large number of proteins. On an even larger scale, Uetz et al. conducted two-hybrid analyses using proteins that represent over 87% of the total gene products in yeast and identified interactions for about 15% of the total yeast proteins. BioEssays 22:503-506, 2000.

Weadick CJ (2020) Genome Biol Evol "Molecular evolutionary analysis of nematode Zona Pellucida (ZP) modules reveals disulfide-bond reshuffling and standalone ZP-C domains."

Weadick CJ

[Genome Biol Evol, 2020]

Zona pellucida (ZP) modules mediate extracellular protein-protein interactions and contribute to important biological processes including syngamy and cellular morphogenesis. While some biomedically-relevant ZP modules are well-studied, little is known about the protein family's broad-scale diversity and evolution. The increasing availability of sequenced genomes from "non-model" systems provides a valuable opportunity to address this issue, and to use comparative approaches to gain new insights into ZP module biology. Here, through phylogenetic and structural exploration of ZP module diversity across the nematode phylum, I report evidence that speaks to two important aspects of ZP module biology. First, I show that ZP-C domains-which in some modules act as regulators of ZP-N domain-mediated polymerization activity, and which have never before been found in isolation-can indeed be found as standalone domains. These standalone ZP-C domain proteins originated in independent (paralogous) lineages prior to the diversification of extant nematodes, after which they evolved under strong stabilizing selection, suggesting the presence of ZP-N domain-independent functionality. Second, I provide a much-needed phylogenetic perspective on disulfide bond variability, uncovering evidence for both convergent evolution and disulfide-bond reshuffling. This result has implications for our evolutionary understanding and classification of ZP module structural diversity and highlights the usefulness of phylogenetics and diverse sampling for protein structural biology. All told, these findings set the stage for broad-scale (cross-phyla) evolutionary analysis of ZP modules and position Caenorhabditis elegans and other nematodes as important experimental systems for exploring the evolution of ZP modules and their constituent domains.

Cohen JD et al. (2020) PLoS Genet "A C. elegans Zona Pellucida domain protein functions via its ZPc domain."

Bermudez JG, Cohen JD, Sundaram MV, Good MC

[PLoS Genet, 2020]

Zona Pellucida domain (ZP) proteins are critical components of the body's external-most protective layers, apical extracellular matrices (aECMs). Although their loss or dysfunction is associated with many diseases, it remains unclear how ZP proteins assemble in aECMs. Current models suggest that ZP proteins polymerize via their ZPn subdomains, while ZPc subdomains modulate ZPn behavior. Using the model organism C. elegans, we investigated the aECM assembly of one ZP protein, LET-653, which shapes several tubes. Contrary to prevailing models, we find that LET-653 localizes and functions via its ZPc domain. Furthermore, we show that ZPc domain function requires cleavage at the LET-653 C-terminus, likely in part to relieve inhibition of the ZPc by the ZPn domain, but also to promote some other aspect of ZPc domain function. In vitro, the ZPc, but not ZPn, domain bound crystalline aggregates. These data offer a new model for ZP function whereby the ZPc domain is primarily responsible for matrix incorporation and tissue shaping.

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.

Cohen JD et al. (2018) Genetics "Epithelial Shaping by Diverse Apical Extracellular Matrices Requires the Nidogen Domain Protein DEX-1 in Caenorhabditis elegans."

Cohen JD, Flatt KM, Schroeder NE, Sundaram MV

[Genetics, 2018]

The body's external surfaces and the insides of biological tubes, like the vascular system, are lined by a lipid, glycoprotein-, and glycosaminoglycan-rich apical extracellular matrix (aECM). aECMs are the body's first line of defense against infectious agents and promote tissue integrity and morphogenesis, but are poorly described relative to basement membranes and stromal ECMs. While some aECM components, such as zona pellucida domain (ZP) proteins, have been identified, little is known regarding the overall composition of the aECM or the mechanisms by which different aECM components work together to shape epithelial tissues. In <i>Caenorhabditis elegans,</i> external epithelia develop in the context of an ill-defined ZP-containing aECM that precedes secretion of the collagenous cuticle. <i>C. elegans</i> has 43 genes that encode at least 65 unique ZP proteins, and we show that some of these comprise distinct pre-cuticle aECMs in the embryo. Previously, the nidogen- and EGF-domain protein DEX-1 was shown to anchor dendrites to the <i>C. elegans</i> nose tip in concert with the ZP protein DYF-7. Here, we identified a new, strong loss-of-function allele of <i>dex-1</i>, <i>cs201</i><i>dex-1</i> mutants die as L1 larvae and have a variety of tissue distortion phenotypes, including excretory defects, pharyngeal ingression, alae defects, and a short and fat body shape, that strongly resemble those of genes encoding ZP proteins. DEX-1 localizes to ZP-containing aECMs in the tissues that show defects in <i>dex-1</i> mutants. Our studies suggest that DEX-1 is a component of multiple distinct embryonic aECMs that shape developing epithelia, and a potential partner of multiple ZP proteins.

Po MD et al. (2012) Neuron "Releasing the inner inhibition for axon regeneration."

Po MD, Calarco JA, Zhen M

[Neuron, 2012]

The adult mammalian central nervous system exhibits restricted regenerative potential. Chen etal. (2011) and El Bejjani and Hammarlund (2012) used Caenorhabditis elegans to uncover intrinsic factors that inhibit regeneration of axotomized mature neurons, opening avenues for potential therapeutics.

Gill HK et al. (2016) PLoS Genet "Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix."

Forman-Rubinsky R, Gill HK, Ayala-Figueroa J, Cohen JD, Bickard K, Parry JM, Poggioli C, Hall DH, Sundaram MV, Pu P

[PLoS Genet, 2016]

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity.

Silverstein NJ et al. (2017) Immunity "Interleukin-17: Why the Worms Squirm."

Huh JR, Silverstein NJ

[Immunity, 2017]

IL-17 is a cytokine known primarily for its role in inflammation. In a recent issue of Nature, Chen etal. (2017) demonstrate that IL-17 plays a neuromodulatory role in Caenorhabditis elegans by acting directly on neurons to amplify neuronal responses to stimuli and produce changes in animal behavior.

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.