Seam Cells Research Articles

The Caenorhabditis elegans epidermis as a model skin. I: development, patterning, and growth

AbstractThe skin of the nematode Caenorhabditis elegans is composed of a simple epidermal epithelium and overlying cuticle. The skin encloses the animal and plays central roles in body morphology and physiology; its simplicity and accessibility make it a tractable genetic model for several aspects of skin biology. Epidermal precursors are specified by a hierarchy of transcriptional regulators. Epidermal cells form on the dorsal surface of the embryo and differentiate to form the epidermal primordium, which then spreads out in a process of epiboly to enclose internal tissues. Subsequent elongation of the embryo into a vermiform larva is driven by cell shape changes and cell fusions in the epidermis. Most epidermal cells fuse in mid‐embryogenesis to form a small number of multinucleate syncytia. During mid‐embryogenesis the epidermis also becomes intimately associated with underlying muscles, performing a tendon‐like role in transmitting muscle force. Post‐embryonic development of the epidermis involves growth by addition of new cells to the syncytia from stem cell‐like epidermal seam cells and by an increase in cell size driven by endoreplication of the chromosomes in epidermal nuclei. WIREs Dev Biol 2012 doi: 10.1002/wdev.79This article is categorized under: Early Embryonic Development > Development to the Basic Body Plan Invertebrate Organogenesis > Worms

Finding a niche for seam cells?

The C. elegans neuroectodermal seam cells provide a tractable and well-established model for studying the stem cell mode of division, due to the reiterative asymmetric divisions occurring during larval development. They are, however, not generally considered to be ‘true’ stem cells, owing to their eventual terminal differentiation and the lack of a defined stem cell niche—a microenvironment that promotes the proliferation and prevents the differentiation of the stem cells that reside within. Here, we discuss the concept of the niche in relation to the seam, with reference to our recent findings suggesting that the stem-like properties of the seam cells are maintained at least in part through protection from differentiation signals emanating from the surrounding hypodermal syncytium. Determining the applicability of the niche concept will require definition of these signals and will have important implications for the status of seam cells in the context of stem cell biology.

Multiple Wnts Redundantly Control Polarity Orientation in Caenorhabditis elegans Epithelial Stem Cells

During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis. However, how extrinsic signals synchronize cell polarization is not understood. In Caenorhabditis elegans, most mitotic cells are polarized along the anterior-posterior axis and divide asymmetrically. Although this process is regulated by a Wnt-signaling pathway, Wnts functioning in cell polarity have been demonstrated in only a few cells. We analyzed how Wnts control cell polarity, using compound Wnt mutants, including animals with mutations in all five Wnt genes. We found that somatic gonadal precursor cells (SGPs) are properly polarized and oriented in quintuple Wnt mutants, suggesting Wnts are dispensable for the SGPs' polarity, which instead requires signals from the germ cells. Thus, signals from the germ cells organize the C. elegans somatic gonad. In contrast, in compound but not single Wnt mutants, most of the six seam cells, V1–V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes. In contrast, in animals in which the functions of three Wnt receptors (LIN-17, MOM-5, and CAM-1) are disrupted—the stem cells are not polarized and divide symmetrically—suggesting that the Wnt receptors are essential for generating polarity and that they function even in the absence of Wnts. All the seam cells except V5 were polarized properly by a single Wnt gene expressed at the cell's anterior or posterior. The ectopic expression of posteriorly expressed Wnts in an anterior region and vice versa rescued polarity defects in compound Wnt mutants, raising two possibilities: one, Wnts permissively control the orientation of polarity; or two, Wnt functions are instructive, but which orientation they specify is determined by the cells that express them. Our results provide a paradigm for understanding how cell polarity is coordinated by extrinsic signals.

CHL-1 provides an essential function affecting cell proliferation and chromosome stability in Caenorhabditis elegans

A family of helicases that are important in maintaining genome stability is the iron–sulfur group. Members of this family include DOG-1/FANCJ, RTEL1, XPD and Chl1p/DDX11. In Caenorhabitis elegans, the predicted gene M03C11.2 has orthology to the CHL1 ( Chromosome loss 1) gene in Saccharomyces cerevisiae and DDX11 (DEAD/H box polypeptide 11) in humans. In this paper, we show that the chl-1 gene in C. elegans is required for normal development and fertility. Mutants have lineage-independent cell proliferation defects that result in a Stu ( sterile uncoordinated) phenotype, characterized by gonadal abnormalities and a reduced number of D motor neurons and seam cells. A chromosome stability defect is present in the germ cells, where an abnormal number of DAPI-staining chromosomes appear in diakinesis. CHL-1 function is required for the integrity of poly-guanine/poly-cytosine DNA in the absence of DOG-1/FANCJ: the loss of CHL-1 alone does not result in the deletion of G-tracts, but it does increase the number of deletions observed in the dog-1; chl-1 double mutant, indicating a role for CHL-1 during replication and repair. In addition, we observed that cohesin defects increased the number of deletions in the absence of DOG-1/FANCJ. Our results demonstrate a role for CHL-1 in cell proliferation and maintaining normal chromosome numbers, and implicate CHL-1 in chromosome stability and repair of unresolved secondary structures during replication.

Regulation of the C. elegans molt by pqn-47

C. elegans molts at the end of each of its four larval stages but this cycle ceases at the reproductive adult stage. We have identified a regulator of molting, pqn-47. Null mutations in pqn-47 cause a developmental arrest at the first larval molt, showing that this gene activity is required to transit the molt. Mutants with weak alleles of pqn-47 complete the larval molts but fail to exit the molting cycle at the adult stage. These phenotypes suggest that pqn-47 executes key aspects of the molting program including the cessation of molting cycles. The pqn-47 gene encodes a protein that is highly conserved in animal phylogeny but probably misannotated in genome sequences due to much less significant homology to a yeast transcription factor. A PQN-47::GFP fusion gene is expressed in many neurons, vulval precursor cells, the distal tip cell (DTC), intestine, and the lateral hypodermal seam cells but not in the main body hypodermal syncytium (hyp7) that underlies, synthesizes, and releases most of the collagenous cuticle . A functional PQN-47::GFP fusion protein localizes to the cytoplasm rather than the nucleus at all developmental stages, including the periods preceding and during ecdysis when genetic analysis suggests that pqn-47 functions. The cytoplasmic localization of PQN-47::GFP partially overlaps with the endoplasmic reticulum, suggesting that PQN-47 is involved in the extensive secretion of cuticle components or hormones that occurs during molts. The mammalian and insect homologues of pqn-47 may serve similar roles in regulated secretion.

Cell shape and Wnt signaling redundantly control the division axis ofC. elegansepithelial stem cells

Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight regulation of the orientation and plane of cell division is crucial in this process. Here, we study the reproducible pattern of anterior-posterior-oriented stem cell-like divisions in the Caenorhabditis elegans seam epithelium. In a genetic screen, we identified an alg-1 Argonaute mutant with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of the microRNA-induced silencing complex (miRISC) and was previously shown to regulate the timing of postembryonic development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively interferes with Wnt signaling, cell adhesion, cell shape and the orientation and timing of seam cell division. We found that Wnt inactivation, through mig-14 Wntless mutation, disrupts tissue polarity but not anterior-posterior division. However, combined Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. Our findings reveal additional alg-1-regulated processes, uncover a previously unknown function of Wnt ligands in seam tissue polarity, and show that Wnt signaling and geometric cues redundantly control the seam cell division axis.

The Caenorhabditis elegans GATA Factor ELT-1 Works through the Cell Proliferation Regulator BRO-1 and the Fusogen EFF-1 to Maintain the Seam Stem-Like Fate

Seam cells in Caenorhabditis elegans provide a paradigm for the stem cell mode of division, with the ability to both self-renew and produce daughters that differentiate. The transcription factor RNT-1 and its DNA binding partner BRO-1 (homologues of the mammalian cancer-associated stem cell regulators RUNX and CBFβ, respectively) are known rate-limiting regulators of seam cell proliferation. Here, we show, using a combination of comparative genomics and DNA binding assays, that bro-1 expression is directly regulated by the GATA factor ELT-1. elt-1(RNAi) animals display similar seam cell lineage defects to bro-1 mutants, but have an additional phenotype in which seam cells lose their stem cell-like properties and differentiate inappropriately by fusing with the hyp7 epidermal syncytium. This phenotype is dependent on the fusogen EFF-1, which we show is repressed by ELT-1 in seam cells. Overall, our data suggest that ELT-1 has dual roles in the stem-like seam cells, acting both to promote proliferation and prevent differentiation.

Membrane extensions are associated with proper anterior migration of muscle cells during Caenorhabditis elegans embryogenesis

C. elegans body wall muscle is formed after a series of well-orchestrated steps. With the onset of specification embryonic muscle cells accumulate under the hypodermal seam cells at the left and right sides of the embryo. Shortly thereafter they begin to migrate dorsally and ventrally resting beneath the dorsal and ventral hypodermis eventually forming the four muscle quadrants present upon hatching. In this study we describe the plasma membrane dynamics of these migrating cells and observe the extension of filopodia and lamellipodia during dorso-ventral migration but not during the earlier stages of accumulation. We also describe an anterior migration event during embryonic muscle morphogenesis, whereby the anterior-most pair of cells in each of the four muscle quadrants extends long processes to the anterior tip of the developing embryo. Anteriormost muscle cells then follow these extensions into their final positions in the developing embryo. Using RNAi and mutant analysis, we have identified laminin as being involved in mediating the dorsal-ventral muscle migrations. Finally we show that the α-integrin INA-1, the ephrin VAB-2 and its receptor VAB-1 and the Robo receptor SAX-3 indirectly promote the proper extension of the ventral anterior muscle processes by organizing the embryonic neurons so as to provide a clear path for muscle membrane extension.

C. elegans Notch Signaling Regulates Adult Chemosensory Response and Larval Molting Quiescence

The conserved DOS-motif proteins OSM-7 and OSM-11 function as coligands with canonical DSL (Delta, Serrate, and LAG-2) ligands to activate C. elegans Notch receptors during development. We report here that Notch ligands, coligands, and the receptors LIN-12 and GLP-1 regulate two C. elegans behaviors: chemosensory avoidance of octanol and quiescence during molting lethargus. C. elegans lacking osm-7 or osm-11 are defective in their response to octanol. We find that OSM-11 is secreted from hypodermal seam cells into the pseudocoelomic body cavity and acts non-cell autonomously as a diffusible factor. OSM-11 acts with the DSL ligand LAG-2 to activate LIN-12 and GLP-1 Notch receptors in the neurons of adult animals, thereby regulating octanol avoidance response. In adult animals, overexpression of osm-11 and consequent Notch receptor activation induces anachronistic sleep-like quiescence. Perturbation of Notch signaling alters basal activity in adults as well as arousal thresholds and quiescence during molting lethargus. Genetic epistasis studies reveal that Notch signaling regulates quiescence via previously identified circuits and genetic pathways including the egl-4 cGMP-dependent kinase. Our findings indicate that the conserved Notch pathway modulates behavior in adult C. elegans in response to environmental stress. Additionally, Notch signaling regulates sleep-like quiescence in C. elegans, suggesting that Notch may regulate sleep in other species.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

DHS-21, a dicarbonyl/ l-xylulose reductase (DCXR) ortholog, regulates longevity and reproduction in Caenorhabditis elegans

Dicarbonyl/ l-xylulose reductase (DCXR) converts l-xylulose into xylitol, and reduces various α-dicarbonyl compounds, thus performing a dual role in carbohydrate metabolism and detoxification. In this study, we identified DHS-21 as the only DCXR ortholog in Caenorhabditis elegans. The dhs-21 gene is expressed in various tissues including the intestine, gonadal sheath cells, uterine seam (utse) cells, the spermathecal-uterus (sp-ut) valve and on the plasma membrane of spermatids. Recombinant DHS-21 was shown to convert l-xylulose to xylitol using NADPH as a cofactor. Dhs-21 null mutants of C. elegans show defects in longevity, reproduction and egg-laying. Knock-down of daf-16 and elt-2 transcription factors affected dhs-21 expression. These results suggest that DHS-21 is a bona fide DCXR of C. elegans, essential for normal life span and reproduction.

The type II poly(A)-binding protein PABP-2 genetically interacts with the let-7 miRNA and elicits heterochronic phenotypes in Caenorhabditis elegans

The type II poly(A)-binding protein PABP2/PABPN1 functions in general mRNA metabolism by promoting poly(A) tail formation in mammals and flies. It also participates in poly(A) tail shortening of specific mRNAs in flies, and snoRNA biogenesis in yeast. We have identified Caenorhabditis elegans pabp-2 as a genetic interaction partner of the let-7 miRNA, a widely conserved regulator of animal stem cell fates. Depletion of PABP-2 by RNAi suppresses loss of let-7 activity, and, in let-7 wild-type animals, leads to precocious differentiation of seam cells. This is not due to an effect on let-7 biogenesis and activity, which remain unaltered. Rather, PABP-2 levels are developmentally regulated in a let-7-dependent manner. Moreover, using RNAi PABP-2 can be depleted by >80% without significantly impairing larval viability, mRNA levels or global translation. Thus, it unexpectedly appears that the bulk of PABP-2 is dispensable for general mRNA metabolism in the larva and may instead have more restricted, developmental functions. This observation may be relevant to our understanding of why the phenotypes associated with human PABP2 mutation in oculopharyngeal muscular dystrophy (OPMD) seem to selectively affect only muscle cells.

Glycosylation genes expressed in seam cells determine complex surface properties and bacterial adhesion to the cuticle of Caenorhabditis elegans.

The surface of the nematode Caenorhabditis elegans is poorly understood but critical for its interactions with the environment and with pathogens. We show here that six genes (bus-2, bus-4, and bus-12, together with the previously cloned srf-3, bus-8, and bus-17) encode proteins predicted to act in surface glycosylation, thereby affecting disease susceptibility, locomotory competence, and sexual recognition. Mutations in all six genes cause resistance to the bacterial pathogen Microbacterium nematophilum, and most of these mutations also affect bacterial adhesion and biofilm formation by Yersinia species, demonstrating that both infection and biofilm formation depend on interaction with complex surface carbohydrates. A new bacterial interaction, involving locomotory inhibition by a strain of Bacillus pumilus, reveals diversity in the surface properties of these mutants. Another biological property--contact recognition of hermaphrodites by males during mating--was also found to be impaired in mutants of all six genes. An important common feature is that all are expressed most strongly in seam cells, rather than in the main hypodermal syncytium, indicating that seam cells play the major role in secreting surface coat and consequently in determining environmental interactions. To test for possible redundancies in gene action, the 15 double mutants for this set of genes were constructed and examined, but no synthetic phenotypes were observed. Comparison of the six genes shows that each has distinctive properties, suggesting that they do not act in a linear pathway.

Identification of a Spatio-Temporal Enhancer Element for the Alzheimer’sAmyloid Precursor Protein-Like-1Gene in the NematodeCaenorhabditis elegans

During the larva-to-adult transition of the nematode Caenorhabditis elegans, expression of the Alzheimer's amyloid precursor protein-like gene, apl-1, is temporally regulated in seam cells. This regulation requires several transcription factors, including nuclear receptor NHR-25. Here we describe a critical cis-regulatory element of apl-1 transcription. The element is conserved among Caenorhabditis sister species and is similar to nuclear receptor binding consensus.

Regulation of C. elegans Fat Uptake and Storage by Acyl-CoA Synthase-3 Is Dependent on NR5A Family Nuclear Hormone Receptor nhr-25

Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting thechallenge of understanding metabolic pathwayswithin intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fattyacyl-CoAs, perhaps incorporated into complexligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C.elegans.

Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

BackgroundThe spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.ResultsWe have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.ConclusionOur results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2–L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Stem cells, from the ground up (Caenorhabditis elegans as a Model for Stem Cell Biology by Pradeep M. Joshi, Misty R. Riddle, Nareg J.V. Djabrayan, and Joel H. Rothman, Dev Dyn 239:1539–1554) C. elegans, may not be the first model system that comes to mind when considering questions of stem cell biology. The organism's only true stem cells are the germline, and it cannot be maintained in cell culture. Nevertheless the amenity of C. elegans to genetic, cell, and molecular manipulations have allowed scientists to use innovative approaches to milk additional information from other cell populations with stem-cell like properties. Epidermal seam cells balance proliferation with differentiation, embryonic progenitor cells progress from a state of pluripotency to cell fate commitment, and the “Y” rectal epithelial cell naturally transforms into a neuron during larval development. This review illustrates how the different cell populations have illuminated mechanisms behind stem cell-niche interactions, self-renewal, cell fate restriction, and transdifferentiation. Although discovered in the lowly worm, many of these themes are proving to be conserved in mammalian systems. Developing a human embryo atlas (Developmental Atlas of the Early First Trimester Human Embryo by Shigehito Yamada, Rajeev R. Samtani, Elaine S. Lee, Elizabeth Lockett, Chigako Uwabe, Kohei Shiota, Stasia A. Anderson, and Cecilia W. Lo, Dev Dyn 239:1585–1595) Many believe that the goal of developmental biology research is to inform human health. However, because our knowledge of human embryogenesis is based largely on drawings and wax models, it lags far behind that of popular model systems, making accurate comparisons impossible. Here, Yamada and colleagues present a much-needed, updated Human Embryo Atlas (http://apps.devbio.pitt.edu/humanatlas/; login: “Human”, password: “Embryo”) encompassing most of the first trimester, when development of major organs and tissues take place. The high-resolution images captured by 21st century techniques—magnetic resonance (MR) imaging and episcopic fluorescence image capture (EFIC)—can be virtually sectioned from any angle, and used to make three-dimensional (3D) reconstructions. The versatile images will help identify potential differences between development of humans and the model organisms that have so far informed our current understanding of vertebrate development, and will facilitate clinical assessments of embryonic health. dalmatian's new trick (Staying Alive: Dalmatian Mediated Blocking of Apoptosis Is Essential for Tissue Maintenance by Bilal E. Kerman and Deborah J. Andrew, Dev Dyn 239:1609–1621) The novel Drosophila nuclear protein Dalmatian (Dmt) was already known for one trick: regulating mitotic spindle assembly. Here, Kerman and Andrew show that dmt also keeps specific cell types from playing dead. In dmt mutants, trachea and salivary gland tubulogenesis is spotty—with networks displaying frequent gaps and breaks. The mutants also show increased expression of the apoptotic activators reaper (rpr) and head involution defective (hid), and the trachea displays enhanced apoptosis during latter stages of morphogenesis (st. 12–14). Dmt salivary gland, and trachea defects are rescued in the background of a deficiency that removes apoptotic inhibitors, and by tissue-specific expression of an anti-apoptotic protein, p35. Of interest, st. 12–14 dmt mutants are sensitized to radiation-induced apoptosis. Because Dmt localizes to heterochromatin, the authors speculate that the protein participates in known epigenetic silencing of rpr and hid during these stages. Dmt's role repressing apoptotic genes probably occurs after cells have ceased dividing, and thus is independent from it's role in mitotic spindle assembly. Always eager to please, Dmt can perform two different tricks.

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3'-phosphoadenosine 5'-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.

C. elegans CAND-1 regulates cullin neddylation, cell proliferation and morphogenesis in specific tissues

Cullin-RING ubiquitin ligases (CRLs) are critical regulators of multiple developmental and cellular processes in eukaryotes. CAND1 is a biochemical inhibitor of CRLs, yet has been shown to promote CRL activity in plant and mammalian cells. Here we analyze CAND1 function in the context of a developing metazoan organism. Caenorhabditis elegans CAND-1 is capable of binding to all of the cullins, and we show that it physically interacts with CUL-2 and CUL-4 in vivo. The covalent attachment of the ubiquitin-like protein Nedd8 is required for cullin activity in animals and plants. In cand-1 mutants, the levels of the neddylated isoforms of CUL-2 and CUL-4 are increased, indicating that CAND-1 is a negative regulator of cullin neddylation. cand-1 mutants are hypersensitive to the partial loss of cullin activity, suggesting that CAND-1 facilitates CRL functions. cand-1 mutants exhibit impenetrant phenotypes, including developmental arrest, morphological defects of the vulva and tail, and reduced fecundity. cand-1 mutants share with cul-1 and lin-23 mutants the phenotypes of supernumerary seam cell divisions, defective alae formation, and the accumulation of the SCF LIN-23 target the glutamate receptor GLR-1. The observation that cand-1 mutants have phenotypes associated with the loss of the SCF LIN-23 complex, but lack phenotypes associated with other specific CRL complexes, suggests that CAND-1 is differentially required for the activity of distinct CRL complexes.