Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

CBD-1 organizes two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans.

Metazoan eggs have a specialized coat of extracellular matrix that aids in sperm-egg recognition. The coat is rapidly remodeled after fertilization to prevent polyspermy and establish a more permanent barrier to protect the developing embryo. In nematodes, this coat is called the vitelline layer, which is remodeled into the outermost layer of a rigid and impermeable eggshell. We have identified three key components of the vitelline layer structural scaffold - PERM-2, PERM-4 and CBD-1, the first such proteins to be described in the nematode C. elegans. CBD-1 tethered PERM-2 and PERM-4 to the nascent vitelline layer via two N-terminal chitin-binding domains. After fertilization, all three proteins redistributed from the zygote surface to the outer eggshell. Depletion of PERM-2 and PERM-4 from the scaffold led to a porous vitelline layer that permitted soluble factors to leak through the eggshell and resulted in embryonic death. In addition to its role in vitelline layer assembly, CBD-1 is also known to anchor a protein complex required for fertilization and egg activation (EGG-1-5/CHS-1/MBK-2). We found the PERM complex and EGG complex to be functionally independent, and structurally organized through distinct domains of CBD-1. CBD-1 is thus a multifaceted regulator that promotes distinct aspects of vitelline layer assembly and egg activation. In sum, our findings characterize the first vitelline layer components in nematodes, and provide a foundation through which to explore both conserved and species-specific strategies used by animals to build protective barriers following fertilization.

Hierarchical assembly of the eggshell and permeability barrier in C. elegans.

In metazoans, fertilization triggers the assembly of an extracellular coat that constitutes the interface between the embryo and its environment. In nematodes, this coat is the eggshell, which provides mechanical rigidity, prevents polyspermy, and is impermeable to small molecules. Using immunoelectron microscopy, we found that the Caenorhabditis elegans eggshell was composed of an outer vitelline layer, a middle chitin layer, and an inner layer containing chondroitin proteoglycans. The switch between the chitin and proteoglycan layers was achieved by internalization of chitin synthase coincident with exocytosis of proteoglycan-containing cortical granules. Inner layer assembly did not make the zygote impermeable as previously proposed. Instead, correlative light and electron microscopy demonstrated that the permeability barrier was a distinct envelope that formed in a separate step that required fatty acid synthesis, the sugar-modifying enzyme PERM-1, and the acyl chain transfer enzyme DGTR-1. These findings delineate the hierarchy of eggshell assembly and define key molecular mechanisms at each step.

Acute drug treatment in the early C. elegans embryo.

Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.

Evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling.

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.

lin-35/Rb and the CoREST ortholog spr-1 coordinately regulate vulval morphogenesis and gonad development in C. elegans.

Using a genetic screen to identify genes that carry out redundant functions during development with lin-35/Rb, the C. elegans Retinoblastoma family ortholog, we have identified a mutation in spr-1. spr-1 encodes the C. elegans ortholog of human CoREST, a protein containing Myb-like SANT and ELM2 domains, which functions as part of a transcriptional regulatory complex. CoREST recruits mediators of transcriptional repression, including histone deacetylase, and demethylase, and interacts with the tumor suppression protein REST. spr-1/CoREST was previously shown in C. elegans to suppress defects associated with loss of the presenilin sel-12, which functions in the proteolytic processing of LIN-12/Notch. Here we show that lin-35 and spr-1 coordinately regulate several developmental processes in C. elegans including the ingression of vulval cells as well as germline proliferation. We also show that loss of lin-35 and spr-1 hypersensitizes animals to a reduction in LIN-12/Notch activity, leading to the generation of proximal germline tumors. This defect, which is observed in lin-35; spr-1; lin-12(RNAi) and lin-35; spr-1; hop-1(RNAi) triple mutants is likely due to a delay in the entry of germ cells into meiosis.

Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2.

Vertebrates produce multiple chondroitin sulfate proteoglycans that play important roles in development and tissue mechanics. In the nematode Caenorhabditis elegans, the chondroitin chains lack sulfate but nevertheless play essential roles in embryonic development and vulval morphogenesis. However, assignment of these functions to specific proteoglycans has been limited by the lack of identified core proteins. We used a combination of biochemical purification, Western blotting, and mass spectrometry to identify nine C. elegans chondroitin proteoglycan core proteins, none of which have homologues in vertebrates or other invertebrates such as Drosophila melanogaster or Hydra vulgaris. CPG-1/CEJ-1 and CPG-2 are expressed during embryonic development and bind chitin, suggesting a structural role in the egg. RNA interference (RNAi) depletion of individual CPGs had no effect on embryonic viability, but simultaneous depletion of CPG-1/CEJ-1 and CPG-2 resulted in multinucleated single-cell embryos. This embryonic lethality phenocopies RNAi depletion of the SQV-5 chondroitin synthase, suggesting that chondroitin chains on these two proteoglycans are required for cytokinesis.

Caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis.

Defects in glycosaminoglycan biosynthesis disrupt animal development and can cause human disease. So far much of the focus on glycosaminoglycans has been on heparan sulphate. Mutations in eight squashed vulva (sqv) genes in Caenorhabditis elegans cause defects in cytokinesis during embryogenesis and in vulval morphogenesis during postembryonic development. Seven of the eight sqv genes have been shown to control the biosynthesis of the glycosaminoglycans chondroitin and heparan sulphate. Here we present the molecular identification and characterization of the eighth gene, sqv-5. This gene encodes a bifunctional glycosyltransferase that is probably localized to the Golgi apparatus and is responsible for the biosynthesis of chondroitin but not heparan sulphate. Our findings show that chondroitin is crucial for both cytokinesis and morphogenesis during C. elegans development.

The Caenorhabditis elegans genes sqv-2 and sqv-6, which are required for vulval morphogenesis, encode glycosaminoglycan galactosyltransferase II and xylosyltransferase.

In mutants defective in any of eight Caenorhabditis elegans sqv (squashed vulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization of sqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAbeta1,3Galbeta1, 3Galbeta1,4Xylbeta-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans.