Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis

The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pulldown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C. elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs.     

rib-2, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes encodes a novel alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate

The proteins encoded by the EXT1, EXT2, and EXTL2 genes, members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the heparan sulfate biosynthesis. Only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes were identified in the Caenorhabditis elegans genome. Although heparan sulfate is found in C. elegans, the involvement of the rib-1 and rib-2 proteins in heparan sulfate biosynthesis remains unclear. In the present study, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The present findings revealed that the rib-2 protein was a unique alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. In contrast, the findings confirmed the previous observations that both the EXT1 and EXT2 proteins were heparan sulfate copolymerases with both alpha1,4-N-acetylglucosaminyltransferase and beta1,4-glucuronyltransferase activities, which are involved only in the elongation step of the heparan sulfate chain, and that the EXTL2 protein was an alpha1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These findings suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that reported for the mammalian system.     

C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans

The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.     

Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.     

Embryonic fibroblasts with a gene trap mutation in Ext1 produce short heparan sulfate chains

Mutational defects in either EXT1 or EXT2 genes cause multiple exostoses, an autosomal hereditary human disorder. The EXT1 and EXT2 genes encode glycosyltransferases that play an essential role in heparan sulfate chain elongation. In this study, we have analyzed heparan sulfate synthesized by primary fibroblast cell cultures established from mice with a gene trap mutation in Ext1. The gene trap mutation results in embryonic lethality, and homozygous mice die around embryonic day 14. Metabolic labeling and immunohistochemistry revealed that Ext1 mutant fibroblasts still produced small amounts of heparan sulfate. The domain structure of the mutant heparan sulfate was conserved, and the disaccharide composition was similar to that of wild type heparan sulfate. However, a dramatic difference was seen in the polysaccharide chain length. The average molecular sizes of the heparan sulfate chains from wild type and Ext1 mutant embryonic fibroblasts were estimated to be around 70 and 20 kDa, respectively. These data suggest that not only the sulfation pattern but also the length of the heparan sulfate chains is a critical determinant of normal mouse development.     

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.     

Transgenic expression of the EXT2 gene in developing chondrocytes enhances the synthesis of heparan sulfate and bone formation in mice

Hereditary multiple exostoses (HME), a dominantly inherited disorder characterized by multiple cartilaginous tumors, is caused by mutations in the gene for, EXT1 or EXT2. Recent studies have revealed that EXT1 and EXT2 are required for the biosynthesis of heparan sulfate and exert maximal transferase activity as a complex. The Drosophila homologue of EXT1 (tout-velu) regulates the movement and signaling of Hedgehog protein, which plays an important role in the regulation of chondrocyte differentiation and bone development. In this study, to investigate the biological role of EXT2 in bone development in vivo and the pathological role of HME mutations in the development of exostoses, we generated transgenic mice expressing EXT2 or mutant EXT2 in developing chondrocytes. Histological analyses and micro-CT scanning showed that the biosynthesis of heparan sulfate and the formation of trabeculae were upregulated in EXT2-transgenic mice, but not in mutant EXT2-transgenic mice. The expression of EXT1 is concomitantly upregulated in EXT2-transgenic and even mutant EXT2-transgenic mice, suggesting an interactive regulation of EXT1 and EXT2 expression. These findings support that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation.     

Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice

Mutations in the EXT1 gene are responsible for human hereditary multiple exostosis type 1. The Drosophila EXT1 homologue, tout-velu, regulates Hedgehog diffusion and signaling, which play an important role in tissue patterning during both invertebrate and vertebrate development. The EXT1 protein is also required for the biosynthesis of heparan sulfate glycosaminoglycans that bind Hedgehog. In this study, we generated EXT1-deficient mice by gene targeting. EXT1 homozygous mutants fail to gastrulate and generally lack organized mesoderm and extraembryonic tissues, resulting in smaller embryos compared to normal littermates. RT-PCR analysis of markers for visceral endoderm and mesoderm development indicates the delayed and abnormal development of both of these tissues. Immunohistochemical staining revealed a visceral endoderm pattern of Indian hedgehog (Ihh) in wild-type E6.5 embryos. However, in both EXT1-deficient embryos and wild-type embryos treated with heparitinase I, Ihh failed to associate with the cells. The effect of the EXT1 deletion on heparan sulfate formation was tested by HPLC and cellular glycosyltransferase activity assays. Heparan sulfate synthesis was abolished in EXT1 -/- ES cells and decreased to less than 50% in +/- cell lines. These results indicate that EXT1 is essential for both gastrulation and heparan sulfate biosynthesis in early embryonic development.     

Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.     

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases,in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.     

pha-2 encodes the C. elegans ortholog of the homeodomain protein HEX and is required for the formation of the pharyngeal isthmus

The pha-2 mutant was isolated in 1993 by Leon Avery in a screen for worms with visible defects in pharyngeal feeding behavior. In pha-2 mutant worms, the pharyngeal isthmus is abnormally thick and short and, in contrast to wild-type worms, harbors several cell nuclei. We show here that pha-2 encodes a homeodomain protein and is homologous to the vertebrate homeobox gene, Hex (also known as Prh). Consistent with a function in pharyngeal development, the pha-2 gene is expressed in the pharyngeal primordium of Caenorhabditis elegans embryos, particularly in pm5 cells that form the bulk of the isthmus. We show that in the pha-2 mutant there is a failure of the pm5 cells to elongate anteriorly while keeping their nuclei within the nascent posterior bulb to form the isthmus during the 3-fold embryonic stage. We also present evidence that pha-2 regulates itself positively in pm5 cells, that it is a downstream target of the forkhead gene pha-4, and that it may also act in the isthmus as an inhibitor of the ceh-22 gene, an Nkx2.5 homolog. Finally, we have begun characterizing the regulation of the pha-2 gene and find that intronic sequences are essential for the complete pha-2 expression profile. The present report is the first to examine the expression and function of an invertebrate Hex homolog, that is, the C. elegans pha-2 gene.     

Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2

Background

Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling.

Mitochondrial genome content is regulated during nematode development

Growth and development rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. We measured the mitochondrial DNA (mtDNA) copy number of each of the Caenorhabditis elegans developmental stages. Embryos, L1, L2, and L3 larvae all have approximately 25,000 copies of maternally derived mtDNA. The copy number increases fivefold in L4 larvae and a further sixfold in adult hermaphrodites, but only twofold in adult males. The majority of mtDNA in adult worms is germline associated, and germline-deficient mutants show markedly reduced mtDNA contents. With sperm-deficient or oocyte-deficient mutants, we confirm that mtDNA amplification is primarily associated with oocyte production. When mtDNA replication is inhibited, a quantitative and homogeneous arrest as L3 larvae occurs. Thus, mtDNA amplification is a necessary component of normal development and its regulation may involve an energy-sensing decision or checkpoint that can be invoked when mitochondrial energy generation is impaired.     

Hereditary multiple exostoses and heparan sulfate polymerization

Hereditary multiple exostoses (HME, OMIM 133700, 133701) results from mutations in EXT1 and EXT2, genes encoding the copolymerase responsible for heparan sulfate (HS) biosynthesis. Members of this multigene family share the ability to transfer N-acetylglucosamine to a variety of oligosaccharide acceptors. EXT1 and EXT2 encode the copolymerase, whereas the roles of the other EXT family members (EXTL1, L2, and L3) are less clearly defined. Here, we provide an overview of HME, the EXT family of proteins, and possible models for the relationship of altered HS biosynthesis to the ectopic bone growth characteristic of the disease.     

A new putative cyclic nucleotide-gated channel gene, cng-3, is critical for thermotolerance in Caenorhabditis elegans

Cyclic nucleotide-gated (CNG) channels encoded by tax-4 and tax-2 genes are required for chemo- and thermo-sensation in Caenorhabditis elegans. Here we report the identification and the characterization of cng-3, a new CNG channel gene, found in C. elegans. CNG-3 contains six putative transmembrane regions and a cyclic nucleotide-binding domain that show high homology with CNG channels of higher animals as well as TAX-4. The expression of cng-3 is detected from early stages in worm development and restricted in five sensory neurons of amphid including AFD neuron. While a cng-3 null mutant displays normal chemotaxis to volatile odorants, the mutant worms exhibit impaired thermal tolerance. These results indicate that CNG-3, a new member of CNG channel subunits, may play a critical role in sensation or response of thermal stress in C. elegans.     

A sperm-supplied product essential for initiation of normal embryogenesis in Caenorhabditis elegans is encoded by the paternal-effect embryonic-lethal gene, spe-11

Loss-of-function mutations in the spe-11 gene in Caenorhabditis elegans result in a paternal-effect embryonic-lethal phenotype: fertilization of wild-type oocytes by sperm from homozygous spe-11 mutant males leads to abnormal zygotic development, whereas oocytes from homozygous spe-11 hermaphrodites when fertilized by wild-type sperm develop normally. Embryos fertilized by sperm from homozygous spe-11 worms fail to complete meiosis and show defects in eggshell formation, mitotic spindle orientation, and cytokinesis. Genetic analysis suggests that the spe-11 gene is expressed before the completion of spermatogenesis and that the wild-type locus encodes a product that is present in sperm and participates, directly or indirectly, in initiating the correct program of early events in C. elegans embryos. Such an ontogenetic role of the spe-11+ gene product in early embryogenesis distinguishes spe-11 mutations from the two paternal-effect mutations identified in Drosophila, ms(3)K81 and pal, which primarily affect chromosome behavior. Analysis of spe-11 provides the first step toward genetic dissection of the functions of the sperm in early embryogenesis in C. elegans.     

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using "optical tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.