ppXyl-T RNAi impairs cytokinesis and leads to an egg-laying defect, multinucleated embryos, and embryonic arrest. A time-lapse panel demonstrates normal cell division in the early C. elegans wild-type embryo (A) and the development of multinucleated embryos after ppXyl-T RNAi treatment (B). (C) N2 oviduct and uterus of a gravid wild-type adult. (D) ppXyl-T RNAi results in an egg-laying defective adult, which is packed with multinucleated embryos that exhibit defects in early embryo cytokinesis.

ppXyl-T RNAi of embryos with GFP-tagged marker proteins demonstrates normal spindle behavior and partitioning properties at the first and second nuclear divisions. (A) Histone::GFP shows normal karyokinesis and a normal initiation of cytokinesis. The spindle is asymmetrically positioned (blue arrows). When cytokinesis is nearly complete, the cleavage furrow begins to regress (green arrows) and an intercellular bridge forms. The daughter cells then loose their rounded shape, whereas the cleavage furrow completely disappears (red arrows). Significantly more cell membrane disappears from the cleavage plane, whereas the spindle apparatus duplicates and begins to initiate the next round of nuclear division. (B) tubulin::GFP shows that the spindle is actively duplicating, migrating and initiating karyokinesis, in the absence of proteoglycans in ppXyl-T RNAi-treated embryos. Centrosome duplication occurs and the microtubule arrays interdigitate (C) PIE-1::GFP of a normal two-cell embryo (top) and in ppXyl-T RNAi-treated embryos (bottom two panels). The PIE-1::GFP signal is present in the most posterior nucleus. Embryos are orientated so that anterior is to the left.

Y110A2AL.14/SQV-2 is a member of the β1,3 Gal-T family in C. elegans. (A) Amino acid sequence alignments of the sugar-donor substrate-binding site of 21 putative C. elegans β1,3 Gal-T proteins and the human Gal-T2 protein. (B) Phylogenetic analysis based on this catalytic domain, shown above. Annotations list the glycosylation pathway of the most closely conserved mammalian sequence homologue for each worm gene. Type-1 oligos refers to type-1 Galβ1,3GlcNAc-R oligosaccharides. The actual sugar structures that are synthesized by most of these enzymes are not known, but they fall in a family of sugars with a β1,3-linkage. (C) Gal-T2 is in the proteoglycan biosynthesis pathway. The pathway begins with an initiation reaction, the transfer of xylose to a serine residue in a proteoglycan, by ppXyl-T. A tetrasaccharide linker (GlcA-Gal-Gal-Xyl-serine) is built through the sequential action of individual glycosyltransferases that add one sugar at a time, after the xylose modification of peptide sequences has occurred. C. elegans Gal-T2 is encoded by Y110A2AL.14 or sqv-2 and is a galactoslytransferase that specifically adds the third sugar residue in the linker tetrasaccharide. Gal-T1 and GlcA-T are encoded by the sqv-3 and sqv-8 genes, respectively. Glycosaminoglycan (GAG) chains chondroitin and heparan biosynthesis require the tetrasaccharide linker.

Gal-T2 localization and detection in C. elegans. (A) Anti-Gal-T2 scFv antibody staining alone (top) or with DAPI nuclear counterstain (bottom) in an eight-cell embryo. (B) High magnification of Golgi apparatus staining and nuclei from embryo in A. (C) Anti-Gal-T2 scFv antibody staining in a 28-cell embryo shows perinuclear staining pattern. (D) GFP fluorescence staining and DIC of intestinal cells from transgenic animals expressing a fusion of the N terminus and stem of Gal-T2 (1–60 aa) to a GFP reporter. Two nuclei are present in the GFP-positive cell. The GFP fluorescence surrounds the left nucleus and forms a patch next to the right nucleus. The nuclei themselves do not exhibit a GFP signal. (E) Structure and design of the wild-type Gal-T2 gene and the Gal-T2 stem::GFP reporter. GFP is fused to amino acid codon 60 in the second exon, such that GFP replaces the C-terminal catalytic domain of Gal-T2. (F) Western blot detection of Gal-T2 with anti-Gal-T2 scFv antibody. Lanes 1 and 2, 50 and 20 ng of recombinant Gal-T2, lacking the N-terminal transmembrane domain. In place of the transmembrane domain, a 38-amino acid purification tag is genetically fused to the stem and catalytic domain of Gal-T2 (see Materials and Methods for details). Lanes 3 and 4, 40 and 15 ng of recombinant Gal-T2 after peptide:N-glycanase and O-glycanase treatment.

Gal-T2 depletion impairs embryonic development. (A) Quantitative reverse transcription-PCR on wild-type and Gal-T2 RNAi-treated embryos shows a 18-fold reduction in Gal-T2 mRNA. RNA levels from 20 embryos in the two preparations are normalized against an ama-1 control mRNA. (B) Q-RT-PCR of Gal-T2 message from the following staged C. elegans RNA preparations: oocytes, embryos, day 1 (L1) larva, day 2 (L2/L3) larva, day 3 (L4 larva and young adults), and day 4 (gravid adults). Identical quantities of total RNA from each staged preparation are used in Quantitative RT-PCR. (C–F) Induction of Gal-T2 RNAi on lactose plates. (G and H) Induction of Gal-T2 RNAi on IPTG plates. (C) Bacterial feeding RNAi at 23°C on lactose plates for two generations. Cytokinesis defects were examined in the embryos laid on the plate and also while embryos were still in the uterus of gravid adults. (D) The uterus of three gravid adults from first generation Gal-T2 lactose-induced RNAi plates show multi-nucleated embryos, variation in egg size, and degrading embryos. (E) Embryos from second generation Gal-T2 lactose-induced RNAi plates. Bottom, DIC of the most common embryos after two generations of RNAi. Top, DAPI staining of nuclei. Middle, merges the DAPI stain (converted to black) over the DIC image. (F) Multinucleated embryos from second generation of RNAi feeding on lactose plates are deficient in cytokinesis. After a few hours, these multinucleated embryos begin to degrade and resemble embryos shown in Figure 4, E and G. (G) Egg size and shape defects and embryonic arrest in second generation of Gal-T2 RNAi treatment on IPTG plates. (H) Gal-T2 RNAi also produces partially developed embryos and larva, displaying a shortened or NOB (no backside) phenotype on IPTG plates.