XB-ART-55795Mech Dev January 1, 2019; 156 20-31.
Developmental regulation of Wnt signaling by Nagk and the UDP-GlcNAc salvage pathway.
In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFβ, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.
PubMed ID: 30904594
PMC ID: PMC6574174
Article link: Mech Dev
Genes referenced: chrd.1 dpagt1 dusp6 dvl2 fzd5 hes1 lrp6 nagk nodal nodal3.1 nodal3.2 notch1 odc1 pgm3 psmd6 ptch1 shh tbxt uap1 wnt8a
Morpholinos: dpagt1 MO1 nagk MO1 pgm3 MO1 uap1 MO1
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|Figure S1. Schematic for generating transcribed PCR products containing poly (A) tails (TPATs) for injecting Xenopus embryos. The Harvard Institute of Proteomics FLEXGene human kinase cDNA collection was used to amplify kinase coding regions. The 5’ primer was designed to introduce a T7 promoter to the 5’ end of the amplified PCR product. To increase the stability of the kinase mRNAs upon their injection into Xenopus embryos, a poly(A) sequence from the pCS2+ plasmid was also amplified by PCR. The products from the kinase and poly(A) reactions were then stitched together in a third PCR reaction to generate kinase cDNAs containing a 5’ T7 promoter and a 3’ poly (A) tail. These DNAs were subsequently used for in vitro transcription reactions to generate TPATs.|
|Figure S2. TPAT mRNAs demonstrate robust expression in Xenopus embryos. (A) Uninjected Xenopus embryos. (B) Xenopus embryo injected with GFP TPAT without the poly(A) tail demonstrates no observable expression of GFP compared to background. Xenopus embryos injected with GFP TPAT without (C) or with (D) purification of final PCR products show robust GFP expression. Each dorsal blastomere of a 2-cell embryo was injected with 1 ng mRNA and allowed to develop to neurula stages (20-21) prior to imaging. Results are aggregates of n≥3 replicates.|
|Figure S3. Schematic of the UDP-GlcNAc salvage pathway (A) Free GlcNAc is released from glycosylated proteins in lysosomes and transported to the cytosol. (B) In a series of reactions, the salvage pathway converts the free pool of cytosolic GlcNAc into UDP-GlcNAc. (C) UDP-GlcNAc is used in a reaction that covalently attaches GlcNAc to dolichol phosphate in the cytosol. The product, GlcNAc-PP-dolichol, is subsequently flipped into the ER lumen to initiate the process of N-glycosylation. GlcNAc, N-acetyl-D-glucosamine; GlcNAc-1-P, N-acetyl-D-glucosamine-1-Phosphate; GlcNAc-6-P, N-acetyl-D-glucosamine-6-phosphate; UDP-GlcNAc, Uridine diphosphate- N-acetyl-D-glucosamine; Nagk, N-acetylglucosamine kinase; Pgm3, N-acetylglucosamine-phosphate mutase; Uap1, UDP-N-acetylglucosamine pyrophosphorylase 1; Dpagt1, Dolichyl-phosphate N-acetylglucosamine phosphotransferase 1.|
|Figure S4. Representative Xenopus phenotypes caused by perturbation of the UDP-GlcNAc salvage pathway. (A) Representative images of posteriorized embryos due to DNagk mRNA injection and anteriorized embryos resulting from NagkT128M mRNA or 3-OMe-GlcNAc injection. (B) Representative images of posteriorized embryos due to injection of UDP-GlcNAc salvage pathway enzyme mRNAs. (C) Representative images of anteriorized embryos due to injection of MOs against the enzymes of the UDP-GlcNAc salvage pathway. (D) Representative images of embryos posteriorized by injection of sugars of the UDP-GlcNAc salvage pathway. (E) Representative images of embryos posteriorized or anteriorized by Dpagt1 mRNA or MO injections, respectively. (G) Representative image of an anteriorized embryo injected with Ngly1 mRNA. For injections, 1.5 ng mRNA, 1 pg MO, or 125 pmol compounds were used.|
|Figure S10. Each qRT-PCR primer set amplifies a single product. Final products from qRT-PCR reactions electrophoresed on a 2% agarose gel. Products are from whole, wild-type embryos. The sizes of each product are indicated (base pairs; bp). 1 kb plus DNA ladder (Invitrogen) is shown to the left of each sample. Ornithine decarboxylase 1, Odc; Patched 1, Ptch1; Dual specificity phosphatase 6, Dusp6. Hes family bHLH transcription factor 1, Hes1; T-box transcription factor T, Tbxt; Xenopus nodal homolog 3 gene 1, Xnr3; Chordin gene 1, Chordin.|
|Figure S14. Model for the regulation of Wnt signaling by the UDP-GlcNAc pool. See text for more detail.|