Neitzel LR , Spencer ZT , Nayak A , Cselenyi CS , Benchabane H , Youngblood CQ , Zouaoui A , Ng V , Stephens L , Hann T , Patton JG , Robbins D , Ahmed Y , Lee E .

R01 GM121421 NIGMS NIH HHS , R01 GM122222 NIGMS NIH HHS , R35 GM122516 NIGMS NIH HHS , R01 CA105038 NCI NIH HHS , UL1 TR000445 NCATS NIH HHS , R01 CA219189 NCI NIH HHS , R01 EY024354 NEI NIH HHS , P30 DK058404 NIDDK NIH HHS

	Fig. 1. Xenopus embryos are posteriorized by Nagk overexpression and anteriorized by Nagk downregulation, respectively. (A) Injection of Nagk mRNA (1.5 ng) posteriorizes Xenopus embryos, and can be suppressed by coinjecting Nagk MO (1 pg) or 3-OMe-GlcNAc (125 pmol). (Right) Representative embryo posteriorized upon injection of NAGK mRNA is shown. (B) Injection of Nagk MO (1 pg), NAGKT128M mRNA (1.5 ng), or 3-OMe-GlcNAc (125 pmol) anteriorizes Xenopus embryos. (Right) Representative anteriorized embryo injected with Nagk MO. (C) Nagk protein (20 pg) and Drosophila Nagk (DNagk) mRNA (1.5 ng) posteriorizes Xenopus embryos (A–C) Aggregated from n ≥ 3 replicates. Embryos were scored for anteriorization or posteriorization according to the dorsal-anterior index (DAI) as previously described (Kao and Elinson). DAI of ventralized embryos ranged from 4 to 2, whereas dorsalized embryos ranged from 6 to 8. Absolute numbers are indicated above bars. Significance was calculated using Fisher's exact test with Bonferroni correction. **ρ < 0.00334, ***ρ < 0.000334, and *ρ < 0.0253.
	Fig. 2. The anterior-posterior patterning defect caused by altered Nagk activity in Xenopus embryos is phenocopied upon disruption of other UDP-GlcNAc salvage pathway components. (A) Overexpression of enzymes from the UDP-GlcNAc glycosylation salvage pathway posteriorizes Xenopus embryos. (B) In contrast, downregulating enzymes of the salvage pathway by MO anteriorizes Xenopus embryos. (C) Injection of the sugars from the UDP-GlcNAc salvage pathway, similar to overexpression of their enzymes, results in posteriorized embryos. (D) Injection of mRNA encoding Ngly1, which removes N-linked glycans from glycoproteins, posteriorizes Xenopus embryos. 1.5 ng of mRNA, 1 pg MO, or 125 pmol compounds were used per injection. Results are aggregates of n ≥ 3 replicates. Absolute numbers are indicated above bars. Significance was calculated using Fisher's exact test with Bonferroni correction. (A–C) *ρ < 0.0127, **ρ < 0.00250, ***ρ < 0.000250. (D) **ρ < 0.01.
	Fig. 7. The UDP-GlcNAC salvage pathway acts at the level of the Wnt ligand and/or receptors. (A–C, Left) Activation of the Wnt pathway by Xwnt8, Wnt8-Fz5, and Lrp6 is inhibited by co-injection with MOs against salvage pathway enzymes as indicated by expression of the Wnt target gene, Xnr3. (A–C; Right) Conversely, co-injection with mRNAs encoding pathway enzymes enhanced Xnr3 expression. (D) In contrast, activation of the Wnt pathway by Dsh was unaffected by inhibition (Left) or overexpression (Right) of salvage pathway enzymes. Data shown are representative of n ≥ 3 biological replicates using n = 3 technical replicates. Each replicate was a pool of n = 5 animal caps. Each dorsal blastomere was injected or co-injected with 1 ng mRNA or 1 pg MO. Expression was normalized to Odc. Graphs display fold change calculated using the 2−ΔΔCt method. Significance was calculated using t-tests with equal variance with Bonferroni correction on the ΔCt values. *ρ < 0.0127, **ρ < 0.00251, ***ρ < 0.000251.
	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 S5. Soaking Xenopus embryos with GlcNAc or tunicamycin is sufficient to disrupt anterior-posterior patterning. (A) Soaking Xenopus embryos in 2.5% (w/v) GlcNAc results in posteriorized embryos. (B) Conversely, soaking embryos in 2.5% (w/v) tunicamycin resulted in anteriorized embryos. (C) Images of representative embryos soaked in GlcNAc or tunicamycin. Absolute numbers are indicated above bars. Significance was calculated using Fisher’s exact test. WT, wild-type. ***ρ<.001.
	Figure S6. Rescue of overexpression phenotypes of individual salvage pathway enzymes in Xenopus embryos by inhibiting downstream components of the UDP-GlcNAc pathway. (A) Co-injecting Nagk MO, Pgm3 MO, Uap1 MO, or Dpagt1 MO rescues the posteriorization phenotype of Nagk mRNA injections. (B) Co-injecting Nagk MO, Pgm3 MO, Uap1 MO, or Dpagt1 MO rescues the posteriorization phenotype of Pgm3 mRNA injections. (C) Co-injecting Nagk MO, Pgm3 MO, Uap1 MO, or Dpagt1 MO rescues the posteriorization phenotype of Uap1 mRNA injections. (D) Co-injecting Nagk MO, Pgm3 MO, Uap1 MO, or Dpagt1 MO rescues the posteriorization phenotype of Dpagt1 mRNA injections. Absolute numbers are indicated above bars. Results are aggregates of n≥3 replicates. Each dorsal blastomere was injected with 1.5 ng mRNA and/or 1 pg MO. Significance was calculated using Fisher’s exact test with Bonferroni correction. *ρ< 0.0127, **ρ< 0.00251, ***ρ< 0.000251.
	Figure S7. The phenotype cause by disrupting UDP-GlcNAc salvage pathway components in Xenopus embryos is rescued by injecting downstream sugar intermediates. (A) Co-injecting GlcNAc-6-P, GlcNAc-1-P, or UDP-GlcNAc rescues the anteriorization phenotype caused by NagkT128M mRNA injection. (B) Co-injecting GlcNAc-6-P, GlcNAc-1-P, or UDP-GlcNAc rescues the anteriorization phenotype caused by 3-OMe-GlcNAc injection. (C) Co-injecting GlcNAc-6-P, GlcNAc-1-P, or UDP-GlcNAc rescues the anteriorization phenotype caused by Nagk MO injection. (D) Co-injecting GlcNAc-1-P or UDP-GlcNAc rescues the anteriorization phenotype caused by Pgm3 MO injection. (E) Co-injecting UDP-GlcNAc rescues the anteriorization phenotype caused by Uap1 MO injections. Absolute numbers are indicated above bars. Results are aggregates of n≥3 replicates. Each dorsal blastomere was injected with 1.5 ng mRNA, 1 pg MO, and/or 125 pmol sugar. Significance was calculated using Fisher’s exact test with Bonferroni correction. (A-C) *ρ< 0.0170, **ρ< 0.00334, ***ρ< 0.000334. (D) *ρ< 0.0253. (E) *ρ< 0.05.
	Figure S8. The phenotype caused by injecting UDP-GlcNAc salvage pathway sugars in Xenopus embryos is rescued by downregulating salvage pathway enzymes. (A) Co-injecting NagkT128M mRNA or 3-OMe-GlcNAc rescued the posteriorization phenotype caused by GlcNAc injection. (B) Co-injecting Nagk MO, Pgm3 MO, Uap1 MO, or Dpagt1 MO rescued the posteriorization phenotype caused by GlcNAc injection. (C) Co-injecting Pgm3 MO, Uap1 MO, or Dpagt1 MO rescued the posteriorization phenotype caused by GlcNAc-6-P injection. (D) Co-injecting Uap1 MO or Dpagt1 MO rescued the posteriorization phenotype caused by GlcNAc-1-P injections. (E) Co-injecting Dpagt1 MO rescued the posteriorization phenotype caused by UDP-GlcNAc injections. Absolute numbers are indicated above bars. Each dorsal blastomere was injected or co-injected with 1.5 ng mRNA, 1 pg MO, and/or 125 pmol sugar. Significance was calculated using Fisher’s exact test with Bonferroni correction. (A and D) *ρ< 0.0253, **ρ< 0.00501. (B) *ρ< 0.0127, **ρ< 0.00251. (C) *ρ< 0.0170, ***ρ< 0.00334. (E) *ρ< 0.05.
	Figure S9. The UDP-GlcNAc salvage pathway selectively regulates Wnt signaling in the early Xenopus embryo. (A) (Top) Expression of Xnr3, a Wnt target gene, in whole embryos. (Left) Inhibition of the UDP-GlcNAc salvage pathway suppresses Xnr3 expression. (Middle) Overexpression of UDP-GlcNAc salvage pathway enzymes or (Right) injection of salvage pathway sugars increases expression of Xnr3 in whole embryos. (Bottom) Similar effects are observed on the expression of Chordin, another Wnt target gene. (B) Fgf, Bmp, Notch, and Shh target genes are unaffected by alterations in the UDP-GlcNAc salvage pathway in the early Xenopus embryo. Dusp6, Tbxt, Hes1, and Ptch are target genes of the Fgf, Bmp, Notch, and Shh pathways, respectively. Data shown is representative of n≥3 biological replicates using n=3 technical replicates. Each replicate was a pool of n=5 whole embryos. Both cells of a 2-cell embryo were injected or co-injected with 2.5 ng mRNA, 2.5 pg MO, or 125 pmol sugar and embryos analyzed at stage 10.5 (Wnt, Fgf, and Bmp), stage 13 (Notch), or stage 16 (Shh). Expression was normalized to Odc. Graphs display fold change calculated using the 2-ΔΔCt method. Significance was calculated using T-test with equal variance and Bonferroni correction on the ΔCt values. (A) (left) *ρ<0.008512, **ρ<0.001674, ***ρ<0.000167. (Middle) and (Right) *ρ<0.012741, **ρ<0.002509, ***ρ<0.000251. No significant ρ values were found for (B).
	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 S13. Expression of Chordin in Xenopus ectodermal explants suggests the UDP-GlcNAc salvage pathway works at the level of the ligand and/or receptor. (A) (Left) Activation of the Wnt pathway by injection of Xwnt8 mRNA (as indicated by expression of the Wnt target gene, Chordin) is inhibited when co-injected with MOs against salvage pathway enzymes. (Right) Conversely, co-injection with mRNA encoding salvage pathway enzymes enhances Chordin expression. Similarly, induction of Chordin by Wnt8-Fz5 (B) or Lrp6 (C) was inhibited or enhanced upon co-injection with MOs or mRNA for salvage pathway enzymes, respectively. (D) In contrast, activation of the Wnt pathway by Dsh was unaffected by inhibition (Left) or overexpression (Right) of the salvage pathway enzymes. Data shown is representative of n≥3 biological replicates using n=3 technical replicates. Each replicate was a pool of n=5 animal caps. Each dorsal blastomere was injected with 1 ng mRNA and/or 1 pg MO. Expression was normalized to Odc. Graphs display fold change calculated using the 2-ΔΔCt method. Significance was calculated using T-tests with equal variance and Bonferroni correction on the ΔCt values. *ρ< 0.0127, **ρ<0.00251, ***ρ<0.000251.
	Figure S14. Model for the regulation of Wnt signaling by the UDP-GlcNAc pool. See text for more detail.

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