Kim YJ et al. (2025), Protein-O-fucosylation of coreceptors may be re...

XB-ART-61276

Mol Cells 2025 May 03;485:100207. doi: 10.1016/j.mocell.2025.100207.

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Protein-O-fucosylation of coreceptors may be required for Nodal signaling in Xenopus.

Kim YJ , Nho SJ , Lee SY , Yeo CY .

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Nodal-related ligands of TGF-β family play pivotal roles for mesoderm induction and body axis formation during vertebrate early embryogenesis. Nodal ligands are distinct from most other TGF-β ligands family as they require EGF-CFC factors as coreceptors for signaling, in addition to their cognate type I and type II TGF-β receptors. In amphibian Xenopus laevis embryos, 5 Nodal-related genes (Xnr1/2/4/5/6) and 2 EGF-CFC genes (XCR1, XCR3) play roles in mesoderm induction and the accumulation of phosphorylated Smad2, while in mammalian embryos, 1 Nodal gene and 1 EGF-CFC gene (Cripto) play roles during mesoderm induction. Mammalian EGF-CFC factors are reported to be O-fucosylated at a conserved threonine residue of the EGF-like motif by protein-O-fucosyltransferase 1 (Pofut1), but this O-fucose modification is shown to be dispensable for Nodal signaling in mammalian embryos. In this study, we investigated the developmental roles of Xenopus laevis Pofut1 (XPofut1) and its potential function in Nodal signaling. We found that morpholino antisense-mediated knockdown of XPofut1 causes reduction of Smad2 phosphorylation in late blastula and axial truncation in neurula. We also found that the O-fucosyltransferase activity of XPofut1 is important in the marginal zone, but not in the vegetal pole region, of blastula. Interestingly, XPofut1 is necessary for Smad2 phosphorylation induced by Xnr1 or Xnr2, but not by Xnr5 or Xnr6. Among the Nodal signaling components, only EGF-CFC factors are known to be modified by Pofut1. Therefore, based on our current observation, we propose that XPofut1 regulates signaling of a subset of nodal ligands in pregastrulation embryos possibly through modulating the function of EGF-CFC factors.

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Species referenced: Xenopus laevis
Genes referenced: acvr1b acvr1c acvr2a acvr2b pofut1 smad2 smad3 xcr1
GO keywords: embryo development [+]

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	Fig. 1. Expression patterns of XPofut1. Total RNAs from embryos or regions of embryos at indicated stages were isolated, the transcript levels of XPofut1 were analyzed by RT-PCR and normalized with those of EF1α. (A) Comparison of the levels of XPofut1 transcripts in unfertilized egg, blastula (stages 7 and 9), gastrula (stages 10+ and 12), neurula (stages 15 and 20), tailbud (stages 25 and 30), and tadpole (stage 35). (B) Stage 9 embryos were divided into the animal (A), marginal (M), and vegetal (V) regions. The levels of VegT (a ventral marker) transcripts are compared to confirm the accuracy of dissection. (C) Stage 10 embryos were divided into the dorsal (D) and vegetal (V) halves. The levels of Gsc (a dorsal marker) and XVent1 (a ventral marker) transcripts are compared to confirm the accuracy of dissection.
	Fig. 2. Spatial expression patterns of XPofut1. Whole-mount in situ hybridization was performed with embryos of indicated stages. Photographs of representative embryos are shown. (A) Stage 12, vegetal view, dorsal side is toward the top. (B-D) Stage 18, anterior view, dorsal side is toward the top (B), lateral view, dorsal side is toward the top (C), and dorsal view, anterior side is toward the top (D). (E and F) Stage 20, anterior view, dorsal side is toward the top (E) and dorsal view, anterior side is toward the top (F). (G and H) Stage 24, lateral view (G), and dorsal view (H). (I-K) Stage 30, lateral view (I), higher magnification of anterior region (J), and dorsal view (K). (L and M) Stage 37, lateral view (L) and higher magnification of dorsal region (M). ap, auditory placode; ba, branchial arch; bp, blastopore; e, eye; mc, mesencephalon, ng, neural groove; np, neural plate; ns, nephrostome; op, optic placode; ov, optic vesicle; pd, pronephric duct; pn, pronephros; pt, pronephric tubule; rc, rhombencephalon; s, somite.
	Fig. 3. XPofut1 knockdown causes axial truncation. (A) Indicated amounts of XPofut1 (2.5-10 nmole/embryo) or control random MO (10 nmole/embryo) were injected into the marginal regions at 2-cell stage and the embryos were cultured until stage 27. Photographs of representative embryos are shown. (B) Indicated combinations of XPofut1 or control random MO (cMO) (5 nmole/embryo), Sec-hPOFUT1 or Sec-XPofut1 mRNA (ng/embryo) were coinjected into the marginal regions at 2-cell stage. The embryos were cultured until stage 27 and scored for phenotypes. Photographs of representative embryos for each category of phenotype are shown, and percentages of embryos to each category are tabulated.
	Fig. 4. XPofut1 is involved in Xnr1 signal transduction. (A) A3-Luc reporter plasmid (50 pg/embryo) was injected into animal regions at 2-cell stage with indicated combinations of control random or XPofut1 MO (5 nmole/embryo) and Xnr1 mRNA (125 pg/embryo). Animal caps were explanted at stage 8.5, cultured until stage 9.5, and harvested to analyze luciferase activities. (B) Indicated combinations of control random (cMO) or XPofut1 MO (5 nmole/embryo) and Xnr1 mRNA (125 pg/embryo) were injected into animal regions of 2-cell-stage embryos. Animal caps were explanted at stage 8.5, cultured until stage 9.5, and harvested for RT-PCR analysis. EF1α is used as a loading control.
	Fig. 5. XPofut1 is important for Smad2 phosphorylation in early embryos. (A) Two-cell-stage embryos were injected into marginal regions with random MO (Co, 10 nmole/embryo) or indicated amounts of XPofut1 MO (nmole/embryo), and cultured until stage 9.5 or 10. The levels of phosphorylated Smad2 (pSmad2) are compared by immunoblotting. Actin is used as loading control. (B-D) Microinjected embryos were cultured until stage 9.5 and harvested to analyze the levels of phosphorylated Smad2 (pSmad2) by immunoblotting. Levels of total Smad2 (Smad2) are also compared and actin is used as a loading control. (B) Random control (Co) or XPofut1 MO (5 nmole/embryo) was injected into marginal regions at 2-cell stage, and then indicated amounts (pg/embryo) of hPOFUT1 mRNA were injected into either 4 animal blastomeres (Animal) or 4 vegetal blastomeres (Vegetal) at 8-cell stage. (C) Random control or XPofut1 MO (5 nmole/embryo) was injected into the marginal region at 2-cell stage, and then mRNA encoding wild-type (WT) or glycosyltransferase-defective mutant form (RA) of hPOFUT1 (100 pg/embryo) was injected into 4 animal blastomeres at 8-cell stage. (D) hPOFUT1 mRNA (100 pg/embryo) was injected into 4 animal blastomeres at 8-cell stage (A), into marginal regions at 2-cell stage (M), or into 4 vegetal blastomeres at 8-cell stage (V).
	Fig. 6. XPofut1 regulates Xnr1 and Xnr5/6 signaling differently. Embryos were injected with indicated combinations of MO and mRNA. Animal caps were explanted at stage 8.5, cultured until stage 9.5, and harvested to analyze the levels of phosphorylated Smad2 (pSmad2) by immunoblotting. Levels of total Smad2 (Smad2) are also compared and actin is used as a loading control. (A) Embryos were injected into marginal regions with indicated combinations of XPofut1 MO (5 nmole/embryo) and mRNAs encoding Xnr1 (125 pg/embryo), Xnr5 (40 pg/embryo), or Xnr6 (300 pg/embryo) at 2-cell stage, and then injected into 4 animal blastomeres with hPOFUT1 mRNA (100 pg/embryo) at 8-cell stage. (B) Embryos were injected into marginal regions with indicated combinations of XPofut1 MO (5 nmole/embryo) and mRNAs encoding Xnr1 (125 pg/embryo), Xnr2 (20 pg/embryo), Xnr4 (100 pg/embryo), Xnr5 (40 pg/embryo), Xnr6 (300 pg/embryo), or XCR1 (250 pg/embryo) at 2-cell stage.
	Fig. 7. A schematic model for XPofut1 functional in Xnr signaling. (A) XPofut1 is necessary for Xnr1/2-induced Smad2 phosphorylation. XPofut1 may control Xnr1/2 signaling by O-fucosylation (dashed arrow) of XCRs, the Xenopus laevis EGF-CFC factors. XPofut1 is not necessary for Xnr5/6-induced Smad2 phosphorylation. (B) Spatial pattern of XPofut1 activity and XCR expression in stages 8 to 10 Xenopus laevis embryos. In the vegetal pole region (Vegetal), XPofut1 transcripts are present but XPofut1 function is not necessary for Smad2 phosphorylation. In the marginal zone (Marginal), XPofut1 function is necessary for Smad2 phosphorylation. In the animal cap region (Animal), XPofut1 is functional as shown in animal cap assays (Fig. 6), yet there are no Xnr ligands or Smad2 phosphorylation in this region. See “Discussion” for details.