Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

Abstract

L-fucose, a monosaccharide widely distributed in eukaryotes and certain bacteria, is a determinant of many functional glycans that play central roles in numerous biological processes. The molecular mechanism, however, by which fucosylation mediates these processes remains largely elusive. To study how changes in fucosylation impact embryonic development, we up-regulated N-linked fucosylation via over-expression of a key GDP-Fucose transporter, Slc35c1, in zebrafish. We show that Slc35c1 overexpression causes elevated N-linked fucosylation and disrupts embryonic patterning in a transporter activity dependent manner. We demonstrate that patterning defects associated with enhanced N-linked fucosylation are due to diminished canonical Wnt signaling. Chimeric analyses demonstrate that elevated Slc35c1 expression in receiving cells decreases the signaling range of Wnt8a during zebrafish embryogenesis. Moreover, we provide biochemical evidence that this decrease is associated with reduced Wnt8 ligand and elevated Lrp6 coreceptor, which we show are both substrates for N-linked fucosylation in zebrafish embryos. Strikingly, slc35c1 expression is regulated by canonical Wnt signaling. These results suggest that Wnt limits its own signaling activity in part via up-regulation of a transporter, slc35c1 that promotes terminal fucosylation and thereby limits Wnt activity.

Keywords: Fucosylation; GDP-Fucose transporter; Wnt signaling; Zebrafish patterning; slc35c1.

Figure 1. slc35c1 enhances the level of N-linked fucosylation expression in zebrafish embryos

A) Schematic illustration of fucose biosynthetic pathways. Slc35c1 transports GDP-Fuc into the late secretary pathway (mainly the Golgi apparatus). B) slc35c1 mRNA expression during zebrafish development as quantified by qPCR. The blue bars show slc35c1 relative expression during development and the red dots show the qPCR cycle number of the control gene odc1. C) Western blot analysis of the expression of mouse Slc35c1-myc fusion protein of 5hpf zebrafish embryos. D) Western blot analysis of global fucosylation levels at shield stage (6hpf) in embryos microinjected with gmds MO or mouse slc35c1-myc mRNA in the presence or absence of GDP-Fuc before and after PNGase F digestion. Proteins were resolved by SDS-PAGE and IB with AAL followed by anti-biotin-HRP in D) or by streptavidin-Alexa488 for the quantification in E). N-fucosylation: N-linked fucosylation; O-fucosylation: fucose in mucin and O-fucosylated proteins.

Figure 2. Elevated N-fucosylation dorsalizes zebrafish embryos

A) Variable degrees of dorsalization in embryos over-expressing mSlc35c1. B) Quantification of the severity and penetrance of dorsalization in each treatment. C–D) qPCR of the ventral transcriptional factors vox and vent in WT, GDP-Fuc injected or mSlc35c1 expressing embryos. E–L) in situ hybridization with dorsal (chordin and gsc) and ventral (eve1) specific markers at shield stage (6hpf). E–H) chordin expression in treated embryos I–L) eve1 and gsc expression. The expression angles of chordin and gsc of embryos with each treatment are summarized in M) and N) respectably. The sample sizes for each treatment are as follows: wt (N=15), GDP-Fuc(N=13), mSlc35c1(N=18) and mSlc35c1 plus GDP-Fuc (N=21) for M); and wt (N=19), GDP-Fuc (N=15), mSlc35c1(N=21) and mSlc35c1 plus GDP-Fuc (N=22) for N). For chordin angle, ordinary One-way ANOVA between sample group means, p=0.0002. For gsc angle, ordinary One-way ANOVA between sample group means, p=0.0001. Note: ** Tukey’s procedure after ANOVA p<0.01. Null hypothesis for χ² between embryos injected with 1ng mSlc35c1 and with GDP-Fuc plus 1ng mSlc35c1 p=1.41E-04; for χ² between embryos injected with 2ng mSlc35c1 and with GDP-Fuc plus 2ng mSlc35c1 p=8.99E-03.

Figure 3. BMP and Notch signaling are intact in mSlc35c1 expressing embryos

A) Phospho-Smad1/5/8 antibody blot indicates BMP signaling activity in each treatment. B) Quantification of Phospho-Smad1/5/8 levels, normalized to α-Tubulin protein levels. C) Molecular interaction between BMP signaling and Slc35c1 function. D) Dorsal view of a transgenic zebrafish larva expressing a GFP reporter of Notch activity. Graph shows the GFP expression levels determined by the fluorescence intensities of transgenic notch reporter embryos injected with the specified molecules. The data represent the average of GFP reporter intensity from individual treated embryos, with WT set as 100%.

Figure 4. GDP-Fuc or mSlc35c1 overexpression inhibits maternal Wnt signaling

A–D) in situ hybridization of a direct Wnt signal target gene bozozok(dharma) in sphere stage (4hpf) embryos; lateral views. E) Quantification of bozozok domain angle (e.g. dashed lines in A–D) in each group. The sample sizes for each treatment were as follows: gmds MO (N=35), wt (N=18), GDP-Fuc(N=54) and mSlc35c1(N=26). Ordinary One-way ANOVA between sample group means, p<0.0001. F) qPCR of bozozok, a direct Wnt target gene in WT, GDP-Fuc injected or mSlc35c1 expressing embryos. bozozok expression is normalized to expression of the eef1a1a house keeping gene. G) Schematic illustration of an embryo demonstrates the boxed regions shown in H–K) for β-catenin protein localization at 512 cell stage (3hpf). View of the dorsal margin, as indicated by the nuclei of the yolk syncitial layer, which lack membranes (red arrowhead) at the margin, and the strong nuclear label (white arrowhead) in cells within the “core” region (i.e. closest to the Wnt8a source). The nuclear localization of β-catenin in the core region of (H) WT (n=6) and (I) mSlc35c2 (n=5) injected embryo, but is faint in either (J) GDP-Fuc (n=7) or (K) mSlc35c1 (n=9) injected embryos. Note: ** Tukey’s procedure after ANOVA ** p<0.01; * p<0.05. Bar=100μm.

Figure 5. mSlc35c1 and GDP-Fuc can suppress Wnt8a induced patterning defects

A–D) in situ hybridization of bozozok expression in injected embryos at sphere stage (4hpf). E) Quantification of bozozok angle (area outlined by dashed lines in A–D). F) qPCR of bozozok, a direct Wnt target gene in wnt8a alone or combined with GDP-Fuc injected, gmds MO injected or mSlc35c1 expressing embryos. bozozok expression was normalized to the expression of the house keeping gene eef1a1a. G) Posteriorization caused by over-expression of wnt8a. All embryos shown are 18hpf. P1–P4 denote the degree of posterioization, as determined by the marker gene pax2a-krox20-myoD expression patterns. The P1 class lacks the telencephalon as indicated by loss of anterior pax2a expression, (red arrow). P2 embryos lack regions anterior to the MHB domain of pax2a, (red arrowhead). P3 embryos lack MHB expression of pax2a. In the P4 class krox20 expression indicates loss of anterior hindbrain segments. H) Quantification of each treatment. Note: ** Tukey’s procedure after ANOVA p<0.01. Null hypothesis for χ² between embryos injected with wnt8a and with wnt8a plus mSlc35c1 p= 6.05E-08.

Figure 6. mSlc35c1 non-cell-autonomously limits Wnt8a activity

A–B) Schematic depicts the procedure for making chimeras and the quantification of the Wnt responses in host embryos. Donor embryos were injected with tracer dye and wnt8a mRNA, with or without mSlc35c1 mRNA. At the 1000-cell stage, cells were extracted from donors and transplanted into dome-stage host embryos. Confocal images of chimeric embryo were taken with the same setting for all embryos. Then 8 sections (equal to 2.4μm of embryo volume) were projected to a single image, as shown in B–C). B) Merged images (Red: donor; blue: nuclei and green: β-catenin staining) regions to be quantified were determined in Photoshop CS5 by manually circling the nuclear region of target cells in nuclei channel and measure in the β-catenin channel. Cells are divided accordingly: D, donor cells; N1, cells directly adjacent to donors; N2, cells 1- cell away from donors; B, background cells (those at least 4 cell diameters away from donor cells). C–D) nuclear β-Catenin abundance in the chimeras, normalized to the staining in background cells. The numbers in each column indicate the number of cells analyzed. C) The response of WT embryos to donor cells expressing mSlc35c1 mRNA. D) The response of WT embryos to donor cells expressing wnt8a or wnt8a plus mSlc35c1 mRNA. F) The response of WT or mSlc35c1 expressing embryos to donor cells expressing wnt8a mRNA. Note: ** Tuckey’s procedure after ANOVA p<0.01; * p<0.05.

Figure 7. Excess fucosylation changes the response pattern of Wnt signaling

AB) Projections of stacks of individual confocal image sections show the nuclear localization of β-catenin in early zebrafish embryos (512 cell stage). a-f) Single confocal sections. A) β-catenin in a representative wild-type embryo. a) Peripheral blastomeres at the animal pole. b) medial region of the blastoderm. c) The “core” region of highest Wnt activity at the dorsal margin. B) The Wnt gradient of a representative mSlc35c1 expressing embryo. d) The edge, e) the medial and f) the core and margin regions. C–D) Schematic illustration shows Wnt activities based on β-catenin nuclear localization. In wt embryos, with normal level of N-fucosylaiton, Wnt protein creates a morphogen gradient. In Slc35c1 over-expressing embryos, with excess N-fucosylation, Wnt protein levels are diminished, which reduces the highest Wnt activity response region; Lrp6 protein level is increased, which increases the basal level of the Wnt response, creating an extended zone of intermediate Wnt response. Note: Bar=200μm.

Figure 8. Excess fucosylation is associated with decreased Wnt8 and enhanced Lrp6

A) AAL Immuno-blot (IB) DN-Wnt8-Flag is modified by fucosylation. Top panel AAL IB; Lower panel, α-Flag IB shows DN-Wnt8-Flag input. Each lane represents pull-down of DN-Wnt8-Flag from a pool of 33 embryos. B) IB shows Lrp6 is fucosylated, which is recognized by bioorthogonal labeling. Top panel: GDP-FucAl incorporated bands. FucAl was detected by linking to Azido-biotin through a click reaction, which is recognized by anti-biotin IB. Lower panel: α-Lrp6 IB shows the endogenous Lrp6 input. Each lane represents Lrp6 pull-down from a pool of 45 embryos. C) anti-Flag IB showing Wnt8a-Flag protein in the presence or absence of mSlc35c1. Each lane represents an IP from a pool of 67 embryos. D) anti-Flag IB shows the DN-Wnt8-Flag (DNW-F) protein level in the presence or absence of mSlc35c1. Each lane represents pull-down of DN-Wnt8-Flag from a pool of 27 embryos. The lower panel is α-Actin IB (2 embryo equivalents for each treatment). E) IB shows mSlc35c1 expression has opposite effects on DN-Wnt8-Flag and Flag-Lrp6 protein abundance. Top panel: α-Flag IB reveals reduced DN-Wnt8-Flag in mSlc35c1 expressing embryos. Middle panel: α-Flag IB shows enhanced Flag-Lrp6 in mSlc35c1 expressing embryos. Each lane represents pull-down of Flag tagged protein from a pool of 64 embryos. The lower panel is the loading control with each lane representing the lysate from 2 embryo equivalents. Blue arrowhead indicates DN-Wnt8-Flag; red arrowhead marks Wnt8-Flag-Mid; orange arrowhead indicates Lrp6-Flag or endogenous Lrp6. F) Quantification of the penetrance and severity of dorsalization in embryos injected with mSlc35c1, lrp6 or both, reveals interaction between mslc35c1 and lrp6. 500pg of mslc35c1 and 300pg lrp6 mRNA was injected per embryo. Null hypothesis for χ² between embryos injected with mSlc35c1 and with lrp6 plus mSlc35c1 p= 9.29E-09.

Figure 9. The interplay of Wnt8a signaling, slc35c1-mediated GDP-Fuc transport and N-linked fucosylation

A) qPCR results show the relative expression of slc35c1 and slc35c2 in zebrafish embryos treated with wnt8a or DN-wnt8a mRNA and untreated control sphere stage (4hpf) embryos:. All results are normalized to odc1. Expression in WT is defined as 100%. B–C) Confocal images of slc35c1 fluorescent in situ shows the positive correlation of slc35c1 expression to β-catenin richness. Red: β-catenin protein staining. Green: slc35c1 probe in situ hybridization in B) or slc35c1 sense probe in situ hybridization in C). Blue: nuclei. D) A schematic model for the relationship between Slc35c1 and Wnt8a. Wnt8a activated canonical Wnt signaling enhances slc35c1 expression. Increased slc35c1 enhances N-inked fucosylation, which inhibits Wnt/beta-catein/TCF signaling during both blastula and gastrula stages. Therefore, these components comprise a negative regulatory loop to control Wnt signaling through N-fucosylation. Note: Bar =300μm.