SWS-derived fibroblasts show altered HSPGs and glypicans after heparinase III (H’ase III) digestion. (A) Western blotting of ΔHS-stub using 3G10 antibody following heparinase III digestion of three SWS-derived fibroblasts and two control fibroblasts. C1 and C2 are control fibroblasts. C1, GM08429; C2, GM08680. P1.1, P4.1, and P5.1 are SWS-derived fibroblasts. (B) Quantitation assay of glypican band density in (A) and two other replicates. The data are presented as mean ± SEM. An unpaired two-tailed t-test was used. ^*⁣*⁣**p < 0.0001. (C) Agarose gel of six human glypicans after reverse-transcript PCR. (D) qPCR of three dominant glypicans in SWS-derived fibroblasts. The relative glypican level was normalized to GAPDH. The graphs represent the 2^–ΔΔCt values. Experiments were performed in triplicates with similar results.

Expression of human COG4^p.G516R in zebrafish increases the protein level of glypicans. (A–C) Expression of human COG4^WT and COG4^p.G516R in zebrafish after mRNA or DNA injection. (A) The scheme of COG4 constructs for in vitro transcription (top) and DNA injection (bottom). (B) Western blot at 24 hpf to detect the presence of COG4 after mRNA injection. (C) Western blot at 48 hpf to detect COG4 after DNA injection; heat shock was performed at 24 hpf for 2 h at 38°C. (D–G) Glypican analysis in zebrafish. (D) Western blotting of ΔHS-stub using 3G10 antibody following heparinase III digestion of control and embryos injected with COG4^WT or COG4^p.G516R mRNA at 3 dpf. (E) Quantitation assay of glypican band density in (D) and two more replicates. The data are presented as mean ± SEM. An unpaired two-tailed t-test was used. ^∗p < 0.05. (F) mRNA expression of glypican genes by RT-PCR using cDNA from control embryos at 6 hpf. (G) qPCR analyses of highly expressed glypicans in control and zebrafish embryos injected with human COG4^WT or COG4^p.G516R mRNA. The relative glypican level was normalized to β-actin. The graphs represent the 2^–ΔΔCt values. One-way ANOVA with Tukey’s multiple comparison tests was applied. ns, not significant; ^∗p < 0.05. Experiments were performed in triplicates with similar results.

Expression of human COG4^p.G516R impairs zebrafish early development and chondrocyte intercalation. (A,B) Expression of COG4^p.^G516R in zebrafish causes gastrulation defects and shortened body axis. (A) Lateral view of representative embryos. Anterior to the top. Compared to the control construct hsp70l:mCherry and COG4^WT, embryos expressing COG4^p.G516R show an axis extension defect at 10 hpf. White arrow points to the head region, and the black arrow points to the tailbud. Expression of COG4^p.G516R mRNA causes similar results. (C) Graphs show the measured body length of each group at 3 and 6 dpf. The data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests was applied. ^****p < 0.0001; ns, not significant. (D) Expression of COG4^p.G516G causes craniofacial abnormalities. Ventral view of representative Meckel’s cartilage of zebrafish larvae at 4 dpf after WGA staining and imaged by a confocal microscope. Dotted circular lines highlight chondrocyte cell shape and their relative configuration with each other. (E) Graphical representation of the length-to-width ratio of chondrocytes in the region of interest, yellow box in (D). Individual cell length-to-width ratio was measured in three representative Meckel’s cartilage images of each group. The data are presented as mean ± SEM. One-way ANOVA with Tukey’s multiple comparison tests was applied. ^****p < 0.0001; ns, not significant. Experiments were performed in triplicates with similar results.

Expression of human COG4^p.G516R elevates the wnt4 transcript in zebrafish embryos. (A) Quantitative PCR analyses of selective non-canonical Wnt pathway ligands in zebrafish embryos injected with human COG4^WT or COG4^p.G516R mRNA. At 6 hpf, wnt4 expression is significantly upregulated in embryos injected with COG4^p.G516R mRNA, but not in those injected with COG4^WT mRNA. Similar upregulation of wnt4 transcripts is observed at 10 hpf as well. Bar graphs represent average gene expression relative to the housekeeping gene β-actin. The data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests was used. ^****p < 0.0001; ns, not significant. (B) Representative images of whole-mount in situ hybridization from control and treated embryos. Whole-mount in situ hybridization analyses demonstrate that wnt4 expression is elevated in embryos injected with COG4^p.G516R mRNA (a″, at 6 hpf; b″ at 12 hpf), but there are no obvious changes in embryos injected with human COG4^WT mRNA (a′ and b′). (b, b’) Arrows point to the restricted wnt4 expression domain in the hindbrain of zebrafish embryos at 12 hpf; (b″) arrows point to expanded expression of wnt4 in the hindbrain region. At 12 hpf, wnt11f2 expression intensity is not significantly changed in embryos injected with COG4^p.G516R mRNA (c″, d″), compared to either control siblings (c, d) or human COG4^WT mRNA-injected embryos (c′, d′). The dotted black color lines landmark the degree of angle between the head and tail, which is much more increased in embryos injected with COG4^p.G516R mRNA, suggesting defects in extension movement during gastrulation. Arrows in c, c′ and d, d″ indicate that wnt11f2 is restricted to the dorsal midline; however, its expression pattern is dispersed in COG4^p.G516R mRNA-injected embryos, suggesting that the convergence movement is impaired (c″, white dotted line and arrow; d″, there is no clear midline expression). Scale bars: 200 μm. Experiments were performed in triplicates with similar results.

Overexpression of wnt4 phenocopies zebrafish embryos injected with the COG4^p.G516G mRNA. (A) Graphs show the measured body length of each group after wnt4 mRNA injection at 3 and 7 dpf. The data are presented as mean ± SD. An unpaired two-tailed t-test was used. ^****p < 0.0001; ^∗∗p < 0.01; ^∗p < 0.05; ns, not significant. (B) Overexpression of wnt4 causes abnormal chondrocyte stacking and intercalation at Meckel’s cartilage. Ventral view of representative Meckel’s cartilage of zebrafish larvae after wnt4 injection at 4 dpf following WGA staining and imaged by a confocal microscope. (C) Overexpression of wnt4 causes cyclopia as expression of the COG4^p.G516G variant. Dorsal view of representative images of cyclopia compared to control. Two hundred picograms of wnt4 or COG4^p.G516G mRNA was used per embryo. Experiments were performed in triplicates with similar results.

LGK974 treatment suppresses shortened body length and chondrocyte defect caused by of COG4^p.G516G expression in zebrafish. (A) Scheme of the LGK974 treatment procedure. (B) Graphs show the measured body length of each group at 4 dpf. The data are presented as mean ± SEM. An unpaired two-tailed t-test was used. ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05; ns, not significant. (C) Ventral view of representative Meckel’s cartilage of control and COG4^p.G516G-injected embryos with or without LGK974 treatment at 4 dpf following WGA staining and imaged by a confocal microscope. (D) Graphs show the length-to-width ratio of chondrocytes in Meckel’s cartilage in (C) and two more representative Meckel’s cartilage images of each group. The data are presented as mean ± SEM. One-way ANOVA with Tukey’s multiple comparison tests was applied. ^****p < 0.0001; ^∗∗p < 0.01; ns, not significant. Experiments were performed in two biological replicates with similar results.

Non-canonical WNTs and related component level in SWS-derived cells. (A) qPCR analyses of selective non-canonical Wnt pathway components in SWS-derived fibroblasts. GAPDH was used as an internal control. The graphs represent the 2^–ΔΔCt values. Unpaired two-tailed t-test was applied for comparison of each SWS fibroblast with two controls. ^****p < 0.0001. Experiments were performed in triplicates with similar results. (B) Western blotting of a few Wnt pathway components. GM05565 and GM09503 are control fibroblasts. P1.1, P4.1, and P5.1 are SWS-derived fibroblasts. (C) Quantitation assay of WNT4 and pJNK band density in (B) and the other replicates. The data are presented as mean ± SEM. An unpaired two-tailed t-test was used. ^∗p < 0.05.