Fig 2: Gcm1 overexpression activates the Wnt/β-catenin pathway in normal C57BL/6 mESCs and NE-4C.a Increased expression of Axin2 in mESCs with Gcm1 overexpression. The western blots show that the Gcm1 overexpression plasmid increased Axin2 expression in normal C57BL/6 mESCs. PGMLV-Gcm1, Gcm1 plasmid. mESCs were transfected with either control siRNA or pGMLV-Gcm1, and the expression levels were determined 72 h post transfection by western blotting. b A Co-IP assay was used to identify the interaction between Gcm1 and β-catenin in cells transfected with pGMLV-Gcm1 overexpression plasmid in C57BL/6 mESCs. An anti-Gcm1 antibody was used for IP, and IB was performed using an anti-β-catenin antibody. c A Co-IP assay was used to identify the interaction between Gcm1 and TCF4 in cells transfected with pGMLV-Gcm1 overexpression plasmid in C57BL/6 mESCs. An anti-Gcm1 antibody was used for IP, and IB was performed using an anti-TCF4 antibody. d A Co-IP assay identified increase interactions between TCF4 and β-catenin in cells transfected with 0.5, 2, and 4 µg of pGMLV-Gcm1 for 72 h in C57BL/6 mESCs. e A Co-IP assay was used to identify the interaction between Gcm1 and TCF4, β-catenin in NE-4C cells transfected with the pGMLV-Gcm1 overexpression plasmid. An anti-Gcm1 antibody was used for IP, and IB was performed using an anti-TCF4 antibody and anti-β-catenin antibody. f The mRNA expression of Fzd5, Lrp, Wnt6, Ccnd1, Nfat5, Lef1, Tcf4, Tcf12, Bcl9L, and Axin2 in C57BL/6 mESCs transfected with the overexpression vector pGMLV-Gcm1. The mRNA was extracted from the mESCs after transfection with pGMLV-Gcm1 for 72 h. g Analysis of luciferase activity by TOP/FOP Flash assays in C57BL/6 mESCs transfected with pGMLV-Gcm1 overexpression plasmid relative to those transfected with scramble plasmid in normal and LiCl medium. LiCl (10 mmol) was added to the control medium before 72 h of transfection. Data a–g represent the mean ± SEM (n = 3). The p value was calculated by Student’s t-test, ns was for no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Folate deficiency upregulates Gcm1 to promote formation of the Gcm1/β-catenin/TCF4 complex.a Differentially expressed NTD-related genes in the microarray expression profiles of C57BL/6 mESCs cultured in folate-deficient conditions for six generations. The microarray data were obtained from three replicates for both control and folate-deficient cells. The X-axis represents the Log2 fold change. bThe mRNA expression of Gcm1 in C57BL/6 mESCs after six generations of folate deficiency. **P = 0.004. c Increased expression of Gcm1 in mESCs with folate deficiency. Top panel: western blots of Gcm1 in control and folate-deficient cells; bottom panel: bar graph showing the quantification of the western blot signal intensities. d Efficiency of siRNA-mediated Gcm1 and siRNA-mediated TCF4 depletion and the expression of TCF4, Gcm1 and Axin2 at the protein level in C57BL/6 mESCs. mESCs with folate deficiency were transfected into either scramble siRNA control or Gcm1 siRNA and TCF siRNA, and post 72 h of transfection, the expression was determined by western blotting. e Efficiency of siRNA-mediated Gcm1 and TCF4 depletion and the expression of TCF4 and Axin2 at the protein level in NE-4C. NE-4C with folate deficiency were transfected into either scramble siRNA control or Gcm1 siRNA, TCF4-siRNA, and the expression was determined 72 h post transfection by western blotting. f A coimmunoprecipitation (Co-IP) assay was used to identify the interaction between Gcm1, β-catenin, and TCF4 in C57BL/6 mESC. Immunoprecipitation (IP) was performed using an anti-Gcm1 antibody and TCF4 antibody, and immunoblotting (IB) was performed using an anti-β-catenin antibody. g A Co-IP assay was used to identify the interactions between Gcm1 and TCF4, β-catenin in NE-4C. An anti-Gcm1 antibody and anti-TCF4 antibody was used for IP, and an anti-TCF4 antibody and anti-β-catenin was used for IB. Data b–f represent the mean ± SEM (n = 3). The p value was calculated by Student’s t-test, ns was for no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Gcm1 and Axin2 are significantly upregulated in NTDs models.a Morphology of CD-1 mouse embryos with low-folate diet and MTX induced from E9.5, left panel is control mouse fetus, right panels are NTDs mouse fetuses. The arrowhead is where the NTDs located; the left panel of NTDs embryos is schizencephaly. Scale bars, 100 μm. b The mRNA expression of Gcm1 in the brains and spines of NTD model mice. *P = 0.0003(brains) and *P = 0.01 (spines). c The mRNA expression of Axin2 in the brains and spines of NTD model mice. *P = 0.002(brains) and **P = 0.02 (spines). d Increased expression of Gcm1 and Axin2 in NTD mouse brains. Left panel: western blots of Gcm1 in normal and NTD brains; right panel: bar graph showing the quantification of the western blot signal intensities. e Increased expression of Gcm1 and Axin2 in NTD mouse spines. Left panel: western blots of Gcm1, Axin2 in normal and NTD brains; right panel: bar graph showing the quantification of the western blot signal intensities. f A Co-IP assay was used to identify the interaction between Gcm1 and TCF4, β-catenin in control and NTDs mouse brains. An anti-Gcm1 antibody was used for IP, and an anti-TCF4 antibody and anti-β-catenin was used for IB, Con was for Control. g A Co-IP assay was used to identify the interaction between Gcm1 and TCF4, β-catenin in control and NTDs mouse spines. An anti-Gcm1 antibody was used for IP, and an anti-TCF4 antibody and anti-β-catenin was used for IB, Con was for Control. h Folate concentrations in control human fetal brain tissue and spina bifida human fetal brain tissue. The data were obtained from 40 human fetus brains. i Increased expression of Gcm1, Axin2, and Atoh1 in human NTD brain samples. Left panel: western blots of Gcm1 in brains of normal humans (n = 20) and human NTD (n = 20) samples; right panel: bar graph showing the quantification of the western blot signal intensities. j The mRNA expression of Gcm1, Axin2, and Atoh1 in the NTDs fetuses, determined by Nanostring (n = 12). k Correlation analysis of folate concentration and Gcm1 mRNA expression (left panel); Gcm1 mRNA expression and Axin2 mRNA expression, Atoh1 mRNA expression (right panel) n = 12. l A Co-IP assay identified an interaction between Gcm1 and β-catenin in randomly selected low-folate fetal brains. Data a–h, l represent the mean ± SEM (n = 3). Data i represent the mean ± SEM (n = 20), Data j, k represent the mean ± SEM (n = 12), The p value was calculated by Student’s t-test, ns was for no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Nanog upregulates Gcm1 promoter activity.a Enrichment scores of transcription factor binding motifs in the mouse Gcm1 promoter. TF transcription factor. The size of the circle represents the −log10 (P value). b Nanog recognizes the Gcm1 promoter. C57BL/6 mESCs were subjected to ChIP analysis using Nanog antibodies. The immunoprecipitated complexes were analyzed by PCR. The TBE sites are located within 1 kb upstream of the Gcm1 promoter; the arrows represent the start and end sites of the PCR amplicons (bottom panel). c Efficiency of siRNA-mediated Nanog depletion and the expression of Gcm1 at mRNA level. C57BL/6 mESCs with folate deficiency were transfected with either control scramble siRNA or Nanog-siRNA, and the expression levels were determined 24 h post transfection by real-time RT-PCR. Left panel: quantification of Nanog in control scramble siRNA and si-Nanog cells; right panel: quantification of Gcm1 in control scrambles siRNA and si-Nanog cells. d Analysis of Gcm1 promoter-driven luciferase reporter activity in folate-deficient mESCs cotransfected with control plasmid or Nanog plasmid. Experimental mESCs were transfected with 1.5 µg of pGcm1-promoter, and control mESCs were transfected with 5.5 µg of Nanog-HA. The structures of the Gcm1 promoter reporter construct and the Nanog-HA construct are shown in the left panel. At 48 h post transfection, the cells were harvested for luciferase reporter assays. e Correlation analysis of folate concentration and Nanog mRNA expression in low-folate NTDs (n = 12). f Correlation analysis of Nanog mRNA expression and Gcm1 mRNA expression in low-folate NTDs, determined by Nanostring (n = 12). Data b–d represent the mean ± SEM (n = 3). Data e, f represent the mean mean ± SEM (n = 12). The p value was calculated by Student’s t-test, ns was for no significance, *P < 0.05, ** P < 0.01, ***P < 0.001.