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SC_11-1204_sm_supplFigure1.tif2527KFigure S1. The Gpc4 hypomorphic alleles. (A) Schematic representation of the mouse Gpc4 gene locus. (B) Schematic representation of gene trap alleles carried by the EX194 (left) and the Pst132 ESC lines (right). The top schemes show the gene trap vectors used to generate the two Gpc4 hypomorphic alleles. Both gene trap vectors contain the betageo (β-geo) reporter. Note that the gene trap vectors used to generate the Pst132 ESCs also contains the alkaline phosphatase reporter (PLAP). Red arrows indicate the insertion points in the first Gpc4 intron. The number 1 corresponds to the 1st exon/intron junction and the red numbers indicate the distance in nucleotides where the trap vectors were inserted. The genomic insertion sites of the gene trap vectors were identified by using the genome walk approach performed with the Universal Genome Walker kit (Clontech). PCR amplification of the targeted region was performed using the trap specific reverse primers p3, p6, and p7. Note that both ESC lines harbor a single trap insertion within the first intron of the Gpc4 gene. The trap insertion sites are at 13,641bp and 26,496bp distant from exon1 in the Ex194 and in the PST132 ESC lines, respectively. (C) Schematic representation of the Gpc4 fusion transcripts present in EX194 and Pst132 ESC lines. The fusion between Gpc4 exon1 and the trap cassette was confirmed by sequencing RT-PCR products obtained using exon1 and trap-specific primers (p1+p3). The enlargement at the bottom shows the nucleotide sequence of the Gpc4 cDNA from base pair 275 to 310 and the corresponding amino acid sequence is in violet. Red arrows and numbers indicate the nucleotide position where Gpc4 transcripts are disrupted in the two ES mutant cell lines. Note that in the Ex194 ESCs, this insertion disrupts Gpc4 transcript at the nucleotide 286, which is upstream of an aspartic acid codon. Instead, the insertion occurred at nucleotide position 308 present in a glycine codon in the PST132 ESC line.
SC_11-1204_sm_supplFigure2.pdf183KFigure S2. Gpc4 regulates ESC differentiation without affecting expression of other glypican family members. (A) Western blot analysis showing the glycanated Gpc4 protein in control, Gpc4gt-1, and Gpc4gt-2 ESCs. Actin protein levels were used as loading controls. Bottom panel shows the expression of β-Gal-fusion proteins in Gpc4gt-1 and Gpc4gt-2 ESCs. Cell extracts were immuno-precipitated by using anti β-Gal antibodies and products were revealed by western blot with the same antibodies. (B) Semi-quantitative RT-PCR analysis showing no changes in the expression levels of glypican family members in total RNA purified from control, Gpc4gt-1, Gpc4over1, and Gpc4over2 cells. (C, D) Semiquantitative RT-PCRs showing Gpc4 expression during neural (C) and EB (D) differentiation of ESCs at different time points. (E) Semi-quantitative RT-PCR analysis of pluripotency molecular markers from ESCs grown in optimal cultured conditions. ctl: control cells.
SC_11-1204_sm_supplFigure3.pdf239KFigure S3. Gpc4 tempers the speed of cell lineage entry. (A) Semi-quantitative RT-PCR analysis of total RNA from control and Gpc4gt-1 ESCs grown in suboptimal conditions, namely on gelatin without inactivated embryonic mouse fibroblasts (EMFI) for 4days. Note that Gpc4gt-1 cells de-repressed early markers of ectoderm, mesoderm, and endoderm differentiation, such as Fgf5, Brachury, and Gata6, when grown in sub-optimal conditions for 4days. The mRNA levels of Oct4 and Nanog remain unchanged, whereas Sox2 expression is down regulated. (B) Morphological analysis of differentiating EBs derived from control (ctl) and Gpc4gt-1 ESCs at different time points. Left panels: brightfield images of EBs at day5 (4X magnification; scale bars: 500 μm). Middle and right panels: immunofluorescence analysis of differentiating EBs showing the distribution of Laminin1 at day6 and day10 (confocal images: 40X and 20X magnifications, respectively; scale bars: 50 μm). (C) Immunofluorescence analysis of differentiating EBs showing the distribution of Actinin-positive cells at day9 and of CD31-positive cells at day7 in control and Gpc4gt-1 ESCs. Note that differentiation is enhanced in Gpc4 mutant cells (20X magnification; scale bars: 100 μm). (D) Semi-quantitative RT-PCR analysis of total RNA extracted from neural differentiating control and Gpc4 Δex3 cells at different time points. Hprt expression (bottom panel) was used to normalize total RNA levels between different samples. (E) Immunofluorescence of Tuj1-positive cells in control and Gpc4 Δex3 cells (20X magnification; scale bars: 100 μm). ctl: control cells.
SC_11-1204_sm_supplFigure4.pdf112KFigure S4. Distribution of Gpc4 transcripts in early mouse embryos. (A) Whole mount in situ hybridization showing the spatial and temporal distribution of Gpc4 transcripts at E7.5. The inset shows Gpc4 expression in the developing brain (arrow). a, anterior; p, posterior. Scale bars: 200 μm. (B, C) Whole mount in situ hybridization showing the spatial and temporal distribution of Gpc4 transcripts at E8.5 and E9.5. Scale bars: 500 μm. Note the high expression levels in the developing forebrain (fb(, presomitic mesoderm (ps(, tail bud (tb(, somites (s(, 1st and 2nd branchial arches (I and II, respectively). mb: midbrain; hb: hindbrain; h: heart.
SC_11-1204_sm_supplFigure5.pdf156KFigure S5. Teratoma assays and regulation of signaling pathways in Gpc4- mutant ESCs. (A) Hematoxylin/eosin staining of cryostat sections from dissected ectopic tissues derived from injected control and Gpc4 Δex3 ESCs. Figure shows two independent sections for each sample. Note that control cells develop tissues containing teratoma-derived structures. Mucus-producing epithelium (endoderm; arrow) and neuroepithelium (ectoderm; arrowheads) are indicated. In contrast, tissue masses derived from Gpc4 Δex3 ESCs lack teratoma-derived structures. Scale bars: 200 μm. (B) Hematoxylin/eosin staining of paraffin sections from dissected ectopic tissues derived from injected control and Gpc4over1 ESCs. Figure shows two independent sections for each sample. Note that both cell types develop tissues containing teratoma-derived structures. Endoderm (arrow(, mesoderm (asterisk(, and neuroepithelium (ectoderm; arrowheads) are indicated. The light blue staining in the bottom right panel shows the distribution of injected Gpc4over1 cells as revealed by β- Gal staining. Scale bars: 200 μm. (C) Immunofluorescence analysis of Tuj1-positive cells in neural differentiating control and Gpc4gt-1 ESCs treated or not with 10ng/ml of Bmp4. Note that Bmp4 impairs neuronal differentiation in both ESC types (20X magnification; scale bars: 100 μm.). (D) Western blot analysis of p-Erks protein levels in control and Gpc4tg-1 cells stimulated with increasing concentration of Fgf2, Fgf8, and Fgf4 (indicated). 2x106 cells were plated on gelatin-coated dishes in neural differentiation media for 12hrs. Thereafter, human recombinant Fgf2, Fgf8c, and Fgf4 were applied for 10-30min at the indicated concentrations in the presence of 1μg/ml heparin. Note that untreated control and Gpc4gt-1 cells had the same basal level of Erks phosphorylation. Moreover, Fgf2, Fgf8, and Fgf4 induced Erks phosphorylation in both control and Gpc4gt-1 cells, although this event was attenuated in mutant cells. ctl: control cells.
SC_11-1204_sm_supplFigure6.pdf224KFigure S6. Gpc4 regulates ESC response to Wnt/β-catenin signaling. (A) Quantitative analysis of beating foci derived from control and Gpc4gt-1 differentiating EBs exposed to Wnt3a from day0 to day5. Analysis was done at different time points and Wnt3a concentrations are indicated. Note that Wnt3a treatment does not rescue the Gpc4gt-1 cell phenotype (mean±SEM; n=3). (B) Quantitative analysis of control and Gpc4gt-1 beating foci exposed to Wnt3a at 150ng/ml at indicated days. Note that Wnt3a does not rescue the differentiation phenotype of mutant cells even at higher concentration. (C) Dual luciferase assay demonstrating that the activation of TOPFlash reporter activity as induced by BIO treatment (1μM) for 24hrs in control and Gpc4-mutant cells. Note that TOPFlash activity significantly occurs in control and Gpc4-mutants (mean±SEM; n=2; *** P<0,001). (D) Quantitative analysis of beating foci in control and Gpc4gt-1 differentiating EBs exposed to the GSK3 inhibitor, LiCl, from day0 to day5. Analysis was done at different time points and LiCl concentrations are indicated. Gpc4gt-1 cells treated with LiCl behave as controls (mean±SEM; n=3). (E) Quantitative analysis of control and Gpc4-mutant beating foci exposed to the GSK3 inhibitor, CHIR 99021 (1 and 3μM(, from day0 to day5. Analysis was done at different time points. Gpc4- mutant cells treated with CHIR 99021 behave as controls (mean±SEM; n=2). (F) Quantitative analysis of control and Gpc4-mutant beating foci exposed to the GSK3 inhibitor, AR A014418 (30μM(, from day0 to day5. Analysis was done at different time points. Gpc4-mutant cells treated with AR A014418 behave as controls (mean±SEM; n=2). (G) Semi-quantitative RT-PCR analysis of Oct4 and Hprt expression levels in total RNA extracted from undifferentiated control (wt) and Gpc4gt-1 ESCs and from the corresponding differentiating EBs at day10. Note that Oct4 transcript levels become drastically reduced in differentiating EBs. Hprt expression (bottom panel) was used to normalize total RNA levels between different samples. (H) Semi-quantitative RT-PCR analysis of total RNA extracted from differentiating EBs at day10 showing that Wnt3a treatment of Gpc4gt-1 EBs does not restore normal expression levels of Oct4 and a- Mhc. Wnt3a concentrations are indicated. (I) Semi-quantitative RT-PCR analysis of total RNA extracted from differentiating EBs at day10 showing that LiCl treatment of Gpc4gt-1 EBs restores normal expression levels of Oct4 and a-Mhc. LiCl concentrations are indicated. (J) Semi-quantitative RT-PCR analysis of total RNA extracted from differentiating EBs at day10 showing that CHIR 99021 treatment of Gpc4gt-1 EBs restores normal expression levels of Oct4 and a-Mhc. CHIR 99021 concentrations are indicated. Hprt expression (bottom panel) was used to normalize total RNA levels between different samples. ctl: control cells.
SC_11-1204_sm_supplFigure7.pdf118KFigure S7. Gpc4 regulates neural stem cell maintenance in vivo. (A) In situ hybridization on sagittal sections of P1 mouse brains using a Gpc4 probe. Left panel shows restricted Gpc4 expression in the lateral ventricle (lv(, rostral migratory stream (rms(, and in ventral regions. The middle panel is an enlargement of the lv. The arrow indicates the presence of Gpc4 transcripts. The left panel shows Gpc4 expression in the rms. In situ hybridization were done either on whole embryos or floating sagittal sections cut at 50μm using a microtome as previously described. Scale bars: left, 500 μm; middle, 500 μm; right, 250 μm. (B) Western blot analysis of protein extracts from NIH3T3 cells either transfected with a mouse Gpc4-Myc tag expressing construct (Gpc4) or co-transfected with the Gpc4-Myc tag expressing construct and shRNA targeting Gpc4 (sh1, sh2, sh3 and sh5). Expression levels of the Myc-tagged Gpc4 protein were assessed by using cMyc antibodies. Note that sh1, sh2 and sh5 impair expression of the Myc-tagged Gpc4 protein whereas sh3 has not effect. The first lane corresponds to mock transfected cells (-). (C) Quantification analysis of the Myc-tagged Gpc4 protein levels in the transfected NIH3T3 cells shown in panel (B). Western blot analysis revealed that one of those, sh1, efficiently inhibited the translation of the Gpc4 protein, whereas sh2 and sh5 were significantly less effective (Gpc4 protein down regulation: sh1: 76%; sh2: 43%; sh5: 40%; normalized by using Actin as loading control). All values are means±SEM from duplicate experiments. (D) Quantitative analysis of radial glia cells and C cells plus neuroblasts in the the SVZ of mouse brain at P7 co-electroporated at P1 with shRNA control and a GFP expression vectors (control) or co-electroporated with the shRNA targeting Gpc4 above described and the GFP expression vectors (sh1, sh2, sh3, and sh5). The percentage of GFP-positive radial glia cells and of GFP-positive C cells plus neuroblasts was identified following the intensity of GFP staining and morphological criteria. Cells were counted and reported as percentage of radial glial cells and of C cells plus neuroblasts over the total number of GFP expressing cells. Note that the reduction into radial glial cell numbers correlates with down regulation of Gpc4 level (mean±sem; n=2).
SC_11-1204_sm_supplTable1.pdf44KSupplemetary Table 1
SC_11-1204_sm_supplTable2.xls30072KSupplemetary Table 2
SC_11-1204_sm_supplinfor.pdf99KSupplemetary Data

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