Xenbase Image ID: 86887

Supplemental Figure 4. Non-canonical Wnt ligands in mouse and Xenopus embryos. (A) Gene expression levels of non-canonical Wnt ligands in the foregut (fg), liver (lv) and pancreas (vpa and dpa) progenitor datasets. FPKM values (y-axis) were plotted against the different progenitors cell types (x-axis). (B) RT-qPCR validation of Wnt5a and Wnt5b gene expression in foregut, dorsal pancreas and liver progenitor cells. Data were normalized to that of SDHA and represented as fold change compared to the E8.5 8 foregut sample (set to 1 as calibrator). Error bars represent ± SEM. (C) In situ hybridization analysis validated the expression of Wnt5a in the E8.5 foregut endoderm (see red arrowheads) and adjacent lateral plate mesoderm (lpm). At E10.5, Wnt5a transcript was abundant in the limbs (Yamaguchi et al. 1999) and mesenchyme surrounding the midgut and pancreatic rudiments (demarcated by dotted yellow line), whereas its expression in pancreatic epithelial cells became weaker. Scale bars, 50 μm. (D) RT-qPCR of Xenopus Wnt5a, Wnt5b and Wnt7b gene expression in endodermal explants. Both anterior (AE) and posterior endoderm (PE) explants were dissected at stage 10, cultured in isolation until stage 28 and assayed for expression of the indicated genes by RT-qPCR analysis. Data were normalized to that of ODC and represented as fold changes compared to AE sample (set to 1 as calibrator). Error bars represent ± SEM. Wnt5a and Wnt5b were detected in both endoderm populations without any significant regionalized expression, whereas Pdx1 was expressed only in AE derivatives, including the pancreas. (E) Analysis of cell proliferation at early tadpole stages (stages 32-34) revealed no significant differences between control (CTRL) and Wnt5a-injected embryos. Wnt5a mRNA was co-injected with β-gal mRNA into AE cells (dorsal vegetal cells) of 8- cell stage Xenopus embryos. Embryos were fixed at stages 32-34, stained for pHH3 and cleared with benzyl alcohol/benzyl benzoate. Subsequently, the number of pHH3+ cells relative to the anterior gut area (LacZ-staining positive area) was measured in transparent embryos. All results are expressed as means ± SEM. n=8 CTRL embryos; n=15 Wnt5a- injected embryos. Abbreviations: fg, foregut; dpa, dorsal pancreas; lpm, lateral plate mesoderm; lv, liver; vpa, ventral pancreas; AE, anterior endoderm; PE, posterior endoderm; ns, not significant. Image published in: Rodríguez-Seguel E et al. (2013) Copyright © 2013. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article. Larger Image Printer Friendly View

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