September 1, 2013;
Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence.
Understanding how distinct cell types arise from multipotent progenitor cells is a major quest in stem cell
biology. The liver
share many aspects of their early development and possibly originate from a common progenitor. However, how liver
cells diverge from a common endoderm
progenitor population and adopt specific fates remains elusive. Using RNA sequencing (RNA-seq), we defined the molecular identity of liver
progenitors that were isolated from the mouse embryo
at two time points, spanning the period when the lineage decision is made. The integration of temporal and spatial gene expression profiles unveiled mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the noncanonical Wnt pathway as a potential developmental regulator of this fate decision and capable of inducing the pancreas
program in endoderm
cells. Our study offers an unprecedented view of gene expression programs in liver
progenitors and forms the basis for formulating lineage-reprogramming strategies to convert adult hepatic cells into pancreatic cells.
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Figure 4. Noncanonical Wnt5a activity promotes pancreatic versus hepatic fate in the anterior endoderm. (A) Xenopus embryos injected with Wnt5a mRNA showed a shortened and mildly bent body at the tailbud stage, as previously described (Kim et al. 2005). (B) Endodermal explants were cultured in the presence of Wnt5a recombinant protein from stage 10, collected at the tadpole stage, and assayed for expression of the indicated pancreatic, hepatic, and duodenum/stomach genes by RT-qPCR analysis. Untreated anterior endodermal explants were used as control. Data were normalized to that of ornithine decarboxylase (ODC) and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). Error bars represent ±SEM. (C) Whole-mount double in situ hybridization analysis of Hex (light blue) and Ptf1a (purple) in control and Wnt5a-injected Xenopus embryos at the tadpole stage. The arrow indicates Hex expression in the liver bud, and arrowheads indicate Ptf1a expression in the two pancreatic buds (dorsal and ventral buds). Dashed lines mark expanded Ptf1a expression in the injected embryos. Total number of injected embryos = 61; 41% showed visible expansion of Ptf1a. (AE) Anterior endoderm; (PE) posterior endoderm. (D) RT-qPCR analysis of endodermal explants treated with Wnt5b (200 ng/mL) recombinant protein. Data were normalized to that of ODC and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). (E) RT-qPCR analysis of endodermal explants treated with 500 ng/mL Wnt3a recombinant protein. Data were normalized to that of ODC and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). (F) RT-qPCR analysis of direct downstream target genes of the Wnt/β-catenin pathway in endodermal explants treated with 500 ng/mL Wnt5a or 500 ng/mL Wnt3a recombinant protein. Data were normalized to that of ODC and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). (G) TOPFLASH and ATF2-luc reporter assays in Xenopus embryos. Four-cell stage embryos were injected into the vegetal blastomeres with 50 pg of TOPFLASH or 100 pg of ATF2-luc plus 25 pg of Renilla luciferase reporter plasmids. Endodermal explants were dissected at stage 9 and either left untreated as control (CTRL) or exposed to 500 ng/mL Wnt5a or 500 ng/mL Wnt3a recombinant protein, as indicated. Luciferase reporter assays were carried out in explants lysed at gastrula and early tailbud stages. (H) Western blot analysis of dissected anterior endodermal explants either left untreated as control (CTRL) or exposed to Wnt5a or Wnt3a recombinant protein. The relative ABC/tubulin levels in the treated explants compared with the control, which was set to 1.0, are indicated. (β-cat) Total β-catenin; (tub) α-tubulin. (*) P < 0.05; (**) P < 0.01, as determined by the REST program statistical analysis (Pfaffl et al. 2002).
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
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.