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First published online 30 August 2006
doi: 10.1242/dev.02551

Development 133, 3787-3796 (2006)
Published by The Company of Biologists 2006
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Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm

R. Coleman Lindsley1, Jennifer G. Gill1, Michael Kyba2, Theresa L. Murphy1 and Kenneth M. Murphy1,3,*

1 Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA.
2 Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9133, USA.
3 Howard Hughes Medical Institute, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA.


Figure 1
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  		Fig. 1. Activation of canonical Wnt signaling during early ES cell differentiation. (A) Transient expression of Wnt8a and Wnt3 during ES cell differentiation. Semi-quantitative RT-PCR was carried out as described in the methods for Wnt8a, Wnt3 and Gapdh. Analysis was performed on ES cells (ESC) and on ES cells differentiated in SCM for the indicated number of days. (B) ES cells bearing a stably integrated SUPER8xTOPFlash luciferase reporter were differentiated in SCM either in the presence or absence of Dkk1, as indicated. Luciferase activity in undifferentiated ES cells (ESC) or in ES cells differentiating for the indicated number of days in SCM or SCM with addition of Dkk1 (SCM + Dkk1) was measured. SUPER8xTOPFlash luciferase activity is presented as a percentage of the activity measured in the same conditions treated with LiCl (20 mM) for 18 hours prior to harvesting. (C) SUPER8xTOPFlash transfected cells in B were differentiated in serum replacement medium alone (SRM) or with the addition of Dkk1 (SRM + Dkk1) for 2 and 4 days, and luciferase activity measured as in B.

 

Figure 2
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  		Fig. 2. Wnt signaling is required for generation of Flk1+ mesoderm. (A) Differentiating ES cells in SCM were treated either with Dkk1 or Noggin/Fc as indicated, or left unmanipulated (no treatment). Embryoid bodies were harvested at day 3, 4 and 5, and analyzed for Flk1 expression as described in the Materials and methods. Shown are dot plots gated on live cells. Numbers represent the percentage of Flk1+ within the indicated region. (B) ES cells were differentiated in SCM supplemented with the indicated protein, and Flk1 expression determined on day 4 as described above. (C) Dkk1 was added to differentiating cultures at day 0, 1.0, 1.5, 2.0, 2.5 or 3.0. The frequency of Flk1-expressing cells at day 4 was determined as described in A. The data are represented as the percentage of inhibition, measured by dividing the frequency of Flk1+ cells in each Dkk1 treated culture by the frequency of Flk1+ cells in parallel untreated cultures.

 

Figure 3
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  		Fig. 3. Wnt-dependent expression of genes associated with primitive streak, EMT, endoderm and mesoderm. ES cells were differentiated in SCM alone or in the presence of Dkk1 (SCM + Dkk1). RNA was isolated at indicated times and analyzed using custom DNA microarrays. Normalized and modeled expression values for the indicated genes in either SCM (solid line) or SCM + Dkk1 (broken line) are expressed in arbitrary units.

 

Figure 4
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  		Fig. 4. Early inhibition of canonical Wnt signaling enhances long-term neuronal differentiation. (A) ES cells were differentiated in SCM alone or with addition of 1x Dkk1. At day 4, cells were washed and transferred to SCM without inhibitor. At days 6-8, RNA was isolated and examined for expression of indicated genes by RT-PCR. (B-D) Dkk1-treated cells described in A were transferred at day 9 to fibronectin-coated slides in SRM and stained for neuronal ßIII-tubulin (green), nestin (red) or Pax6 (D, red) expression at day 13. Nuclei are stained with Hoechst 33342 (blue) and images were acquired using 10x (B,D) and 20x (C) objective magnification. Untreated cells exhibited negligible staining for neuronal markers.

 

Figure 5
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  		Fig. 5. Canonical Wnt signaling is required for generation of ES cell-derived mesoderm. (A) ES cells were differentiated in SCM alone (no treatment) or treated with either Dkk1 or Noggin/Fc for 4 days as indicated. After 4 days, cells were washed and transferred to SCM without inhibitor for an additional 2 days. On day 6, cells were assayed for hematopoietic precursor potential using methylcellulose colony-forming assays as described in the methods. (B) ES cells described in A were treated as indicated, harvested at day 6 and assessed for expression of the indicated genes by RT-PCR. (C) ES cells were differentiated as in A, except that at day 4, cells were washed and transferred to gelatinized plates in SRM without inhibitor. At days 6, 7 and 8, cells were harvested and analyzed by RT-PCR for the indicated genes. (D) Cells were differentiated as in C and analyzed by fluorescence microscopy for expression of E-cadherin (green) or fibronectin (red) as described in the Materials and methods. Images of representative colonies, acquired using 10x objective magnification, are shown for unmanipulated (no treatment) and Dkk1-treated conditions.

 

Figure 6
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  		Fig. 6. Stabilized ß-catenin is insufficient for induction of primitive streak-associated gene expression. (A) A2lox.sßcat ES cells transiently transfected with the SUPER8xTOPFlash (TOP) or SUPER8xFOPFlash (FOP) reporters were left untreated (no treatment), or treated with either 1 µg/ml doxycycline (DOX) or LiCl for 18 hours. Whole cell lysates were prepared and luciferase activity measured. Shown is the luciferase activity normalized to a co-transfected Renilla luciferase construct driven by the CMV promoter as previously described (Ranganath et al., 1998Go). (B) A2lox.sßcat ES cells were differentiated in SRM alone or in the presence of Dkk1 (+Dkk1) and of the indicated concentration of doxycycline (DOX). Dkk1 was added at the initiation of differentiation. Doxycycline was added at day 1 of differentiation. Cells were harvested at day 3.5 of differentiation and evaluated for expression of the indicated genes by RT-PCR. Expression found in ES cells differentiated in SCM without doxycycline represent positive controls.

 

Figure 7
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  		Fig. 7. Soluble factors can restore mesoderm generation in the presence of Dkk1. (A) ES cells were differentiated in SCM (no treatment), SCM with Dkk1 alone, or in SCM + Dkk1 and one of the following factors: TGFß1, Bmp2, Bmp4, Nodal, Fgf8, cripto or FST. On day 4 of differentiation, cells were examined for Flk1 expression by FACS. Numbers indicate the percentage of Flk1+ cells in the indicated region. (B) ES cells were treated as described in A, and on day 4, embryoid bodies were washed, cultured for an additional 2 days in SCM and then analyzed for hematopoeitic potential as described in Fig. 5A. (C) Cells were treated as described in A, embryoid bodies washed and transferred to gelatinized dishes in serum-free medium for 4 days. Cultures were analyzed at day 8 for cardiac gene expression by RT-PCR. (D) ES cells were differentiated in SCM alone (SCM) or SCM + Dkk1 in the absence (-) or presence of Bmp4 or cripto, as indicated. Bmp4 and cripto were added on day 1, and cells were analyzed on days 3 and 4 as indicated.

 

Figure 8
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  		Fig. 8. Wnt and Bmp signaling act cooperatively to regulate brachyury expression. (A) ES cells were differentiated in SRM and the indicated concentration of Bmp4 and Dkk1. RNA was prepared at day 3.5 and analyzed by RT-PCR for brachyury and Gapdh expression. (B) A2lox.sßcat ES cells were differentiated in SRM alone or SRM plus the indicated concentration of Bmp4 and/or doxycycline. RNA was prepared at day 3.5 and analyzed by RT-PCR for brachyury, Nodal and Gapdh expression.

 

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