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1.
Figure 1

Figure 1. Axin2LacZ/+ reporter is active in the region of the primitive streak and derived cell populations. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

No expression is visible in E5.5 and E6.0 embryos, before primitive streak formation. The E6.5 and E7.5 embryos are oriented with their posterior pointing right.

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.
2.
Figure 6

Figure 6. Tissue organization in 7xTCF-eGFP embryoid bodies. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

Cryosections through 7xTCF-eGFP (green in A, C and E) embryoid bodies stained for laminin (A,B), phalloidin (C) or Oct4 (D,E) (red). Nuclei were stained with dapi (blue). (A) No basal lamina could be detected in 4 day old embryoid bodies (n>60). (B) Basal lamina were readily detectable around the periphery of 7 day old embryoid bodies. (C) Phalloidin staining (red) revealed no epithelial polarity in cells prior to activating 7xTCF-eGFP. (D) Oct4 was expressed throughout 3.5 day (shown) and earlier (not shown) embryoid bodies, before expression of the Wnt reporter became apparent. (E) Oct4 expression was down regulated in cells activating 7xTCF-eGFP (green). No GFP was imaged in (B) and (D).

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.
3.
Figure 7

Figure 7. Induction of Wnt signaling in embryoid bodies requires external signals. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

(A) When 7xTCF-eGFP embryoid bodies were cultured in serum- and growth factor-free medium (initial aggregation was performed in serum-containing medium for 2.5 days), they failed to induce Wnt signaling (white) (>90%, n=48). Addition of Wnt3a (10 ng/ml), Bmp4 (12.5 ng/ml) or ActivinA (12.5 ng/ml) activated the reporter (83%, 50%, and 79%, respectively, n=24). This could be repressed by Dkk1 (60 ng/ml), showing that it is the result of the induction of endogenous Wnt proteins (100%, n=24). Noggin (12.5 µg/ml) was unable to inhibit induction of the reporter by Wnt3a or ActivinA (n=8). (B) The Bmp antagonist noggin (12.5 µg/ml) inhibited activation of the Wnt-reporter in 7xTCF-eGFP embryoid bodies cultured in serum-containing medium (83%, n=12). Scale bar 100 µm (A, B).

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.
4.
Figure 2

Figure 2. Localized Wnt signaling in embryoid bodies. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

(A) Axin2LacZ/+ embryoid bodies spontaneously activated the reporter in a localized fashion after 3 days in culture (n>200). (B) Wnt signaling expanded overtime throughout the entire Axin2LacZ/+ embryoid body. Addition of recombinant Wnt3a (200 ng/ml) led to accelerated activation of the reporter, whereas Dkk1 (80 ng/ml) delayed reporter activation by 2 days (n>200). (C) 7xTCFeGFP embryoid bodies displayed similar expression of the reporter as Axin2LacZ/+ embryoid bodies, but with a delay of approximately one day. The delay is probably due to the larger size of the embryoid bodies, and time required for GFP maturation (n>200). Scale bar 500 µm (A, B), 100 µm (C).

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.
5.
Figure 3

Figure 3. Wnt signaling regulates the local expression of markers for the primary germ layers in embryoid bodies. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

(A) 7xTCF-eGFP embryoid bodies were cultured for 4 days, separated into GFP-positive and GFP-negative cell pools using a cell sorter, and the pools were analyzed by real-time PCR. Expression of Brachyury, FoxA2, Flk1 and Mixl1 was increased in the GFP-positive cells compared to the GFP-negative cells, whereas Otx2, Pax6 and Sox1 were repressed (mean +/− s.e.m., n=3). (B) Real time PCR analysis of RNA collected from embryoid bodies cultured in presence of the indicated factors. Factors were added 2.5 days after the start of aggregation. Addition of Wnt3a (200 ng/ml) resulted in early peaking levels of Brachyury, Foxa2 and Flk1, whereas the neurectodermal markers Otx2 and Pax6 became rapidly down regulated, and induction of Sox1 was prevented. Addition of Dkk1 (80ng/ml) resulted in delayed induction of Brachyury, Foxa2 and Flk1, and promoted expression of Otx2, Pax6 and Sox1 (mean +/− s.e.m., n=3).

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.
6.
Figure 5

Figure 5. Wnt signaling controls epithelial-to-mesenchymal transition in embryoid bodies. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

(A) 7xTCF-eGFP embryoid bodies were cultured for 4 days, separated into GFP-positive and GFP-negative cell pools using a cell sorter, and the pools were analyzed by real-time PCR. Expression of Snail and fibronectin was enriched in the GFP-positive cells compared to the GFP-negative cells, whereas E-cadherin was repressed (mean +/− s.e.m., n=3). (B) Real time PCR analysis of RNA collected from embryoid bodies cultured in presence of the indicated factors. Addition of Wnt3a (200 ng/ml) resulted in accelerated induction of Snail and fibronectin, whereas E-cadherin was rapidly down regulated. Addition of Dkk1 (80ng/ml) resulted in delayed induction of Snail and fibronectin, and maintained expression of E-cadherin (mean +/− s.e.m., n=3). (C) Cryosections through 7xTCF-eGFP (green) embryoid bodies were immunostained for Brachyury or E-cadherin (red). Brachyury and GFP expression overlap to a large extent (80%, n>25), whereas E-cadherin and GFP were mutually exclusive except at the edges of the GFP domain (arrows) (>90%, n>25). Scale bar 100 µm.

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.
7.
Figure 4

Figure 4. Wnt signaling regulates the local formation of mesendoderm in embryoid bodies. From: Wnt signaling mediates self-organization and axis formation in embryoid bodies.

(A) Whole mount immunostaining of 7xTCF-eGFP embryoid bodies showed co-localization (yellow) of primitive streak markers Brachyury and Foxa2 (red) with the Wnt reporter (green). Expression of Otx2 (red) was mutually exclusive with the Wnt reporter. Treatment with Wnt3a (200 ng/ml) accelerated the upregulation of the Wnt reporter, Brachyury, and FoxA2, whereas it repressed Otx2. Dkk1 treatment (80 ng/ml) had the opposite effect, repressing Wnt signaling, Brachyury, and FoxA2 expression, and promoting Otx2 expression throughout the embryoid body. Factors were added 2.5 days after the start of aggregation. (B) In embryoid bodies derived from a Brachyury-GFP;7xTCF-mCherry cell line, both reporters expressed in close proximity or overlapping domains (GFP-green, mCherry-red; 80%, n>120). The side population of small embryoid bodies that formed during aggregation was excluded from subsequent analyses. (C) Wnt3a treatment induced expression of both reporters throughout Brachyury-GFP;7xTCF-mCherry embryoid bodies (50%, n>60), whereas Dkk1 repressed expression of both reporters (>90%, n>60). Scale bar 100 µm (A, C), 200 µm (B).

Derk ten Berge, et al. Cell Stem Cell. ;3(5):508-518.

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