Wnt signaling specifies and patterns intestinal endoderm

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Fig 1. Cute little mouse
http://www.myshared.ru/slide/212462/

Contents

1. Introduction
1.1. Mouse intestinal endodermal development
1.2. Mammal intestine
1.3. Wnt signaling pathway and Cdx2 protein
2. Results
2.1. Wnt signaling is active in posterior endoderm between E 7.5 ~ E 8.5
2.2. Activation of Wnt signaling induces Cdx2 expression
2.3. Characterization of intestinal induction of Wnt pathway
2.4. Wnt signaling induces intestinal differentiation in mouse/human embryonic stem cell derived endoderm
2.5. Comparison of Wnt intestinal induction and Cdx2 induction
2.6. Wnt signaling affects anterior-posterior intestinal domain specification
3. Summary
4. Critique

1. Introduction

The intestine is an essential organ in our bodies that is responsible for digestion and absorption of food; thus, when colorectal cancers became the second leading cause of cancer-related mortality, intestine became an important research topic. While considerable insight has been gained about adult intestinal stem cell self-renewal, differentiation, and dysfunction in disease, relatively little is known about how different cell types within the intestine arise during embryogenesis (Barker et al., 2008; Sherwood et al., 2011).

Sherwood et al. explored what molecular distinctions and mechanisms lead to various structures of intestines during embryogenesis. The remaining introduction sections will briefly cover mouse intestinal endodermal development and mammal intestine while the main portion of this article will review the major results discovered by Sherwood et al. For more detailed experimental methods and entire results, click the hyperlink to access the actual paper.

1.1. Mouse Intestinal Endodermal Development 

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Fig 2. Endoderm gastrulation of mouse
http://physrev.physiology.org/content/87/4/1343/F6

During mouse development, the endoderm forms between E6.5 and E7.5 as a flat sheet on the outside of the cup-shaped embryo. By E7.5, the mouse embryo has developed “inside-out” with an internal layer of ectoderm that is surrounded by mesoderm and endoderm.

Fig 3. Formation of AIP and CIP, and head turnover event http://physrev.physiology.org/content/87/4/1343/F6

Fig 3. Formation of AIP and CIP, and head turnover event. Blue is ectoderm; pink is mesoderm; yellow is endoderm
http://physrev.physiology.org/content/87/4/1343/F6

Formation of the gut tube is initiated by the formation of the anterior intestinal portal (AIP). The AIP is an endodermal invagination at the anterior end of the embryo. Formation of the AIP is closely followed by the formation of a second endodermal invagination at the caudal end of the embryo, the caudal intestinal portal (CIP). At E8.5, a complex turning process of the embryo is initiated that folds the endoderm to the inside of the embryo and elongates the AIP and CIP while bringing their openings together around the yolk sac stalk upon completion of the turning process at E9.5. It forms an internal single-cell-thick gut tube which proliferates and begins to become stratified between E8.75 and E14.5. At that point, intestine deforms in the radial plan, eventually forming fingerlike protrusions into the lumen. The adult intestine is composed of these protrusions, known as villi (Brink, 2007). 

 

 

 1.2. Mammal intestine

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Fig 4. small intestine
http://www.proprofs.com/quiz-school/upload/yuiupload/1670740173.jpg

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Fig 5. large intestine
http://3.bp.blogspot.com/_bAYLalavZ6w/TULw4EPNJUIs/uTWl8g_4k6Y/s1600/large+intestine.jpg

 When you study intestines, various morphogenetic, enzymatic, and absorptive properties can be observed. The small intestine is traditionally divided into three segments, the duodenum, jejunum, and ileum, by morphological criteria. The large intestine is also similarly divided into the cecum, ascending, transverse, and descending colon, and rectum. Villous architecture and Paneth cells at the base of crypts are present only in small intestine, while the large intestine is characterized by a flat epithelium with deep crypts. Many digestive enzymes are exclusive to the duodenum and pancreas. Enteroendocrine cells display segment-specific expression. For instance, cholecystokinin is primarily produced in anterior small intestine and peptide yy primarily in distal small intestine and large intestine (Whitcomb and Lowe, 2007; Ratineau et al., 2003).

1.3. Wnt signaling pathway and Cdx2 protein

The two things that this paper focuses on are Wnt signaling pathway and Cdx2 protein. Cdx2 protein is expressed from Cdx2 gene which is the mammalian homologue of the Drosophila posterior determinant. Cdx2 is found to be required for posterior development in all germ layers. It is the first expressed in the posterior endoderm at E7.5, and has been shown to be necessary for intestinal specification in the endoderm (Gao et al., 2009).

Involvement of Wnt pathway in intestinal development was implicated when researchers found out Tcf1/Tcf4 mutants have embryonic intestinal defects (β-catenin, the signal transducer of Wnt pathway, is the transcription factor for Tcf family). Other studies have found that inhibition of Wnt signaling appears necessary for proper differentiation of foregut endoderm derivatives which are anterior to the intestines. Sherwood et al. suspected relationship between Wnt pathway and Cdx2, and studied the effects of their interaction on the specification and anteroposterior patterning of intestinal enderderm (Sherwood et al., 2011).

 

2. Results

2.1. Wnt signaling is active in posterior endoderm

To verify the role of Wnt signaling in intestinal specification and regionalization, first, whether Wnt signaling is active in early definitive endoderm and where in endoderm it is active had to be determined. Sherwood et al. started out by looking at an entire mouse embryo under confocal microscope. The embryo they chose was from BAT-gal mouse strain. BAT-gal mouse is a transgenic mouse that reports canonical Wnt activity through expression of nuclear β-galactosidase. The results were the following:

1. E7.5:

fig 1abc

Fig 6. (A) Wholemount immunofluorescence image of E7.5 BAT-Gal embryo stained for β-gal. Boxed region imaged in higher detail in (B) and (C).

i. β-galactosidase activity observed in posterior endoderm (figure 6 B).

ii. β-galactosidase expressing cells also expressed Cdx2, the posterior endodermal and future intestinal transcriptional regulator (figure 6 C).

iii. β-galactosidase expressing cells are anterior than Cdx2 expressing cells

2. E8.25:

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Fig 7. (E) Wholemount immunofluorescence image of E8.25 BAT-Gal embryo stained for β-gal. Boxed region imaged in higher detail in (F) and (G).

i. It’s hard to see but notice how β-galactosidase expressing cells occur as swath of cells spanning the anterior border of Cdx2 expression (figure 7 F,G).

Fig 5. Wholemount immunofluorescence image of E8.75 BAT-Gal embryo stained for β-gal.

Fig 8. Wholemount immunofluorescence image of E8.75 BAT-Gal embryo stained for β-gal.

3. E8.75:

i. β-galactosidase is no longer detected in posterior endoderm (figure 8).

To make sure Wnt signaling is active in posterior endoderm, Sherwood et al. also looked into expression of Wnt target gene, Axin2. Axin 2 expression followed the patterns of Wnt signaling. It was also expressed in E7.5~E8.5, but down-regulated by E9.5. They also found out that Axin2 expression is weaker in anterior endoderm than posterior endoderm.

2.2. Activation of Wnt signaling induces Cdx2 expression 

After observing apparent correlation between the anterior border of Cdx2 expression and Wnt activity, authors tested whether Wnt signaling could directly induce Cdx2 or not. A whole embryo electroporation strategy was used to introduce DNA into the E8.25 foregut endoderm. The two vectors that were introduced were GFP-expressing DNA, which helps locating the transfected cells, and constitutively active β-catenin (CA β-catenin)-expressing DNA, which activates the Wnt signaling pathway. The results were the following:

1. E8.25 foregut endoderm electroporation experiment:

fig 2 ab

Fig 9. Wholemount confocal immunofluorescence images of E8.25 foregut electroporated with nuclear GFP

i. Control: GFP-expressing DNA ONLY The electroporated cells were detected specifically in the foregut endoderm, but they did NOT express Cdx2 (figure 9 A,B)

fig 2 de

Fig 10. Wholemount confocal immunofluorescence images of E8.25 foregut electroporated with nuclear GFP+CA β-catenin

ii. Experimental: BOTH GFP + CA β-catenin DNA →  The electroporated cells were detected specifically in the foregut endoderm. Notice how Figure 10 E has more GFP tagged cells compared to Figure 9 B. This indicates that Cdx2 is expressed when Wnt is activated (figure 10 D,E).

To eliminate the possible effects of endogenous Cdx2-expressing cells, Sherwood et al. tested whether Wnt signaling can induce Cdx2 in the absence of endogenous Cdx2 endoderm. They took anterior half of the embryo and explanted after foregut was electroporation with CA β-catenin vector.

2. Mouse embryo anterior half electroporation experiment:

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Fig 11. Wholemount confocal immunofluorescence images of E8.25 anterior foregut explant electroporated with nuclear GFP+CA β-catenin

i. Control: Without CA β-catenin → NO Cdx2 expression was observed (Authors didn’t provide figures for this).

ii. Experimental: With CA β-catenin → Cdx2 was successfully induced for E7.5~8.5, and Sox2, anterior endoderm marker, was reduced (figure 11 G,H,I); however, Cdx2 induction failed for E9.5.

These results support that Wnt signaling can directly induce Cdx2, without the presence of Cdx2. The fact that Cdx2 induction failed for E9.5 matches up with previous experiments where Axin2 expression was down-regulated by E9.5. It reinforces the previous observation of transient effects of Wnt signaling on endoderm.

2.3. Characterization of intestinal induction of Wnt pathway

After determining that Wnt can directly induce Cdx2, Sherwood et al. went further to determine what other gene changes are induced by Wnt pathway. They performed microarray analysis on mouse embryo anterior explants that were treated with GSK3 inhibitor XV (GSK3iXV). GSK3iXV is another signaling molecule that could activate Wnt pathway by preventing β-catenin proteolysis. Beads soaked with GSK3iXV were placed in foregut cavity. Gene changes were measured at two time points, after 6hrs and 24hrs. The result was the following:

table 1

1. Cdx2 was up-regulated as expected. On the other hand, anterior endoderm development transcription factors, such as Hhex, Otx2, and Sox2, were down-regulated. Refer to Table 1 for more detailed list.

This up-regulation of posterior intestinal genes, combined with the down-regulation of genes involved in anterior endodermal development, provides strong evidence that Wnt pathway activation induces an intestinal gene expression program.

2.4. Wnt signaling induces intestinal differentiation in mouse embryonic stem cell derived endoderm

The authors then demonstrated that activation of Wnt signaling is sufficient enough to induce intestinal differentiation of embryonic stem cell-derived endoderm. The authors differentiated ES cells into definitive endoderm, and with the derived endoderm, they tested how ES cells derived-endoderm responses to Wnt pathway.

1. Gene changes of ES cell-derived endoderm experiment

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Fig 12. Immunofluorescence analysis of mouse ES-derived endoderm treated with GSK3iXV and stained for Cdx2

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Fig 13. Immunofluorescence analysis of mouse ES-derived endoderm treated with DMSO and stained for Cdx2

If you compare Figure 12 and 13, you can see that ES-derived endoderm treated with GSK3iXV can induce Cdx2 while ES-derived endoderm, not treated with GSK3iXV, yielded less than 1% ES cells expressing Cdx2.

2. Microarray analysis was performed on ES-derived endoderm, treated with GSK3iXV. As expected induces transcripts of intestinal marker genes such as Cdx2 and down-regulate anterior endodermal genes such as Hhex. (Refer to Table 2 for detailed list)

table 2

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Fig 14. Immunofluorescence analysis of human ES-derived endoderm treated with GSK3iXV and stained for Cdx2

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Fig 15. Immunofluorescence analysis of human ES-derived endoderm treated with DMSO and stained for Cdx2

 3. Similar results from human embryonic stem cell-derived endoderm (figure 14, 15). hES-derived endoderm was capable of inducing Cdx2 when Wnt was activated.

 The correlation between gene expression changes in E8.25 embryonic endoderm and ES derived endoderm indicates that Wnt signaling plays a similar role in embryonic and ES derived endoderm.

2.5 Comparison of Wnt intestinal induction and Cdx2 induction

Since Cdx2 is the first known molecular marker for the future intestinal eptihelium, Sherwood et al. suspected that Cdx2 alone could be sufficient enough to induce an intestinal gene program. To test it, they used ES cell line that has tetracycline-responsive promoter integrated into the locus upstream to the gene of interest. This way the gene of interest can be expressed in the presence of tetracycline and ensures uniform induction of the gene. Microarray analysis was performed on ES cell-derived endoderm, treated with either GSK3iXV alone or tetracycline-inducible Cdx2 alone. The followings are the results:

1. Tetracycline-inducible Cdx2 ES cell experiment

i. Control: GSK3iXV alone

ii. Experimental: Tetracycline-induced Cdx2 alone Only 18% of the genes induced by GSK3iXV alone were induced (27/154). On the other hand, larger degree of overlap (42%) was observed between genes down-regulated by GSK3iXV and Cdx2 (93/221)

This analysis indicated that the role of Wnt in intestinal induction is not simply the induction of Cdx2.

2.6. Wnt signaling affects anterior-posterior intestinal domain specification

Lastly, Sherwood et al. tested how Wnt signaling affect different regions of intestines. They divided the intestine into 4 regions, listed below. Each 4 region has unique molecular markers (documented by previous studies), and Sherwood et al. observed how those molecular markers are affected by Wnt signaling pathway.

1. 4 endodermal populations (figure 16):

fig 5a

Fig 16. Wholemount image of E14.5 gut with different anterior-posterior divisions dissected for microarray analysis labeled.

i. Large intestine:
molecular markers: Hoxd10, Ly6a, Xpnpep2 

ii. Anterior half of small intestine:
molecular markers: Pdx1, Onecut2, Anpep, Tm4sf4 

iii. Posterior half of small intestine:
molecular markers: Cib2, Fzd10, Osr2 

iv. Stomach:
molecular markers: Ly6h, Sox21

 To determine whether Wnt signaling specifies a particular intestinal domain,  gene changes in anterior endoderm treated with GSK3iXV beads were tested. The results were the following:

i. Large intestine:
molecular markers: Hoxd10, Ly6a, Xpnpep2 → up-regulated

ii. Anterior half of small intestine:
molecular markers: Pdx1, Onecut2, Anpep, Tm4sf4 → down-regulated

iii. Posterior half of small intestine:
molecular markers: Cib2, Fzd10, Osr2 → up-regulated

iv. Stomach:
molecular markers: Ly6h, Sox21 → down-regulated

The authors concluded that Wnt pathway specifically induces posterior small intestine and large intestine fate.

3. To investigate the role of Wnt in intestinal subtype specification in more detail, E8.25 anterior endoderm explant was treated with different concentration of GSK3iXV.

fig 5 ij

Fig 17. Wholemount confocal immunofluorescence images of E8.25 foreguts, treated with 1uM GSK3iXV stained for Ly6a. (J) Green is Ly6a; Red is Foxa2)

i. 1uM GSK3iXV Induced large intestine specific Ly6a. This suggests that higher level of Wnt signaling preferentially induce more posterior intestinal genes (figure 17)

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Fig 18. Wholemount confocal immunofluorescence images of E8.25 foreguts, treated with 10nM GSK3iXV stained for Ly6a. (J) Green is Ly6a; Red is Foxa2)

 ii. 10nM GSK3iXV  Compared to Figure 14, posterior intestinal gene expression is almost non-existent (figure 18)

3. Summary

In conclusion, Sherwood et al. established a clear, direct, and multifaceted role for Wnt signaling in intestinal specification and patterning. Wnt signaling activates Cdx2 in the early endoderm and is capable of inducing an intestinal gene program in anterior endoderm cells. Wnt signaling not only specifies intestinal fate from undifferentiated endoderm, it appears to have concentration-dependent effects on anterior–posterior intestinal segments. They have transferred these findings to the in vitro differentiation of embryonic stem cells and show that Wnt signaling efficiently specifies intestinal fate in endoderm derived from mouse and human ES cells.

4. Critique

Sherwood et al. did truly superb work in this paper. They have thoroughly convinced me that Wnt signaling induces intestinal endoderm specification. They did not overstate their claims – they never suggested that Cdx2 induction by Wnt signaling is the only mechanism that patterns intestinal endoderm; rather, they proved shortcoming of Cdx2 and suggested other functions of Wnt, such as down-regulating anterior endoderm development transcription factors. As far as I can understand, their claims were perfectly within the bounds of the data presented.

The only thing I found odd was that there were some experiment plans mentioned in the paper but the results were neither presented nor discussed why they were omitted. They weren’t included in supplementary materials either. Also, one experimental design flaw that I’ve noticed was when the authors were testing whether Cdx2 alone is capable of inducing intestinal gene program. They chose ES-derived endoderm treated with GSK3iXV alone as a control group and ES-derived endoderm treated with Cdx2 alone as the only experimental group, but I think there should have been an experimental group that treated ES-derived endoderm with both GSK3iXV and Cdx2 to compare the outcome to the result from Cdx2 alone group.

5. Citation

1. Barker, N., van de Wetering, M., Clevers, H., 2008. The intestinal stem cell. Genes Dev. 22: 1856–1864.

2. Gao, N., White, P., Kaestner, K.H., 2009. Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2. Dev. Cell 16: 588–599.

3. Ratineau, C., Duluc, I., Pourreyron, C., Kedinger, M., Freund, J.N., Roche, C., 2003. Endoderm- and mesenchyme-dependent commitment of the differentiated epithelial cell types in the developing intestine of rat. Differentiation 71: 163–169.

4. Sherwood, R.I., Chen, D.Y., Melton, D.A. 2001. Wnt signaling specifies and patterns intestinal endoderm. Mechanism of Development. 128: 387-400

5. van den Brink, G.R. 2007. Hedgehog Signaling in Development and Homeostasis of the Gastrointestinal Tract. Physiological Reviews 87: 1343-1375

6. Whitcomb, D.C., Lowe, M.E., 2007. Human pancreatic digestive enzymes. Dig. Dis. Sci. 52, 1–17.

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