Sox17-mediated deletion of Setdb1 leads to two different phenotypes

As described before, we generated Setdb1 EGFP reporter mice and the EGFP reporter gene could help us to trace Sox17 lineage cells. By analyzing the expression of EGFP in the embryos, we found that not all of the EGFP staining was confined to the endoderm region and also a few embryos showed EGFP staining in other regions of the embryo. This EGFP expression pattern could be detected both in mutant and control embryos. Activation of EGFP reporter and deletion of Setdb1 is due to the expression of Sox17. So far, the fate of the Sox17 expressing cells in the morula is still elusive.

Because in late stage blastocysts Sox17 is restricted to primitive endoderm, it is unclear that if these early Sox17 expressing cells in the morula are maintained in the blastocyst to contribute to the formation of other organs during development. Interestingly, the mutant embryos which show the overall EGFP staining in our experiment are severely retarded (Figure 2.15). This finding corresponded to our observation that there are two different kinds of phenotypic defects in Setdb1^END mutants (Figure 2.14). We assumed that the type1 mutant embryos are generated by the specific deletion of Setdb1 in the endoderm during development. In contrast, type2 mutant embryos are generated by an overall deletion of Setdb1.

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Figure 2.15 Two different EGFP expression patterns in control and mutant embryos. In type1 embryos the GFP signal is restricted to the endoderm region of the epiblast derivatives (A, B, C and D represent control embryos; I, J, K and L represent mutant embryos). There is no EGFP staining in the neural tube (C and K) in type1 embryos. The whole embryos show EGFP staining in type2 embryos (E, F, G and H represent control embryos; M, N, O and P represent mutant embryos). The EGFP signal can be detected in the neural tube of the type2 embryos (G and O).

2.2.4 Structural changes in the hindgut region in Setdb1^END mutant embryos

The Setdb1^END embryos show a clear developmental defect. Next, we wanted to specify marker changes by in situ experiments. In these experiments we focused on the function of Setdb1 in the definitive endoderm cells and only analyzed type1 mutant embryos which show the specific deletion

37 RESULTS

of Setdb1 in endoderm cells. We use different probes for Pyy, Trh, Nepn and Sox17 to detect these key markers of different embryonic regions. Pyy is a foregut region marker which is expressed in the foregut invagination. Trh is mainly expressed in neural tissues at roughly E9.0 (McKnight et al. 2007).

Nepn is a midgut region marker and could only be recognized at the open region of the gut tube at the E8.5 embryos. Sox17 could be recognized in the hindgut region which is the closed region of the gut tub as well as in hemangioblasts and visceral endoderm (McKnight et al. 2010).

Comparing the Setdb1^END (mutant) and wild type embryos with these different probes (Figure 2.16), the expression of Pyy, Sox17, Trh does not show a clear difference between mutant and wild type embryos. As Sox17 is also expressed in the visceral endoderm, it is hard to judge the precise expression of Sox17 in definitive endoderm. According to Pyy, Trh and Nepn expression in the embryos we could judge that the development of the gut region and neural tube in the mutant embryos is not affected. The expression of these three markers is the same among mutant and control embryos.

Thus deletion of Setdb1 in definitive endoderm does not affect the formation of the gut region.

Figure 2.16 No difference in gut region and neural tissue marker expression between control and Setdb1^END (mutant) embryos at E8.5 by in situ hybridization. Pictures are taken from the lateral and dorsal side of the embryos. There is no difference that could be detected using these four different in situ markers.

Based on the expression of these markers, there is no difference in gut tube formation between control and mutant embryos. Further, we intended to identify any structural alterations of the gut region in mutant embryos. We collected mutant and control embryos from different developmental stages at E8.0, E8.5 and E9.5. These embryos were embedded in paraffin and used to generate sections of different layers of these embryos to identify structural changes. The structure of the mutant and control embryos at E8.0 is almost the same and the shape and the position of the endoderm cells is also very similar (Figure 2.17). These analyses revealed that endoderm could be properly formed in the mutant embryos at E8.0.

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Figure 2.17 No structural change of the hindgut region upon deletion of Setdb1 at E8.0. A, B, C and D) H&E staining of paraffin sections of control and Setdb1^END (mutant) embryos at E8.0. E) The schematic of the position of the sections shown in A-D.

We chose another critical time point at which the mutant embryos show the axis turning problem at around E8.5. During mouse development, the hindgut diverticulum initiates to be closed from the hindgut region to the foregut region. We could see the hindgut diverticulum is already closed in both control and mutant embryos but the structure of that region is altered in the mutants. In control embryos the hindgut diverticulum is closed and in contact with the splanchnopleura (Figure 2.18 A, B, C and D). In mutant embryos the hindgut diverticulum could be formed but it is smaller and loses the contact with the splanchnopleura (Figure 2.18 E, F, G and H). Moreover the size of the dorsal aorta is much bigger in mutant embryos as compared to control embryos. Notably, the development of anterior parts of the mutant embryos such as neural tube and heart is normal.

As the splanchnopleura and is not derived from the endoderm lineage, we assumed the loss of the contact between diverticulum and splanchnopleura is caused by non-cell autonomous effects. This means lack of Setdb1 in endoderm cells does not only affect the development of the endoderm derived tissue but also the tissues close to endoderm.

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Figure 2.18 Defective gut development upon deletion of Setdb1 in the Setdb1^END (mutant) embryos at E8.5. A, B, E and F) H&E staining of paraffin sections at E8.0 from different layers. C, D, G and H) Enlarged picture from the square region. I) Schematic indicates the position of the different sections. Red arrows point to the defected region.

Immunohistochemistry was further conducted for later stages when the embryos already showed strong phenotypes at E9.5. We found that the anterior part of the mutant embryos showed a similar structure as the wild type embryos but the posterior gut region between mutant and control embryos was not comparable. At this developmental stage the mutant embryos already showed severe defects and the overall structure was severely affected. The hindgut diverticulum is surrounded by the splanchnopleura in control embryos but the hindgut diverticulum could not properly interact with other tissues and was stuck in the dorsal aorta in mutant embryos (Figure 2.19 arrow). This is consistent with our findings in E8.5 embryos. We thus conclude that deletion of Setdb1 in endoderm results in defective development of the posterior part of the mutant embryos.

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Figure 2.19 The posterior part of the Setdb1^END (mutant) embryos is strongly defective upon deletion of Setdb1 at E9.5. A) The gut region is surrounded by the splanchnopleura in the control embryo. B) The gut is stuck in the dorsal aorta in the mutant embryo and the posterior region is underdeveloped. Arrows show hindgut diverticulum.

The histological analysis at E8.5 revealed that the diverticulum in the mutant embryos cannot be well connected to the mesoderm derivatives. Furthermore the dorsal aortal is much bigger in the mutant embryos. As Sox17 lineage tracing experiments showed that Sox17 progenitor cells contribute to the formation of blood vessels we tested if the formation of the vasculature is affected upon deletion of Setdb1.

Platelet endothelial cell adhesion molecule (Pecam-1) is proved to be related to the formation of the vasculature and mediates cell-cell adhesion. Pecam1 is expressed in all endothelial cells and is first detected in the yolk sac and subsequently within the embryo itself (Baldwin et al. 1994). By staining of Pecam1 in the mutant embryos we found that the expression of pecam1 is not affected but the blood vessels are bigger in the mutant embryos in the comparison with the control embryos. This is consistent with our histological analysis. Based on this result we conclude that the formation of the vasculature is not generally affected by loss of Setdb1.

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Figure 2.20 Whole mount immunostaining of control and Setdb1^END (mutant) embryos at E9.0 for Pecam1 and Caspase3. In mutant embryos, the blood vessels which are stained with Pecam1 (red) are wilder than in control embryos. The squares on the left side show the position of the images in the embryos.