The role of SOX2 during murine development

SOX2 is not only an important factor for pluripotency of embryonic stem cells but also known to be highly expressed in endoderm derived tissues of vertebrates, thus likely to contribute to stomach development and upholding of adult gastric stem cells.

SOX2 expression during mouse embryonic development was confirmed in the central nervous system including the retina as reported by Stevanovic and Collignon [244, 245], in the hair follicles [246], in the lungs [247] as well as in stomach and esophagus [147, 148].

Deletion of Sox2 using a conditional knock out mouse resulted in different phenotypes depending on dose and time point of injection of tamoxifen. Early Sox2 depletion at day E 7.5 resulted in deformed embryos at day E 19.5 showing especially degeneration of the stomach. It was assumed that deletion of Sox2 leads to an intestinalization of the stomach due to a shift in pathway regulation. Sox2, together with Cdx2, plays a crucial role during early gut development. It is important for regionalization of the primitive streak influencing anterior and posterior gut formation. Later on these compartments become subdivided into foregut, midgut and hindgut and then differentiate into stomach and esophagus where SOX2 is expressed, or duodenum,

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jejunum, ileum caecum and colon which express CDX2 but not SOX2 [248]. In correlation to these data it has been shown recently that expression of Sox2 in the prospective intestinal part redirects intestinal epithelium towards a premature gastric phenotype during gut development [249]. These data strengthen the findings of the present work that loss of Sox2 in the gastric part of the gut on the other hand might induce an intestinal phenotype. A more severe macroscopic phenotype was observed with increasing dosage of tamoxifen injections. Injecting four times during pregnancy led to stagnation of development of knock-out embryos. This severe phenotype might occur due to several reasons. As mentioned above Sox2 is not only important in development of the stomach but also contributing to formation of several other tissues. Therefore, degeneration of embryos could not further be curtailed to a specific reason and has to be investigated in more detail. Thus, to have a more specific view on Sox2 deletion in embryonic stomach development and to avoid too many off-targets effects due to the expression of Sox2 in other tissue we additionally worked with a Sox17iCre mouse. Sox17 is expressed in the definite endoderm, which gives rise to lung, heart, liver, pancreas, stomach and GI tract. Since breeding generated no Sox17iCre x Sox2^fl/fl offspring it is assumed that expression of Sox2 in the definite endoderm is crucial for the viability of embryos. Sox17iCre is expressed as early as day E 6.0 in the extraembryonic visceral endoderm [198]. An expression of Sox2 in the same part of the embryo was reported by Avilion et al. at day E 6.5 [127]. Hence the role of Sox2 during this stage of embryonic development is pivotal, so that deletion leads to complete malformation and therefore resorption of k.o. embryos. Generation of a stomach specific Cre-mouse-line would be fundamental for further investigations of the role of Sox2 during stomach development.

Furthermore, data of Avilion et al. could be confirmed by analyzing Sox2^geo mice.

As reported earlier [144] no differences in number of blastocytes, late morulae or unfertilized eggs could be found, obtained from Sox2^geo/wt inbred compared to Sox2^wt/wt inbred embryos, however, at E 7.5 about 1/4 of embryos of Sox2^geo/wt inbred mice where degenerated. These results confirm the essential role of Sox2 during early mouse development since it is already expressed in the inner cell mass (ICM) of blastocytes, although only at the stage of implantation a disruption of Sox2 leads to lethality. In the growing oocyte there is much maternal SOX2 protein left which might rescue the k.o.

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blastocytes. This protein is diluted during rapid growth of the embryo and hence is not able to further rescue k.o. embryos after implantation.

During early postnatal development of mice, k.o. of Sox2 by induction of CreER^T2 via breast feeding led to retardation in development which was measurable after one week to 10 days. K.o. pups showed significantly lower weight compared to their wt littermates (Fig. 46), did not develop proper fur (Fig. 47) and died within two to three weeks of age. Since Sox2 is an important factor in development of dermal papillae [246]

this explains the improper growth of fur. However, further experiments should be conducted to investigate how Sox2 regulates generation of fur. Furthermore, Sox2 is important in stomach development of mice [148] and loss of Sox2 leads to a growth defect and moreover might also influence hormonal regulation in the stomach [147].

Negative effects of Sox2 k.o. in pups get more severe over time, since the daily uptake of breast milk increases the tamoxifen dosage, thus increasing the k.o. of Sox2, and resulted in weight loss and eventually in their death. Combined with the findings that SOX2 is co-expressed at proliferation sites in the assumed stem cell zones of the adult mouse stomach and Sox2 k.o. in adult mice leads to less proliferating cells (Fig. 49) it is suggesed that Sox2 k.o. in pups influences the growth of the stomach by suppression of proliferating cells, hence leading to malformations. Additionally, a hormonal dysregulation is also likely to influence weight loss in k.o. pups.

A recent publication by Arnold et al. showed that Sox2 positive cells are indeed potential markers for adult stem cells in the stomach [37]. They could also observe that adult Sox2 k.o. resulted in death of mice, which they explained by the development of gastric ulcer. However, in this study adult mice did not die after Sox2 k.o. This might be due to the different k.o. systems. Excision of Sox2 in the system applied in the present study is strongly dependent on the dosage of tamoxifen reaching the cells, thus this k.o.

occurs generally only partially and leaves the cells alive. The ganciclovir/delta thmyidine kinase (DTK) system applied by Arnold at al. is not only more sensitive but also kills every cell expressing DTK, thus every Sox2 positive cell, after ganciclovir induction. Hence, a more severe phenotype is expected. Nevertheless, findings of this study confirm the results of Arnold et. al. that Sox2 is influencing proliferation of cells in the adult mouse

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stomach. Besides, these data are consistent with earlier findings shown in this work that Sox2 is also expressed at proliferation sites in gastric tumor samples, again accrediting Sox2 an essential role in controlling proliferation.

The observed phenomenon that Sox2^fl/fl x Rosa-CreER^T2 mothers induced with tamoxifen were not able to do proper lactation might be due to the fact that SOX2, among other stem cell factors, was recently found to be expressed in breast milk cells which were derived from the breast epithelium during lactation [250]. Hence Sox2 might be essential to initiate proper lactation of mothers.

In summary it can be concluded that Sox2 is essential for proper murine development as early as gastrulation. Embryonic k.o. after E 7.5 led to malformations of the stomach and was becoming more severe in a dose-dependent manner influencing the whole embryo. During early post-natal mouse development, depletion of Sox2 resulted in death of pups which may be due to problems in nutrient uptake and hormonal regulations in the stomach. Sox2 was also seen to be essential to sustain proliferation in gastric crypts of adult mice, suggesting that it indeed might be a potential factor for the maintenance of adult gastric stem cells.

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