Differentiation of the endoderm lineage in the murine system.

The endoderm is one of the three principle germ layers that arise during gastrulation in early embryonic development. In the course of development it gives rise to functionally diverse organs, e.g. thymus, lung, liver, pancreas, stomach and the gastrointestinal tract. The main research topic of the thesis was the analysis of the endoderm differentiation in murine embryonic stem cells (ES cells) and embryos. The generation and analysis of two knock-in Cre mouse lines for two endodermally expressed transcription factors are described here. The analysis comprises studies of Cre recombination pattern as well as studies regarding the influence of the Foxa2 hypomorhic mutation on metabolism. On the basis of the obtained data the existence of two different promotors could be shown and the tissue-specific expression of the resulting mRNA isoforms could be confirmed. Furthermore an in vitro endoderm differentiation system for ES cells was established and characterized. Using this system the influence of different micro RNAs (miRNAs) on ES cell differentiation was investigated. This way two miRNAs (miR335 and miR194) were identified that regulate Foxa2 translation and thereby endoderm differentiation

A mouse line expressing Foxa2-driven Cre recombinase in node, notochord, floorplate, and endoderm.

Foxa2 is a forkhead transcription factor expressed in the node, notochord, floorplate, and definitive endoderm and is required in the foregut endoderm for the normal development of the endoderm-derived organs, such as the liver, lung and pancreas. To conditionally inactivate genes in these tissues and organs, we have targeted a Cre recombinase into Exon 1 of the Foxa2 gene. We show, upon crossing to the ROSA26 reporter mice, that Cre expression from the Foxa2(iCre) knock-in allele specifically activates beta-galactosidase expression in the node, notochord, floorplate, and endoderm. In addition, we detect Cre recombination activity in the endoderm-derived organs including lung, liver, pancreas, and gastrointestinal tract throughout development. These results demonstrate that the Foxa2(iCre) knock-in mice are a valuable tool to analyze gene function in endoderm progenitors and endoderm-derived organs. Moreover, the widespread beta-galactosidase reporter activity in the endoderm suggests that Foxa2 marks a progenitor cell population, which gives rise to the majority of cells in endoderm-derived organs

Generation of a mouse line expressing Sox17-driven Cre recombinase with specific activity in arteries.

The HMG-box transcription factor Sox17 has been shown to play important roles in both endoderm formation and cardiovascular development. To conditionally inactivate genes in these domains, we have targeted a codon improved Cre Recombinase (iCre) into exon 1 of the Sox17 gene. Surprisingly, Cre-mediated recombination in the Rosa26 reporter mouse line revealed largely specific activity within the vasculature rather than in endoderm-derived tissues. Here we report a new Cre knock-in mouse line, Sox17(iCre) with activity in the vascular endothelial cells of arteries in the cardiovascular system but not in veins. Cre-mediated recombination was also strongly detected in the liver and spleen, the two organs associated with hematopoiesis. Thus, these results indicate that the Sox17(iCre) would be an appropriate tool for conditional mutagenesis of genes in the vasculature and could be used in studies of blood vessel development and angiogenesis. Additionally, we provide evidence that two different promoters drive Sox17 expression in the endodermal and vascular system

miR-335 promotes mesendodermal lineage segregation and shapes a transcription factor gradient in the endoderm.

Transcription factors (TFs) pattern developing tissues and determine cell fates; however, how spatio-temporal TF gradients are generated is ill defined. Here we show that miR-335 fine-tunes TF gradients in the endoderm and promotes mesendodermal lineage segregation. Initially, we identified miR-335 as a regulated intronic miRNA in differentiating embryonic stem cells (ESCs). miR-335 is encoded in the mesoderm-specific transcript (Mest) and targets the 3'-UTRs of the endoderm-determining TFs Foxa2 and Sox17. Mest and miR-335 are co-expressed and highly accumulate in the mesoderm, but are transiently expressed in endoderm progenitors. Overexpression of miR-335 does not affect initial mesendoderm induction, but blocks Foxa2- and Sox17-mediated endoderm differentiation in ESCs and ESC-derived embryos. Conversely, inhibition of miR-335 activity leads to increased Foxa2 and Sox17 protein accumulation and endoderm formation. Mathematical modeling predicts that transient miR-335 expression in endoderm progenitors shapes a TF gradient in the endoderm, which we confirm by functional studies in vivo. Taken together, our results suggest that miR-335 targets endoderm TFs for spatio-temporal gradient formation in the endoderm and to stabilize lineage decisions during mesendoderm formation