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.

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.

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.KEY WORDS: Foxa2, Sox17, Endoderm, Mesendoderm, miR-335, Mir335, Gastrulation, Mouse INTRODUCTIONThe first lineage decision during mouse development occurs when the morula develops to the blastocyst stage at embryonic day (E) 2.5-3.5 (Rossant and Tam, 2009). During this time, the inner cell mass (ICM) of the blastocyst segregates from the trophectoderm (TE) that will form the placenta. The ICM further develops to the epiblast epithelium that will give rise to all differentiated cell types in the mammalian body. At E6.5, gastrulation starts and pluripotent Oct4 + epiblast cells undergo epithelial-mesenchymal transition (EMT) to ingress into the posterior primitive streak (PS) region to form mesoderm and definitive endoderm (DE), whereas the remaining epiblast cells will form the ectoderm (Beddington and Robertson, 1999;Tam and Loebel, 2007;Zorn and Wells, 2009). Both EMT and mesendoderm differentiation are induced by Wnt/β-catenin and Nodal/TGFβ signaling in the mouse embryo Arnold and Robertson, 2009). Wnt/β-catenin signaling leads to the activation of target genes in the epiblast, such as the Forkhead transcription factor a2 (Foxa2) (Sasaki and Hogan, 1993;Monaghan et al., 1993;Sawada et al., 2005) and the T-box transcription factor Brachyury (T; Herrmann, 1991;Yamaguchi et al., 1999;Arnold et al., 2000), which mark distinct mesendodermal progenitor cell populations in the posterior epiblast (Burtscher and Lickert, 2009). Genetic lineage tracing experiments revealed that Foxa2 + and T + mesendoderm progenitors give rise to anterior and posterior mesendoderm populations, respectively (Uetzmann et al., 2008;Horn et al., 2012; Kumar et al., 2007;Verheyden et al., 2005); however, how lineageinappropriate TF expression is prevented after...