Global endoderm targeting in the early embryo

Stem cells are a potential source of differentiated cells for use in tissue replacement therapies. Along with colleagues, I investigated published protocols claiming to produce beta cells from the directed differentiation of ES cells. I show that these protocols are the result of culture artifact and do not represent bona fide beta cells. Currently there is no reliable or robust protocol for the generation of beta cells from ES cells. A logical approach to accomplish this goal would be an in vitro recapitulation of in vivo beta cell development. The first step in this process would be to generate definitive endoderm from ES cells. This challenges one to understand the molecular signals responsible for its specification, proliferation, and patterning.; I describe my efforts at identifying a global marker of definitive endoderm, Claudin-6 (Cldn6). I report its expression in early development with particular attention to definitive endoderm derivatives. I also explain my strategy for targeting the endogenous locus with a cassette that allows for the generation of mice for loss of function or gain of function studies in the endoderm using Cre recombinase. These mice are used to perform a lineage analysis to further characterize the fate of these cells later in development. Cldn6 cells adopt epithelial fates in the early embryo, including the entire definitive endoderm.; Finally, I perform two cursory functional analyses. First, I examined if Cldn6 played a role in development of the early mouse embryo, paying particular attention to the definitive endoderm. Cldn6 null mutants are viable and show no obvious phenotypic abnormalities, possibly due to redundant function of other Claudin family members. Second, I used these mice as Cre-driver lines for misexpression of different signaling pathways throughout the endoderm. This proof of principle experiment shows that ectopic expression of Cre responder lines are possible using the Cldn6CIHV+ driver.