Retinoic Acid Induces Pdx1 -Positive Endoderm in Differentiating Mouse Embryonic Stem Cells Suzanne J. Micallef 1 , Mary E. Janes 1 , Kathy Knezevic 2 , Richard P. Davis 1 , Andrew G. Elefanty 1 and Edouard G. Stanley 1 1 Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia 2 Department of Hematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, U.K Address correspondence and reprint requests to Edouard G. Stanley, Centre for Early Human Development, Monash Institute of Reproduction and Development, 27-31 Wright St., Clayton, Victoria 3168, Australia. E-mail: ed.stanley{at}med.monash.edu.au Abstract We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene ( Pdx1 ). Analysis of chimeric embryos derived from blastocyst injection of Pdx1 GFP/w ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1 + pancreatic endoderm. RA was most effective at inducing Pdx1 expression when added to cultures at day 4 of ES differentiation, a period corresponding to the end of gastrulation in the embryo. RT-PCR analysis showed that Pdx1 -positive cells from day 8 cultures expressed the early endoderm markers Ptf1a , Foxa2 , Hnf4α , Hnf1β , and Hnf6 , consistent with the notion that they corresponded to the early pancreatic endoderm present in the embryonic day 9.5 mouse embryo. These results demonstrate the utility of Pdx1 GFP/w ES cells as a tool for monitoring the effects of factors that influence pancreatic differentiation from ES cells. EB, embryoid body E-cad, E-cadherin ES, embryonic stem GFP, green fluorescent protein RA, retinoic acid Footnotes Posted on the World Wide Web at http://diabetes.diabetesjournals.org on 7 December 2004. Accepted November 16, 2004. Received September 28, 2004. DIABETES