Disorder aniridia that manifests as cataracts, corneal opacification, and retinal anomalies, while compound heterozygosity for PAX6 loss-of-function causes anophthalmia. Thus, Pax6 appears to function as a key regulatory gene for metazoan eye development, acting as one of several ‘eye specification’ genes that function in an interconnected, non-linear GRN with feedback and autoregulatory circuits. A second eye specification gene is the Drosophila homeobox gene sine oculis ; its presumptive vertebrate orthologue is Six3. Ectopic expression of mouse Six3 in Medaka fish results in ectopic lentoid formation, presumably by activation of Pax6 expression in the presumptive lens ectoderm, while Six3 deficiency in mice results in defective lens induction. Collectively these observations support a key, evolutionarily conserved regulatory function of Pax6 and Six3 in metazoan eye development that extends to vertebrate lens induction. Given the conserved role for these two ocular developmental regulators, we hypothesized that ES cells might provide an attractive system to investigate early vertebrate ocular and lens regulatory mechanisms in vitro. Previous studies have shown that both mouse and primate ES cells possess the ability to differentiate into lentoids upon prolonged culture in vitro. In these studies, the induction of lentoid formation, defined by a characteristic 3-D morphology and the expression of lens markers, involved the upregulation of Pax6-expression in differentiating ES cells co-cultured with a stromal cell feeder layer. For example, these cells have been reported to provide stromal cell-derived inducible factors that promote the differentiation of pluripotent stem cells to neuronal pigmented epithelial cell fates. Two additional SCH727965 reports describe the induction of lens progenitors and lentoids from hES cells and from iPS cells derived from cataract patients using chemically defined protocol. These investigations used a three-step protocol that was based on known signaling requirements in lens development, and achieved efficient induction of lentoid bodies. Collectively, these studies show that ES cells from at least three species – rodent, human, and non-human primate – possess lens forming potential, and suggest a clear role for extrinsic signals in this process. In the case of rodent and non-human primate cells, culture with a stromal feeder layer resulted in increased Pax6 expression in differentiating cells and in the development of lentoid like structures, while in the hES cell protocol, PAX6 and SIX3 expression were documented as key early responses in lentoid induction. Given these results, we sought to investigate whether Pax6 itself, alone or in combination with Six3, could directly induce the expression of lens fate in mES and hES cells. We further sought to determine whether this process occurred in a cell autonomous or non-cell autonomous fashion. The differentiation potential of ES cells makes these cells attractive candidates for cell-based therapies and for unraveling the in vivo mechanisms of tissue-specific differentiation.