By way of comparison, the induction of ES cells to lens fate has also been efficiently achieved by a three step manipulation of signaling pathways known to act in endogenous lens development. Considered together, these complementary results indicate that specific aspects of the endogenous lens forming gene regulatory network are recapitulated in the ES cell lens differentiation system. A novel aspect of the present work was the generation of a mES cell line that expresses a GFP reporter under the control of the Pax6 P0 promoter and upstream lens ectoderm enhancer. This mES cell line should facilitate our understanding of the inductive mechanisms involved in lens progenitor cell differentiation. For example, when Pax6-GFP reporter mES cells are transduced with Pax6 or Six3, directed differentiation along the lens pathway appears to commence as early as 3 days post treatment, when GFP reporter expression is detected. In vivo, the mouse Pax6 ectoderm enhancer directs Pax6 expression as early as E8.5 during lens placode Reversine specification and thereafter in the AEL, and it is positively autoregulated by the Pax6 gene product. Hence, the early appearance of GFP expression following introduction of Pax6 into Pax6-GFP reporter mES cells is consistent with the known positive autoregulation of the Pax6 EE. In Pax6 or Six3 transfected Pax6-GFP or G4 mES cells, differentiation ensues with expression of cA-crystallin and of additional lens differentiation markers. Ultimately, by 30 days posttransduction, some aggregates coalesce to form lentoid bodies. The lens marker genes expressed during differentiation in the in vitro ES cell system are normally expressed in distinct spatial and temporal patterns during in vivo lens development. Specifically, the developing lens involves a single progenitor cell lineage with multiple states of differentiation. Therefore, the significant degree of non-overlapping expression of lens markers in differentiating ES cells may reflect the emergence of distinct lens cell phenotypes via normal developmental regulatory mechanisms. Alternatively, the discordant expression of the lens markers in differentiating cells in these cultures could reflect a high degree of cellular and molecular heterogeneity due to variable micro-environmental cues, nor are these two mutually exclusive. Both early markers as well as late markers of lens cell development, are identified and described in mESC and hESC cultures using immunolabeling while concurrently demonstrating up regulation of Pax6 expression in both Pax6 and Six3 transduced ES cultures. These findings further lend support to the recapitulation of physiologically relevant differentiation pathways in vitro.