We examined the functional role of PI3K in the developing lens using a cell culture system that closely mimics lens differentiation in vivo.
28 Undifferentiated primary lens epithelial cells were isolated from embryonic quail and plated on a laminin substrate. Initially, these undifferentiated cells form colonies of well-spread epithelial cells (
Fig. 1A , left). Within 5 days, these epithelial cells compacted into a cobblestone arrangement (
Fig. 1A , center), the earliest morphologic indication for the initiation of lens cell differentiation.
28 These cells then further differentiated in culture, resulting in the formation of multicellular, multilayered, lens-like structures known as lentoids (
Fig. 1A , right). To examine whether PI3K has a requisite role in lens cell differentiation, undifferentiated lens epithelial cells were exposed to the specific PI3K inhibitor LY294002 for 72 hours beginning at day 2 in culture. This period of treatment encompasses the time during which these primary lens epithelial cells begin to differentiate. In contrast to control cultures, lens epithelial cells treated with LY294002 were unable to form a compacted epithelial monolayer. They remained as a well-spread epithelium, typical of undifferentiated lens cells throughout the culture period
(Fig. 1B) . The suppression of lens cell differentiation by treatment with LY294002 was paralleled at the biochemical level. At the initiation of differentiation, high levels of expression of the cell cycle inhibitor p27(KIP1) and the lens cell differentiation-specific proteins filensin, CP49, and δ-crystallin indicated that control lens cell cultures had both withdrawn from the cell cycle and begun their differentiation program (
Fig. 1C , control). In the presence of LY294002, cultured lens cells continued to proliferate, as evidenced by their expression of PCNA and their failure to induce expression of the cell cycle inhibitor p27(KIP1;
Fig. 1C ). Previous studies from our laboratory and others have shown that cell cycle withdrawal is essential for initiation of the lens differentiation program,
29 30 31 and therefore, the failure to induce p27(KIP1) expression is a good indication that LY294002-treated lens cells did not differentiate. Further supporting a role for PI3K in lens differentiation, we found that LY294002 suppressed expression of filensin and CP49, two differentiation-specific intermediate filament proteins of the lens
(Fig. 1C) . In contrast, LY294002 treatment caused a small but significant increase in the expression of δ-crystallin, in agreement with a previous report.
32 Although δ-crystallin has been used as a differentiation marker, this result suggests that the function of δ-crystallin in the absence of PI3K signaling is not linked to differentiation.