Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

Pax‐6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1‐(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax‐6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax‐6‐positive cells were also K3 keratin‐positive, suggesting that Pax‐6 is expressed in differentiating cells. Pax‐6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT‐qPCR. The raise in Pax‐6 expression preceded for 9 h the increase in LDH‐H and LDH‐M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full‐length mRNAs encoding for the two major Pax‐6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax‐6 mRNA was 2‐ to 2.5‐fold more abundant than Pax‐6(5a) mRNA. The ectopic expression of Pax‐6 or Pax‐6(5a) decreased proliferative ability leading to the formation of abortive, non‐proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax‐6. Collectively, results show that Pax‐6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax‐6 as the main driver of the differentiation of corneal epithelial cells. J. Cell. Physiol. 220: 348–356, 2009. © 2009 Wiley‐Liss, Inc.