| Our lab has previously implicated transcription factor, activating protein-2alpha (AP-2alpha), in the some of the key events leading to vertebrate lens morphogenesis. However, as a result of the overt craniofacial defects that these mice also possess, the possibility of secondary effects arising from these defects could not be ruled out. Thus, the focus of this study is to determine the cell-autonomous requirement(s) for AP-2alpha during lens development.;Thus, the data presented here demonstrate that AP-2alpha-mediated disregulation of cadherin molecules negatively impacts lens vesicle separation and leads to an alteration in the lens epithelial cell phenotype.;To address this specific aim, I have conditionally inactivated AP-2alpha in the developing mouse surface ectoderm (Le-AP-2alpha mutants) using the Cre/loxP recombination approach. Embryonic and adult Le-AP-2alpha mutants exhibited defects confined to presumptive lens ectoderm derivatives, including a persistent adhesion of the lens to the overlying corneal epithelium (or lens stalk). This failure in lens vesicle separation from the embryonic surface ectoderm was correlated with reduced expression of the calcium dependent cell adhesion molecule, E-cadherin. Microarray analysis confirmed this finding and also suggested that AP-2alpha also plays an important role in maintaining the lens epithelial cell phenotype. Furthermore, other cadherin molecules expressed within the developing lens (N and P) were found to be misexpressed in the lens stalk of Le-AP-2alpha mice. In order to implicate a loss of E-cadherin with a deregulation in cell sorting leading to failure in lens vesicle separation, E-cadherin was also conditionally inactivated within the developing embryonic mouse lens. Embryonic and adult E-cadherin conditional mutant mice failed to exhibit any corneal-lenticular adhesions, suggesting that E-cadherin alone does not mediate lens vesicle separation. However, these conditional mutants did exhibit severe structural deficits, including microphthalmia, persistent vacuolization within the fibre cell region, and eventual deterioration of the anterior lens epithelium. Additionally, an abnormal disruption in zonula occludens-1 expression and an increase in alpha-smooth muscle actin expression were observed indicative of defects in cell polarity and differentiation. These studies indicate that a loss of E-cadherin alone does not disregulate lens vesicle separation during development but does have an impact on the differentiative state of lens epithelial cells. Considering, N-cadherin was still expressed in the E-cadherin conditional knockout mice, the possibility of cadherin compensation during lens separation could not be ruled out. To determine the co-requirement of both E-cadherin and N-cadherin within the developing lens, a double conditional knockout of these cadherins was performed. These mice possessed distinct defects in lens separation characterized by the retention of P-cadherin expression within the cells lining the lens stalk region. These double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival, as lens epithelial cells underwent detachment-induced apoptosis (anoikis). The data presented in this study suggest that both E- and N-cadherin are required for normal lens vesicle separation and that the number of functional cadherin alleles has a significant impact on the growth of the lens. |
