Molecular analyses of carbonic anhydrase‐II expression and regulation in the developing chicken lens

The expression of carbonic anhydrase‐II (CA‐II) in the developing chicken lens was examined and compared with that in the retina of the chicken embryo. CA‐II expression was measured by immunohistochemistry and radioimmunoassay during development, and CA‐II mRNA was quantified by Northern blot and densitometric scanning and localized by in situ hybridization. A functional promoter of the chicken CA‐II gene was identified by transfection of primary embryonic chicken lens epithelial cells and analyzed in deletion mutants. The results establish that CA‐II makes up about 0.1% of the total soluble protein of the embryonic chicken lens, an amount insufficient to make it a candidate for an enzyme crystallin in this species. Lens fiber differentiation coincided with a loss of CA‐II mRNA and protein; by contrast, CA‐II persisted in the epithelial cells of the embryonic and mature lens. This and previous studies showed that CA‐II amounts to as much as 3% of the protein of the embryonic chicken retina and follows a different developmental time course of expression; like the lens, CA‐II decreases until day 10 in the embryonic retina, but, unlike the lens, it increases thereafter and plateaus at hatching. Progressive deletions of the 5′ flanking regions (from position −1314 to +32) of the CA‐II gene fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene resulted in a gradual loss of promoter activity, consistent with an additive effect of putative cis‐regulatory elements found in many crystallin genes. These experiments provide the foundation for a molecular analysis of the developmental and differential regulation of the CA‐II gene in lens and retina. © 1992 Wiley‐Liss, Inc.