Molecular cloning, expression and characterization of TOP(AP): A marker of cell position in the developing visual system

Abstract

TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} is a 40 kD protein expressed in a position-specific, continuously graded, fashion in the developing chick visual system. Antibody binding and immunocytochemical studies have shown that TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} is preferentially expressed in the more posterior region of the retina and in the more anterior region of the optic tectum. This pattern of expression mirrors and precedes that of ganglion cell axonal projections, thus TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} may play some role in the generation of these position-specific neuronal connections. In an attempt to learn more about the structure, function and expression of this protein, a cDNA clone encoding TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} was isolated from a posterior quadrant chick retinal library. Analysis of the 3.2 kb cloned sequence revealed a near full length cDNA containing an open reading frame encoding a novel 359 amino acid protein. The deduced amino sequence contained large regions of predicted coiled coil structure, with a putative transmembrane domain present at the extreme carboxyl terminus. Genomic cloning, Southern and northern blot analyses determined that a single gene and a single 3.4 kb mRNA encode the chicken TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} protein. Polyclonal antisera raised against the cloned protein recognized authentic TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} on immunoblots, thereby confirming the identity of the cloned protein. A combination of standard biochemical and histological assays performed on retinal cells and TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} transfected cells suggested that TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} is an integral membrane protein present both intracellularly and on the cell surface. Tissue distribution studies performed by RNase protection and immunoblot analysis demonstrated that TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} is present in a variety of chick tissues with the highest levels of expression seen in the developing retina, heart, and intestine. In addition, the temporal expression of TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} varied with tissue type, with some tissues increasing and others decreasing expression levels over developmental time. Further experimentation performed on dissociated primary retinal cultures showed that the temporal and differential expression of TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} by posterior or anterior retinal cells is reiterated in vitro. This implies that TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar} expression is largely controlled by intrinsic cellular mechanisms. In addition, a positionally graded 46 kD TOP{dollar}\sb{lcub}\rm AP{rcub}{dollar}-related protein has been identified in the chick, the identity of which is unknown.