Fiz1, a Protein that Attenuates Transactivation of the Rhodopsin Promoter, is Expressed During Maturation of Photoreceptors in Developing Neural Retina

Abstract

Abstract: : Purpose: Fiz1 is a zinc–finger (Zf) protein that can attenuate NRL–mediated activation of the rhodopsin promoter. (Hum Mol Genet, 2003) We hypothesize that Fiz1 is a Zf–type repressor and that it is integrated into regulatory networks that control the expression level of several retinal–specific genes. (ie. rhodopsin in photoreceptors). We investigated the expression pattern of Fiz1 relative to the timing of photoreceptor maturation in developing mouse retina. Methods: Expression of Fiz1 protein was examined at several stages of postnatal development in the mouse neural retina, including the period of retinal maturation from P–5 to P–21. Detection of Fiz1 in frozen sections of mouse retina was improved using the avidin–biotin–complex (ABC) immunostaining protocol. Relative levels of Fiz1 protein, in neural retina protein extracts, were measured by electrophoresis and immunobloting. Retinal sections were probed with rabbit polyclonal anti–sera prepared against a GST–bovine–Fiz1 fusion protein. Pre–immune sera served as a negative control. Immunoblots were probed with an affinity–purified version of the same antibody. Total relative levels of Fiz1 mRNA and rhodopsin mRNA were determined using quantitative PCR (real–time, Sybr–green). Results: Fiz1 protein levels were below detection in sections of immature retina (age P–0). In constrast, mature mouse retina displayed an intense staining for Fiz1 in photoreceptors and the ganglion cell layer. In agreement with these immunochemical observations, immunoblot analysis indicated that Fiz1 protein levels were extremely low prior to the onset of photoreceptor maturation. Protein levels then elevated sharply from P–5 to P–38. Real–time PCR analysis indicated that Fiz1 and rhodopsin mRNA levels show a sharp and continuing elevation over the same time–period of photoreceptor maturation. Conclusions: The first examination of Fiz1 expression during mouse retinal development indicated that Fiz1 gene expression is increased dramatically during the time–period of photoreceptor maturation and the onset of photoreceptor–specific gene expression (ie. rhodopsin). ABC detection of Fiz1 in retinal sections confirmed intense staining in mature photoreceptors. Fiz1 is expressed in the right place, and at the right time, to have a role in the precise control of photoreceptor specific gene expression.

Keywords: retinal development • gene/expression • transcription factors