Identification of Rx Promoter Elements in Vertebrates

Abstract

Abstract: : Purpose: The retinal homeobox (Rx) gene product is necessary for specification of the presumptive eye fields from the anterior neural plate of developing vertebrates. We are investigating Rx regulation by comparing the regulatory regions of Rx1A and Rx2A in Xenopus laevis. The previously characterized Rx1A promoter is being utilized as a model to study the Rx2A promoter. We are characterizing a 2.8 KB fragment of the Rx2A promoter to identify cis– and trans–acting elements involved in Rx regulation. Elucidation of the molecular events underlying the initiation of Rx expression will provide a basis for understanding vertebrate eye specification. Methods: Rx2A/GFP transgene constructs were introduced into X. laevis embryos via intracytosolic sperm injections (ICSI). Candidate trans–acting factors were tested for binding specificity using electrophoretic mobility shift assays (EMSA). The Rx2A/GFP transgene was introduced into zebrafish embryos by microinjection, cultures of embryonic chick retina by transfection, and mouse embryos by pronuclear injection. GFP expression was visualized by fluorescent microscopy. Results:Deletion analysis reveals that Rx2A promoter contains a proximal region necessary for late expression, and a distal region necessary for early expression. This organization is shared with the Rx1A promoter. The Rx2A sequence contains a putative forkhead binding site necessary for late promoter activity. Rx and FoxN4, a forkhead transcription factor, are expressed in developing X. laevis retinas. We tested and confirmed that FoxN4 binds the putative Rx2A forkhead binding element. This data is consistent with a role for FoxN4 in the regulation of Rx promoter activity. In primary culture of embryonic chick retina, the full–length Rx2A/GFP transgene is expressed in retinal progenitor cells (RPCs). The transgene is expressed in retinas of zebrafish, frogs and mice. These data demonstrate that the Rx2A regulatory region is active in four vertebrate species, suggesting a common mechanism for Rx regulation among vertebrates.

Keywords: transcription • transcription factors • retinal development