Abstract
Purpose: :
NRL (Neural Retina Leucine zipper) is a bZIP transcriptional regulator that controls rod versus cone cell fate decision during retinal development. It is essential and sufficient for rod differentiation. NRL is also required for maintenance of rod photoreceptor function as it regulates the expression of most rod-specific genes. Hence, regulation of NRL expression should provide an excellent model for understanding the genetic programs controlling photoreceptor development. This study aims at understanding how expression of the mouse Nrl gene is regulated during retinal development.
Methods: :
Putative mouse Nrl promoter/enhancer sequences were identified by inter-species conservation of genomic region upstream of the Nrl transcription start site. Various promoter/enhancer fragments were cloned in the pEGFP-N1 vector. The plasmid constructs were electroporated in newborn CD1 mouse retina. The GFP reporter gene expression was assessed 14 days after in vivo electroporation. In silico analysis was performed to identify transcription factor binding sites on Nrl promoter fragments. The binding of candidate transcription factors is being assessed by EMSA using nuclear extracts from P0-2 and adult mouse retina. Promoter sequences are being investigated by luciferase reporter assays.
Results: :
Four different lengths of Nrl promoter region (from 0.4 kb to 3.9 kb) were electroporated in P0 mouse retina. Except for the 0.4 kb construct, the three larger constructs faithfully produced GFP expression. The 2.8 kb sequence upstream of the Nrl transcription initiation site was sufficient to drive high GFP expression specifically in the rod photoreceptors. In silico study revealed binding sequences for bHLH proteins and several transcription factors that are predicted to play a role in photoreceptor development. Identity of specific cis-regulatory elements and DNA binding proteins is being determined by EMSA and luciferase assays.
Conclusions: :
Our data show that the regulatory sequences necessary for the rod photoreceptor specific expression of Nrl are located between 0.4 kb and 2.8 kb of upstream region. Further analysis of the promoter/enhancer regions points to multiple putative transcription factor that may control Nrl expression. The identity of specific Nrl regulators will lead to better understanding of regulatory networks that dictate the genesis of photoreceptors from multipotent retinal progenitors.
Keywords: transcription factors • photoreceptors • gene/expression