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. This study aims at understanding how expression of the mouse Nrl gene is regulated during retinal development.
Methods: :
Putative mouse Nrl promoter/enhancer regions were identified by sequences homology of genomic region upstream of the Nrl transcription start site. Relevant genomic regions were cloned in the pEGFP-N1 vector. The plasmid constructs were electroporated in newborn CD1 mouse retina. The fluorescent 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 segments. The binding of candidate regulators was assessed by EMSA using nuclear extracts from P0-2 and adult mouse retina. HEK293 cells were transiently transfected to assess promoter activation with luciferase reporter gene.
Results: :
From 0.4 kb to 3.9 kb lengths of Nrl promoter region were electroporated in P0 mouse retina. Except for the 0.4 kb construct, the three larger constructs faithfully produced GFP expression in rod photoreceptors. The 0.9 kb sequence upstream of the Nrl transcription initiation site was sufficient to drive high reporter gene expression. In silico study revealed binding sequences for bHLH proteins and several candidate transcription factors. EMSA studies confirm the binding of regulatory proteins on predicted conserved sequences. Amongst the transcription factors tested by luciferase assay, MASH1 shows high activation of the Nrl promoter.
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 factors that may control Nrl expression in developing and mature retina. The identification of specific Nrl regulators will lead to better understanding of regulatory networks that dictate the genesis of photoreceptors from multipotent retinal progenitors.
Keywords: photoreceptors • retinal development • gene/expression