Abstract
Purpose: :
The homeodomain transcription factor Chx10 is the earliest characterized specific marker for retinal progenitor cells (RPCs) and is known to play an important role in RPC proliferation. To better understand the evolutionarily conserved molecular mechanism of RPC maintenance, we attempted to identify the Chx10 promoter and upstream factor using zebrafish and mice as animal models.
Methods: :
To identify evolutionarily conserved enhancer elements of Chx10 gene, we applied bioinformatic analysis and sought to characterize the Chx10 promoter using zebrafish as an animal model. Using a mouse animal model, we assessed the evolutionarily conserved activity of the cis-regulatory region. To identify the upstream protein, the minimal cis-regulatory motif was analyzed by an electrophoretic mobility shift assay, and binding proteins were identified by proteomic analysis. The transactivation activity of the gene was examined using luciferase as a reporter gene. In order to elucidate the function of the upstream protein, we electroporated the shRNA expressing plasmid into retinal explants prepared from an E17.5 mouse eye
Results: :
By computer-based sequence analysis, we identified 4 evolutionarily conserved non-coding sequences (CNS) in the Chx10 locus. Among them, using a series of transient GFP reporter gene injections into zebrafish embryos, we found that the cis-regulatory motif located 35 kb upstream of mouse Chx10 can control GFP expression in RPCs in both zebrafish and mice. From the proteomic analysis of the minimal cis-regulatory motif binding proteins, we identified NPM1 as a novel upstream factor of Chx10. Both mouse and zebrafish homologs of NPM1 were strongly expressed in RPCs during retinal development. Interestingly, NPM1-knockout mice were previously reported to die around E11.5 with a complete loss of the eyes. When we assessed the transactivation activity using luciferase as a reporter gene, we found that NPM1 directed reporter gene expression in a C-terminal DNA-binding motif-dependent manner. Moreover, the suppression of NPM1 expression by shRNA decreased the number of Chx10-positive cells, down-regulated cell proliferation, and induced apoptosis in RPCs.
Conclusions: :
These results suggest that NPM1 plays a pivotal role in RPCs maintenance by regulating the Chx10 gene expression during retinal development.
Keywords: retinal development • development • proteomics