Abstract
Purpose :
The adaptation process of rod photoreceptors following intense and/or prolonged light exposure is not yet completely understood, but is critical for optimal function and homeostasis of these cells. To gain further insights into the molecular mechanisms contributing to this process, we examined the potential role of a novel candidate gene (Frmpd1) in modulating rod adaptation using a retina-specific knockout mouse model.
Methods :
Tissue-specific transcriptional start sites of Frmpd1 were identified by 5’-rapid amplification of cDNA ends (5’-RACE) and its promoter identified by in vivo electroporation of mouse retina with putative regulatory regions driving GFP expression. Promoter regulation was further investigated using electrophoretic mobility shift (EMSA) and luciferase assays. A loss-of-function mouse model was generated by a CRISPR/Cas9-mediated genomic deletion of the retina-specific promoter of Frmpd1, and validated by in situ hybridization and immunoblot. Functional and morphological consequences of Frmpd1 knockdown in these mice was assessed by immunohistochemistry and electroretinography.
Results :
An investigation of the regulatory mechanisms of Frmpd1 revealed that transcription of this gene is initiated from a unique retina-specific alternative promoter, which is modulated by key rod transcription factors Nrl and Crx. Interestingly, a CRISPR/Cas9-mediated genomic deletion of this alternative promoter resulted in a retina-specific deletion of Frmpd1 at both the RNA and protein levels. Frmpd1-/- mice exhibit a delayed return of transducin to outer segments following light adaptation, and a delay in the recovery of rod photoresponse after a moderate visual pigment bleach.
Conclusions :
We provide evidence that Frmpd1 aids in the homeostasis and functional optimization of rod photoreceptors by mediating adaptation of these cells following light exposure. These studies also demonstrate the potential use of CRISPR technologies to generate tissue-specific knockdown models by targeting untranslated regulatory, rather than protein-coding, regions of genes. This approach could potentially be used in various tissues of the central nervous system, which characteristically express tissue-specific isoforms of genes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.