Abstract
Purpose:
The differentiation of rod photoreceptors is stringently regulated by a number of transcription factors, which include NRL (neural retina leucine zipper) and CRX (cone-rod homeobox). We hypothesized that genes dramatically increasing in expression during rod photoreceptor development and regulated by NRL and CRX would play a substantial role in the functional maturation of rods, and that cellular adhesion genes involved in this process would be important for establishing and stabilizing proper organization of photoreceptors within the outer nuclear layer.
Methods:
RNA-seq data from FACS-sorted murine rod photoreceptors (from transgenic mice expressing GFP under the control of the Nrl promoter) was used to select for candidate cell adhesion genes differentially upregulated in rods from P2-P28. The rod-enriched protein expression of one such gene, Frmpd1, was confirmed by Western immunoblotting. To determine the effect on retina morphology upon Frmpd1 knockdown, a variety of commercially available shRNA constructs were used to transfect murine retinal cells at P1 via in vivo electroporation. Eyes were harvested at various ages (P7, P10, P15, P21) and processed for immunohistochemistry to assess morphological consequences of shRNA-mediated Frmpd1 knockdown.
Results:
There was a noticeable decrease in Frmpd1 expression in the Nrl-/- cone-dominated retina and enrichment in the rod-dominated Nrl+/+ retina at both the RNA and protein levels. ShRNA-mediated knockdown of Frmpd1 resulted in severe disruption of retina organization and photoreceptor morphology. Many of the Frmpd1 knockdown photoreceptors were abnormally distributed within the inner and outer nuclear layers and appeared to have shortened outer segments with reduced expression of rhodopsin; in some cases, the outer segments clustered together to form “puckered” regions and rosettes in the outer nuclear layer.
Conclusions:
Frmpd1 is likely to play a substantial role in the functional maturation of rods, and particularly in establishing and stabilizing proper organization of photoreceptors within the outer nuclear layer.