Nrl expression and promoter activity in developing cone photoreceptors

The MAF family transcription factor Nrl is a key regulator of vertebrate rod photoreceptor formation/differentiation. The expression of Nrl in nascent photoreceptors has been proposed to act as a master regulator to initiate a rod program and to repress a default cone photoreceptor sister fate. The evidence for rod-specific expression of Nrl has been most strongly supported by mouse transgenic models that use a transcriptional promoter element for Nrl. However, this element has not been rigorously evaluated to have rod lineage-specific activity or as a validated proxy for endogenous Nrl expression, despite its wide use under this assumption. Here we identify that the Nrl promoter is not exclusively active in rod photoreceptors but is also transiently active within a high proportion of the differentiating cone photoreceptor population. Thus, the Nrl promoter cannot be used to distinguish rods from cones at developmental timepoints. Furthermore, multiple lines of evidence using immunofluorescence and reanalysis of published single cell RNA-seq/ATAC-seq datasets support the presence of endogenous Nrl gene expression in early differentiating cone photoreceptors. Taken together, these results suggest that proposed gene regulatory networks that position Nrl as a master regulator of rod versus cone fate are inadequate as currently constructed. This impacts our understanding of visual system evolution and has implications for the development of therapeutic strategies of cell replacement for human blindness.