CRX Regulates RdCVF Figure 4. Temporal activity of the murine and human NXNL1 and NXNL2 promoter constructs in electroporated retinal explants. GFP fluorescence was monitored daily from DIV1 to DIV9. The RFP expression driven by bovine rhodopsin promoter was used as positive control. Quantification of the fluorescence of the retinal explants. GFP expression NXNL1, Nxnl1, NXNL2 and Nxnl2, and RFP for rhodopsin at DIV2, 3, 4, 6 and 8. Values represent the average of at least three independent experiments done in triplicate. Error bars represent SEM. doi:10.1371/journal.pone.0013075.g004 photoreceptor cell bodies (Figure 5A). We also observed the co- localization of GFP and RFP in a substantial number of cells, indicating that the NXNL1 promoter construct is active in rods. The construct might also be active in cones as seen by the two GFP-positive/RFP-negative cells at the upper right corner of the panel, but this issue remains to be more definitively resolved. The results were quite similar with the mouse Nxnl1 construct 2351/ +51 (Figure 5B), but by examining more extensively the labeled slices, we could identify some GFP positive cells in the inner part of the retina as represented in the merged portion of the panel. These non-photoreceptors cells where the Nxnl1 promoter is active are likely to be bipolar cells, because endogenous RdCVF mRNA is also expressed by bipolar cells [10]. These observations demon- strate that the 2205 to +57 bp human NXNL1 sequence and the 2351 to +51 bp murine Nxnl1 sequence are each sufficient, at least in retinal explants, to drive the expression of RdCVF specifically in photoreceptors. The results were strikingly different when retinal explants were electroporated with GFP driven by the mouse and human NXNL2 promoter 59-upstream regions. With the human -393/+27 NXNL2 promoter construct, GFP-positive cells were observed mostly in the inner part of the retina (Figure 5C) - the difference from the rhodopsin promoter can be easily appreciated by the absence of co-localization in the merged image. A similar situation was observed with the mouse Nxnl2 2382/+70 construct (Figure 5D). The GFP pattern of expression resembles that of Muller glial cells. The absence of expression of these Nxnl2 reporters in photore- ceptor cells cannot be attributed to the lack of transfection of the plasmids into photoreceptors since we could visualize RFP-positive rods in the same retinal explants. Similar results were obtained whatever the length of the upstream regions for both the human and mouse NXNL2 genes (data not shown), demonstrating that important photoreceptor-specific elements regulating the expres- sion of Nnxl2 in photoreceptor cells are likely localized outside the 4.5 kb 59-upstream region that we tested. The NXNL2 -393 to -315 bp region contains a positive transcriptional regulator and the NXNL1 277 to +57 bp region contains a photoreceptor-restricting element Our mapping analysis of the human NXNL2 promoter indicated the presence of a positive regulatory element located between 2393 and 2315 bp that is active in the retina but that is not sufficient to provide photoreceptor specificity (Figure 3C and data not shown). To determine whether 79 bp sequence requires other elements of the NXNL2 promoter not included in the constructs for its activity, we separated it from the rest of the RdCVF2 promoter, by cloning it into pGL4.17 immediately upstream of the NXNL1 promoter sequence from 277 to +57 bp (79+134, Figure 6A). We chose the 277/+57 NXNL1 fragment as the scaffold for these experiments because this minimal promoter sequence contains needed core RNA polymerase II binding elements, since it is active in Y79 and HEK 293 cells (Figure 2A), but it is by itself insufficient for transcriptional activity in the more stringent and biologically relevant environment of retinal explants (Figures 3A and 4). When transfected into HEK 293 cells, the 79+134 fusion construct stimulated luciferase activity approximately 5-fold over the basic 277/+57 construct (Figure 6B). When both constructs were tested using the retinal explant assay, the 277/+57 human NXNL1 construct was found to be inactive as observed earlier (Figures. 3A and 4). However, the 79+134 fusion construct was active, and perhaps surprisingly, its activity was found to be photoreceptor- specific (Figure 6C-D). The merged image confirmed that the GFP-positive cells, which were only observed within the ONL, were also rhodopsin (RFP) expressing cells. A CRX element controls the expression of RdCVF and is responsible for its photoreceptor-specific expression We took a bioinformatics approach, using Genomatix promoter analysis software, to try to identify elements within the NXNL1 277/+57 region that could account for its biological activity. The analysis identified four binding elements corresponding to transcription factors reported to be expressed specifically or preferentially in the eye (CRX, MAF, POU1F1, and POU3F4). A similar analysis was performed on the mouse Nxnl1 2352/+51 promoter sequence and two transcription factors specific to the eye were found to be common between the two analyses: a MAF- response element half site and a putative CRX binding element. Although MAF-response element half sites can show activity, usually less than that observed with full sites [36], we chose to concentrate our efforts on the putative CRX sites, located at position 256 to 250 bp in the human gene and at 254 to 248 bp in the murine gene. The contribution of the identified CRX binding elements to NXNL1 promoter activity was assessed by mutating the TAA in the TAAT core recognition site to CCG in the human and mouse 2205/+57 and 2353/+51 promoter constructs, respectively. We choose the CCG mutation because this change has been previously reported to abolish CRX binding to DNA [37]. The wild-type and mutated human constructs were tested in Y79 cells, which express CRX as indicated by RT-PCR analysis (result not shown). As expected, the wild-type -205/+57 NXNL1 construct was active, but when the CRX site was mutated promoter activity dropped to that of the empty luciferase control vector (Figure 7A). We then tested the effect of CRX mutation of the human and murine constructs in a time-course analysis in the retinal explant system (Figure 7B). Mutation of the CRX site led to complete elimination of promoter activity for both the human and murine constructs. These results demonstrate that the proximal CRX binding element present in the human and mouse Nxnl1 upstream regions is necessary for their promoter activity. We also tested the effect of the mutation of the proximal CRX binding element in the 79+134 NXNL2/NXNL1 fusion construct. In the Y79 assay, the NXNL1 CRX mutation within the fusion construct led to loss of promoter activity, just as observed with the NXNL1 -205/+57 construct (Figure 7A). When tested in the ex vivo retinal explant assay, the CRX mutation in the context of the fusion construct did not lead to loss of promoter activity (Figure 8A-B). However, the CRX mutation did lead to a major change, a loss of photoreceptor specificity, as shown by the presence of multiple GFP positive cells within the inner part of the electroporated retina (Figure 8C). Discussion Our objective in this study was to begin to elucidate the cis- elements and trans-factors that control the retinal expression of the trophic factors RdCVF and RdCVF2, which are encoded by PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13075