CRX Regulates RdCVF Figure 6. NXNL1 proximal promoter region, which itself lacks promoter activity, confers photoreceptor-specificity to a NXNL2 promoter region with positive regulatory activity. (A) Schematic diagram of the 79+134 construct, which consists of the NXNL1 sequence extending from 277 to +57 bp fused to the NXNL2 sequence extending from 2393 to 2315 bp. In terms of the sequence variants mentioned above, it has a C at position -30 and a G at position -8. (B) Transient transfection analysis performed in HEK 293 cells and normalized to control (pRL-TK). The results are expressed as fold induction compared to the NXNL1 277/+57 construct. Values represent the average of at least three independent experiments done in triplicate. Error is shown as SEM. (C) Mouse retinal explants electroporated at P0 and maintained in culture for 8 days. (D) Retinal explants were sectioned at DIV8, fixed and stained with anti-GFP (green) and anti-RFP (red) antibodies. ONL: outer nuclear layer; INL: inner nuclear layer; and GCL: ganglion cell layer. ***p,0.001. doi:10.1371/journal.pone.0013075.g006 found that regulatory regions identified, using the same promoter constructs, differed depending upon whether we performed the assays with the cell lines versus the retinal explants, with the explants giving stronger evidence of cell-type specificity. For example, with the Y79 cell assay the NXNL1 -77 to +57 bp construct retained considerable activity compared to the 2205 to +57 bp construct, while in the more stringent explant system the 59 deletion from 2205 to 277 bp led to loss of all detectable promoter activity, indicating that the deleted region contained a sequence with regulatory function that was essential in retinal cells but not in the cell lines tested. This suggests that the activity of the Nxnl1 promoter, like that of other genes that show highly cell-type restricted expression patterns, is regulated by transcription factors that are sensitive to the cellular context of the cell. Of even greater significance, as demonstrated with the NXNL1 and NXNL1/2 fusion gene studies, the explant system allows analysis of cell type PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13075