A coordinated interplay of intrinsic factors and extrinsic cues dictates the generation of retinal neurons. Extracellular signaling molecules modulate the synergistic (or antagonistic) action of a limited number of transcription factors that guide the expression of cell-type specific genes (52
). NRL is the key transcriptional regulatory protein, essential for rod photoreceptor differentiation (27
). NRL expression in cone photoreceptor precursors transforms their fate to functional rods, suggesting that NRL initiates the cascade of molecular events required for rod differentiation.4
It is however unclear as to how NRL expression is initiated in specific neuroepithelial progenitors when they are exiting cell cycle. RA has previously been implicated as a mediator of rod differentiation (37
). In this study, we provide evidence in support of RA being one of the signaling molecules that can induce NRL expression. Our data come from studies in Y79 cells and dissociated rod photoreceptors of newborn rat and adult porcine retina. We also show that the effect of RA is mediated by RA receptors and cis-sequence elements present within the Nrl
The lack or reduction of NRL transcripts and protein in the absence of serum suggests that one or more soluble factors regulate its expression at the level of transcription. Serum contains a complex mixture of growth factors, cytokines and other signaling molecules that stimulate the expression of several genes including c-fos
, cyclin D1
, and VEGF
, in cultured cells (53
). While we have identified RA as one of the molecules, it is likely that additional pathways exist. Although NRL levels are decreased in normal rod photoreceptor in vitro
upon withdrawal of serum, they remain detectable. Additionally, NRL contains a number of consensus phosphorylation sites; hence, it is possible that growth factor signaling through the extracellular signal-related kinase (ERK) pathway plays an important role in modulating NRL activity and/or stability. Induction of NRL expression occurs within 2 h of treatment with serum, whereas a gradual increase in NRL expression was observed when cells were treated with RA. This suggests that RA-mediated effect requires de novo
protein synthesis, a phenomenon observed previously for the expression of human cone-arrestin gene (45
). Treatment of Y79 cells with RA is reported to cause an increase in the levels of RARs and RXRs (45
). Therefore, we propose that RA stimulates the expression of its own receptors, which in turn act on the Nrl
promoter, leading to a time delay in inducing NRL expression.
The amount and activity of transcription factors is critical for regulation of their downstream targets (54
). Vertebrate rod photoreceptors are highly metabolically active post-mitotic neurons; ~9 billion opsin molecules are synthesized every second in each human retina and transported to the outer segments, the site where phototransduction occurs (55
). The expression of opsins and other phototransduction proteins must be stringently controlled because over- or underexpression of rhodopsin leads to photoreceptor degeneration (56
). The expression of NRL has to be continuously maintained at transcriptional and/or post-transcriptional levels; missense mutations that affect the activity of NRL lead to photoreceptor degeneration (28
). It is therefore expected that amount and activity of NRL are critical determinants of normal rod photoreceptor function. Our serum-depletion data suggest that NRL has a relatively short half-life. In this respect, RA could be a critical signaling molecule in up-regulating NRL expression.
RA-mediated signal transduction occurs through its interaction with two classes of nuclear receptors: retinoic acid receptor (RARα, RARβ, and RARγ) and retinoid X receptor (RXRα, RXRβ, and RXRγ). 9-cis
RA is a ligand for RXRs, whereas the RAR subtype binds both at
RA and 9-cis
). Given that RARα, RXRα, and RXRγ are expressed in the outer nuclear layer of the developing mouse retina (58
), our results suggest that RA receptors play a significant role in activating NRL expression during retinal development. Because RXRs form heterodimers with RARs we cannot rule out the possibility of the binding of such heterodimers on the Nrl
We observe high induction of endogenous levels of NRL by RA; however, transient transfection experiments using a 2.5-kb fragment of Nrl
promoter show a relatively weaker (2–2.5-fold) effect of RA. These data indicate that whereas RAREs are important in mediating RA-dependent up-regulation of the Nrl
promoter, the 2.5-kb promoter fragment is not in the right context of chromatin in Y79 cells and therefore, may not be able to bind to or recruit other transcription factors necessary for NRL expression. Furthermore, RA may not be the only soluble factor that can affect NRL expression. A number of other factors have been shown to influence rod photoreceptor differentiation; these include taurine and FGF (60
). We have observed an increase in NRL expression in the presence of FGF,5
whereas taurine had no detectable effect in the same experiment (data not shown). These results reveal that either a combination of some of these factors is required for optimal activity, or their effect on rod differentiation is mediated by a pathway distinct from the one studied here.
Although our studies have been performed using cell culture models to demonstrate RA-mediated regulation of NRL expression, the data obtained using Y79 retinoblastoma cells and cultured photoreceptors can be extrapolated to the in vivo
situation. Y79 cells are childhood intraocular tumors of photoreceptor origin and express a number of photoreceptor-specific genes, including NRL
, all RA receptors, and can be maintained under standard conditions with serum (22
). Our studies offer convenient in vitro
model systems of using serum-deprived cells to study the role of soluble factors in photoreceptor development and maintenance.
In summary, we demonstrate a previously undescribed functional link between an environmental factor involved in rod development (RA) and a key transcriptional regulator (NRL). Given that retinal progenitors express RA receptors throughout rod development (59
), we hypothesize that RA directs these cells toward photoreceptor cell fate and influences rod differentiation by up-regulating NRL. A detailed analysis of NRL expression in RA receptor knock-out mice (63
) may facilitate understanding of the role of RA receptors in rod photoreceptor development.