Modulation of Neurod Expression and Photoreceptor Production by bFGF

Abstract

Abstract: : Purpose: To learn how the genetic program of photoreceptor production is modulated by extrinsic factors. Previously we showed that photoreceptor genesis in the chick retina employs 2 transcriptional pathways that involve ngn2 and ath5 and converge at neuroD. This study tests the hypothesis that the photoreceptor transcriptional hierarchies receive input from bFGF, an extrinsic factor known to potentiate retinal ganglion cell fate. Methods: RPE cells lack endogenous expression of proneural genes, including ngn2, ath5, and neuroD. Furthermore, cultured RPE cells can be induced with an appropriate proneural gene to transdifferentiate along the photoreceptor pathway. Thus, cultured RPE cells can be an excellent medium to examine how photoreceptor production is modulated by extrinsic factors, which can be readily applied to the culture medium. RPE cells were dissociated and cultured in the presence or absence of bFGF. Ectopic expression of proneural genes in cultured RPE cells was achieved by infecting the culture with a replication–competent retrovirus RCAS expressing ngn2, ath5, neuroD, or GFP as a control. The cultures were analyzed for RPE transdifferentiation along the photoreceptor pathway. Semi–quantitative RT–PCR was used to evaluate the induction of neuroD and visinin in the cultures. Results: We found that RPE transdifferentiation along the photoreceptor pathway induced by neuroD appeared unaffected by bFGF. Similar transdifferentiation induced by ath5 was significantly enhanced by bFGF. On the other hand, ngn2–induced RPE transdifferentiation along the photoreceptor pathway was significantly reduced by bFGF. We observed that ngn2–induced visinin expression decreased when the RPE cells were cultured in the presence of bFGF. Notably, the induction of neuroD by ngn2 also decreased in the presence of bFGF. Conclusions: Our data suggest that the photoreceptor pathway is modulated by bFGF. Since neuroD appeared "immune" to bFGF, the modulation likely takes place prior to neuroD expression. This is consistent with the observation that bFGF negatively modulated ngn2–induced neuroD expression. The differential effect of bFGF on ngn2– and ath5–induced neuroD expression and photoreceptor production implies that photoreceptor production is governed by an intricate network involving interplays of intrinsic and extrinsic factors.

Keywords: photoreceptors • gene modifiers • growth factors/growth factor receptors