Abstract
Abstract: :
Purpose: To delineate the transcription network underlying photoreceptor cell genesis and differentiation. RaxL has been shown to play a role in the initiation of photoreceptor differentiation (Chen and Cepko, 2002, Development, 129:5363). Published studies from our laboratory indicate that photoreceptor genesis may involve bHLH genes neuruogenin2 (ngn2) and neuroD. Our unpublished data suggest that cath5 may represent a different pathway leading to neuroD expression and photoreceptor genesis. This study focuses on the hierarchal relationship between the three bHLH genes and RaxL. Methods: Replication–competent retrovirus RCAS was used to express genes in cultured RPE cells derived from day 6 chick embryos. Induction of gene expression in the retroviral–transduced RPE cells was assayed with semi–quantitative RT–PCR. Specifically, RCAS–ngn2, RCAS–neuroD, RCAS–cath5, RCAS–cNSCL1 (as a bHLH gene control), RCAS–cath5+cNSCL1 (co–expressing cath5 and cNSCL1 through an internal ribosomal entry site), RCAS–RaxL, or RCAS–GFP (as a viral infection control) was added to primary RPE cell cultures. Total RNA was isolated from the virus infected cells using the guanidine isothiocyanate method. First–strand cDNA was synthesized using oligo–dT as a primer and was diluted to 20– 40 fold before 1 µl was used in each PCR reaction. Results: While ngn2, neuroD, and cath5 induced RPE transdifferentiation into neural cells that resembled developing photoreceptor cells, no such transdifferentiation was observed with RaxL, indicating that RaxL was insufficient to guide an RPE cell to differentiate along the photoreceptor pathway. Induction of RaxL expression was detected in RPE cell cultures infected with RCAS expressing ngn2, neuroD, cath5, and the construct co–expressing cath5 and cNSCL1, but only background level of RaxL expression was present in cultures infected with RCAS–cNSCL1 or RCAS–GFP. None of the bHLH genes was induced by RaxL. Conclusions: RaxL is among the downstream targets in the photoreceptor pathway that can be induced by ngn2, neuroD, or cath5, and RaxL is insufficient to induce RPE transdifferentiation towards photoreceptor cells.
Keywords: retinal development • regeneration • transcription factors