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L. Liang, R.–T. Yan, S.–Z. Wang; Superior Neural Differentiation by Co–Expression of NSCL2 With Ath5 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5370.
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Purpose: Transcription factors of the basic helix–loop–helix (bHLH) family play important roles in cellular differentiation. This study investigates the role of NSCL2, a bHLH gene, in neuronal differentiation using an in vitro RPE transdifferentiation assay through co–expression with ath5, also a bHLH gene. Methods: Since NSCL2 does not, by itself, bring about RPE transdifferentiation into retinal neurons, we designed its co–expression with ath5, through an internal ribosomal entry site (IRES). Dissociated RPE cells were cultured in the presence of bFGF and infected with retrovirus RCAS expressing ath5–IRES–NSCL2. Subsequently, cells in the cultures were fixed and analyzed using immunocytochemistry with markers that label specific types of retinal neurons. Morphological changes from RPE to neuron were also considered in judging whether neural transdifferentiation had occurred. Results: Ath5 alone is able to induce cultured RPE cells to transdifferentiate along the photoreceptor pathway, but the number of cells positive for visinin, a photoreceptor marker, increased when the RPE cells were cultured in the presence of bFGF. With co–expression of ath5 and NSCL2, the number of Visinin+ cells was much greater than that with ath5 alone. Morphologically, individual transdifferentiating cells in RPE cultures infected with RCAS–ath5–IRES–NSCL2 exhibited elegant cellular differentiation typical of young photoreceptor cells. Nonetheless, a significant portion of the Visinin+ cells appeared in clusters of many round cells. The neural transdifferentiation–promoting effect of NSCL2 was also manifested upon ath5–enhanced RPE transdifferentiation along the retinal ganglion cell pathway. The number of cells that molecularly and morphologically resembled ganglion cells was increased to a much greater extent with the co–expression of ath5 and NSCL2 than with ath5 alone. Conclusions: NSCL2 promoted neural transdifferentiation either initiated or enhanced by ath5 from cultured RPE cells, even though NSCL2 alone is unable to initiate such transdifferentiation. We propose that NSCL2 regulates the expression of genes involved in cellular differentiation of neurons.
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