neuroD induces photoreceptor cell overproductionin vivo andde novo generationin vitro

Abstract

The molecular mechanisms underlying the generation of the various types of cells in the vertebrate retina are largely unknown. We investigated the possibility that genes belonging to the basic helixloop-helix (bHLH) family of transcriptional factors participate in cell-type specification during retinal neurogenesis. Chick neuroD was isolated from an embryonic cDNA library and its deduced amino acid sequence showed 75% identity with mouse neuroD. In situ hybridization showed that neuroD was expressed in cells located at the outer portion of the developing retinal neuroepithelium, the location where prospective photoreceptors reside. Misexpression of neuroD in retinal neuroepithelium through replication-competent, transformation-deficient retroviruses produced a retina with three, instead of two, layers of photoreceptor cells; the number of cells that express visinin, a marker for cone photoreceptors, increased over 50% compared to control embryos misexpressing the green fluorescent protein. No significant changes were observed in the number of other retinal neurons, including those that express RA4 (ganglion cells), pax6 (ganglion cells and amacrine cells), and chx10 (bipolar cells). Retroviral-driven misexpression of neuroD in monolayer cultures of retinal pigment epithelium yielded de novo production of photoreceptor cells with no other types of retinal neurons detected. We propose that neuroD is important for photoreceptor cell production in the vertebrate retina.

neurogenin2 elicits the genesis of retinal neurons from cultures of nonneural cells

“…To avoid potential cross hybridization with other bHLH genes, the coding sequence for the CЈ terminal nonconserved 61-aa and 3Ј untranslated sequence was used to synthesize digoxigenin-labeled antisense RNA probes (788 nucleotides long) against ngn2. Preparations of digoxigenin-labeled probes against other genes and the details of in situ hybridization with retinal cryosections have been described (16,17). In situ hybridization of cultured cells was also as described (32).…”

“…Recombinant viral DNA was introduced into chick embryonic fibroblast cells through transfection. Retrovirus particles were harvested as described (16). The titer of the concentrated viral stock was 1-2 ϫ 10 8 pfu͞ml.…”

“…RPE tissues were dissected from E6 chick embryos, and dissociated RPE cells were cultured as described (16). After 3-4 days in culture, when Ϸ50% confluence was reached, 5 l of the retrovirus stock was added to each 35-mm dish, and the culture was kept for an additional 8-12 days.…”

“…6). Recent reports indicate that the intrinsic factors may include a number of basic helix-loop-helix (bHLH) genes with proneural activities, such as ash-1 (7), nurogenin1 (ngn1) (8), ath3 (8,9), ath-5 (10)(11)(12)(13)(14)(15), and neuroD (16)(17)(18).…”

“…For example, ectopic expression of neuroD triggers de novo production of photoreceptor cells (16,17). In the presence of basic fibroblast growth factor, on the other hand, cultured RPE cells will transdifferentiate into cells that express RA4 (32), which is a marker for ganglion cells in the chick retina (33).…”