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E.F. Moreira, R. Adler; Transcription Factor Changes Associated With Inhibition of Amacrine Cell Differentiation in Activin Loss–Of–Function Experiments . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4025.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: This laboratory previously reported that activin, a TGF ß family member, regulates the differentiation of retinal amacrine neurons. The present studies sought to determine whether such effects are correlated with changes in the expression of transcriptional regulators of the bHLH and homeobox families. Methods: Loss–of–function experiments were done by injecting chick embryos intra–vitreally on embryonic day (ED) 2.5 with an RCAS retrovirus encoding follistatin (FS), an activin binding protein and inhibitor; RCAS–alkaline phosphatase (AP) was used as control. Embryos were harvested on ED6 or ED8, and processed for immunocytochemistry (ICC) or in situ hybridization (ISH) for candidate transcription factors (e.g. NeuroD, Prox1, Pax6, and Chx10), either in sections or as dissociated cells. Levels of retroviral infection were assessed by ICC for viral antigens. Quantitative analysis was done with Image J (NIH). Additional retinas were lysed for RNA isolation and real time PCR. Results: As we previously reported, follistatin over–expression caused a marked decrease in amacrine cells on ED8. NeuroD ICC appeared unchanged by ED 6, but its expression domain in the outer nuclear layer (ONL) was broader in FS than control retinas by ED 8. A clear change in distribution of Prox1 (+) cells was also observed, with many such cells residing in the INL region closest to the ONL in control retinas, but predominating near the vitreal surface in FS– treated samples. Although the thickness of the INL region occupied by Pax6 (+) cells was somewhat reduced, their total number did not appear significantly reduced. However, the intensity of Pax6 immunofluorescence signals was lower in FS than in control samples. Lower levels of Pax6 expression in FS–treated retinas were also observed at the mRNA level by real time PCR. No differences in mRNA levels for Chx10 or Prox1 have been observed between FS and control samples. Conclusions: These results suggest that activin may regulate cell differentiation at least in part by modulating the expression levels of bHLH and homeobox genes. These studies are currently being extended to other candidate transcription factors.
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