Purchase this article with an account.
T. L. Belecky-Adams, R. Sehgal, N. Shebani, S. J. Rhodes; Bone Morphogenetic Protein 7 (BMP7) and Sonic Hedgehog (SHH) Regulate Pax2 in Retinal Astrocytes by Relieving Tlx Repression.. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2528.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that BMP7 and SHH regulate the differentiation of optic nerve astrocytes by directly regulating Pax2 expression.
Retinal astrocytes were isolated and purified from 4 week old immortomice and grown in vitro. Cells treated with BMP7 and/or SHH were analyzed by one of the following methods to determine the mechanism by which these factors regulate Pax2 expression; 1) immunoblotting to determine expression levels of PAX2 and transcription factors involved in each of the signaling pathways, 2) co-immunoprecipitation to establish direct interactions of BMP and SHH pathway members, SMADs and GLIs, with the repressor protein TLX, 3) electrophoretic mobility shift assay (EMSA) to determine whether SMADs and/or GLIs change the binding affinity of TLX for the Pax2 promoter, 3) avidin-biotin conjugated DNA binding (ABCD) assays to verify that SMADs and GLIs do not interact directly with the proximal region of the Pax2 promoter, and 4) luciferase assays in which 900bp of the Pax2 proximal promoter was co-transfected with combinations of SMADs, GLIs, and TLX to confirm that repression of Pax2 expression is modulated by SMAD and GLI interactions with TLX.
Treatment of mouse retinal astrocytes with BMP7 and/or SHH led to marked increases in both PAX2 protein and mRNA levels. SHH appeared to be upstream of BMP7 in retinal astrocytes, such that when BMP7 signaling was blocked, the effects of SHH on PAX2 levels were diminished. Both transcription factors SMAD1 (BMP pathway) and GLI2 (SHH pathway) directly interacted with the transcriptional repressor TLX and relieved TLX repression of Pax2 expression in retinal astrocytes.
Together these data reveal a novel mechanism for the cooperative actions of signaling pathways in astrocyte determination and differentiation and suggest interactions of regulatory pathways that are applicable to other developmental programs.
This PDF is available to Subscribers Only