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A. Onishi, U. Alexis, C.-D. Hsu, G.-H. Peng, S. Chen, S. Blackshaw; Pias3 Promotes Rod Differentiation in Postnatal Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4472.
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© ARVO (1962-2015); The Authors (2016-present)
Protein Inhibitor of Activated Stat3 (PIAS3) is a transcription co-regulator that modulates the function of target proteins both by direct protein-protein interaction and by post-translational modification by catalyzing site-specific SUMOylation. We have demonstrated that PIAS3 is selectively expressed in developing mouse photoreceptors. We aim to further clarify the cellular localization of PIAS3, analyze the role in retinal cell fate specification by gain and loss of function, and determine which domains of PIAS3 are crucial for its role in retinal cell fate specification.
Monoclonal antibodies against PIAS3 were used in combination with antibodies for major mature cell types in postnatal retina, along with a number of transcription factors that are selectively expressed in developing rod photoreceptors, such as Crx and Nr2e3. In vivo electroporation was used to introduce full-length PIAS3 expressed from the ubiquitous CAG promoter in conjunction with a bicistronic GFP into neonatal mouse retina. Mutant forms of PIAS3 that are deficient in SUMOylation were also overexpressed. Knockdown of PIAS3 was achieved by in vivo electroporation of appropriate shRNA constructs driven from either the H1 or U6 promoter. Retinas were analyzed at postnatal days 4, 7, 10 and 14 by section immunohistochemistry, and also by disassociated cell immunocytochemistry in conjunction with specific markers of retinal cell subtypes.
We have found that PIAS3 is predominantly expressed in postmitotic, immature rods. It is coexpressed with both Crx and Nr2e3. PIAS3 overexpression results in a significant increase in the number of rhodopsin-positive transfected cells at all time points tested. This effect is much less pronounced when the SUMOylase-deficient form of PIAS3 is overexpressed. Instead an increase in amacrine and Müller glial markers is observed in transfected cells. In cells transfected with anti-PIAS3 shRNA constructs expressed under the strong U6 promoter, a dramatic increase in cone-specific markers is observed, while cells transfected with the same shRNA constructs expressed under the weaker H1 promoter are converted to a Müller glial fate.
PIAS3 is selectively expressed in immature rods and positively regulates rod development. This rod-promoting function of PIAS3 requires its E3 SUMO ligase function. Loss of function of PIAS3, however, results in a conversion of developing rods to a Müller glia or cone photoreceptor fate.
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