July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Siah E3 ubiquitin ligase indirectly regulates Pax2 expression by targeting Nlz2 for proteosomal degradation during retinal morphogenesis.
Author Affiliations & Notes
  • Warlen Piedade
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Jakub Famulski
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Footnotes
    Commercial Relationships   Warlen Piedade, None; Jakub Famulski, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2593. doi:
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      Warlen Piedade, Jakub Famulski; Siah E3 ubiquitin ligase indirectly regulates Pax2 expression by targeting Nlz2 for proteosomal degradation during retinal morphogenesis.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2593.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The purpose of this study is to shed light on the post-translational mechanisms that regulate eye development by analyzing Siah E3 ligase expression, protein localization and interaction with its targets during key stages of zebrafish eye morphogenesis, in particular choroid fissure fusion.

Methods : We screened the zebrafish proteome for Siah “degron” motifs and focused on Nlz2, a postulated repressor of Pax2 expression involved in choroid fissure fusion. We used whole mount in situ hybridization (WISH) and Immunohistochemistry (IHC) to provide a spatial and temporal profile of Siah and Nlz2 gene expression and protein localization during early embryonic development. In order to track endogenous Siah activity, we have also developed a GFP reporter construct containing the Siah “degron” motif found in Nlz2 thus enabling real time readout of Siah activity. To study Siah ubiquitin ligase function during eye morphogenesis, we employed gain and loss-of-function approaches using Siah or dominant negative SiahDRING mRNA, respectively. Immunostaining (IHC) for laminin was performed as a readout of basement membrane integrity and fissure fusion while WISH for Pax2, served as an indicator of Nlz2 activity.

Results : Siah1 and Siah2l genes are expressed in the nervous system including the eye during early embryonic development. The expression of their predicted target, Nlz2, overlaps with the expression of both Siah genes. IHC confirmed Siah protein is present in cells of the nervous system and eyes, during early development, 24-72hpf. Using our GFP-degron reporter assay, we confirmed endogenous Siah activity in the developing eye between 20-48hpf. Western blotting, confirmed that Siah actively targets Nlz2 for proteosomal degradation. Siah and SiahDRING mRNA injections both inhibited disassembly of the basement membrane and ultimately optic fissure fusion up to 72hpf. Siah gain-of-function resulted in an increase in Pax2 expression, while the dominant negative DRING construct resulted in decreased Pax2 expression. We predict this phenomenon was directly related to levels of Nlz2 protein which has been shown to repress Pax2 expression.

Conclusions : Taken together, our results suggest that Siah ubiquitin ligases may control Nlz2 stability and therefore indirectly regulate Pax2 gene expression in order to modulate timing of choroid fissure closure.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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