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Heithem M. El-Hodiri, Yi Pan; Neil3 is Genetically Downstream of Rx and Essential for Normal Retinal Development. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6013.
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© ARVO (1962-2015); The Authors (2016-present)
NEIL3 is a member of the nei family of DNA glycosylases. Unlike other family members, the NEIL3 gene encodes two putative DNA-binding zinc fingers in addition to the canonical DNA glycosylase domain found in all nei family members. We identified NEIL3 in a microrarray screen for genes dependent on expression of the Retinal Homeobox (Rx) gene. The purpose of this study is to define the genetic relationship between Rx and NEIL3 and to investigate the involvement of NEIL3 in retinal development.
Gene expression was visualized by non-radioactive in situ hybridization using wholemount or sectioned embryos and antisense riboprobes or quantified by quantitative RT-PCR. NEIL3 antisense morpholino oligonucleotides (MOs) were designed by and procured from Gene Tools LLC. For phenotypic analysis and phenotype rescue experiments, NEIL3 and/or Rx antisense MOs or RNA were microinjected into 2 - 4 celled Xenopus laevis embryos. For histology and immunohistochemistry, embryos were fixed, paraffinized, and sectioned at 8 micron thickness.
We verified that NEIL3 expression is dependent on Rx: (1) NEIL3 is expressed in a subset of the Rx expression pattern during retinal development; (2) NEIL3 expression is diminished upon Rx knockdown; and (3) NEIL3 expression is induced by Rx expression in naïve ectoderm. Notably, exogenous expression of NEIL3 largely rescues the Rx knockdown phenotype. Rescue is diminished by perturbation of the canonical NEIL3 domains, the DNA glycosylase domain and DNA-binding (GRF-type) zinc fingers. NEIL3 knockdown results in small or no eye (microphthalmia or anophthalmia). Partial knockdown of both Rx and NEIL3 results in a more severe phenotype than knockdown of either gene product alone. Small eyes generated by NEIL3 knockdown exhibited highly disorganized retinas that contained few and sporadically distributed differentiated cells and persistent proliferating cells.
NEIL3 is genetically downstream of Rx and is necessary for normal retinal development. All putative functional domains are necessary for NEIL3 function. Our data is consistent with a role for NEIL3 in gene regulation, downstream of Rx, in the maturation of retinal progenitor cells during normal retinal development.
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