June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
The functional significance of zinc-finger protein Nlz2 in optic fissure closure
Author Affiliations & Notes
  • Grace Shih
    National Eye Institute, Bethesda, MD
    Howard Hughes Medical Institute, Bethesda, MD
  • Ramakrishna Alur
    National Eye Institute, Bethesda, MD
  • Felix Onojafe
    National Eye Institute, Bethesda, MD
  • Brian Brooks
    National Eye Institute, Bethesda, MD
  • Footnotes
    Commercial Relationships Grace Shih, None; Ramakrishna Alur, None; Felix Onojafe, None; Brian Brooks, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3065. doi:
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      Grace Shih, Ramakrishna Alur, Felix Onojafe, Brian Brooks; The functional significance of zinc-finger protein Nlz2 in optic fissure closure. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3065.

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

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Purpose: Currently, the genetic networks underlying the closure of the optic fissure during vertebrate eye development are poorly understood. Failure of optic fissure closure leads to a potentially blinding congenital ocular malformation called uveal coloboma. Profiling of global gene expression during optic fissure closure has suggested a role for the C2H2 zinc finger proteins Nlz1 and Nlz2 in early eye development. Knockdown of either gene in zebrafish using a morpholino strategy results in optic fissure closure defects, perhaps via dysregulation of the critical transcription factor, Pax2. The aim of this project was to determine the role of the Nlz2 gene in optic fissure closure in the mammalian eye.

Methods: Long range PCR and southern blotting confirmed the homologous recombination of the knockout mouse construct. Beta-galactosidase staining was used to study Nlz2 gene expression in knockout mice. Histopathological and immunohistochemical stains of Nlz2 mouse embryo sections at E11.5 and E13.5 days were evaluated for ocular and systemic morphological differences. Fundus and slit lamp photography were employed in adult mice. The two exons of NLZ2 (ZNF503) were amplified and sequenced (Sanger dideoxynucleotide sequencing) from genomic DNA of 174 patients with clinically observed coloboma.

Results: Nlz2 demonstrates homozygous lethality in knockout mice by E17.5 days. Beta-galactosidase staining of Nlz2 mice demonstrated widespread expression, particularly in the eyes, hindbrain, facial prominences, and limbs. Gross and histopathological sections of the developing eye revealed a failure of the optic fissure to close in homozygous knockout mice by E13.5-E14.5 days, but not in heterozygous or wild-type mice. Fundus and anterior segment evaluation of adult mice revealed no overt phenotypic differences between heterozygotes and wild-type mice. No NLZ2 sequence changes or SNPs were found in our coloboma patient population.

Conclusions: Knockout of Nlz2 in mice leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. Additionally, the gene product appears to function in the fusion of the closely apposed edges. As no direct NLZ2 sequence mutations were found in a cohort of patients with coloboma, further genetic studies will be conducted to elucidate the mechanism by which Nlz2 interacts with other genes or players causative of uveal coloboma.

Keywords: 497 development • 539 genetics • 740 transgenics/knock-outs  

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