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S. Cui, B.A. Link; Expression and Functional Analysis of Lgl1 and Lgl2 During Retinal Lamination in Zebrafish . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4177.
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© ARVO (1962-2015); The Authors (2016-present)
The Lethal Giant Larvae (Lgl) proteins are demonstrated substrates of atypical Protein Kinase C (aPKC). Previously, we have shown that aPKC lamda and zeta are essential for multiple aspects of retinal development. During zebrafish retinal development, loss of both aPKC isoforms results in retinal defects in mitotic cell behaviors (mitotic division orientation and M–phase localization), post–mitotic cell migration, photoreceptor morphogenesis, and overall retinal histology. The cell type positioning defects are non–cell autonomous, indicating that aPKC activity may function by regulating a secreted signal. Lgl proteins have been shown to regulate polarized exocytosis by interacting with exocytic machinery in both yeast and mammalian cells. To begin to test whether Lgl mediates aPKC functions during retinal development, we have isolated the zebrafish Lgl1 homologue and investigated the expression and loss–of–function consequences of Lgl1 and Lgl2 within the developing retina.
Zebrafish Lgl1 was isolated by database analysis, RT–PCR, and RACE. Antibodies against Lgl1 were generated in rabbits using KLH–conjugated peptides as antigens. Protein and mRNA expression analysis was conducted by immunohistochemistry, western blotting, and in situ hybridization. Loss–of–function experiments utilized translation blocking anti–sense oligonucleotides injected into embryos expressing cell type specific GFP–tagged transgenes. Histology and GFP markers were used to judge retinal lamination.
Zebrafish Lgl1 coding sequence is 3123bp with 68% identity to human Lgl1 and 53% identity to zebrafish Lgl2 (Clustal W Alignment). The zebrafish Lgl1 and Lgl2 genes are both expressed within the developing retina during the period of lamination. Subcellularly, the Lgl1 protein showed a punctuated membrane localization within retinal neuroepithelial cells. Loss of Lgl1 or Lgl2 alone produced phenotypes including smaller eye, heart edema and curly tail. Histological analysis showed relatively normal retinal lamination. However, combined loss of Lgl1 and Lgl2 disrupted retinal lamination in a manner similar to loss of aPKC.
Zebrafish Lgl1 and Lgl2 are expressed within retinal neuroepithelial cells during development. Within the retina their function appears to be redundant and essential for retinal lamination. We are currently exploring the mechanisms by which loss of Lgl activity results in laminar defects.
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