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Celso H. Alves, Bokyung Park, Alicia Sanz Sanz, Susanne Beck, Gesine Huber, Naoyuki Tanimoto, Jan Wijnholds; CRB2 In Retinal Development - Part 1. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4346.
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© ARVO (1962-2015); The Authors (2016-present)
In Leber congenital amaurosis vision is lost within the first year of life. In about 10% of the cases this is caused by mutations in the Crumbs homolog 1 or CRB1 gene. The function of the CRB1 protein is not well known. Mice lacking CRB1 show retinal degeneration in up to one quadrant of the retina, which might be due to overlapping functions with CRB2 and/or CRB3 in the other quadrants. To answer to that question we generated and analyzed Crb2 conditional knockout mice. The aim of this project is to analyze the phenotype of the Crb2 conditional knockout and study the functions of CRB2 in the retina.
The Crb2 conditional knockout mice were generated and crossed with Chx10-Cre transgenic mice.Animals from embryonic day 16 till 1 year of age were analyzed. In order to investigate the possible ectopic localization of proteins and cells we performed immunohistochemistry (IHC) on the retinas of these animals, using different antibodies. Moreover, a morphological analysis was performed using toluidine blue staining in Technovit sections. Optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO) and electroretinography (ERG) will be presented in Part 2.
Retinal disorganization starts at embryonic day 18, in the periphery. At post-natal days 6 and 10, progressive disorganization was detected in all four quadrants of the retina, characterized by gaps in the outer limiting membrane (OLM) and ectopic cell nuclei in the sub retinal space. At a later stage, retinal degeneration of the outer and inner retina was detected.IHC experiments performed on adult animals, showed loss of localization of members of the Crumbs complex at regions with disrupted OLM. We detected unusual localization of MPP4 and PSD95 in the outer nuclear layer suggesting ectopic synapses or misplaced cells. We also observed increased levels of GFAP and CD11b in these animals suggesting gliosis and activated microglia.
Loss of CRB2 results in a more severe retinal degeneration than loss of CRB1. CRB2 seems be necessary for proper retinal development, especially at a late time point. The lack of the protein leads to early retinal disorganization and at a later stage to retinal degeneration. However, the mechanism behind the phenotype needs to be elucidated in order to reveal the functions of CRB proteins in the retina.
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