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Ke Ning, Tia Kowal, Vinit B Mahajan, Yang Hu, Yang Sun; Characterization of primary cilia in mouse amacrine cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1269.
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© ARVO (1962-2015); The Authors (2016-present)
Primary cilia are hair-like organelles that protrude from the apical surface of cells into the extracellular space. They are found on nearly all differentiated cell types in the human body, including most neurons throughout the brain and eyes. Defects in primary cilia result in ciliopathies with symptoms ranging from renal disease, cerebral anomalies and retinal disorders. Our preliminary data indicate that amacrine cells (ACs) form cilia in mouse and primate retinas. However, it is unknown which of the 33 subtypes of ACs contain cilia. The purpose of this study is to describe the distribution of primary cilia in subtypes of ACs in mouse.
To study the distribution of primary cilia in ACs in vivo, 6 retinas were collected from perfused adult transgenic mice, Arl13b-mCherry::Centrin2-GFP, in which the primary cilia is fluorescently labeled. Each retina was cut into six fragments to facilitate whole mount staining and analysis. Fragments from each retina were stained using Glutamic Acid Decarboxylase 67 (GAD67), Calbindin, Calretinin, choline acetyltransferase (ChaT), tyrosine hydroxylase (TH) and Prox1 antibodies to determine the fraction of ciliated cells that express various markers. The inner nuclear layer was viewed by confocal microscopy and three images (500um from edges) per retinal segment were captured using a 63x objective. 50 marker-positive cells were analyzed. Arl13b-mCherry signal was counted as a cilium if one end of the signal was directly adjacent to the Centrin2-GFP signal.
The TH-positive subtype was the most common ciliated subtype in adult mouse retina (54.17±31.50%), which was followed by the Calbindin-positive subtype (57.46 ±3.42%). Less than half of the GAD67-positive subtype ACs (43.76 ±6.61%), Calretinin-positive subtype ACs (34.61 ±13.07%) and ChaT-positive subtype ACs (35.67±2.03%) expressed primary cilia. For Prox1-positive ACs, only 3.33 ±4.71% are ciliated. All data were analyzed by ANOVA and post-hoc t-test with Tukey correction, with P value of <0.05.
We showed a novel distribution of primary cilia in subtypes of ACs in the mouse retina. These findings highlight possible roles primary cilia could play in retinal homeostasis and disease.
This is a 2020 ARVO Annual Meeting abstract.
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