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Suraj P Bhat, Rajendra K Gangalum, Dennis Mock; Gene Expression in Single Fiber Cells of the Ocular Lens: A window into molecular heterogeneity in a functional tissue. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
The multiplicity of the known cataract phenotypes suggests pervasive molecular and physiological heterogeneity in the fiber cells that generate transparency in the ocular lens. Here we investigate the molecular basis of this phenotypic heterogeneity at the level of gene expression.
Our motivation was to work with the fiber cells without culture or excessive ex-vivo manipulations. Unlike most other tissues the ocular lens lends itself to such near - in vivo studies seamlessly. Based on earlier studies in literature we developed a modified methodology to isolate single fiber cells from 2-day old postnatal mouse lenses. The lenses were incubated with a protease to gently remove the capsule and along with the epithelium. The fiber mass was transferred to a new buffer without the protease and incubated with shaking. Single fiber cells were collected manually. Individual fiber cells were sized before RNA extraction. This RNA was used for cDNA synthesis followed by multiplexed qRT-PCR in a 48 or 96 well microfluidic chip (Biomark, Fluidigm Inc., Palo Alto CA). We interrogated each fiber with 48 genes that included crystallins, MIP26 and non-crystallin genes and various transcription factors.
We report heterogeneity of gene expression from one lens to the other and also between fiber cells from various lens regions. Melting temperature profiling of a significant number of amplicons revealed masking of the individual gene expression preferences seen when interrogated in the total RNA from individual lenses. Analysis of the data, including unsupervised hierarchical clustering (absolute Pearson correlation) of 48 genes reveals stark differences in the highly expressed crystallin transcripts (gene products that represent terminal differentiation) and the non-crystallin genes. Almost all the fiber cells analyzed showed expression of the crystallins while appreciable heterogeneity was revealed in the expression of non-crystallins. Interestingly almost all crystallin gene products showed bimodal transcriptional bursts as characterized by violin plots.
Our data presents evidence that every fiber cell of the ocular lens is an unique unit sustained by its specific gene expression profile(s) and therefore, must display fiber cell-specific, unique physiology.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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