Abstract
Purpose :
We have recently shown that highly variable transcriptional activity, in single cortical fiber cells, mediates the transition from nascent to terminally differentiated fiber cells, in the developing ocular lens. We have now investigated the status of this heterogeneity in cataractogenesis, with a motivation to probe the earliest molecular events that presage the visible appearance of the cataract pathology. We have used the postnatal inherited opacities in a transgenic model of the most prevalent childhood cataract, the Lamellar cataract, as a paradigm.
Methods :
Single fiber cells were manually isolated from post-natal day 2 lens, in a fashion that retains their spatial context. Biomark microfluidics RT-PCR (Fluidigm Inc.) was used for quantitative determinations of gene expression. We present a cumulative merge of the data obtained from five Biomark IFC (Integrated fluidic circuits) chips (5 lenses x 96 samples=480 samples). In one chip, we interrogate 92 single fiber cells (from one lens). SINGuLAR software (Fluidigm) was used to analyze the transcriptional repertoire in single fibers isolated from the wild type (WT) lens and the transgenic cataract paradigm.
Results :
Unsupervised clustering and Principal component analysis (PCA) sifts gene activity data (obtained with single fiber cells isolated from the Lamellar cataract), into clusters of spatially mixed populations of cells (representing equatorial, cortical and nuclear fibers). Unlike in the WT, we find that there is little differential gene activity in fiber cells of different regions of the mutant lens. The pivotal observation is that the molecular heterogeneity, seen in the wild type cortical lens fiber cells, is absent in the mutant lens fiber cells, suggesting absence of a cell type (s) in the developing transgenic lens. This absence of heterogeneity seems to accompany the derangement of the developmental program, which finally proves pathological (appearance of a cataract).
Conclusions :
The data demonstrates remarkable absence of transcriptional heterogeneity in single fiber cells, concomitant with the absence of terminal differentiation in the cataract lens, indicating that homogenization of gene activity may represent a prelude to impending pathology, when a mutation disrupts normal development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.