Abstract
Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens. To understand the role of transcriptional heterogeneity in terminal differentiation and the functional phenotype (transparency) of this tissue, here we present a single-cell analysis of the developing lens, in a transgenic paradigm of an inherited pathology, known as the lamellar cataract. Cataracts hinder transmission of light into the eye. Lamellar cataract is the most prevalent bilateral childhood cataract. In this disease of early infancy, initially, the opacities remain confined to a few fiber cells, thus presenting an opportunity to investigate early molecular events that lead to cataractogenesis. We used a previously established paradigm that faithfully recapitulates this disease in transgenic mice. About 500 single fiber cells, manually isolated from a 2-day-old transgenic lens were interrogated individually for the expression of all known 17 crystallins and 78 other relevant genes using a Biomark HD (Fluidigm). We find that fiber cells from spatially and developmentally discrete regions of the transgenic (cataract) lens show remarkable absence of the heterogeneity of gene expression. Importantly, the molecular variability of cortical fiber cells, the hallmark of the WT lens, is absent in the transgenic cataract, suggesting absence of specific cell-type(s). Interestingly, we find a repetitive pattern of gene activity in progressive states of differentiation in the transgenic lens. This molecular dysfunction portends pathology much before the physical manifestations of the disease.
Highlights
Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens
In the ocular lens, the functional phenotype of fiber cells is transparency and the disease phenotype is a cataract, it would be meaningful to examine the status of transcriptional heterogeneity [7] in the individual fiber cells of a cataract
The mutant lens physiognomy looks normal, a cross section of the lens at PND02 reveals the persistence of nuclei (Fig. 1B, cataract PND02), an abnormal phenotype, known to be associated with the loss of Hsf4 gene activity [21] [19]; the nuclei are seen in the anterior as well as in the posterior of the lens (Fig. 1B, cataract PND02) [19]
Summary
Cataract is a disease of the ocular lens where the fiber cells, instead of remaining transparent, become opaque, which inhibits light transmission. In the ocular lens, the functional phenotype of fiber cells is transparency and the disease phenotype is a cataract (opacification), it would be meaningful to examine the status of transcriptional heterogeneity (which is characteristic of the WT transparent fiber cells) [7] in the individual fiber cells of a cataract (lens). For these investigations, we have used the transgenic model of a genetic cataract known as the lamellar cataract. We worked with the postnatal 2-day-old (PND02) transgenic mutant lens, which allows us to probe early molecular changes, much before the cataract pathology is visible after the eyes open (Fig. 1A)
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