RESUMO
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.
Assuntos
Catarata/genética , Cristalinas/genética , Animais , Animais Geneticamente Modificados , Catarata/metabolismo , Diferenciação Celular/genética , Cristalinas/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Cristalino/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Retina/embriologia , Análise de Célula Única/métodos , Transcriptoma/genéticaRESUMO
The developing eye lens presents an exceptional paradigm for spatial transcriptomics. It is composed of highly organized long, slender transparent fiber cells, which differentiate from the edges of the anterior epithelium of the lens (equator), attended by high expression of crystallins, which generates transparency. Every fiber cell, therefore, is an optical unit whose refractive properties derive from its gene activity. Here, we probe this tangible relationship between the gene activity and the phenotype by studying the expression of all known 17 crystallins and 77 other non-crystallin genes in single fiber cells isolated from three states/regions of differentiation, allowing us to follow molecular progression at the single-cell level. The data demonstrate highly variable gene activity in cortical fibers, interposed between the nascent and the terminally differentiated fiber cell transcription. These data suggest that the so-called stochastic, highly heterogeneous gene activity is a regulated intermediate in the realization of a functional phenotype.