Investigation of the Multiple Roles of Notch Signaling in Embryonic Retinal Development Using Novel High-Throughput Techniques

Abstract

The process by which cell diversity is created during development is a central question in developmental biology. One of the most diverse groups of cells, the cells of the nervous system, exemplify this diversification with a dizzying array of cell types that are still being identified. The retina, a part of the central nervous system, has served as a powerful model with which to study the process of cell type diversity generation in the nervous system. During development of the embryonic mouse retina, retinal progenitors can be broadly classified into three large classes, each fated to produce different cell types. The Notch signaling pathway, a pathway used throughout development, is known to regulate these fate decisions. However, the mechanism by which this pathway, often thought to be one of binary fate decisions, can regulate three different fates is unknown. In order to investigate this process, we used single-cell RNA sequencing to profile progenitors in the embryonic retinas. Using known markers for two of these progenitor classes, we identified all three. This analysis revealed a potential three-tiered model of Notch signaling, with each tier specifying one progenitor class through the regulation of two sequential fate decision points. We provide further evidence for this model using experimental simulation of a middle-level Notch signaling state, and add to our model mechanisms for how this middle state is achieved. This model is further supported with data collected using high-throughput enhancer discovery and analysis techniques that we developed, which also allow for the investigation of upstream transcriptional regulation in other systems.