Why is stochastic cell fate specification important?
“I, at any rate, am convinced that He does not play dice.” These famous words were spoken by Albert Einstein about the existence of an underlying reality and the predictability of the universe. A simple look at a pair of twins would back the notion that nature and biology can be amazingly reproducible. However, one would only have to look into the eyes of these twins to see something remarkable: the different types of cells that detect light are randomly distributed and thus each twin is unique. Only recently have we begun to understand the mechanisms controlling such stochastic developmental events. It appears that noise in molecular processes creates variation, and this variation can be exploited to diversify the functions of cells. Stochastic specification is critical for generating a wide range of cell types, from human cone cells that detect colors, to olfactory neurons that sense odors, to B cells required for immune responses. Beyond its role in normal development, stochastic gene regulation can determine whether a person with a mutated gene will suffer from disease. Despite its importance, very little is understood about how this fundamentally different strategy operates.
The random mosaic of photoreceptors in the fly eye
We are using the fly eye as a paradigm to elucidate the mechanisms controlling stochastic gene expression during development. Similar to the human color vision system, the photoreceptors of the fly eye randomly express several light-detecting Rhodopsin proteins (Rhodopsin3 =blue; Rhodopsin4 = red). The fly eye is an ideal system to study this phenomenon because it provides a simple binary output for stochastic gene expression, the general mechanisms of cell-fate specification are well-understood, and a vast array of genetic and transgenic tools are available to manipulate cis-regulatory inputs and upstream trans-acting factors.
The transcription factor Spineless is the critical regulator controlling the random mosaic pattern of photoreceptor subtypes in the fly eye. Each allele of spineless makes its own random expression choice independent of the other. Stochastic on/off expression of spineless is determined by general activation coupled with random repression requiring combinatorial inputs from cis-regulatory elements acting at long range. Through interchromosomal communication, the two alleles coordinate their expression state. The next goal is to determine the molecular mechanisms controlling intrinsically stochastic expression decisions and interchromosomal gene regulation:
Project 1. Random expression decisions (Caity Anderson, Alexandra Neuhaus-Follini, Liz Urban, Luke Voortman, Mini Yuan): How does a gene randomly decide to be on or off? To address this question, we are studying how DNA looping, nuclear architecture, chromatin state, and transcriptional regulators control stochastic expression of spineless.
Project 2. Nuclear architecture and interchromosomal gene regulation (Kayla Viets, Chaim Chernoff, Jeong Han, Adrienne Chen, Maha Sarfraz): Very little is known about how long range interactions of regulatory DNA elements control stochastic gene expression. We are studying how copies of the spineless gene come into physical proximity to mediate interchromosomal communication.
The random mosaic of photoreceptors in the human eye
The human retina is composed of three cone cell subtypes that are stochastically distributed. These cone cell subtypes are defined by expression of color-detecting opsin proteins: L/red, M/green, and S/blue opsins. Despite their importance in color and daytime vision, very little is understood about how these three photoreceptor subtypes are generated. Since mice and fish have dramatically different patterns of cone cells, we must address this question in developing human tissue. We are studying the question of stochastic cone cell specification by differentiating human induced pluripotent stem cells (iPSCs) into retinal cups. These retinal cups contain all the cell types of the human retina including opsin-expressing cone photoreceptors (green = L/M opsins; blue = S opsin).
Project: Stochastic human opsin choice (Kiara Eldred, Sarah Hadyniak, Kasia Hussey): Human stem cell-derived retinal cups are an ideal system to study stochastic opsin expression due to their limited number of cone cell subtypes, arrangement in a monolayer for simple quantitative analysis, thin tissue depth allowing live visualization, and tractability for CRISPR-mediated genome engineering. To address the mechanisms controlling stochastic cone cell subtype specification, we are generating reporter lines for the three opsins that we will use for live imaging and to sort cells for genomics analyses. We are testing the roles of upstream transcriptional regulators, subnuclear positioning, and DNA looping in the regulation of opsin expression.