Generating neuronal diversity in fly eyes and human retinal organoids

A central challenge in developmental neurobiology is to understand how the incredible diversity of neuronal cell types is generated. My lab studies this question in the color vision systems of flies and humans. By studying highly divergent organisms, we aim to identify fundamental mechanisms that diversify neuronal function during development.

The random mosaic of photoreceptors in the fly eye

“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.

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.

061714 website fly eye

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.

Our studies in flies address the mechanisms controlling stochastic photoreceptor patterning during development (Project 1) and the role of nuclear architecture in gene regulation (Project 2). Two subtypes of color-detecting photoreceptors are randomly patterned across the fly retina. We found that a two-step mechanism involving transcriptional priming and chromatin compaction determines stochastic expression of a transcription factor that controls patterning in the retina. This mechanism may represent a general paradigm for gene regulation during development. Our studies of nuclear architecture focus on the pairing of homologous chromosomes in somatic cells, which enables gene regulation between chromosomes. We found that clusters of DNA-looping insulators and chromatin structures called topologically associating domains (TADs) button chromosomes together to promote interchromosomal gene regulation. Our findings highlight how distinct elements in the genome drive physical interactions between chromosomes to regulate gene expression.

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, Liz Urban, Luke Voortman, Mini Yuan, Grace Gu): 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 (Liz Urban, Jeong Han, Adrienne Chen): 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 gene regulation.

Project 3. Innate color preference (Natalie Roberts): Flies have innate attraction to different colors. We are investigating how natural variation in color photoreceptor specification and environmental perturbations impact innate color preference.

The mosaic of photoreceptors in the human eye

In humans, three subtypes of cone photoreceptors enable trichromatic color and high acuity vision. To overcome the challenges associated with studies of human development, we utilized a human organoid system that recapitulates retinal development and photoreceptor specification. We found that spatiotemporal regulation of thyroid hormone and retinoic acid signaling specifies cone subtypes in human retinal organoids. Our studies advanced human retinal organoids as a model for revealing mechanisms of human development, with promising utility for therapeutics and vision repair.

The human retina is composed of three cone cell subtypes. 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 address this question in developing human tissue. We are studying the question of cone cell specification by differentiating human stem cells  into retinal cups. These retinal organoids contain all the cell types of the human retina including opsin-expressing cone photoreceptors (green = L/M opsins; blue = S opsin).


Project: Human photoreceptor choice (Kiara Eldred, Sarah Hadyniak, Kasia Hussey, Christina McNerney, Aki Sogunro): Human stem cell-derived retinal organoids are an ideal system to study cone subtype specification 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 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.

Retinal Ganglion Cell subtype specification