The development and connectivity of starburst amacrine cells in human retinal organoids

Project Summary: The direction-selective (DS) circuits in the retina detect the direction of motion in the visual field. Starburst Amacrine Cells (SACs) are specialized interneurons in DS circuits that respond to increases (ON) or decreases (OFF) in light and asymmetrically synapse onto direction-selective ganglion cells (DSGCs). Very little is known about the development and specification of SACs and their downstream connections with DSGCs and other SACs in the human retina. In this project, I will explore mechanisms of SAC fate specification and characterize the development of SAC connectivity in human retinal organoids. The mechanisms controlling when and how SACs develop in the human retina are poorly understood. In my preliminary studies, I found that SACs arise early during retinal organoid development. In Aim 1, I will establish a timeline of SAC birth using IHC, live imaging, and EdU birthdating in organoids. The transcription factor ISL1 is required for SAC specification in rodents8. To assess the role of ISL1 in human SAC generation, I will differentiate organoids from an ISL1∆ null mutant stem cell line9 and assess the density and timing of SAC generation. SACs are classified as ON or OFF subtypes based on their responses to light intensity, soma positions, and gene expression. In Aim 2, I will establish a timeline of SAC subtype generation in human fetal retinas and organoids using IHC, RNA FISH and live imaging. The transcription factor FEZF1 regulates ON SAC cell fate in mice. Preliminary data shows that FEZF1 is expressed in a subset of SACs in the human fetal retina. I will test the role of FEZF1 in the specification of ON SACs by generating and analyzing a FEZF1∆ null mutant stem cell line. SACs synapse onto other SACs and DSGCs in DS circuits. While the identification of SAC synaptic partners has not been established in humans, my preliminary data show that SACs form close associations with retinal ganglion cells. In Aim 3, I will differentiate organoids carrying a VACHT-Cre/tdTomato reporter and infect with a virus harboring a WGA-GFP monosynaptic tracer. In this method, the presynaptic SAC will express tdTomato and GFP, and the postsynaptic cell will express GFP only. To examine homotypic SAC-SAC connections, I will use a chimeric organoid strategy. Successful completion of this project will identify mechanisms of SAC specification and DS circuitry in the human retina, providing insights will inform the development of therapies to treat vision disorders.