Abstract
Purpose :
The numerous neuronal types of the retina are generated at different overlapping time points, yet a high resolution timeline of cell birth, maturation, and death in the developing human retina has not been achieved, due to the experimental inaccessibility of human fetal retinal tissue. Moreover, the spatiotemporal mechanisms controlling neuronal development in the human retina are poorly understood. It is unclear if the temporality of cell fate specification reflects the birth timing or maturation rates of these cells. The main goal of this study is to identify temporal mechanisms governing the generation and maturation of neurons in the human retina using human retinal organoids.
Methods :
To assess the timing of neuronal type birth, maturation, and death, I am using nucleotide analogs (i.e. EdU) to label newly replicated DNA for 48 hours every 7-days across organoid development. Whereas dividing progenitor cells will dilute the label over time, post-mitotic cells will retain this label, enabling me to track cells born at distinct times. The Johnston lab found that retinoic acid (RA) acts early and thyroid hormone (TH) acts late during retinogenesis to specify photoreceptor fates. To test how these signals affect the timing of cell generation, I am examining changes in cell birth dates upon changes in signaling environments.
Results :
Preliminary experiments showed that EdU is toxic when administered in >48-hour pulses, limiting the length of the temporal windows. In a parallel approach, I am developing a transgene-based strategy to birth date cells with H2B-GFP in wider windows. Mitotic cells will divide and dilute the label while neurons born in the window will retain the H2B-GFP. This system allows more fine-tuned control and adaptability for later experiments.
Conclusions :
This project will generate a timeline of human retinal neuron development at an unprecedented temporal and cell-type resolution, characterize new neuronal subtypes, and determine how signals affect the timing of their development. In addition, these studies will determine how signaling pathways govern the timing of cell type generation and cell fate specification decisions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.