DIO3 coordinates photoreceptor development timing and fate stability in human retinal organoids

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. DIO3 mutant organoids display precocious development of S cones, L/M cones, and rods; increased photoreceptor density; and subpopulations of photoreceptors that coexpress different opsin proteins. Our multiomics and chimeric organoid experiments show that cell-autonomous and non-cell-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared with a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass-like mechanism in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, triggering development of photoreceptor subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.