Abstract
Purpose :
During normal embryonic development, cell fate determination is critical for the success of a healthy embryo. Cell specificity during development results from differences in spatial and temporal regulation of transcription factor-encoding genes. The PRDM family is a group of transcriptional regulators that drive and maintain cell state transitions in many tissues and cell types. Of interest, PRDM13 is expressed in the developing retina and neural tube. In the rare ocular disease North Carolina Macular Dystrophy (NCMD), PRDM13 is duplicated in some patients. NCMD patients exhibit a loss of central and color vision at birth or within the first decade of life, suggesting a developmental defect. However, how PRDM13 is involved in photoreceptor degeneration is not yet known. In this study, we sought to determine the effect of overexpression (OE) of PRDM13 on the human retina at different stages of development.
Methods :
To address this objective, we generated a novel human embryonic stem cell (hESC) line containing a Doxycycline (Dox) inducible OE of PRDM13. We differentiated these hESCs into 3D retinal organoids that mimic human retinal development in culture and induced PRDM13 OE at different stages during development. To monitor transcriptomic changes in the heterogeneous cell population, we performed single cell RNA (scRNA) sequencing of control and PRDM13 OE retinal organoids at days 35 and 45, the start of retinal cell formation in the organoids. We also characterized and validated what retinal cell types were present by examining retinal cell mRNA and protein markers using qRT-PCR and immunostaining, respectively.
Results :
Excitingly, we found that inducing PRDM13 OE at day 30 of organoid differentiation results in morphology changes, such as an increase in budding, growth, and a rearrangement of layers. At day 35, after 5 days of PRDM13 OE, most of the unique retinal cell subtypes were able to be formed and clustered in our scRNA-sequencing data. However, we did see a change in some retinal cell type formations, including the loss of the developing photoreceptor cell population.
Conclusions :
Based on our results, temporal regulation of PRDM13 at specific time points during development affects retinal cell type specification. When PRDM13 is overexpressed as retinal progenitor cells are beginning differentiation, this can lead to a loss of the photoreceptor cell population, imitating NCMD’s degenerative phenotype.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.