Abstract
Purpose :
Previous work has identified cell-type specific gene regulatory networks (GRNs) that control cell-fate specification in developing mice and human retinas (Lyu, et al., 2021; Thomas et al., 2021). Transcription factors Zbtb7a and Zbtb7b were identified within GRNs specific to photoreceptor (PR) specification and were predicted to regulate rod PR fate. We have designed overexpression constructs for Zbtb7a and Zbtb7b to test their predicted phenotypes in mouse retinal explants. Functional analysis of TFs regulating cell-fate specification of PRs will potentially help to drive therapies such as Muller glia (MG) reprogramming that are aimed at replacing retinal neurons lost to disease.
Methods :
Candidate TFs were expressed in a pCAGIG construct downstream of a CAG promoter, with eGFP downstream under the control of a bicistronic IRES. Retinal explants were electroporated with overexpression constructs ex-vivo during late-stage retinal development at postnatal day 0 (P0) to functionally analyze the TFs ability to alter the developmental trajectory of murine retina. Retinal explants were collected for immunohistochemical analysis at P11, and explants overexpressing Zbtb7a were collected at P6 for sc-RNA sequencing analysis.
Results :
Retinal explants electroporated with Zbtb7a and Zbtb7b, TFs predicted to regulate rod PR fate, did not show phenotypes consistent with their predicted function. Overexpression of Zbtb7a showed an increase in cells expressing retinal progenitor cell/ MG marker Sox9 and cone PR markers, with an overall reduction in rod cells. This was consistent with Zbtb7b overexpression analysis, which showed an increase in cells co-expressing GFP and cone marker Arrestin-C with a decrease in cells expressing rod marker Nr2e3. Further analysis of Zbtb7b overexpression using scRNA-sequencing is currently underway.
Conclusions :
These results demonstrate that overexpression of Zbtb7a and Zbtb7b is sufficient to severely shift the developmental trajectory of the murine retina. This highlights the importance of these TFs as positive regulators of cone PR fate. These results also suggest that manipulation of Zbtb7a or Zbtb7b, individually or in combination, could help to promote the reprogramming of competent MG cells into photoreceptors, the neurons primarily lost in retinal degenerative diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.