Abstract
Purpose :
Previous work identified cell-type specific gene regulatory networks (GRNs) that control cell-fate specification in developing mice and human retinas. Transcription factors Zbtb7a and Zbtb7b were identified within GRNs specific to photoreceptor (PR) specification and were predicted to regulate PR fate. We have designed overexpression (OE) constructs for Zbtb7a/b to test their predicted phenotypes in mouse retinal explants and have designed conditional knockout mouse models using CHX10 and Rax Cre lines. Functional analysis of TFs regulating cell-fate specification of PRs will potentially help to drive therapies aimed at replacing retinal neurons lost to disease.
Methods :
Candidate TFs were expressed in a pCAGIG construct downstream of a CAG promoter. Retinal explants were electroporated with OE constructs ex-vivo during late-stage retinal development at postnatal day 0 (P0) to analyze the TFs ability to alter the developmental trajectory of mouse retina. Retinal explants were collected at P6 for sc-RNA sequencing analysis. Zbtb7a lox/lox and Zbtb7b lox/lox mice were bred to CHX10-Cre and Rax-Cre;Sun1GFP mice to induce knock out of these TFs in RPCs. Individual knockout mutants were collected for IHC analysis, and analysis of double knockout mutants are still underway.
Results :
Overexpression of Zbtb7a in retinal expants showed an increase in cells expressing retinal progenitor cell/ MG marker Sox9 and cone photoreceptor markers, with an overall reduction in rod cells. This was consistent with Zbtb7b overexpression analysis, which showed an increase in cells expressing cone marker Arrestin-C, with a decrease in cells expressing rod marker Nr2e3. The phenotype of CHX10-Cre; Zbtb7a lox/lox and CHX10-Cre; Zbtb7b lox/lox also showed alterations in the cellular distribution of the retina.
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 paralog TFs as positive regulators of cone photoreceptor 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 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.