Abstract
Purpose :
Sox8 and Sox9 are highly and specifically expressed in late-stage retinal progenitor cells (RPCs) and mature Muller glia (MG) and are predicted to repress proliferative and neurogenic competence in these cell types. We have developed dominant negative (DN) constructs of Sox8 and Sox9 to test their ability to alter the developmental trajectory in the murine retina. Constructs that promote an early-stage RPC cell fate and/or production of early-born retinal cell types will be used to reprogram adult Muller glia to early-stage RPCs that are competent to produce cone photoreceptors upon retinal injury in vivo.
Methods :
DNs for Sox8 and Sox9 consisting of the DNA binding domain (DBD) only, DBD fused with a KRAB repressor (DBD+KRAB), and DBD fused with a VP48 activator (DBD+VP48) were designed. DNs were expressed in a pCAGIG construct downstream of a CAG promoter and upstream of an eGFP under the control of a bicistronic IRES element. Retinal explants were electroporated ex vivo to screen the DN constructs for their ability to alter the developmental trajectory in the retina. Retinal explants were electroporated with the DN constructs during late-stage retinal development (postnatal day(P)0-P8). The resulting phenotypes were analyzed using both scRNA-seq and immunohistochemistry.
Results :
Retinal explants electroporated with the DBD and DBD+KRAB DN constructs at P0 showed phenotypes at P8 that were consistent with the known or predicted genetic loss of function of each cluster of paralogous genes, including a reduction in MG, bipolar and amacrine cell generation, and an increase in photoreceptor generation. Notably, these constructs also induce formation of a small number of cells expressing markers of cone photoreceptors.
Conclusions :
These results suggest that suppression of Sox8 and Sox9 function is sufficient to shift the developmental trajectory of the retina to promote photoreceptor specification and induce expression of cone-specific markers. Since adult MG express many of the same TFs as late-stage RPCs, these DN constructs are potentially promising candidates for reprogramming MG to early-stage RPCs that are competent to produce cone photoreceptors upon retinal injury in vivo.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.