Effects of the OTX2ECR2 enhancer & PROX1 overexpression in murine retina development

There is a large subset of retinal progenitor cells that upregulate the OTX2 gene in the developing retina of the mouse. OTX2 is a homeobox transcription factor important for the development of photoreceptors and bipolar cells. If OTX2 is knocked out, photoreceptors and bipolar cells fail to differentiate. In order for OTX2 to be activated in certain retinal populations, it requires the activity of an enhancer called OTX2ECR2. Past studies demonstrated that retinal progenitor cells that had activity of OTX2ECR2 turned into horizontal cells, which require OTX2 for cell genesis and survival, and photoreceptors which require OTX2 for cell genesis, survival, and its continuous expression post differentiation. Regulation of OTX2 by OTX2ECR2 in the past had only been studied by means of electroporation of reporters. In this study however, we used the 07 transgenic mouse line created by our lab which contains the construct OTX2ECR2::GFP::IRES::CRE integrated into its genome. We then crossed this mouse to the Ai14 transgenic mouse by Jackson Laboratories which contains the construct ROSA::CAG::LSL::tdTomato, and proceeded to genotype the new born pups. The pups positive for both constructs have constitutive expression of tdTomato in any cells showing a history of expression of OTX2ECR2 due to CRE recombinase doing a cis recombination of LoxP-stop-LoxP cassette, and therefore extracting a LoxP site with the stop codon located in this construct. P30 mice showed a history of expression of OTX2ECR2 in both rod photorceptors and amacrine cells. Since in the past there have been studies of retinal progenitor cells that are OLIG2+ that give rise to both rod photoreceptors and amacrine cells, we decided to test if these same cells would have a history of OTX2ECR2. In this study we report that OLIG2+ retinal progenitor cells showing activity of the OTX2ECR2 enhancer differentiate into rod photoreceptors and AII amacrine cells. Our study is the first to use a stable transgenic mouse. Prox1 is a prospero related homeobox transcription factor essential for the development of the central nervous system. In the eye Prox1 is seen the most in the region where the lens fibers elongate in the mouse. It has also been observed in the outer neuroblastic layer at E12.5, E14.5, and E17.5, which contain proliferating retinal progenitor cells. At later stages Prox1 serves as a marker for AII amacrine cells, for all horizontal cells, and weakly for bipolar cells. In this study, misexpression of Prox1 in P0 pups allowed to grow 2 weeks demonstrated electroporated cells that appeared to have a cone photoreceptor morphology. Under further experimentation, it was not clear if these cells were positive for RXRGamma and CRX, both of which are photoreceptor markers. More studies need to be conducted to fully decipher the identity of these newly produced cells by either looking for more photoreceptor markers, or by doing single cell RNA analysis to further elucidate the genetic profile of these cells