• Mitochondrial dynamics
• reactive oxygen species
• retinal stem cells
• retinogenesis
• zebrafish

Mitochondria are highly dynamic and versatile organelles which are key regulators of several cellular processes. Moreover, mitochondria have emerged as fundamental contributors of cell fate acquisition, given their dynamic ability to interconnect environment with cellular homeostasis. In neural stem cells (NSCs), several studies have shown that the processes of mitochondrial fusion and fission coordinate the passage from self-renewal to commitment and differentiation. In the retina, based on previous findings from the team (Albadri et al. 2019), we hypothesized that mitochondria could serve as key intermediaries between metabolism and transcriptional programs, regulating as such retinal stem cell fate. This is the question that my project aims to investigate. To assess the role of mitochondrial dynamics and intracellular ROS modulation in retinal development, we have used zebrafish larvae (Danio rerio) as a model organism as one of its unique features is the presence of a stem cell niche called the ciliary marginal zone (CMZ), which harbors a pool of quiescent and proliferating retinal stem cells and committed progenitors spatially well-compartmentalized. Firstly, a clear characterization of mitochondrial morphology has been performed to highlight differences in proliferative versus differentiated cells of the epithelium, by different approaches. Taking advantage of the Gal4/UAS bipartite system, micro-injections have been performed in both tg(rx2:gal4) and tg(atoh7:gal4) transgenic embryos at 1-cell stage with a UAS construct (14UAS-UBC:RFP-caax-E2A-phb-GFP) to label distinct cell types at different stages of differentiation. At the organelle level, we have further assessed mitochondrial architecture architecture in distinct cells of the CMZ and differentiated retina through serial electron microscopy (sEM) using a readily available retinal dataset from a 6 days post-fertilization (dfp) zebrafish larva (Fulton KA et al., 2024). To gain deeper insight into how mitochondrial dynamics and ROS production influence RPCs fate, we have developed genetic tools for both the characterization and manipulation of the mitochondrial fission and fusion machinery. In parallel, since mitochondria are major contributors to reactive oxygen species (ROS) regulation, we focused on examining the role of one of the key mitochondrial antioxidants, Peroxiredoxin 3 (Prdx3), which is expressed in the CMZ. To assess whether Prdx3 play a role for retinal development and RPC differentiation, we performed a Crispan approach using CRISPR/Cas9 to target prdx3. To evaluate the impact of prdx3 loss of function on cell fate, we followed by immunostainings on the F0 injected embryos using proliferative and differentiation markers.
Exploring mitochondrial dynamics and mitochondrial ROS modulation could be fundamental in achieving a more comprehensive understanding of retinogenesis. Furthermore, several data have demonstrated that mitochondrial related dysfunctions are involved in several ocular disorders, including the Dominant Optic Atrophy where OPA1 function is altered. As such, these results could contribute to better understanding the impact of mitochondrial dysfunction on retinal cell differentiation, paving the way for innovative therapies.