DescriptionTopoisomerase II beta (Top2b) is an enzyme that assists with DNA transcription by controlling the topological state of DNA to prevent supercoiling of the DNA and is widely expressed in postmitotic neurons. It is known to play a role in neuronal migration, cell adhesion, voltage-gated calcium channel activity, synaptic transmission, and cytoskeleton formation. In the retina, Top2b contributes to the late-stage differentiation and maturation of photoreceptors and affects the expression of key genes linked to retinopathies. Top2b deficiency has shown to lead to degeneration of the plexiform layers and outer segment of photoreceptors and reduction of cell number in the retina. However, the specific morphological differences between Top2b deficient retinal cells and normal retinal cells are not well documented. Additionally, the role of Top2b in interkinetic nuclear migration during retinal development has not been well defined. In this study, the role of Top2b in the developing retina was studied in two model systems: 1) the chick model was used to perform shRNA-based gene knockdown on cultured cells and retinal explants at embryonic day 10 (E10) to determine the function of Top2b at the embryonic stage; and 2) the gene knockout mouse model was used to examine tissue sections with Top2b deficiency at postnatal day 7 (P7), P14, and P21. In vitro shRNA knockdown was conducted to examine the role of Top2b in morphological development of the retina at the cellular level, while ex vivo and in vivo studies were used to examine cellular migration and morphology at the tissue level. Results show that i) after 3 days of Top2b knockdown in vitro, E10 retinal cells exhibit less surface area (3-fold reduction, p < 0.05) and shorter cellular processes (2-fold reduction, p < 0.05); ii) after 3 days of Top2b knockdown in the developing E10 chick retina, cells have shortened cellular processes but do not exhibit any migratory delays; iii) Top2b knockout delays interkinetic nuclear migration at P7 in the developing mouse retina, but does not inhibit migration of cells into the ONL in the long-term. This study helps to further characterize the role of Top2b in the developing retina and may provide insights into pathogenesis of various retinal disorders.