Abstract
Topoisomerase II beta (Top2b) is an enzyme that alters the topologic states of DNA during transcription. Top2b deletion in early retinal progenitor cells causes severe defects in neural differentiation and affects cell survival in all retinal cell types. However, it is unclear whether the observed severe phenotypes are the result of cell-autonomous/primary defects or non-cell-autonomous/secondary defects caused by alterations of other retinal cells. Using photoreceptor cells as a model, we first characterized the phenotypes in Top2b conditional knockout (cKO). Top2b deletion leads to malformation of photoreceptor outer segments (OS) and synapses accompanied with dramatic cell loss at late-stage photoreceptor differentiation. Then, we performed mosaic analysis with shRNA-mediated Top2b knockdown in neonatal retina using
in vivo
electroportation to target rod photoreceptors in neonatal retina. Top2b knockdown causes defective OS without causing a dramatic cell loss, suggesting a Top2b cell-autonomous function. Furthermore, RNA-seq analysis reveals that Top2b controls the expression of key genes in photoreceptor gene-regulatory network, e.g., Crx, Nr2e3, Opn1sw, and Vsx2, and retinopathy-related genes, e.g., Abca4, Bbs7, and Pde6b. Together, our data establish a combinatorial cell-autonomous and non-cell-autonomous role for Top2b in late-stage of photoreceptor differentiation and maturation.
Three major stages are well defined in photoreceptor cell development: 1) cell proliferation and cell fate determination, during which the multipotent retinal progenitors proliferate and their competence being restricted as photoreceptor cell precursors; 2) early differentiation stage, during which genes for morphogenesis and phototransduction are expressed; and 3) late differentiation stage, which includes the axonal growth, synapse formation and outer segment (OS) biogenesis (
Swaroop et al. 2010
). Top2b functions in both cell-autonomous and non-cell-autonomous manners in regulating late stage photoreceptor development by controlling key genes in the photoreceptor transcriptional network.