Characterizing the disarrayed Zebrafish Mutant – Insights Into Cell Cycle Regulation in the Development of the Retina

Abstract

Abstract: : Purpose: We are interested in understanding cell cycle regulation and proliferative cell behaviors and their relationships to development of the retina. During retinogenesis cell cycle exit and differentiation are coordinated in spatially and temporally regulated waves suggesting these events occur through local cell–to–cell signaling. We have isolated and are studying a zebrafish mutation, disarrayed, which exhibits defects in both retinal cell cycle control and retinal cell differentiation. Methods: Cell type marker analyses, genetic mosaic experiments and in vivo time–lapse imaging were used to assess retinal–specific cell cycle defects in a new zebrafish mutant, disarrayed. Positional cloning is currently underway to identify the disarrayed gene. Results: The disarrayed mutant can be identified early in development by its small eyes and reduced forebrain while other structures appear normal. Histology and marker analyses suggest that retinal development is delayed by approximately 12 hours in mutants; however, mutant eyes eventually show all the major cell types and synaptic connectivity. While overall retinal organization develops, photoreceptor morphogenesis remains awry: outer segment formation is compromised. Examination of the disarrayed mitotic cycle using the S–phase marker BrdU and by direct observation via time–lapse imaging revealed an extended cell cycle period. Mutant retinoblasts also showed delayed cell cycle exit: proliferating cells were not restricted to the marginal zone until 96hpf in the mutant whereas wild–type precursors are restricted by 72hpf. In addition, a wave of elevated cell death began at 42hpf which peaked at 60hpf. Time–lapse imaging revealed defects in interkinetic nuclear migration, a cell behavior exhibited in mitotic retinoblasts. Interestingly, genetic mosaic experiments indicate that the disarrayed mutation is non–cell autonomous for both morphology and cell cycle exit. Conclusions: The disarrayed mutant offers insights into cell cycle regulation as well as an avenue to examine extrinsic signaling pathways that affect cell cycle control and development of the retina.

Keywords: retinal development • cell–cell communication • proliferation