Supplementary Material for: Arl6ip1 Plays a Role in Proliferation during Zebrafish Retinogenesis

Objective: Arl6ip1 has been reported to play a role in ocular development, but its regulatory function as it relates to proliferation is unclear. Therefore, this study aimed to evaluate how Arl6ip1 may regulate the proliferative behavior of retinal progenitor cells during zebrafish embryogenesis. Method: Arl6ip1 was specifically knocked down by introducing morpholino nucleic acid oligomers. The DNA content of cells dissociated from morphant eyes was analyzed by fluorescence-activated cell sorting (FACS). Retinal cells in the S- and late G2/M-phase were detected by labeling with BrdU and then immunostaining with anti-BrdU antibody and by immunostaining with phospho-histone H3 antibody, respectively. We also examined the expressions of shh,p57kip2, and cyclin D1 in retinas of experimental animals. The bidirectional plasmid pGFP:HSE:p57kip2 was used to rescue the defect in cell cycle exit. Results: FACS analysis showed that the >2C DNA content per cell in the eyes of arl6ip1 morphants was 2-fold greater than that of the wild type. Following functional Arl6ip1 knockdown, anti-BrdU- and anti-phospho-histone H3-positive signals were higher and the retinal progenitors kept expressing cyclin D1 but not shh or p57kip2, suggesting that eye progenitor cells remained in an early progenitor state and could not exit the cell cycle to progress to differentiation. Interestingly, overexpression of p57kip2, which enables exit from the cell cycle, led to a reduction of anti-BrdU-positive signals in the retinas of arl6ip1 morphants. Conclusion: Arl6ip1 not only affects signals controlling eye development but also plays an important role in the proliferation of retinal progenitor cells.