Abstract
Abstract: :
Purpose: Spontaneous mutations in Pax6, a homeobox gene, cause a severe and complex phenotype (human aniridia, mouse small eye), that includes abnormalities in several eye components. The precise timing and sequence of Pax6 involvement in various aspects of eye development, however, are not yet known. We report here their investigation using antisense morpholinos against Pax6, which allow blocking its expression at defined developmental stages. Methods: Fluorescein–labeled morpholinos against Pax6, or control morpholinos, were microinjected into chick embryo brain vesicle at Hamburger–Hamilton (HH) stages 9 to 14. After electroporation, the embryos were allowed to develop for 24 to120 additional hrs, fixed, examined in toto, cryosectioned, and processed for immunocytochemistry or in–situ hybridization. Results: Control morpholinos caused no detectable changes in Pax6 expression or eye development at any of the stages studied. Pax6 morpholinos, on the other hand, altered eye development in a stage–dependent manner. Embryos treated at HH stages 9–10 either developed no optic cup and lens, or had the optic vesicle replaced by a rudimentary, pigmented, club–shaped structure, accompanied by a very small lens. A dramatic change in expression of cell–specific molecular markers was observed in morpholino–containing cells, including downregulation of Pax6, ectopic expression of Pax2 in the rudimentary optic vesicle, and lack of L–Maf expression in the rudimentary lens. Pax6 morpholino effects were considerably milder when treatment was applied at somewhat later stages (HH 11–14), resulting in smaller but otherwise normal optic cup and an underdeveloped lens. Conclusions: These experiments suggest that there is a narrow time window (of only a few hours) during which Pax6 must act to ensure the normal fate of optic vesicle cells. The molecular events that occur during this narrow time window are now being further investigated.
Keywords: retinal development • transcription factors • gene/expression