Link B A, Kainz P M, Ryou T, Dowling J E
Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Dev Biol. 2001 Aug 15;236(2):436-53. doi: 10.1006/dbio.2001.0340.
To identify and study genes essential for vertebrate retinal development, we are screening zebrafish embryos for mutations that disrupt retinal histogenesis. Key steps in retinogenesis include withdrawal from mitosis by multipotent neuroepithelial cells, specification to particular cell types, migration to the appropriate laminar positions, and molecular and morphological differentiation. In this study, we have identified two recessive mutations that affect the transition of proliferating neuroepithelial cells to postmitotic retinal cells. Both the perplexed and confused mutant phenotypes were initially detectable when the first retinal neuroepithelial cells began to leave the cell cycle. At this time, each mutant retina showed increased cell death and a lack of morphological differentiation. Cell death was found to be apoptotic in both perplexed and confused retinas based on TUNEL analysis and activation of caspase-3. TUNEL-phosphoRb-BrdU colocalization studies indicated that the perplexed mutation caused death in cells transitioning from a proliferative to postmitotic state. For the confused mutation, TUNEL-phosphoRb-BrdU analysis revealed that only a subset of postmitotic cells were induced to activate apoptosis. Mosaic analysis demonstrated that within the retina the perplexed mutation functions noncell-autonomously. Furthermore, whole lens or eye cup transplantations indicated that the retinal defect was intrinsic to the retina. Mosaic analysis with confused embryos showed this mutation acts cell-autonomously. From these studies, we conclude that the perplexed and confused genes are essential at distinct stages during the transition from proliferating to postmitotic cells within the zebrafish retina.
为了鉴定和研究脊椎动物视网膜发育所必需的基因，我们正在筛选斑马鱼胚胎中破坏视网膜组织发生的突变。视网膜发育的关键步骤包括多能神经上皮细胞退出有丝分裂、分化为特定细胞类型、迁移到适当的层状位置以及分子和形态学分化。在本研究中，我们鉴定出两个隐性突变，它们影响增殖性神经上皮细胞向后有丝分裂视网膜细胞的转变。当第一批视网膜神经上皮细胞开始离开细胞周期时，最初就能检测到“困惑”（perplexed）和“混乱”（confused）突变体的表型。此时，每个突变体视网膜都显示出细胞死亡增加和形态学分化缺失。基于TUNEL分析和caspase-3的激活，发现“困惑”和“混乱”视网膜中的细胞死亡均为凋亡性。TUNEL-磷酸化Rb-BrdU共定位研究表明，“困惑”突变导致从增殖状态向后有丝分裂状态转变的细胞死亡。对于“混乱”突变，TUNEL-磷酸化Rb-BrdU分析显示，只有一部分后有丝分裂细胞被诱导激活凋亡。镶嵌分析表明，在视网膜内，“困惑”突变以非细胞自主方式起作用。此外，全晶状体或眼杯移植表明视网膜缺陷是视网膜固有的。对“混乱”胚胎的镶嵌分析表明，该突变以细胞自主方式起作用。从这些研究中，我们得出结论，“困惑”和“混乱”基因在斑马鱼视网膜内从增殖细胞向后有丝分裂细胞转变的不同阶段是必不可少的。
