Molecular and cell biological study of functions and mechanisms of vertebrate gap gene Otx1

采用分子和细胞生物学方法研究了吻侧间隙基因Otx 1在斑马鱼胚胎发育中的功能。(1)错误表达的Otx 1的功能研究：斑马鱼胚胎显微注射体外合成的Otx 1 RNA在其1-细胞阶段显示出显着的原肠胚缺陷的剂量依赖性方式，这表明其抑制作用的细胞运动。当将Otx 1 RNA与GFP RNA沿着注射到16-细胞胚胎的单个细胞中时，其具有GFP荧光的子细胞通常在蛋黄上形成紧密的聚集体。另一方面，当32-细胞期的胚胎被注射到它们的单个细胞中时，荧光细胞通常迁移到未来的前脑-中脑区域并并入正常的喙部结构。用笼状荧光素示踪剂的研究表明，尽管在原肠胚形成期间细胞大量运动，但该区域的细胞仍保持一致性。糖皮质激素配体结合域的otx 1构建体仅在地塞米松激活后诱导细胞聚集。这些结果表明，Otx 1诱导同型细胞粘附，确定未来的脑-中脑区域。(2)Otx 1下游基因和因子的筛选：以钙粘蛋白和原钙粘蛋白为候选基因对斑马鱼文库进行筛选。它产生了一些钙粘蛋白超家族的克隆。未来的计划进行调查的其他类别的下游基因。

Functions of a rostral gap gene Otx 1 in developing zebrafish embryos were studied with molecular and cell biological strategies. (1) Functional studies of mis-expressed Otx1 : Zebrafish embryos micro-injected with in vitro synthesized Otx1 RNA at their 1-cell stage showed significant gastrulation defects in dose-dependent manner, suggesting its inhibitory action on cell motility. When Otx1 RNA was injected along with GFP RNA to a single cell of 16-cell embryos, its daughter cells with GFP fluorescence formed tight aggregates often on the yolk. On the other hand, when 32-cell stage embryos were injected in their a single cell by animal pole, fluorescent cells often migrated to future forbrain-midbrain region and incorporated to normal rostral structures. Studies with caged fluorescein tracer showed cells in this region retain coherence in spite of massive cell movements during gastrulation. Otx1 construct with glucocorticoid ligand-binding domain induced cell aggregation only upon activation with dexamethasone. These results indicated Otx1 induces isotypic cell adhesion by which define future forbrain-midbrain region. (2) Screening for downstream genes and factors of Otx1 : Zebrafish library was screened for cadherin and protocadherin as most-likely candicates. It gave some clones of cadherin superfamilies. Future plans are investigated for other classes of downstream genes.