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

用分子生物学和细胞生物学方法研究了OTX-1基因在斑马鱼胚胎发育中的作用。(1)错误表达Otx1的功能研究：将体外合成的Otx1 RNA显微注射到斑马鱼胚胎的1-细胞期，显示出明显的原肠发育缺陷，并呈剂量依赖关系，表明其对细胞运动的抑制作用。当将Otx1 RNA和GFP RNA一起注射到16-细胞胚胎的单个细胞中时，其带有GFP荧光的子细胞通常在卵黄上形成紧密的聚集体。另一方面，当32-细胞期胚胎通过动物极点注入其单细胞时，荧光细胞往往迁移到未来的脑-中脑区，并结合到正常的吻部结构中。用笼式荧光素示踪剂进行的研究表明，尽管在原肠形成过程中细胞大量移动，但该区域的细胞仍保持一致性。含有糖皮质激素配体结合区的OTX1只有在地塞米松激活时才能诱导细胞聚集。这些结果表明，Otx1诱导同型细胞黏附，从而决定了脑-中脑区的未来。(2)Otx1下游基因和因子的筛选：筛选斑马鱼文库中最可能的钙粘蛋白和原钙粘蛋白。它提供了一些钙粘蛋白超家族的克隆。对其他类别的下游基因的未来计划也进行了调查。

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.