Reugels Alexander M, Boggetti Barbara, Scheer Nico, Campos-Ortega José A
Institut für Entwicklungsbiologie, Universität zu Köln, 50923 Köln, Germany.
Dev Dyn. 2006 Apr;235(4):934-48. doi: 10.1002/dvdy.20699.
In the neural plate and tube of the zebrafish embryo, cells divide with their mitotic spindles oriented parallel to the plane of the neuroepithelium, whilst in the neural keel and rod, the spindle is oriented perpendicular to it. This change is achieved by a 90 degrees rotation of the mitotic spindle. We cloned zebrafish homologues of the gene for the Drosophila cell fate determinant Numb, and analyzed the localization of EGFP fusion proteins in vivo in dividing neuroepithelial cells during neurulation. Whereas Numb isoform 3 and the related protein Numblike are localized in the cytoplasm, Numb isoform 1 is localized to the cell membrane. Time-lapse analyses showed that Numb 1 is distributed uniformly around the cell cortex in dividing cells during plate and keel stages, but becomes localized at the basolateral membrane of some dividing cells during the transition from neural rod to tube. Using in vitro mutagenesis and Numb:EGFP deletion constructs, we showed that the first 196 amino acids of Numb are sufficient for this localization. Furthermore, we found that an 11-amino acid insertion in the PTB domain is essential for localization to the cortex, whereas amino acids 2-12 mediate the basolateral localization in the neural tube stage.
在斑马鱼胚胎的神经板和神经管中，细胞分裂时其有丝分裂纺锤体与神经上皮平面平行，而在神经嵴和神经索中，纺锤体则与之垂直。这种变化是通过有丝分裂纺锤体旋转90度实现的。我们克隆了果蝇细胞命运决定因子Numb基因的斑马鱼同源物，并在神经胚形成过程中对分裂的神经上皮细胞中EGFP融合蛋白的体内定位进行了分析。Numb同工型3和相关蛋白Numblike定位于细胞质中，而Numb同工型1定位于细胞膜。延时分析表明，在神经板和神经嵴阶段，Numb 1在分裂细胞的细胞皮层周围均匀分布，但在从神经索向神经管转变过程中，会定位于一些分裂细胞的基底外侧膜。通过体外诱变和Numb:EGFP缺失构建体，我们发现Numb的前196个氨基酸足以实现这种定位。此外，我们发现PTB结构域中的一个11个氨基酸的插入对于定位于皮层至关重要，而第2至12个氨基酸介导神经管阶段的基底外侧定位。
