Asymmetric localization of Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio rerio.
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.
Institut für Entwicklungsbiologie, Universität zu Köln, 50923 Köln, Germany. alexander.reugels@uni-koeln.de
Alexander M
AM
Reugels
Barbara
B
Boggetti
Nico
N
Scheer
José A
JA
Campos-Ortega
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D007438:Introns; D007605:Juvenile Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046569:Neuroepithelial Cells / Q000166:cytology / Q000502:physiology*; D010802:Phylogeny; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000502:physiology; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; D007605:Juvenile Hormones; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; C559058:hspd1 protein, zebrafish; C060606:numb protein, Drosophila; C513665:numb protein, zebrafish; D049452:Green Fluorescent Proteins