Developmental expression of zebrafish Numb 1 and Numb 3. Total RNAs were treated with DNase I, retrotranscribed using an oligo-dT primer, and amplified by PCR (30 cycles) with isoform-specific primer pairs. Both isoforms are expressed in all stages. A fragment of β-actin was amplified as a control.

A-C: Sequences of time-lapse frames of mitotic cells of embryos injected with numb(PTB^L PRR^L):egfp mRNA. The transparent grey line marks the midline. All photographs are dorsal views. Anterior is towards the top. Scale bar = 20 μm. A: Two mitotic cells in the neural plate of a 5- to 6-somite embryo (arrows in 0'). Cell divisions are planar at this stage. Numb:EGFP is distributed ubiquitously around the cell cortex and segregated to both daughter cells upon cell division (arrows in 5'00"). B: A mitotic cell in the neural rod of a ~16-hpf-old embryo (arrow in 0'). Cells in this stage divide perpendicularly to the midline, due to a 90° rotation of the mitotic spindle. As in the neural plate stage, Numb:EGFP is localized ubiquitously around the cell cortex and, therefore, segregated to both daughter cells (arrows in 3'00"). C: Two mitotic cells during the transition from neural rod to neural tube in a ~17-hpf-old embryo. Whereas one cell (arrow in 0') still divides perpendicular to the plane of the epithelium with Numb:EGFP being ubiquitously localized (arrows in 8'), the other cell (arrowhead in 0') already divides parallel to plane of the epithelium, as is typical for all cell divisions in the neural tube (arrowheads in 10'). Note that this cell starts to localize Numb:EGFP to the basolateral cell cortex when it rounds up at the midline for mitosis (frames 0'- 8'). See also Supplemental Material for the corresponding Supplementary Movies 1 to 3.

A sequence of 15 confocal micrographs of a dorsal view of the neural tube of a ~24-hpf-old embryo injected with numb(PTB^L PRR^L):egfp mRNA. The transparent grey line in the first picture marks the neurocoel. Scale bar = 20 μm. Three cells (arrows, arrowheads, and asterisks in 0') one after another round up at the neurocoel and subsequently undergo mitotic cell division. Note that Numb:EGFP becomes localized to the basolateral cell cortex in all three cells, whereas the apical side is completely devoid of signal. Although Numb:EGFP is clearly asymmetrically localized in these cells, it is distributed to both daughter cells upon division, since the cells divide parallel to the plane of the neuroepithelium. See also Supplemental Material for the corresponding Supplementary Movie 4.

A-H: Comparison of Numb(PTB^L PRR^L):EGFP, Numb(PTB^S PRR^L):EGFP, Nbl:EGFP, and five different Numb:EGFP deletion constructs with respect to their localization in dividing cells. Confocal micrographs showing a dorsal view of mitotic cells in the neural tube of embryos injected with the corresponding mRNA. Anterior is towards the top and the midline/apical surface is approximately in the middle of each photograph. See text for details. B-D: Note that the nucleus in non-dividing cells is free of the signal (arrow), whereas in mitotic cells (arrowhead) the signal is found in the entire cell, after the nuclear membrane has broken down. F: Amino acids 1-196 (including the PTB^L insertion) are efficiently localized to the basolateral cortex. G: Deletion of amino acids 2-12 impairs cortical and abolishes basolateral localization in dividing cells. H: Impairment of cortical localization is enhanced after deletion of amino acids 2-22.

A: Solution structure of the Drosophila Numb PTB domain-Nak peptide complex as described by Zwahlen et al. ([2000]). The PTB domain is coloured in grey, whereas the Nak peptide is coloured in blue. Residues that have direct contacts to target peptides are coloured in green. Alignment of the Drosophila and the zebrafish PTB domain indicates that the additional 11 amino acids of the zebrafish PTB^L domain (arrow) would be inserted between amino acids 111 and 112 (highlighted in red). Figure generated using RasMol Vers. 2.7.2.1. B: Sequence of the PTB^L insertion. Amino acids with acidic or basic side chains (D, R, K) are coloured in black. Amino acids with nonpolar side chains (V, F, G) are coloured in grey. C: Dorsal view of the neural tube of an embryo injected with numb(PTB^L PRR^L):egfp mRNA (compare with Fig. 5). D-I: Alanine-scanning mutagenesis of the 11-amino acid insertion. Confocal micrographs of mitotic cells in the neural tube of embryos injected with mRNA made from different mutagenized numb(PTB^L PRR^L):egfp constructs. See text for details. J: A sequence of five time-lapse frames of two mitotic cells, in the neural tube of embryos injected with numb(DRKAAKAAAKK):egfp mRNA, showing mislocalization of the fusion protein and misorientation of the cell division. The transparent red line marks the midline. Anterior is towards the top. The upper cell (arrowhead in 0′) exhibits a mislocalized Numb(DRKAAKAAAKK):EGFP crescent and subsequently divides with an orientation that is clearly oblique to the plane of the neuroepithelium (arrowheads in 3′00"). The lower cell (asterisks in 0′) divides parallel to the neuroepithelial plane, as is the normal case in neural tube stage (asterisks in 4′30"), but localizes Numb(DRKAAKAAAKK):EGFP ubiquitously around the cell cortex. Note the apical localization of the fusion protein also in the non-dividing cell (arrow in 1′30").

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