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doi: 10.1242/10.1242/dev.00603

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A 90° rotation of the mitotic spindle changes the orientation of mitoses of zebrafish neuroepithelial cells

Institut für Entwicklungsbiologie, Universität zu Köln, 50923 Köln, Germany

View larger version (41K): [in a new window]   Fig. 1. The major steps in neurulation, as discussed in the text.

View larger version (122K): [in a new window]   Fig. 2. A sequence of six confocal micrographs of a planar view of the neural plate of an embryo that had received an injection of mRNA encoding tau:GFP. A cell about to divide (arrow in 0') has left the epithelium and come to be located apically (arrows track its progress). The cell has developed processes oriented to the midline (thin white line in each photograph). During convergence, the dividing cell maintains the same neighbours (the asterisk on the neighbouring cells is for reference).

View larger version (90K): [in a new window]   Fig. 3. Two different embryos (A-D and E-H) that have received an injection of mRNA encoding tau:GFP. The photographs show orthogonal sections (z-scans) obtained from sequences of 250 frames (60 second intervals). The white line above the neural plate cells in each plate corresponds to the section of the coverslip (refer to Materials and Methods). The drawings beneath each photograph illustrate the profile of the epithelial cells. One cell (arrows in A-D) has lost the connections to the basement membrane at time point 0, and divides apically 9 minutes later (B). One of the daughter cells reintegrates into the epithelium (D) in the plane of focus. The embryo at E-H is slightly older. The photographs show one cell (arrows) leaving the epithelium (E-G) to divide apically 60 minutes later (H). not, notochord.

View larger version (161K): [in a new window]   Fig. 4. (A-H) Mitotic divisions in the neural plate stage. (A-D) The neural plate of an embryo carrying the transgenic insertions Tg(H2AF/Z)kca37 and Tg(H2AF/Z)kca66 was photographed from above. The first photograph (A) shows three metaphase plates (arrows). The other frames, taken 3 minutes (B), 5 minutes (C) and 7 minutes (D) later, show anaphase and telophase. Note that the plane of cleavage is perpendicular to the neuroepithelium in all three. (E-H) Time-lapse photographs from a sequence taken at 45 second intervals of a mitotic cell (arrows) in the neural plate of an embryo injected with tau:egfp mRNA. The neural plate was photographed from above. Notice the appearance of the centrosome, its division (F), the migration of the daughter centrosomes to opposite sides of the cell and formation of the spindle (G), which lies parallel to the plane of the neuroepithelium. Planar movements of the metaphase plate (the dark zone between spindle fibres, arrows in F,G) are relatively weak in this particular case. (I-P) Two sequences of four time-lapse confocal photographs each to illustrate mitotic divisions in the neural tube. The photographs in I-L (from 0' to 14') show the neural tube of an embryo carrying the transgenic insertions Tg(H2AF/Z)kca37 and Tg(H2AF/Z)kca66, flanked by somites on either side. Several mitoses are visible, two of which are indicated by the arrows. Notice that the plane of cleavage is perpendicular to the neurocoel (neur). (M-P) An embryo injected with tau:egfp mRNA. The earliest sign of an impending cell division is the duplication of the centrosome (arrows in 0'), and the daughters migrate to opposite poles of the cell. The spindle can be seen in N,O. Note that the mitotic spindle is parallel to the neurocoel (neur). Anterior is towards the top in all photographs.

View larger version (171K): [in a new window]   Fig. 5. (A-R) Sequences of time-lapse photographs to illustrate mitotic divisions in the neural rod. (A-I) The neural rod of an embryo carrying the transgenic insertions Tg(H2AF/Z)kca37 and Tg(H2AF/Z)kca66, surrounded by somites (som) on either side. The oblique white line in A labels the zone of apposition of the two halves of the neural anlage. The photographs show a nucleus (arrow), which is about to enter prophase. Notice that the orientation of the metaphase plate has turned by 90° at 12 minutes 45 seconds (C). The nucleus then divides within 3 minutes and 45 seconds (D), and the two daughter nuclei are incorporated into different sides of the neuroepithelium (arrows). Note that the plane of cleavage in this division, as well as in other divisions shown in the remaining photographs, is parallel to the neuroepithelium. (J-R) A mitosis at the neural rod stage of an embryo that expresses tau:GFP. The arrows in J and L indicate the centrosome before and after division, those in M-P indicate the plane of rotation. Anterior is towards the top in all photographs.

View larger version (120K): [in a new window]   Fig. 6. A sequence of eight time-lapse confocal photographs (time from 0' to 137' 20'') of an embryo carrying the transgenic insertions Tg(H2AF/Z)kca37 and Tg(H2AF/Z)kca66 to illustrate the migration of postmitotic cells to the subventricular region of the neural tube. The embryo is oriented a little obliquely, such that the two halves of the neural tube are not in register. One cell in metaphase is indicated by an arrow at 0'. After division at 12' (arrows indicate both daughter nuclei), one of the nuclei (left arrow) can be seen to move towards the subventricular region to join differentiating cells in the external region of the spinal cord. Notice that the most external nuclei are separated from the others by a dark line, most probably corresponding to an incipient neuropil (at 12' and thereafter). Anterior is towards the left in all photographs.

View larger version (155K): [in a new window]   Fig. 7. Distribution of ASIP:EGFP and aPKC during neurulation. (A-H) Eight confocal photographs of a z-multilevel apicobasal scan, in steps of 0.75 µm (numbers give the distances in µm), through a few cells of the neural plate of an embryo that had been injected with asip:egfp mRNA. Notice that the fluorescence is punctate and localised to the membrane, but it is not restricted to the apical region. (I) A confocal photograph of an embryo in neural keel/rod stage that had been injected with asip:egfp mRNA. Notice that the fluorescence, although weak, is localised mainly to the apical pole of the cells (arrows), at the region where the two halves of the neural primordium are in apposition. (J-L) Three confocal photographs through three different levels of the neural tube of an embryo injected with asip:egfp mRNA. Numbers give the distances in µm. The fluorescent signal is strong and clearly restricted to the apical pole of the neuroepithelial cells. The width of the neurocoel increases towards ventral levels. (M-O) Anti-aPKC antibody staining. Two different levels of the neural keel (nk), separated by 15 µm, are shown (M,N). Notice that immunoreactivity is restricted to ventral levels of the neural keel (arrow in N). som, somites. (O) An optical section through the neural tube. Note the apical localisation of the epitope.

View larger version (188K): [in a new window]   Fig. 8. Anti-ß-catenin antibody stainings of wild-type embryos (A-G). (A-C) Confocal photographs of a z-multilevel apicobasal scan (0-6 indicate 3 µm steps) through the neural plate. No local accumulation of the epitope can be distinguished. (D-F) A similar scan through the neural keel, to illustrate the incipient apical localisation of ß-catenin (arrows) (0-16 indicate the distance in µm). (G) The neural tube, in which the epitope is clearly concentrated apically (arrows).

View larger version (132K): [in a new window]   Fig. 9. (A-C) The neural tube of embryos stained with Sytox (green) and the anti-aPKC antibody (red). Mitotic cells (arrows) in metaphase (A), anaphase (B) and telophase (C) are shown. Note that the epitope is expressed at the apical membrane of the mitotic cells. (D-F) A sequence of three time-lapse frames of a mitotic cell in the neural tube of an embryo injected with asip:egfp mRNA. Note that ASIP:EGFP (arrow in D) is located at the apical pole throughout mitosis (two arrows in F). (G-J) A sequence of four time-lapse frames of a mitotic cell in the neural keel of an embryo injected with asip:egfp mRNA. ASIP:EGFP is located at the apical pole of the neural keel cells (two black arrows in G), including the mitotic cell (white arrow in A). The apical localisation of ASIP:EGFP is lost when the cell enters mitosis, and in metaphase labelling is present in the entire cell membrane (four white arrows in H). After telophase (I), labelling reappears at the apical pole in the daughter cells (arrows, J).

View larger version (92K): [in a new window]   Fig. 10. (A-D) The neural tube of a Tg(H2AF/Z)kca37; Tg(H2AF/Z)kca66 embryo that had received an injection of ASIP morpholinos. The arrows in A and B point to a metaphase plate that forms parallel to the neuroepithelium, but shifts by 90° to divide perpendicularly. Both daughter cells integrate into the same side of the neural tube. (E-H) The neural tube of a hasm129 /hasm129; Tg(H2A.F/Z)kca37/+ embryo. As in A-D, the metaphase plate forms parallel to the neurocoel, but shifts by 90° and the ensuing division is perpendicular. Arrows indicate the dividing cell. Anterior is towards the top.