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Files in this Data Supplement:
Fig. S1. Rate of neuron production in the early neural tube. (A) Total numbers of DAPI-stained cells (blue line), non-neuronal cells (red line), neurons (HuC- and NeuroM-positive cells, purple line) and recently born neurons (NeuroM-positive cells, green line) in 1.5-, 2-, 2.5- and 3-day-old embryos in neural tube flanked by somites 1-3. At each developmental stage, four 16 μm sections from three embryos were analyzed. Bars represent s.d. (B) Proportion of cells that are either recently born neurons (NeuroM-positive cells, pink line) or the total proportion of differentiated cells (HuC- and NeuroM-positive cells, dark-blue line), calculated from data set in A.
Fig. S2. Comparison of cell cycle parameters. (A) Cell cycle time is similar in fixed and live tissue. Cell cycle time was calculated in fixed tissue by comparing BrdU incorporation following exposure for 1 or 4 hours (see Materials and methods) as 16 hours 6±25 minutes (s.d. indicates count variance between embryos). In live tissue cell-cycle time varied from ∼9 to 28 hours, with an average of 16 hours 10 minutes±4 hours (n=87 cells). (B) Time spent in mitosis is similar between fixed and live tissue. Mitosis time in fixed tissue was calculated as 32±8 minutes using cell cycle time (see above) and mitotic index. In live tissue this M-phase was measured directly, 28±9 minutes (n=39 cells). (C) Dot plot showing cell cycle times of all cells measured directly from untreated live experiments (n=87 cells).
Fig. S3. Ngn2 expression in the spinal cord. Transverse section of the chick neural tube at HH10 following in situ hybridization for the neurogenic gene Ngn2. Note that at this stage Ngn2 is only present in a scattering of neuroepithelial cells. Scale bar: 40 μm.
Movie 1. Mitotic spindle movements and midbody contribution to the apical process. Time-lapse movie corresponding to Fig. 1C and Fig. 5C (see legends for details). The mitotic spindle exhibits dramatic rotation and at cytokinesis a microtubule-rich cytoplasmic bridge midbody forms between the two sibling cells, which later gives rise to microtubules in the new apical processes. Dual-labeled cells (Gap43-RFP, red; eYFP-tubulin, green) imaged at 1.5-minute intervals (scale bar: 10 μm).
Movie 2. Asymmetric neuron production and neuron birth. Time-lapse (38 hours) movie corresponding to stills in Fig. 2B (see legend for details). The cell (white arrow) migrates to the apical surface where it undergoes mitosis. Both cells re-establish their basal processes, and at ∼10 hours after mitosis the more basal daughter (red arrow) releases its apical process, which is withdrawn over its sister cell (green arrow). The basal cell body now realigns along the basal surface and puts out an axonal process, whereas its sister undergoes a further round of division. For lineage tree see Fig. 2B′. Scale bar: 10 μm.
Movie 3. Progenitor-generating divisions. Time-lapse movie (38 hours) corresponding to stills in Fig. 3 (see legend for details). A cell undergoes multiple rounds of division resulting in the birth of 16 cells. Cleavage plane orientation varies. For lineage tree see Fig. 3A′. Scale bar: 10 μm.
Movie 4. A division with a perpendicular cleavage plane generates two neurons. Time-lapse movie (38 hours) corresponding to Fig. 4 (see legend for details). The cell (white arrow) migrates to the apical surface where it undergoes division, maintaining the cleavage plane orientation at 81° to the apical surface. Both daughters maintain processes to the apical surface as the nuclei migrate towards the basal surface. These apical processes both release and withdraw (yellow arrows). While one daughter (green arrow) becomes obscured by its sister (red arrow) and surrounding cells, three-dimensional analysis confirms that it withdraws its apical process in the same way as its sister. Scale bar: 10 μm.
Movie 5. A division with a perpendicular cleavage plane generates a neuron and a progenitor cell. Time-lapse movie (38 hours) corresponding to stills in Fig. 6 (see legend for details). A cell divides with a perpendicular cleavage plane to generate a neuron and a progenitor. The daughter cell withdrawing its apical process loses its YFP signal in the last few frames, whereas its sister divides again. For lineage tree see Fig. 6A′. Scale bar: 10 μm.
Movie 6. FGF reduces cell cycle time and disrupts interkinetic nuclear migration. Time-lapse movie (38 hours) of stills in Fig. 7B. An FGF-treated cell undergoes multiple rounds of division resulting in the birth of 16 cells. Interkinetic nuclear migration back to the basal surface is reduced, and with one exception all daughter cells return to the apical surface to divide. Cleavage plane orientation is varied; for lineage tree see Fig. 7B′. Scale bar: 10 μm.
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