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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00825

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An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307, Dresden, Germany

View larger version (45K): [in a new window]   Fig. 1. WEC faithfully reproduces neurogenesis in vivo. Double immunofluorescence for TIS21 (a-f) and MAP2 (a'-f') on sagittal cryosections through the ventral mesencephalon (a-c') and rostral telencephalon (d-f') of embryos developed in utero up to E9.5 (E9.5) or E10.5 (E10.5) or in utero to E9.5 followed by WEC for 24 hours (E9.5+24h WEC). Note the caudal (ca) to rostral (ro) gradient of neurogenesis in the ventral mesencephalon. Filled arrowheads, apical (ventricular) side of the neuroepithelium; arrows, neuronal layer; open arrowheads, auto-fluorescent blood cells (absent in embryos developed in WEC because of the loss of blood cells after opening of the yolk sac); open arrow, reaction of the anti-mouse secondary antibody with the skin, presumably with adsorbed mouse immunoglobulins present in the mouse serum used for WEC. Dotted lines delineate the boundaries of the neuroepithelium. Scale bar: 100 µm.

View larger version (35K): [in a new window]   Fig. 2. Olomoucine induces lengthening of the cell cycle in WEC. (A) Size (crown to rump) of E9.5 embryos developed for 24 hours in WEC in the absence (control) or presence of 80 µM iso-olomoucine (iso-olo) or olomoucine (olo) followed by fixation. Arrows indicate the reported (Kaufman, 1992) size of fixed embryos with 22-26 pairs of somites (E9.5) and 30-35 pairs of somites (E10.25-10.5). Bars represent the s.d.; control, n=5; iso-olo, n=8 and olo, n=7; olo vs. iso-olo P<0.0001. (B) Analyses of NE cells in the rostral telencephalon of E9.5 littermate embryos developed in WEC in the presence of 80 µM iso-olomoucine (iso-olo) or olomoucine (olo). (a,b) TUNEL staining after 24 hours WEC. (c,d) BrdU immunoreactivity after 19 hours WEC, with 50 µM BrdU added at 3 hours. (a-d) Scale bar: 50 µm. (e) Cumulative BrdU labeling, with 50 µM BrdU added at 3 hours (time=0) and WEC being continued for the indicated times. The BrdU labeling index indicates the proportion of DAPI-stained cell nuclei in the neuroepithelium that were stained for BrdU (all nuclei stained for BrdU=1.0). For each time point, one littermate embryo each was analyzed for iso-olo (filled circles) and olo (open squares); data are the mean of two independent litters (except for the 1 hour time point); bars indicate the variation of the duplicate values from the mean; correlation coefficients are r2=0.995 and r2=0.998 for iso-olo and olo, respectively. The dotted line indicates the BrdU labeling index observed after 16 hours of BrdU labeling, which was the same for iso-olo and olo (for clarity, the filled circle and open square symbols at the 16 hours time point are placed next to each other). The extrapolated intercept of the iso-olo and olo best-fit lines with the dotted line is indicated by the solid and dashed arrow on the abscissa, respectively, and provides an estimate of the sum of the lengths of the G2, M, plus G1 phases (plus the fraction of S phase required to detect BrdU incorporation).

View larger version (34K): [in a new window]   Fig. 3. Olomoucine increases the proportion of neuron-generating NE cells. (a,b) Composite showing TIS21 immunofluorescence in sagittal cryosections through the spinal cord (sc), hindbrain (hi), mesencephalon (me) and telencephalon (te) of E9.5 littermate embryos developed for 24 hours in WEC in the presence of 80 µM iso-olomoucine (iso-olo, a) or olomoucine (olo, b). (a',b') Corresponding phase contrast images shown at lower magnification. Filled arrowheads in b indicate areas of the telencephalon prematurely expressing TIS21. Scale bar: 400 µm. (c) Quantitation of TIS21 immunoreactivity. The region of the telencephalon indicated by the dotted lines between the open arrowheads numbered 1 and 24 in a and b was divided into 24 segments of similar area, and the fluorescence in each area was quantitated; a.u., arbitrary units. Filled circles, iso-olo; open squares, olo. Graphs in d and e show a similar quantitation of the TIS21 immunoreactivity shown in Fig. 4a and b (segments 1-22 corresponding to bottom-to-top) and Fig. 4c and d (segments 1-23 corresponding to top-to-bottom), respectively.

View larger version (38K): [in a new window]   Fig. 4. Olomoucine induces premature neurogenesis. Double immunofluorescence for TIS21 (a-d, b', green) and MAP2 (a'-d', red) on sagittal cryosections through the rostral telencephalon of littermate (a vs. b, c vs. d) E9.5 embryos developed for 24 hours in WEC in the presence of 80 µM iso-olomoucine (isoolo; a, a' and c, c') or olomoucine (olo; b, b' and d, d'). Arrowheads, apical (ventricular) side of neuroepithelium. Arrows indicate prematurely generated neurons. The white box in b indicates the area shown at higher magnification in b'. b' and d' show different degrees of premature neurogenesis upon olomoucine treatment, with few (b') and many (d) neurons being generated. Open arrows in a', c' and d' indicate reaction of the anti-mouse secondary antibody with the skin, presumably with adsorbed mouse immunoglobulins present in the mouse serum used for WEC. Dotted lines in a' and c' delineate the boundaries of the neuroepithelium.

View larger version (19K): [in a new window]   Fig. 5. Cell cycle length, asymmetric cell division and cell fate determination – a model. In our model, a molecule capable of inducing a cell fate change (cell fate determinant) is assumed to be distributed unequally upon mitosis, e.g. with daughter cell A receiving 60% and daughter cell B 40% of this determinant (arrows). For a cell fate change to occur, the effect of the cell fate determinant has to reach a threshold (dashed line), i.e. the cell fate determinant needs to act for a certain length of time (for example, a transcription factor activating transcription until a certain amount of gene product has been produced). Given the unequal amounts of cell fate determinant in cells A and B, this threshold will be reached by neither cell A nor cell B after one unit of time, by cell A but not cell B after two units of time, and by both cell A and cell B after three units of time. If the cell fate determinant would, for example, be able to induce the switch of NE cells from a proliferative to a neuron-generating division, neither NE cell would switch to neurogenesis if the length of the critical phase of the cell cycle corresponded to one time unit, NE cell A but not NE cell B would switch upon doubling this phase, and both NE cells would switch upon tripling it. Hence, an unequal distribution of a cell fate determinant upon mitosis will only lead to an asymmetric daughter cell fate if the length of the cell cycle is appropriate. Moreover, if early in G1, the cell fate determinant were to drive the production of a molecule that causes cell cycle arrest upon reaching the threshold, depending on the length of G1 either none, one or both of the daughter cells would acquire a post-mitotic state (i.e. turning G1 into G0), as is characteristic of neurons. Note that the basic principle of the model, i.e. the dependence of cell fate on time, holds true for any cell, irrespective of asymmetric division. Moreover, the model can be adopted to two cells being exposed to different concentrations of an extracellular factor affecting cell fate, or to two cells being exposed to the same concentration of such a factor for different times.

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