The Control of P and Q with Symmetric and Asymmetric Cell Divisions

The output of neurons by the PVE is controlled, in principle, by a variety of factors including the proportional representation of the three possible types of mitotic divisions: (1) symmetric nonterminal cell division (which produces two daughter cells that remain in the PVE and continue to proliferate), (2) symmetric terminal cell division (which produces two daughter cells that both migrate out of the PVE to become young neurons), and (3) asymmetric cell division (which produces one daughter cell that continues to proliferate and one that migrates out of the PVE). Changes in the proportions of these three types of mitotic divisions have been inferred from changes in the proportion of cells that enter vs leave the cell cycle (Takahashi et al., 1996a; Miyama et al., 1997), from time lapse cinematography (O'Rourke et al., 1992; Adams, 1996), from changes in the orientation of the mitotic apparatus (Smart, 1973; Chenn and McConell, 1995; Adams, 1996), and from immunohistochem-istry (Chenn et al., 1995). How are such changes effected within single lineages? How might they be distributed among lineages making various cortical cell types? For example, it has been suggested that there are specific populations "reserved" in the PVE to produce either specific cell types or cells that occupy specific laminae (Dehay et al., 1993; Kennedy and Dehay, 1993; Luskin et al., 1993). Since all of the cells in a given neighborhood of the PVE are proliferating (Takahashi et al., 1995a, 1996a) with a similar cell cycle length (Cai et al., 1997a), in the absence of cell death such a "reserved population" would have a specific pattern of repeated symmetric nonterminal mitoses and would expand for several cell cycles to produce relatively large lineages of a specific and characteristic size (8, 16, 32, 32, etc.) containing only proliferating cells. Similarly, lineages following other specific patterns of proliferation, for example, repeated asymmetric divisions, would produce lineages of other specific characteristic sizes, for example, a preponderance of even-sized or odd-sized lineages, etc. The alternative to such repeated patterns of mitosis-type is the absence of pattern in the sequence of cell divisions within a lineage, in which case no specific and characteristic lineage size distribution will be produced. In general, the size distribution of lineages obtained during defined periods of development will reflect the dynamic changes in the proportions of these three types of cell divisions and will, thus, reveal any extant repeated patterns of mitosis. Note that the presence of cell death to any significant extent (cf. Blaschke et al., 1996; Thomaidou et al., 1997) would modify the specific and characteristic lineage sizes obtained, but would do so in a predictable way.

To estimate the frequency of occurrence of each of these distinct behaviors individual retrovirally labeled lineages were studied; each lineage consisting of proliferating cells in the PVE in the developing neocortex at known numbers of cell cycles after infection with a retrovirus. In contrast to most previously

FIGURE 10. A visualization of the changes shown in the graphs of Fig. 12 and as given by the changes in P and Q per cell cycle (CC) (Nowakowski et al., 2002). At the onset of the neuronogenetic interval (NI) (CC = 0), a single unit of the PVE is shown. At the next cell cycle (CC = 1) the PVE has an increased volume; the output from the first cell cycle is shown in the position of the cortical plate. At CC = 2, the PVE has increased in volume again, and now the output from the first two cell cycles is shown in the position of the cortical plate. At CC = 3, the process is repeated. In the right-hand side of the figure, the diagram shows the final Total Output of all of the 11 cell cycles of the NI. Note that the output from the first three cell cycles corresponds to only a small part of the Total Output, whereas the output of the last three cell cycles comprises about 50% of the Total Output.

FIGURE 10. A visualization of the changes shown in the graphs of Fig. 12 and as given by the changes in P and Q per cell cycle (CC) (Nowakowski et al., 2002). At the onset of the neuronogenetic interval (NI) (CC = 0), a single unit of the PVE is shown. At the next cell cycle (CC = 1) the PVE has an increased volume; the output from the first cell cycle is shown in the position of the cortical plate. At CC = 2, the PVE has increased in volume again, and now the output from the first two cell cycles is shown in the position of the cortical plate. At CC = 3, the process is repeated. In the right-hand side of the figure, the diagram shows the final Total Output of all of the 11 cell cycles of the NI. Note that the output from the first three cell cycles corresponds to only a small part of the Total Output, whereas the output of the last three cell cycles comprises about 50% of the Total Output.

FIGURE 11. The sequence of dynamic changes in the length of the cell cycle (and in P and Q) is initiated in the rostrolateral-most portions of the neopallium and then spreads as a gradient of maturation across the neopallial surface. This wave-like progression of maturation means that at any given time there are "domains" of the PVE that are in different states. This is, in theory, sufficient to provide a basis for cell cycle length to serve as positional information that could be involved in the development of cytoarchitectonic subdivisions.

FIGURE 11. The sequence of dynamic changes in the length of the cell cycle (and in P and Q) is initiated in the rostrolateral-most portions of the neopallium and then spreads as a gradient of maturation across the neopallial surface. This wave-like progression of maturation means that at any given time there are "domains" of the PVE that are in different states. This is, in theory, sufficient to provide a basis for cell cycle length to serve as positional information that could be involved in the development of cytoarchitectonic subdivisions.

published experiments using this method (Price, 1987; Luskin et al, 1988; Walsh and Cepko, 1988, 1992; Williams et al., 1991; Luskin, 1993; Mione et al, 1994, 1997; Lavdas et al., 1996), the resulting labeled lineages were examined after short survivals, that is, during the period that cell proliferation continues to occur, and have focused on the size of the proliferating population, that is, the cells that remain in the PVE, rather than on the cells that migrate to the cortical plate. There are three influences (Fig. 12) that could act at each cell cycle to reduce the number of cells per lineage from the maximum number that would be produced in a pure population of symmetric nonterminally dividing cells. First, some cells of the lineage could leave the cell cycle to migrate and become young neurons (Q-cells, Q in Fig. 1). Second, some PVE cells could die (D in Fig. 12). Cell death could, in theory, occur at any time during development and has been well studied in the maturing neocortex during the postnatal period (Leuba et al., 1977; Finlay and Slattery, 1983; Heumann

FIGURE 12. A schematic diagram depicting the influences in the ventricular zone that could affect lineage size in a single cell cycle. For a cluster of cells present at the beginning of G1 (in this example eight cells are shown) some cells could continue to proliferate (P), leave the proliferative population (Q), die (or lose the marker) in either the proliferative (D, VZ cell in gray with dashed lines) or postproliferative compartment (D, CP cell in gray with dashed lines), or move tangentially within the proliferative population (T). Abbreviations: M, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; V, lateral ventricle.

FIGURE 12. A schematic diagram depicting the influences in the ventricular zone that could affect lineage size in a single cell cycle. For a cluster of cells present at the beginning of G1 (in this example eight cells are shown) some cells could continue to proliferate (P), leave the proliferative population (Q), die (or lose the marker) in either the proliferative (D, VZ cell in gray with dashed lines) or postproliferative compartment (D, CP cell in gray with dashed lines), or move tangentially within the proliferative population (T). Abbreviations: M, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; V, lateral ventricle.

and Leuba, 1983; Crandall and Caviness, 1984; Finlay and Pallas, 1989; Verney et al., 2000). However, estimates of the magnitude of cell death occurring within the proliferative population and during the early period of cortical development vary greatly from <1.0% at any given time (Thomaidou et al., 1997) to over 70% of the progenitor cells (Blaschke et al., 1996). Thus, it remains unclear what role cell death in the proliferative population plays in the regulation of neuron number (Gilmore et al., 2000). Third, some PVE cells could move tangentially (T in Fig. 12), that is, away from their sisters and cousins (Fishell et al., 1993; Tan and Breen, 1993; Walsh, 1993). Such tangential movements would not affect the actual numbers of cells in the proliferative population, but would affect the apparent number of cells identified in a lineage and would concomitantly increase the putative number of lineages identified.

The cells in each retrovirally labeled lineage in the developing VZ reside in clusters (or clades) of varying size (Cai et al., 1997a). The size of these clusters is dependent on the proliferative behavior of the cells in the labeled lineage, and depends on the mixture of symmetric nonterminal, symmetric terminal, and asymmetric cell divisions. There are three hypothetical mixtures of these three types of cell divisions that could occur (Fig. 13). The three Models differ only with respect to their composition of types of cell division, that is, they each have different ratios of asymmetric, symmetric nonterminal, and symmetric terminal cell divisions; however, all three Models are based on the same P/Q values measured using double S-phase labeling methods (Takahashi, 1996b; Miyama et al., 1997). Importantly, the distribution of cluster sizes is best accounted for by the goodness-of-the-fit of the experimentally determined distribution with the distributions obtained from the model which assumes that all three types of cell divisions coexist during the entire NI, i.e., Model 1 of Fig. 13. Thus, these retro-viral experiments: (1) provides evidence for the role of changes in P/Q in regulation of lineage size, (2) indicates that the amount of cell death and tangential movements in the PVE is low, and (3) indicate that the numbers of lineages that undergo a series of cell divisions with a repeated pattern is undetectable. In essence, these data suggest that the two daughter cells from a single cell division have their fate determined independently.

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