Cerebral corticogenesis requires precise spatio-temporal control of the rates of progenitor cell division. Previous research on the developing mouse embryo established that the transcription factor Pax6 is a key regulator of this process (Götz et al., 1998; Warren et al., 1999; Estivill-Torrus et al., 2002; Heins et al., 2002; Haubst, 2004; Manuel et al., 2006, 2015; Quinn et al., 2007; Sansom et al., 2009; Asami et al., 2011; Georgala et al., 2011a,b; Mi et al., 2013). Pax6 is expressed by cortical radial glial progenitors, which are by far the most numerous cortical cell type present at the onset of corticogenesis at around embryonic day 12.5 (E12.5) in mouse. Previous functional studies have shown that Pax6 is an important cell autonomous repressor of radial glial cell cycle progression (Warren et al., 1999; Estivill-Torrus et al., 2002; Manuel et al., 2006; Georgala et al., 2011b; Mi et al., 2013). Loss of Pax6 shortens the overall length of cortical progenitor cell cycles and hence accelerates cell division (Mi et al., 2013). Previously, we identified a number of highly conserved cell cycle genes with actions fundamental to cell cycle progression across cell types and species whose levels of expression are affected by constitutive loss of Pax6 function (Nurse, 2000, 2002). One of these was the cell cycle gene Cdk6 (Mi et al., 2013).

Cdks drive progression through the eukaryotic cell cycle by forming complexes with cyclins (Morgan, 1997; Loog and Morgan, 2005; Hochegger et al., 2008; Coudreuse and Nurse, 2010; Uhlmann et al., 2011). In most natural systems studied so far, levels of Cdks remain relatively constant during the cell cycle whereas levels of the cyclins oscillate, causing Cdk activity to oscillate. In experiments using engineered fission yeast, oscillations in Cdk activity alone, without the numerous additional regulatory mechanisms that normally affect the cell cycle, can be sufficient to drive orderly progression through major cell cycle events (Coudreuse and Nurse, 2010). Cdk6 specifically partners a particular type of cyclin (the D-type cyclins) and primarily regulates the G1 to S phase transition (Hochegger et al., 2008). In cerebral cortical progenitors, Pax6 negatively regulates the expression of Cdk6 (Mi et al., 2013).

Here we addressed several questions. Are the effects of Pax6 on the cell cycle likely to be mediated primarily through actions on Cdk6, or does Pax6 control multiple cell cycle regulators in parallel? Do the effects of Pax6 vary between cell cycle phases? Are levels of Cdk6 normally constant throughout the cell cycle of cortical progenitors? Does Pax6 modulate Cdk6 levels equally or differentially across the phases?

To address the first of these questions, we used quantitative real time polymerase chain reaction (qRT-PCR) to test for changes in the expression of a set of cell cycle regulators following acute cortical deletion of Pax6. The set we tested contained all cell cycle regulators that were shown in previous work to have altered cortical expression levels in constitutive Pax6^-/- mutants (Mi et al., 2013). Here we studied embryos with tamoxifen-induced cortex-specific deletion of Pax6. This allowed us to focus on the effects of Pax6 loss that are more likely to be direct by minimizing the possibility of secondary changes arising as a long-term consequence of cortical deletion or Pax6 removal from non-cortical tissues. We then used flow cytometry to discover how Pax6 affects the phases of the cell cycle and the levels of Cdk6 in each. We found that Pax6 loss increases Cdk6 levels similarly in all cell cycle phases and, particularly interestingly, that Cdk6 levels oscillate with cell cycle phase. This prompted us to extend our analysis to examine the relationship between Pax6 levels and Cdk6 levels across cell cycle phases. Our results indicated that Pax6 sets the level of expression around which Cdk6 oscillates but is unlikely to be responsible for controlling the oscillation itself.

Our new results support a central role for Cdk6 in Pax6’s regulation of cortical progenitor cell cycles. Figure 6 shows a model illustrating how elevated Cdk6 levels across the cell cycle could shorten G1 phase. We propose that the time of G1 and the G1 to S phase transition is determined by the time it takes Cdk6 activity to rise above a threshold; our model proposes that the threshold is similar in both Pax6^+/+ and Pax6^-/- progenitors (Figures 6B,C). Oscillation of Cdk6 activity at abnormally high levels would shorten the length of G1 since it would take less time to achieve the threshold required to exit G1. We also found evidence that other cell cycle phases were slightly longer than normal in Pax6^-/- progenitors, particularly at the later age studied, E14.5. This is reflected in the way Figure 6 is drawn. These changes were not enough to compensate for the shortening of G1 and, overall, cell cycle lengths were reduced. We do not know how these other phases might be affected. At least one possibility is that they are lengthened as a consequence of accelerated progression through G1 phase rather than a direct action of Pax6 on regulators of these phases. Another possibility is that delayed changes in the expression of cell cycle genes other than Cdk6, such as those observed here (Figure 1: Cdca7, Smad3, Smad7, or Sesn1) directly affect the lengths of these phases.

This proposal has similarity to the mechanism proposed to regulate an engineered minimal control network in fission yeast (Coudreuse and Nurse, 2010). This control network is based on oscillating levels of CDK activity, which drive cell cycle progression as they cross thresholds triggering transition between cell cycle phases including G1 to S. As in the fission yeast model, it is possible that the oscillations in Cdk6 in cortical progenitors result from phase-dependent changes in Cdk6 synthesis and degradation.

An alternative possibility is that cyclical changes in Pax6 levels might cause the oscillations in Cdk6 levels, given that Pax6 can directly repress Cdk6 expression (Mi et al., 2013). The oscillations in Pax6 were slightly out of phase with those in Cdk6 (Figures 6A,B) but it is very unlikely that fluctuations in Pax6 protein levels alone cause the oscillations in Cdk6 protein levels. Additional factors would be required to explain the very different rates of change of Pax6 during its rising phase and Cdk6 during its falling phase and vice versa. An additional complexity would be explaining the dynamics of this scenario. Changes in Pax6 protein levels would need a certain time to influence transcription of the Cdk6 gene and the mRNA would then need to be translated to alter Cdk6 protein levels. Although we do not exclude the possibility that fluctuations in Pax6 levels might contribute to the temporal pattern of change of Cdk6 levels, the simplest suggestion is that Pax6 reduces Cdk6 levels overall and that the oscillations in Cdk6 levels during the cell cycle are driven by other mechanisms. Our previous work showed that Pax6 overexpression significantly reduced Cdk6 levels (Mi et al., 2013), and in the future it would be interesting to test how Cdk6 levels oscillate during the cell cycle in these mutants and how cell cycle phases change.

It is likely that post-translational processes are involved in the relatively rapid cyclical changes in Cdk6 and Pax6 levels identified here. Cortical progenitor cells might generate excess amounts of their mRNAs, which might be translated at levels that vary as the cell progresses through the cell cycle. A similar mechanism, termed “translation on demand,” was suggested in a recently published review paper (Liu et al., 2016). It suggests that quick generation of proteins in response to signals could be achieved purely by regulating the translation rate of pre-existing mRNA (Le Roch et al., 2004; Lee et al., 2011; Lackner et al., 2012; Eichelbaum and Krijgsveld, 2014; Jovanovic et al., 2015). Some studies suggested that transcription factors are more likely to be subject to translation on demand regulation, as some rapid cell state transitions require fast synthesis and reactions of transcription factors to modulate their downstream gene networks (Lee et al., 2013; Jovanovic et al., 2015; Liu et al., 2016). Rapid degradation via ubiquitination might also contribute to rapid adjustments of Pax6 and Cdk6 protein levels during the cell cycle. In eye development, for example, a post-translational ubiquitin-mediated proteasomal mechanism degrades Pax6 protein (Pfirrmann et al., 2016). It is possible that Pax6 regulates the overall levels of Cdk6 not only by direct repression of the Cdk6 gene itself (Mi et al., 2013) but also by affecting the post-translational mechanisms that determine Cdk6 protein production and stability.

A Pax6-loss-induced change in the length of G1 phase is likely to have consequences for the subsequent development of progenitors and the neurons that they generate. Previous studies have shown that cell cycle length has a direct impact on a cell’s mode of division, i.e., whether it is neurogenic, producing neurons, or proliferative, producing new progenitor(s). G1 length is increased in neurogenic progenitors compared with proliferative progenitors (Caviness et al., 2003; Lukaszewicz et al., 2005; Dehay and Kennedy, 2007; Salomoni and Calegari, 2010). This is in line with the finding that basal progenitors, which mostly generate neurons through neurogenic division, have a longer G1 phase than apical progenitors (Calegari et al., 2005; Salomoni and Calegari, 2010; Arai et al., 2011). The functional importance of G1 length was demonstrated by manipulating it. An increase in G1 length leads to neurogenic division and premature neurogenesis (Calegari, 2003; Calegari et al., 2005). Shortening G1 increases proliferative divisions, leading to an expansion of the progenitor pool, and affects laminar phenotypes later in development (Lange et al., 2009; Pilaz et al., 2009). This might be one mechanism by which Pax6 affects the later stages of cortical formation despite being downregulated in postmitotic neurons.

DM, MM, and Y-TH performed the experiments. DP and JM helped to design the experiments, obtained the funding, and supervised the work. DP wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.