Nervous decision-making: to divide or differentiate
Authors: Laura J.A. Hardwick, Anna Philpott
Journal: Trends in Genetics (2014)

•Multiple mechanisms coordinate the cell cycle and neuronal differentiation.•Lengthening of G1 phase is functionally important for differentiation.•Cell cycle components can directly and independently affect neurogenesis.•Differentiation factors can directly affect the cell cycle structure and machinery. The intricate balance between proliferation and differentiation is of fundamental importance in the development of the central nervous system (CNS). The division versus differentiation decision influences both the number and identity of daughter cells produced, thus critically shaping the overall microstructure and function of the CNS. During the past decade, significant advances have been made to characterise the changes in the cell cycle during differentiation, and to uncover the multiple bidirectional links that coordinate these two processes. Here, we explore the nature and mechanistic basis of these links in the context of the developing CNS, highlighting new insights into transcriptional, post-translational, and epigenetic levels of interaction. The intricate balance between proliferation and differentiation is of fundamental importance in the development of the central nervous system (CNS). The division versus differentiation decision influences both the number and identity of daughter cells produced, thus critically shaping the overall microstructure and function of the CNS. During the past decade, significant advances have been made to characterise the changes in the cell cycle during differentiation, and to uncover the multiple bidirectional links that coordinate these two processes. Here, we explore the nature and mechanistic basis of these links in the context of the developing CNS, highlighting new insights into transcriptional, post-translational, and epigenetic levels of interaction. Formation of the CNS requires exquisite regulation of precursor proliferation, cell cycle exit, and differentiation to generate the diverse array of neurons and glial cells at the correct time and place. During neurogenesis, the population of precursor cells can undergo three different modes of division (see Glossary): early proliferative divisions are critical for expanding the precursor pool, and the timing of the switch to asymmetric and later symmetric neurogenic divisions ultimately determines differential rates of growth in different regions of the nervous system and, thus, the overall microstructure and function [1Zhong W. Chia W. Neurogenesis and asymmetric cell division.Curr. Opin. Neurobiol. 2008; 18: 4-11Crossref PubMed Scopus (121) Google Scholar]. Neurogenesis follows a temporal pattern, with precursor cells changing their competence and forming different cell types over time [2Desai A.R. McConnell S.K. Progressive restriction in fate potential by neural progenitors during cerebral cortical development.Development. 2000; 127: 2863-2872Crossref PubMed Google Scholar]; therefore, maintenance of the precursor pool is essential to enable the full repertoire of cell types to form [3Hatakeyama J. et al.Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation.Development. 2004; 131: 5539-5550Crossref PubMed Scopus (472) Google Scholar]. Furthermore, this highly regulated temporal production of different cell types is conserved throughout amniote evolution [4Nomura T. et al.Changes in the regulation of cortical neurogenesis contribute to encephalization during amniote brain evolution.Nat. Commun. 2013; 4: 2206PubMed Google Scholar], but modifications to progenitor cell number, location, and proliferative capacity has enabled expansion of the mammalian cortex and the emergence of gyrencephaly that characterises the primate brain [5Borrell V. Reillo I. Emerging roles of neural stem cells in cerebral cortex development and evolution.Dev. Neurobiol. 2012; 72: 955-971Crossref PubMed Scopus (136) Google Scholar, 6Betizeau M. et al.Precursor diversity and complexity of lineage relationships in the outer subventricular zone of the primate.Neuron. 2013; 80: 442-457Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar]. Indeed, cell fate specification throughout embryogenesis is intimately linked with the cell cycle. For example, early lineage determination of proliferating pluripotent stem cells occurs in different phases of the cell cycle, with endodermal versus neuroectodermal specification occurring in early or late G1 phase, respectively [7Pauklin S. Vallier L. The cell-cycle state of stem cells determines cell fate propensity.Cell. 2013; 155: 135-147Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar]. Similarly, the characteristic six-layered architecture of the mammalian cortex is formed by sequential waves of neurogenesis and newborn neurons migrating radially to the cortical plate, with terminal laminar fate determined during the final S or G2 phase of the proliferating precursors [8McConnell S.K. Kaznowski C.E. Cell cycle dependence of laminar determination in developing neocortex.Science. 1991; 254: 282-285Crossref PubMed Scopus (624) Google Scholar]. The coordination between the events of the cell cycle (Figure 1) and the changing modes of precursor cell division has been largely unexplored until relatively recently. Surprisingly, despite the intimate relation between the cell cycle and differentiation, these processes can be experimentally uncoupled, and cell cycle exit is neither a prerequisite for neurogenesis [9Lobjois V. et al.Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord.Neural Dev. 2008; 3: 4Crossref PubMed Scopus (35) Google Scholar], nor always a consequence of neuronal differentiation [10Lacomme M. et al.NEUROG2 drives cell cycle exit of neuronal precursors by specifically repressing a subset of cyclins acting at the G1 and S phases of the cell cycle.Mol. Cell. Biol. 2012; 32: 2596-2607Crossref PubMed Scopus (52) Google Scholar]. Nevertheless, recent advances have characterised the cell cycle dynamics, transcriptome, and proteome accompanying the transition from proliferating precursor cell to differentiating neuron, uncovering the existence of multiple links between components of the cell cycle and differentiation machinery. Here, we focus on exploring these links and their underlying mechanistic basis in the context of the developing CNS. Recent findings indicate that the duration of G1 and S phase may have a crucial role in the precursor maintenance versus differentiation decision, which has been widely studied in the mouse CNS. Early studies in mouse ventricular zone (VZ) precursor cells characterised the progressive lengthening of the cell cycle during the neurogenic period, from 8 h at embryonic day (E)11 up to 18 h by E16, due to a lengthening of the G1 phase from 3 to 12 h [11Takahashi T. et al.The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.J. Neurosci. 1995; 15: 6046-6057Crossref PubMed Google Scholar], but this did not distinguish between precursors undergoing different modes of cell division. More recently, the Tis21-GFP knock-in reporter mouse has been used to express GFP selectively in the precursor cells undergoing neurogenic but not proliferative divisions [12Calegari F. et al.Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development.J. Neurosci. 2005; 25: 6533-6538Crossref PubMed Scopus (294) Google Scholar], and subsequent work has used molecular markers (Pax6 and Tbr2) to further differentiate the apical progenitor (AP) and basal progenitor (BP) populations [13Arai Y. et al.Neural stem and progenitor cells shorten S-phase on commitment to neuron production.Nat. Commun. 2011; 2: 154Crossref PubMed Scopus (275) Google Scholar]. Proliferating precursor cells display a 3.3-fold longer S phase than their neurogenic counterparts, possibly due to a greater investment in fidelity of DNA replication [13Arai Y. et al.Neural stem and progenitor cells shorten S-phase on commitment to neuron production.Nat. Commun. 2011; 2: 154Crossref PubMed Scopus (275) Google Scholar] and similar changes in S phase duration have been reported following experimental manipulation to promote proliferative divisions of precursors [14Spella M. et al.Geminin regulates cortical progenitor proliferation and differentiation.Stem Cells. 2011; 29: 1269-1282Crossref PubMed Scopus (42) Google Scholar]. G1 lengthening is associated with the switch to neuron-generating cell fate [12Calegari F. et al.Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development.J. Neurosci. 2005; 25: 6533-6538Crossref PubMed Scopus (294) Google Scholar], specifically during the transition from AP to BP [13Arai Y. et al.Neural stem and progenitor cells shorten S-phase on commitment to neuron production.Nat. Commun. 2011; 2: 154Crossref PubMed Scopus (275) Google Scholar]. More recent advances have been made using imaging techniques to analyse cell cycle dynamics in live stem cell cultures, with several groups utilising the fluorescence ubiquitination cell cycle indicator (FUCCI) reporter system [15Sakaue-Sawano A. et al.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.Cell. 2008; 132: 487-498Abstract Full Text Full Text PDF PubMed Scopus (1510) Google Scholar] to label live cells in different phases of the cell cycle. These studies demonstrate clear links between cell cycle parameters and the propensity to differentiate. Pluripotency in mouse embryonic stem cells (mESC) is associated with a short G1 phase of approximately 2 h within a cell cycle of approximately 14 h, and cells with faster cell cycles express lower levels of differentiation markers [16Roccio M. et al.Predicting stem cell fate changes by differential cell cycle progression patterns.Development. 2013; 140: 459-470Crossref PubMed Scopus (108) Google Scholar]. Furthermore, pluripotency can be promoted in culture by stimulation of the LIF signalling path, and this may partly be due to an accelerated transit through G1 [17Coronado D. et al.A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency.Stem Cell Res. 2013; 10: 118-131Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar]. Induction of differentiation results in a doubling of G1 length [16Roccio M. et al.Predicting stem cell fate changes by differential cell cycle progression patterns.Development. 2013; 140: 459-470Crossref PubMed Scopus (108) Google Scholar, 17Coronado D. et al.A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency.Stem Cell Res. 2013; 10: 118-131Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar] with similar results reported in human ESCs [18Calder A. et al.Lengthened G1 phase indicates differentiation status in human embryonic stem cells.Stem Cells Dev. 2013; 22: 279-295Crossref PubMed Scopus (104) Google Scholar]. The functional link between G1 length and the decision to proliferate or differentiate has led to 'The cell cycle length hypothesis', based on a model whereby the length of the G1 phase determines whether a fate-determining signal will have sufficient time to produce an effect [19Calegari F. Huttner W.B. An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis.J. Cell Sci. 2003; 116: 4947-4955Crossref PubMed Scopus (259) Google Scholar]. This paradigm is repeatedly seen across multiple different stem cell lineages [20Lange C. Calegari F. Cdks and cyclins link G1 length and differentiation of embryonic, neural and hematopoietic stem cells.Cell Cycle. 2010; 9: 1893-1900Crossref PubMed Scopus (145) Google Scholar] and recent work has demonstrated that G1-phase ESCs have an increased susceptibility to differentiate when compared with equivalent S or G2 phase cells [17Coronado D. et al.A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency.Stem Cell Res. 2013; 10: 118-131Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 21Sela Y. et al.Human embryonic stem cells exhibit increased propensity to differentiate during the G1 phase prior to phosphorylation of retinoblastoma protein.Stem Cells. 2012; 30: 1097-1108Crossref PubMed Scopus (82) Google Scholar]. The past decade has seen the development of multiple different experimental approaches to alter cell cycle parameters and subsequent analysis of the effects on neuronal differentiation (Box 1). The unifying result is that manipulations that prolong the G1 phase of precursors lead to increased neurogenic divisions and premature differentiation, whereas a shortening of G1 favours proliferative divisions and precursor expansion. It should be noted that experiments using in utero electroporation create transient transfection effects due to the short half-life of cyclin/cdks and dilution of plasmids through cell division. Therefore, the manipulated precursor pool then undergoes physiological differentiation 48–72 h later, and a transient shortening of G1 that expands the precursor pool then generates an excess of late-born neurons [22Lange C. et al.Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.Cell Stem Cell. 2009; 5: 320-331Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar].Box 1Manipulation of G1 and effects on neuronal differentiationPharmacological inhibition of cdksEarly work demonstrated that the cdk inhibitor Olomoucine both lengthens G1 and induces a premature switch from proliferative to neurogenic precursor divisions [19Calegari F. Huttner W.B. An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis.J. Cell Sci. 2003; 116: 4947-4955Crossref PubMed Scopus (259) Google Scholar]. Similarly, treatment of adult precursor cells in vitro with a cell permeable cdk4 inhibitor induces an increase in the percentage of cells in G1, and promotes neuronal differentiation under both self-renewing and induced differentiation culture conditions [16Roccio M. et al.Predicting stem cell fate changes by differential cell cycle progression patterns.Development. 2013; 140: 459-470Crossref PubMed Scopus (108) Google Scholar].Cdk/cyclin null phenotypesCyclin-D2 knockout mice show a specific defect in BP proliferation, with a substantial lengthening of G1 and premature terminal differentiation that results in microcephaly [36Glickstein S.B. et al.Cyclin D2 is critical for intermediate progenitor cell proliferation in the embryonic cortex.J. Neurosci. 2009; 29: 9614-9624Crossref PubMed Scopus (92) Google Scholar]. Recent work has created cdk2 and cdk4 double knockout (DKO) mice, also showing a striking reduction in cortical neurons, although DKO cells demonstrate no defects in proliferation in vitro due to compensatory function of cdk1 and upregulation of cyclin-D1 and cdk6. Microcephaly occurs due to a significantly increased G1 length and premature neurogenic divisions of BP cells that deplete the precursor pool and reduce long-term neuronal output [54Lim S. Kaldis P. Loss of Cdk2 and Cdk4 induces a switch from proliferation to differentiation in neural stem cells.Stem Cells. 2012; 30: 1509-1520Crossref PubMed Scopus (58) Google Scholar].Overexpression of cyclin-cdksIn utero electroporation of cyclin-E1 or cyclin-D1 at E14.5 reduces G1 length and markedly expands the BP population; rates of cell cycle re-entry in BP cells are increased 80% compared with AP [55Pilaz L-J. et al.Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 21924-21929Crossref PubMed Scopus (168) Google Scholar]. This differential effect is also seen with acute overexpression of cyclin-D1/cdk4 at E13.5, resulting in a 40% increase in BP cells that undergo proliferative rather than neurogenic divisions, whereas the AP population is unchanged [22Lange C. et al.Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.Cell Stem Cell. 2009; 5: 320-331Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar].Similar results are seen in the adult dentate gyrus. Acute overexpression of cyclin-D/cdk4 in the 6–10-week-old hippocampus cell autonomously increases the expansion of the precursor pool by increasing proliferative divisions at the expense of neurogenic divisions. When overexpression is stopped, physiological differentiation resumes and the neuronal output of the manipulated pool of precursors can be doubled. In both developing and adult brains, it is the cells with the relatively longer G1 phase that are preferentially affected by overexpression of cyclin-cdk complexes, suggesting that it is the relative change in G1 length, rather than the absolute duration, that is important [56Artegiani B. et al.Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse brain.J. Exp. Med. 2011; 208: 937-948Crossref PubMed Scopus (81) Google Scholar]. Early work demonstrated that the cdk inhibitor Olomoucine both lengthens G1 and induces a premature switch from proliferative to neurogenic precursor divisions [19Calegari F. Huttner W.B. An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis.J. Cell Sci. 2003; 116: 4947-4955Crossref PubMed Scopus (259) Google Scholar]. Similarly, treatment of adult precursor cells in vitro with a cell permeable cdk4 inhibitor induces an increase in the percentage of cells in G1, and promotes neuronal differentiation under both self-renewing and induced differentiation culture conditions [16Roccio M. et al.Predicting stem cell fate changes by differential cell cycle progression patterns.Development. 2013; 140: 459-470Crossref PubMed Scopus (108) Google Scholar]. Cyclin-D2 knockout mice show a specific defect in BP proliferation, with a substantial lengthening of G1 and premature terminal differentiation that results in microcephaly [36Glickstein S.B. et al.Cyclin D2 is critical for intermediate progenitor cell proliferation in the embryonic cortex.J. Neurosci. 2009; 29: 9614-9624Crossref PubMed Scopus (92) Google Scholar]. Recent work has created cdk2 and cdk4 double knockout (DKO) mice, also showing a striking reduction in cortical neurons, although DKO cells demonstrate no defects in proliferation in vitro due to compensatory function of cdk1 and upregulation of cyclin-D1 and cdk6. Microcephaly occurs due to a significantly increased G1 length and premature neurogenic divisions of BP cells that deplete the precursor pool and reduce long-term neuronal output [54Lim S. Kaldis P. Loss of Cdk2 and Cdk4 induces a switch from proliferation to differentiation in neural stem cells.Stem Cells. 2012; 30: 1509-1520Crossref PubMed Scopus (58) Google Scholar]. In utero electroporation of cyclin-E1 or cyclin-D1 at E14.5 reduces G1 length and markedly expands the BP population; rates of cell cycle re-entry in BP cells are increased 80% compared with AP [55Pilaz L-J. et al.Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 21924-21929Crossref PubMed Scopus (168) Google Scholar]. This differential effect is also seen with acute overexpression of cyclin-D1/cdk4 at E13.5, resulting in a 40% increase in BP cells that undergo proliferative rather than neurogenic divisions, whereas the AP population is unchanged [22Lange C. et al.Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.Cell Stem Cell. 2009; 5: 320-331Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar]. Similar results are seen in the adult dentate gyrus. Acute overexpression of cyclin-D/cdk4 in the 6–10-week-old hippocampus cell autonomously increases the expansion of the precursor pool by increasing proliferative divisions at the expense of neurogenic divisions. When overexpression is stopped, physiological differentiation resumes and the neuronal output of the manipulated pool of precursors can be doubled. In both developing and adult brains, it is the cells with the relatively longer G1 phase that are preferentially affected by overexpression of cyclin-cdk complexes, suggesting that it is the relative change in G1 length, rather than the absolute duration, that is important [56Artegiani B. et al.Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse brain.J. Exp. Med. 2011; 208: 937-948Crossref PubMed Scopus (81) Google Scholar]. The precise mechanism behind the importance of the G1 phase in controlling neurogenesis has yet to be determined, but several hypotheses can be put forward by considering the events and molecular changes during G1, as discussed below. Firstly, recent work identified G1 as a time of early lineage specification in human ESCs (hESCs). Endodermal specification in response to extrinsic Activin/Nodal signalling occurs only during early G1, and cells become refractory in late G1, instead adopting an alternative neuroectodermal cell fate. Mechanistically, the accumulation of active cyclin-D-cdk4/6 complexes during G1 phase results in inhibitory phosphorylation of smad2 and smad3, preventing the cellular response downstream of Activin/Nodal signalling [7Pauklin S. Vallier L. The cell-cycle state of stem cells determines cell fate propensity.Cell. 2013; 155: 135-147Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar]. Other direct targets of cyclin/cdks may also have key roles in precursor maintenance and neuronal differentiation (see below). Secondly, the responsiveness of the cell during G1 may reflect the complement of transcription factors expressed at that time. Pluripotent stem cells express several key developmental regulators with a cell cycle bias. For example, FoxA2, GATA4, and Pax7 are upregulated during the G1 phase and downregulated as cells transit into S phase; therefore, G1 may represent a time when cells are lineage primed [23Singh A.M. et al.Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.Stem Cell Rep. 2013; 1: 532-544Abstract Full Text Full Text PDF Scopus (98) Google Scholar]. Similarly, there is evidence to suggest that basic helix-loop-helix (bHLH) proneural proteins, such as Neurogenin 2 (Ngn2) and Achaete-Scute Homologue 1 (Ascl1), which are master regulators of the neurogenic machinery (see below), adopt a cell cycle-dependent expression pattern, specifically during mid-corticogenesis (E15.5) in the mouse. Ngn2 is expressed in the late G1 phase nuclei located in the central VZ region and is excluded from the G2/M phase nuclei. By contrast, Ascl1 accumulates in early G1 nuclei. Given that Ngn2 is critical to specification of cortical neuron fate, the longer G1 phase may allow a greater accumulation of Ngn2 protein [24Britz O. et al.A role for proneural genes in the maturation of cortical progenitor cells.Cereb. Cortex. 2006; 16: i138-i151Crossref PubMed Scopus (130) Google Scholar]. Finally, the susceptibility to extrinsic fate determinants during G1 may reflect a more permissive chromatin state. Global epigenetic changes occur in pluripotent stem cells in a cell cycle-dependent manner and this may regulate gene expression to allow a cell to respond specifically during a given cell cycle phase [23Singh A.M. et al.Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.Stem Cell Rep. 2013; 1: 532-544Abstract Full Text Full Text PDF Scopus (98) Google Scholar]. It is likely that multiple mechanisms operate to coordinate cell cycle, cell fate, and overt differentiation, and these may have variable importance in different cell types. For example, two populations of cortical precursor cells exit the cell cycle on E14 in the mouse, and either rapidly (Q-fast) or slowly (Q-slow) leave the VZ; fate choice of the former may be predominantly determined by cell intrinsic mechanisms, whereas the latter are influenced more by extrinsic signals [25Goto T. et al.Altered patterns of neuron production in the p27 knockout mouse.Dev. Neurosci. 2004; 26: 208-217Crossref PubMed Scopus (38) Google Scholar]. Furthermore, recent work in developing chick spinal cord suggests that a shortened G2 phase in spinal precursors undergoing neurogenic divisions may be important to limit the receptive window for pro-proliferative cues from the Notch and Wnt signalling paths [26Peco E. et al.The CDC25B phosphatase shortens the G2 phase of neural progenitors and promotes efficient neuron production.Development. 2012; 139: 1095-1104Crossref PubMed Scopus (29) Google Scholar]. bHLH transcription factors have key roles at multiple points during neurogenesis in the CNS, binding DNA as active heterodimers with ubiquitously expressed E proteins. Indeed, bHLH proneural determination factors, such as Ngn2 and Ascl1, are considered master regulators of neurogenesis, activating a plethora of differentiation genes that coordinate neural commitment, subtype specification, and neuronal maturation [27Wilkinson G. et al.Proneural genes in neocortical development.Neuroscience. 2013; 253C: 256-273Crossref Scopus (89) Google Scholar]. However, these factors are also instrumental in activating expression of the Notch ligand, Delta, and subsequent maintenance of the progenitor phenotype in neighbouring cells via lateral inhibition. Early work established that, at least in some cases, progenitor-associated genes have a more open chromatin state, whereas differentiation-associated genes require additional epigenetic remodelling before activation [28Koyano-nakagawa N. et al.Activation of Xenopus genes required for lateral inhibition and neuronal differentiation during primary neurogenesis.Mol. Cell. Neurosci. 1999; 14: 327-339Crossref PubMed Scopus (65) Google Scholar]. Recently, a mechanism has been described that directly links cell cycle progression in neural precursor cells with their propensity to undergo differentiation, through post-translational modification of Ngn2 [29Ali F. et al.Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis.Development. 2011; 138: 4267-4277Crossref PubMed Scopus (126) Google Scholar]. These findings have allowed the development of a detailed model, whereby cdk-dependent phosphorylation of this key regulator coordinates the cell cycle control of precursor maintenance versus differentiation. Ngn2 can be phosphorylated on up to nine serine residues, found within serine–proline (SP) pairs, and phosphorylation of these multiple sites is dependent on both the level and duration of exposure to cdk activity. Therefore, a functional response to these phosphorylation events gives a rheostat-like response to changes in cyclin-cdk activity during the cell cycle and development [29Ali F. et al.Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis.Development. 2011; 138: 4267-4277Crossref PubMed Scopus (126) Google Scholar]. Indeed, when the cell cycle is active and cyclin-cdk levels are high, Ngn2 is in a (hyper)-phosphorylated form and has a reduced DNA binding affinity that is sufficient only to activate the progenitor-associated target promoters that have open chromatin. As the cell cycle lengthens, cyclin-cdk activity is reduced and Ngn2 phosphorylation decreases, resulting in an increase in DNA-binding affinity. This longer promoter dwell time by hypophosphorylated Ngn2 appears to be necessary to bring about the epigenetic remodelling and activation of downstream target promoters that drive neuronal differentiation. Thus, as cdk levels decrease, the level of progenitor gene expression remains fairly static and the expression of differentiation genes relatively increases to tip the balance in favour of differentiation [30Hindley C. et al.Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation.Development. 2012; 139: 1718-1723Crossref PubMed Scopus (69) Google Scholar]. Experimentally, a phosphomutant form of Ngn2 that has all nine SP sites mutated to serine–alanine (SA) and so cannot be phosphorylated by cdks, shows a significantly enhanced ability to drive neuronal differentiation both in vitro and in vivo, supporting the model presented above [29Ali F. et al.Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis.Development. 2011; 138: 4267-4277Crossref PubMed Scopus (126) Google Scholar]. Finally, Ngn2 undergoes both canonical and noncanonical ubiquitination, which contribute to rapid protein turnover via the proteasome. Ngn2 displays changes in stability at different cell cycle phases, and noncanonical ubiquitination via cysteine residues may contribute to the greater turnover observed during mitosis [31Vosper J.M.D. et al.Ubiquitylation on canonical and non-canonical sites targets the transcription factor neurogenin for ubiquitin-mediated proteolysis.J. Biol. Chem. 2009; 284: 15458-15468Crossref PubMed Scopus (60) Google Scholar]. Moreover, the Xenopus cdk inhibitor p27Xic1 directly stabilises the Ngn2 protein independently of its a