NEUROG2 Drives Cell Cycle Exit of Neuronal Precursors by Specifically Repressing a Subset of Cyclins Acting at the G₁and S Phases of the Cell Cycle

Abstract

bProneural NEUROG2 (neurogenin 2 [Ngn2]) is essential for neuronal commitment, cell cycle withdrawal, and neuronal differentiation. Although NEUROG2's influence on neuronal commitment and differentiation is beginning to be clarified, its role in cell cycle withdrawal remains unknown. We therefore set out to investigate the molecular mechanisms by which NEUROG2 induces cell cycle arrest during spinal neurogenesis. We developed a large-scale chicken embryo strategy, designed to find gene networks modified at the onset of NEUROG2 expression, and thereby we identified those involved in controlling the cell cycle. NEUROG2 activation leads to a rapid decrease of a subset of cell cycle regulators acting at G 1 and S phases, including CCND1, CCNE1/2, and CCNA2 but not CCND2. The use of NEUROG2VP16 and NEUROG2EnR, acting as the constitutive activator and repressor, respectively, indicates that NEUROG2 indirectly represses CCND1 and CCNE2 but opens the possibility that CCNE2 is also repressed by a direct mechanism. We demonstrated by phenotypic analysis that this rapid repression of cyclins prevents S phase entry of neuronal precursors, thus favoring cell cycle exit. We also showed that cell cycle exit can be uncoupled from neuronal differentiation and that during normal development NEUROG2 is in charge of tightly coordinating these two processes.O ne important challenge in neurobiology is to understand how different types of postmitotic neurons, with distinct cellular and physiological properties, are generated in the developing central nervous system (CNS) from a pool of dividing neural progenitors. The embryonic spinal cord is a good model to tackle these issues, because the role of extracellular signals and transcription factors in neuron specification and differentiation is relatively well defined. This structure is derived from the neural tube, a single pseudoepithelium that will sequentially give rise to a large variety of neurons and glial cells dedicated to serve specific functions in the adult. Neurogenesis is achieved via a succession of steps that follow a stereotypic temporal order. A neural progenitor is committed to a neuronal fate at the expense of a glial fate and becomes a neuronal precursor. Concomitantly, this neural progenitor is destined to differentiate into a specific neuronal subtype. Soon after, neuronal precursors stop cycling and initiate their differentiation to give rise to postmitotic differentiated neurons.The main positive regulators of vertebrate neurogenesis are proneural transcription factors of the neural basic helix-loop-helix (bHLH) family, including neurogenins (NeuroG1/2/3) (5, 35). They control different steps of neurogenesis, such as neuronal commitment, cell cycle exit, subtype specification, and neuronal differentiation (5, 35, 42). In the spinal cord, loss-of-function studies have shown that NEUROG2 is involved in the acquisition of motoneuron and interneuron fates (46). Together with NEUROG1, NEUROG2 also controls neuronal differentiation as shown by the loss of neurons in Ne...

A computational model for the coordination of neural progenitor self-renewal and differentiation through Hes1 dynamics

“…Furthermore, Ngn2 has a short half-life and its stabilization by DNA binding is inhibited following its phosphorylation by cyclin-Cdk2/1 (Ali et al, 2011). Although Ngn2 alone can exert a brake on early G1-phase progression by repressing cyclin D, E1 and E2 transcription (Lacomme et al, 2012), concomitant Ngn2 and Cip/Kip overexpression coincident with irreversible cell cycle exit is only observed at a later differentiation stage, in nascent neurons (Gui et al, 2007). The present model accounts for such an intricate collaboration between Cip/Kip and Ngn2, in which early Ngn2 accumulation promotes the expression of neural differentiation markers and early G1-phase lengthening before activating a positive-feedback loop with the emerging Cip/Kip proteins, which sets up an irreversible and robust cell cycle exit associated with terminal differentiation.…”

“…A crucial component of the neurogenic pathway is the proneural protein Ngn2 that not only regulates neuronal differentiation, migration and maturation, but also contributes to cell cycle exit by indirectly repressing a subset of cyclins, notably cyclin D1, E1 and E2, and by activating, directly or indirectly, the Cip/Kip genes (Farah et al, 2000;Lacomme et al, 2012). Multi-site Ngn2 phosphorylation by cyclin-Cdk1,2, in turn, leads to its inactivation as a transcriptional activator and its destabilization (Ali et al, 2011), whereas Ngn2 binding to Cip/Kip proteins (e.g.…”

“…Neurogenesis in vertebrates primarily relies on the activity of proneural bHLH transcription factors, such as Ngn1, Ngn2 and Mash1, which not only are direct Notch targets but also directly regulate G1-phase progression (Farah et al, 2000;Nguyen et al, 2006;Castro et al, 2011;Lacomme et al, 2012) and which exhibit oscillatory expression that inversely correlates with that of Hes1 in mouse neural progenitors Imayoshi et al, 2013). The neurogenic phase of cortical development therefore appears as the most propitious system in which to study how the interactions between Notch-Hes1 signaling, G1-phase regulation and differentiation signals may succeed in dictating proper cell fate decisions.…”

Can the ‘neuron theory’ be complemented by a universal mechanism for generic neuronal differentiation

“…Selection of Ngn2/Neurog2 transfected cells early (6 h) after electroporation and expression profiling using microarrays shows downregulation of the transcripts for core regulators of G1 and S phase such as Cyclin D1, E1, E2 and A2 (Lacomme et al 2012). Additional targets belong to the Notch signaling pathway such as Hes1, Hes5, Dll1, Jag1 and Jag2.…”

Regulação da expressão do gene cScratch2 na embriogênese neural.

“…A Ngn2 é ainda responsável por disparar a expressão de marcadores de diferenciação neural independentemente da saída do ciclo celular, sugerindo que a diferenciação é um evento controlado paralelamente à cessão do ciclo celular (Lacomme et al, 2012). Por fim, Ngn2 confere destino neuronal às células progenitoras através da repressão de genes expressos em progenitores neurais, incluindo Sox1/2/3 e outros envolvidos na especificação do subtipo celular, tais como Pax6 e Olig2 (Lacomme et al, 2012).…”