Cell Cycle Exit Of Progenitors Research Articles

APLP2 regulates neuronal stem cell differentiation during cortical development

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

APLP2 regulates neuronal stem cell differentiation during cortical development

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

Insm1a-mediated gene repression is essential for the formation and differentiation of Müller glia-derived progenitors in the injured retina

In zebrafish, retinal injury stimulates Müller glia (MG) reprograming; allowing them to generate multipotent progenitors that regenerate damaged cells and restore vision. Recent studies suggest transcriptional repression may underlie these events. To identify these repressors, we compared the transcriptomes of MG and MG-derived progenitors and identified insm1a, a transcriptional repressor exhibiting a biphasic pattern of expression that is essential for retina regeneration. Insm1a was found to suppress ascl1a and its own expression and link injury-dependent ascl1a induction with dickkopf (dkk) suppression, which is necessary for MG dedifferentiation. We also found that Insm1a was responsible for sculpting the zone of injury-responsive MG by suppressing hb-egfa expression. Finally, we provide evidence that Insm1a stimulates progenitor cell cycle exit by suppressing a genetic program driving progenitor proliferation. Our studies identify Insm1a as a key regulator of retina regeneration and provide a mechanistic understanding of how it contributes to multiple phases of this process.

A Unilateral Negative Feedback Loop BetweenmiR-200microRNAs and Sox2/E2F3 Controls Neural Progenitor Cell-Cycle Exit and Differentiation

MicroRNAs have emerged as key posttranscriptional regulators of gene expression during vertebrate development. We show that the miR-200 family plays a crucial role for the proper generation and survival of ventral neuronal populations in the murine midbrain/hindbrain region, including midbrain dopaminergic neurons, by directly targeting the pluripotency factor Sox2 and the cell-cycle regulator E2F3 in neural stem/progenitor cells. The lack of a negative regulation of Sox2 and E2F3 by miR-200 in conditional Dicer1 mutants (En1(+/Cre); Dicer1(flox/flox) mice) and after miR-200 knockdown in vitro leads to a strongly reduced cell-cycle exit and neuronal differentiation of ventral midbrain/hindbrain (vMH) neural progenitors, whereas the opposite effect is seen after miR-200 overexpression in primary vMH cells. Expression of miR-200 is in turn directly regulated by Sox2 and E2F3, thereby establishing a unilateral negative feedback loop required for the cell-cycle exit and neuronal differentiation of neural stem/progenitor cells. Our findings suggest that the posttranscriptional regulation of Sox2 and E2F3 by miR-200 family members might be a general mechanism to control the transition from a pluripotent/multipotent stem/progenitor cell to a postmitotic and more differentiated cell.

Regulation of retinal progenitor expansion by Frizzled receptors: implications for microphthalmia and retinal coloboma

Nineteen Wnt ligands and 10 Frizzled (Fz) receptors mediate multiple distinct cellular events during neuronal development. However, their precise roles in cell-type specification and organogenesis are poorly delineated because of overlapping functions and expression profiles. Here, we have explored the role of two closely related Frizzled receptors, Fz5 and Fz8, in mouse retinal development. We previously showed that Fz5(-/-) mice exhibit mild coloboma and microphthalmia at ~50% penetrance. Fz8 expression overlaps with Fz5 in the neural retina and optic fissure/disc. Mice lacking Fz8 show minimal eye and retinal defects. The embryos lacking both Fz5 and Fz8 die early in development, but a majority of triallelic Fz5(-/-);Fz8(+/-) mutants survive until birth. The triallelic mutant develops severe retinal coloboma and microphthalmia with full penetrance. At the cellular level, impaired neurogenesis is indicated by increased early-born retinal neurons that result from accelerated cell cycle exit of progenitors. Deficiency of apical retinal neuroepithelium is indicated by altered localization of apical junction markers, such as atypical protein kinase C, RhoA and β-catenin. Hes1 expression, which is critical for retinal progenitor expansion, is down-regulated in the triallelic mutant mouse. Furthermore, blocking Frizzled receptors in cultured retinal explants led to basally shifted divisions of retinal progenitors. Together, our studies suggest a dose-dependent regulation of signaling by Fz5 and Fz8 in optic fissure/disc formation and progenitor expansion.

Specific Protein Kinase C Isoforms Are Required for Rod Photoreceptor Differentiation

The protein kinase C (PKC) family of enzymes regulates cell physiology through phosphorylation of serine and threonine residues of many proteins in most cell types. Here we identify PKC-β1 and PKC-γ as isoforms that are essential for rod photoreceptor differentiation in mouse retinas. Using ex vivo retinal explants, we found that phorbol ester 12-myristate 13-acetate and insulin-like growth factor 1 (IGF1) induced rod differentiation, as defined by opsin or Crx expression, in a PKC-dependent manner days ahead of rod development in untreated explants. PKC-β1 and PKC-γ were colocalized with proliferating cell nuclear antigen (PCNA)- and STAT3-positive progenitors through the later differentiation period. Pharmacological or genetic inhibition of either isoform resulted in a partial reduction in the appearance of rods, whereas removing both isoforms resulted in their complete absence. Furthermore, a significant decline of STAT3 tyrosine phosphorylation was observed by activation of PKC, while inhibition of PKC resulted in an increase of phosphorylated STAT3 along with a delayed cell cycle exit of progenitors with prolonged PCNA expression. In adult retinas, IGF1 activates PI-3 kinase (PI3K), but in neonatal retinas its action is identical to the action of an PI3K inhibitor. These data unveil a novel signaling cascade that coordinates and regulates rod differentiation through specific PKC isoforms in mammals.

The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.

Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis

The balanced proliferation and cell cycle exit of neural progenitors, by generating the appropriate amount of postmitotic progeny at the correct time and in the proper location, is required for the establishment of the highly ordered structure of the adult brain. Little is known about the extrinsic signals regulating these processes, particularly in the midbrain. Fibroblast growth factor (Fgf) 15, the mouse ortholog of FGF19 and member of an atypical Fgf subfamily, is prominently expressed in the dorsolateral midbrain of the midgestational mouse embryo. In the absence of Fgf15, dorsal midbrain neural progenitors fail to exit the cell cycle and to generate the proper amount of postmitotic neurons. We show here that this is due to the altered expression of inhibitory/neurogenic and proneural/neuronal differentiation helix-loop-helix transcription factor (TF) genes. The expression of Id1, Id3, and Hes5 was strongly increased and ectopically expanded, whereas the expression of Ascl1 (Mash1), Neurog1 (Ngn1) and Neurog2 (Ngn2) was strongly decreased and transcription of Neurod1 (NeuroD) was completely abolished in the dorsolateral midbrain of Fgf15−/− mice. These abnormalities were not caused by the mis-expression of cell cycle regulatory proteins such as cyclin-dependent kinase inhibitors or retinoblastoma proteins. Furthermore, human FGF19 promotes cell cycle exit of murine dorsal neural progenitors in vitro. Therefore, our data suggest that Fgf15 is a crucial signaling molecule regulating the postmitotic transition of dorsal neural progenitors and thus the initiation and proper progression of dorsal midbrain neurogenesis in the mouse, by controlling the expression of neurogenic and proneural TFs.

Duplicate dmbx1genes regulate progenitor cell cycle and differentiation during zebrafish midbrain and retinal development

BackgroundThe Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome.ResultsUsing gene knockdown experiments we examined the function of the dmbx1 gene paralogs in zebrafish, dmbx1a and dmbx1b in regulating neurogenesis in the developing retina and midbrain. Dose-dependent loss of dmbx1a and dmbx1b function causes a significant reduction in growth of the midbrain and retina that is evident between 48-72 hpf. We show that this phenotype is not due to patterning defects or persistent cell death, but rather a deficit in progenitor cell cycle exit and differentiation. Analyses of the morphant retina or anterior hindbrain indicate that paralogous function is partially diverged since loss of dmbx1a is more severe than loss of dmbx1b. Molecular evolutionary analyses of the Dmbx1 genes suggest that while this gene family is conservative in its evolution, there was a dramatic change in selective constraint after the duplication event that gave rise to the dmbx1a and dmbx1b gene families in teleost fish, suggestive of positive selection. Interestingly, in contrast to zebrafish dmbx1a, over expression of the mouse Dmbx1 gene does not functionally compensate for the zebrafish dmbx1a knockdown phenotype, while over expression of the dmbx1b gene only partially compensates for the dmbx1a knockdown phenotype.ConclusionOur data suggest that both zebrafish dmbx1a and dmbx1b genes are retained in the fish genome due to their requirement during midbrain and retinal neurogenesis, although their function is partially diverged. At the cellular level, Dmbx1 regulates cell cycle exit and differentiation of progenitor cells. The unexpected observation of putative post-duplication positive selection of teleost Dmbx1 genes, especially dmbx1a, and the differences in functionality between the mouse and zebrafish genes suggests that the teleost Dmbx1 genes may have evolved a diverged function in the regulation of neurogenesis.

Sumoylation controls retinal progenitor proliferation by repressing cell cycle exit in Xenopus laevis

Precisely controlled progenitor proliferation is essential for normal development. However, molecular mechanisms, which control the correct timing of cell cycle withdrawal during development, have been poorly understood. We show here that ubc9, a sumo-conjugating enzyme, controls the cell cycle exit of retinal progenitors. We found that ubc9 is highly expressed in retinal progenitors and stem cells in Xenopus embryos. Ubc9 physically and functionally associates with Xenopus hmgb3, which is required for retinal cell proliferation, and prolonged expression of ubc9 and hmgb3 results in suppression of the cell cycle exit of retinal progenitors in a sumoylation-dependent manner. Overexpression of ubc9 and hmgb3 decreased expression of the cell-cycle inhibitor p27Xic1. Furthermore, progenitor proliferation is regulated, at least in part, by sumoylation of transcription factor Sp1. These results suggest a significant role of sumoylation for cell cycle regulation in retinal progenitors.

Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation

Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.

Ikaros‐1 couples cell cycle arrest of late striatal precursors with neurogenesis of enkephalinergic neurons

During central nervous system development, several transcription factors regulate the differentiation of progenitor cells to postmitotic neurons. Here we describe a novel role for Ikaros-1 in the generation of late-born striatal neurons. Our results show that Ikaros-1 is expressed in the boundary of the striatal germinal zone (GZ)/mantle zone (MZ), where it induces cell cycle arrest of neural progenitors by up-regulation of the cyclin-dependent kinase inhibitor (CDKi) p21(Cip1/Waf1). This effect is coupled with the neuronal differentiation of late precursors, which in turn is critical for the second wave of striatal neurogenesis that gives rise to matrix neurons. Consistently, Ikaros(-/-) mice had fewer striatal projecting neurons and, in particular, enkephalin (ENK)-positive neurons. In addition, overexpression of Ikaros-1 in primary striatal cultures increases the number of calbindin- and ENK-positive neurons. Our results also show that Ikaros-1 acts downstream of the Dlx family of transcription factors, insofar as its expression is lost in Dlx1/2 double knockout mice. However, we demonstrate that Ikaros-1 and Ebf-1 independently regulate the final determination of the two populations of striatal projection neurons of the matrix compartment, ENK- and substance P-positive neurons. In conclusion, our findings identify Ikaros-1 as a modulator of cell cycle exit of neural progenitors that gives rise to the neurogenesis of ENK-positive striatal neurons.

Transforming Growth Factor β Promotes Neuronal Cell Fate of Mouse Cortical and Hippocampal Progenitors In Vitro and In Vivo: Identification of Nedd9 as an Essential Signaling Component

Transforming Growth Factor β (Tgfβ) and associated signaling effectors are expressed in the forebrain, but little is known about the role of this multifunctional cytokine during forebrain development. Using hippocampal and cortical primary cell cultures of developing mouse brains, this study identified Tgfβ-regulated genes not only associated with cell cycle exit of progenitors but also with adoption of neuronal cell fate. Accordingly, we observed not only an antimitotic effect of Tgfβ on progenitors but also an increased expression of neuronal markers in Tgfβ treated cultures. This effect was dependent upon Smad4. Furthermore, in vivo loss-of-function analyses using Tgfβ2−/−/Tgfβ3−/− double mutant mice showed the opposite effect of increased cell proliferation and fewer neurons in the cerebral cortex and hippocampus. Gata2, Runx1, and Nedd9 were candidate genes regulated by Tgfβ and known to be involved in developmental processes of neuronal progenitors. Using siRNA-mediated knockdown, we identified Nedd9 as an essential signaling component for the Tgfβ-dependent increase in neuronal cell fate. Expression of this scaffolding protein, which is mainly described as a signaling molecule of the β1-integrin pathway, was not only induced after Tgfβ treatment but was also associated with morphological changes of the Nestin-positive progenitor pool observed upon exposure to Tgfβ.

Selective Cortical Layering Abnormalities and Behavioral Deficits in Cortex-Specific Pax6 Knock-Out Mice

The transcription factor Pax6 has been implicated in neocortical neurogenesis in vertebrates, including humans. Analyses of the role of Pax6 in layer formation and cognitive abilities have been hampered by perinatal lethality of Pax6 mutants. Here, we generated viable mutants exhibiting timed, restricted inactivation of Pax6 during early and late cortical neurogenesis using Emx1-Cre and hGFAP-Cre lines, respectively. The disruption of Pax6 at the onset of neurogenesis using Emx1-Cre line resulted in premature cell cycle exit of early progenitors, increase of early born neuronal subsets located in the marginal zone and lower layers, and a nearly complete absence of upper layer neurons, especially in the rostral cortex. Furthermore, progenitors, which accumulated in the enlarged germinal neuroepithelium at the pallial/subpallial border in the Pax6 mutants, produced an excess of oligodendrocytes. The inactivation of Pax6 after generation of the lower neuronal layers using hGFAP-Cre line did not affect specification or numbers of late-born neurons, indicating that the severe reduction of upper layer neurons in Pax6 deficiency is mostly attributable to a depletion of the progenitor pool, available for late neurogenesis. We further show that Pax6(fl/fl);Emx1-Cre mutants exhibited deficiencies in sensorimotor information integration, and both hippocampus-dependent short-term and neocortex-dependent long-term memory recall. Because a majority of the morphological and behavior disabilities of the Pax6 mutant mice parallel abnormalities reported for aniridia patients, a condition caused by PAX6 haploinsufficiency, the Pax6 conditional mutant mice generated here represent a valuable genetic tool to understand how the developmental cortical disruption can lead to a human behavior abnormality.

Shh and Gli3 activities are required for timely generation of motor neuron progenitors

Generation of distinct ventral neuronal subtypes in the developing spinal cord requires Shh signaling mediated by the Gli family of transcription factors. Genetic studies of Shh−/−;Gli3−/− double mutants indicated that the inhibition of Gli3 repressor activity by Shh is sufficient for the generation of different neurons including motor neurons. In this study, we show that although ventral neural progenitors are initiated in normal numbers in Shh−/−;Gli3−/− mutants, the subsequent appearance of motor neuron progenitors shows a ∼20-hour lag, concomitant with a delay in the activation of a pan-neuronal differentiation program and cell cycle exit of ventral neural progenitors. Accordingly, the Shh−/−;Gli3−/− mutant spinal cord exhibits a delay in motor neuron differentiation and an accumulation of a ventral neural progenitor pool. The requirement of Shh and Gli3 activities to promote the timely appearance of motor neuron progenitors is further supported by the analysis of Ptch1−/− mutants, in which constitutive Shh pathway activity is sufficient to elicit ectopic and premature differentiation of motor neurons at the expense of ventral neural progenitors. Taken together, our analysis suggests that, beyond its well established dorso-ventral patterning function through a Gli3-derepression mechanism, Shh signaling is additionally required to promote the timely appearance of motor neuron progenitors in the developing spinal cord.

Distinct Developmental Roles of Cell Cycle Inhibitors p57Kip2 and p27Kip1 Distinguish Pituitary Progenitor Cell Cycle Exit from Cell Cycle Reentry of Differentiated Cells

Patterning and differentiation signals are often believed to drive the developmental program, including cell cycle exit of proliferating progenitors. Taking advantage of the spatial and temporal separation of proliferating and differentiated cells within the developing anterior pituitary gland, we investigated the control of cell proliferation during organogenesis. Thus, we identified a population of noncycling precursors that are uniquely marked by expression of the cell cycle inhibitor p57(Kip2) and by cyclin E. In p57(Kip2-/-) mice, the developing pituitary is hyperplastic due to accumulation of proliferating progenitors, whereas overexpression of p57(Kip2) leads to hypoplasia. p57(Kip2)-dependent cell cycle exit is not required for differentiation, and conversely, blockade of cell differentiation, as achieved in Tpit(-/-) pituitaries, does not prevent cell cycle exit but rather leads to accumulation of p57(Kip2)-positive precursors. Upon differentiation, p57(Kip2) is replaced by p27(Kip1). Accordingly, proliferating differentiated cells are readily detected in p27(Kip1-/-) pituitaries but not in wild-type or p57(Kip2-/-) pituitaries. Strikingly, all cells of p57(Kip2-/-);p27(Kip1-/-) pituitaries are proliferative. Thus, during normal development, progenitor cell cycle exit is controlled by p57(Kip2) followed by p27(Kip1) in differentiated cells; these sequential actions, taken together with different pituitary outcomes of their loss of function, suggest hierarchical controls of the cell cycle that are independent of differentiation.

Patterns of p57Kip2 expression in embryonic rat brain suggest roles in progenitor cell cycle exit and neuronal differentiation

In developing central nervous system, a variety of mechanisms couple cell cycle exit to differentiation during neurogenesis. The cyclin-dependent kinase (CDK) inhibitor p57Kip2 controls the transition from proliferation to differentiation in many tissues, but roles in developing brain remain uncertain. To characterize possible functions, we defined p57Kip2 protein expression in embryonic (E) day 12.5 to 20.5 rat brains using immunohistochemistry combined with markers of proliferation and differentiation. The p57Kip2 was localized primarily in cell nuclei and positive cells formed two distinct patterns including wide dispersion and laminar aggregation that were brain region-specific. From E12.5 to E16.5, p57Kip2 expression was detected mainly in ventricular zone (VZ) and/or mantle zone of hippocampus, septum, basal ganglia, thalamus, hypothalamus, midbrain, and spinal cord. After E18.5, p57Kip2 was detected in select regions undergoing differentiation. The p57Kip2 expression was also compared with regional transcription factors, including Ngn2, Nkx2.1, and Pax6. Time course studies performed in diencephalon showed that p57Kip2 immunoreactivity colocalized with BrdU at 8 hr in nuclei exhibiting the wide dispersion pattern, whereas colocalization in the laminar pattern occurred only later. Moreover, p57Kip2 frequently colocalized with neuronal marker, beta-III tubulin. Finally, we characterized relationships of p57Kip2 to CDK inhibitor p27Kip1: in proliferative regions, p57Kip2 expression preceded p27Kip1 as cells underwent differentiation, though the proteins colocalized in substantial numbers of cells, suggesting potentially related yet distinct functions of Cip/Kip family members during neurogenesis. Our observations that p57Kip2 exhibits nuclear expression as precursors exit the cell cycle and begin expressing neuronal characteristics suggests that the CDK inhibitor contributes to regulating the transition from proliferation to differentiation during brain development.

Selective cortical interneuron and GABA deficits in cyclin D2-null mice

In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.

Homeodomain Transcription Factor Phox2a, via Cyclic AMP-Mediated Activation, Induces p27Kip1 Transcription, Coordinating Neural Progenitor Cell Cycle Exit and Differentiation

Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27(Kip1) is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27(Kip1) transcription have not been identified. We demonstrate the homeodomain transcription factor Phox2a, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27(Kip1) transcription. Phox2a transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27(Kip1) transcription, G(1) arrest, and neuronal differentiation. Small interfering RNA silencing of either Phox2a or p27(Kip1) suppresses p27(Kip1) transcription and neuronal differentiation, suggesting a causal link between p27(Kip1) expression and differentiation. Conversely, ectopic Phox2a expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27(Kip1) transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed Phox2a activated by cAMP signaling binds homeodomain cis-acting elements of the p27(Kip1) promoter in vivo and mediates p27(Kip1)-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link Phox2a activation with p27(Kip1) transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27 Kip1 having the broadest expression, and both p57 Kip2 and p21 Cip1 showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57 Kip2 ;p27 Kip1 double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57 Kip2 in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.