Annie Paquin

An Essential Role for a MEK-C/EBP Pathway during Growth Factor-Regulated Cortical Neurogenesis

Mammalian neurogenesis is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues such as growth factors. Here we have defined a signaling cascade, a MEK-C/EBP pathway, that is essential for cortical progenitor cells to become postmitotic neurons. Inhibition of MEK or of the C/EBP family of transcription factors inhibits neurogenesis while expression of a C/EBPbeta mutant that is a phosphorylation-mimic at a MEK-Rsk site enhances neurogenesis. C/EBP mediates this positive effect by direct transcriptional activation of neuron-specific genes such as Talpha1 alpha-tubulin. Conversely, inhibition of C/EBP-dependent transcription enhances CNTF-mediated generation of astrocytes from the same progenitor cells. Thus, activation of a MEK-C/EBP pathway enhances neurogenesis and inhibits gliogenesis, thereby providing a mechanism whereby growth factors can selectively bias progenitors to become neurons during development.

Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development

Increasing evidence indicates that development of embryonic central nervous system precursors is tightly regulated by extrinsic cues located in the local environment. Here, we asked whether neurotrophin-mediated signaling through Trk tyrosine kinase receptors is important for embryonic cortical precursor cell development. These studies demonstrate that inhibition of TrkB (Ntrk2) and/or TrkC (Ntrk3) signaling using dominant-negative Trk receptors, or genetic knockdown of TrkB using shRNA, caused a decrease in embryonic precursor cell proliferation both in culture and in vivo. Inhibition of TrkB/C also caused a delay in the generation of neurons, but not astrocytes, and ultimately perturbed the postnatal localization of cortical neurons in vivo. Conversely, overexpression of BDNF in cortical precursors in vivo promoted proliferation and enhanced neurogenesis. Together, these results indicate that neurotrophin-mediated Trk signaling plays an essential, cell-autonomous role in regulating the proliferation and differentiation of embryonic cortical precursors and thus controls cortical development at earlier stages than previously thought.

Endogenous microglia regulate development of embryonic cortical precursor cells.

Microglia play important roles in the damaged or degenerating adult nervous system. However, the role of microglia in embryonic brain development is still largely uncharacterized. Here we show that microglia are present in regions of the developing brain that contain neural precursors from E11 onward. To determine whether these microglia are important for neural precursor maintenance or self‐renewal, we cultured embryonic neural precursors from the cortex of PU.1−/− mice, which we show lack resident microglia during embryogenesis. Cell survival and neurogenesis were similar in cultures from PU.1−/− vs. PU.1+/+ mice, but precursor proliferation and astrogenesis were both reduced. Cortical precursors depleted of microglia also displayed decreased precursor proliferation and astrogenesis, and these deficits could be rescued when microglia were added back to the cultures. Moreover, when the number of microglia present in cortical precursor cultures was increased above normal levels, astrogenesis but not neurogenesis was increased. Together these results demonstrate that microglia present within the embryonic neural precursor niche can regulate neural precursor development and suggest that alterations in microglial number as a consequence of genetic or pathological events could perturb neural development by directly affecting embryonic neural precursors.

CCAAT/Enhancer-Binding Protein Phosphorylation Biases Cortical Precursors to Generate Neurons Rather Than Astrocytes In Vivo

The intracellular mechanisms that bias mammalian neural precursors to generate neurons versus glial cells are not well understood. We demonstrated previously that the growth factor-regulated mitogen-activated protein kinase kinase (MEK) and its downstream target, the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, are essential for neurogenesis in cultured cortical precursor cells (Ménard et al., 2002). Here, we examined a role for this pathway during cortical cell fate determination in vivo using in utero electroporation of the embryonic cortex. These studies demonstrate that inhibition of the activity of either MEK or the C/EBPs inhibits the genesis of neurons in vivo. Moreover, the MEK pathway mediates phosphorylation of C/EBPβ in cortical precursors, and expression of a C/EBPβ construct in which the MEK pathway phosphorylation sites are mutated inhibits neurogenesis. Conversely, expression of a C/EBPβ construct, in which the same sites are mutated to glutamate and therefore are “constitutively” phosphorylated, enhances neurogenesis in the early embryonic cortex. A subpopulation of precursors in which C/EBP activity is inhibited are maintained as cycling precursors in the ventricular/subventricular zone of the cortex until early in postnatal life, when they have an enhanced propensity to generate astrocytes, presumably in response to gliogenic signals in the neonatal environment. Thus, activation of an MEK-C/EBP pathway in cortical precursors in vivo biases them to become neurons and against becoming astrocytes, thereby acting as a growth factor-regulated switch.

TAp73 Acts via the bHLH Hey2 to Promote Long-Term Maintenance of Neural Precursors

Increasing evidence suggests that deficits in adult stem cell maintenance cause aberrant tissue repair and premature aging [1]. While the mechanisms regulating stem cell longevity are largely unknown, recent studies have implicated p53 and its family member p63. Both proteins regulate organismal aging [2-4] as well as survival and self-renewal of tissue stem cells [5-9]. Intriguingly, haploinsufficiency for a third family member, p73, causes age-related neurodegeneration [10]. While this phenotype is at least partially due to loss of the ΔNp73 isoform, a potent neuronal prosurvival protein [11-16], a recent study showed that mice lacking the other p73 isoform, TAp73, have perturbations in the hippocampal dentate gyrus [17], a major neurogenic site in the adult brain. These findings, and the link between the p53 family, stem cells, and aging, suggest that TAp73 might play a previously unanticipated role in maintenance of neural stem cells. Here, we have tested this hypothesis and show that TAp73 ensures normal adult neurogenesis by promoting the long-term maintenance of neural stem cells. Moreover, we show that TAp73 does this by transcriptionally regulating the bHLH Hey2, which itself promotes neural precursor maintenance by preventing premature differentiation.

p63 Antagonizes p53 to Promote the Survival of Embryonic Neural Precursor Cells

The molecular mechanisms that regulate survival of embryonic neural precursors are still relatively ill-defined. Here, we have asked whether the p53 family member p63 plays any role during this developmental window, focusing on the embryonic cerebral cortex. We show that genetic knockdown of p63 either in culture or in the embryonic telencephalon causes apoptosis of cortical precursors and newly born cortical neurons, and that this can be rescued by expression of ΔNp63, but not TAp63 isoforms. This cortical precursor apoptosis is the consequence of deregulated p53 activity, since both basal precursor apoptosis and that induced by loss of p63 are rescued by coincident genetic silencing of p53. Finally, we demonstrate that the third p53 family member, ΔNp73, does not regulate survival of cortical precursor cells, but that it collaborates with ΔNp63 to ensure the survival of newly born cortical neurons. Thus, the balance of ΔNp63 versus p53 determines the life versus death of embryonic cortical precursors, a role that these p53 family members may well play in other populations of developing and/or adult neural precursors.

Costello syndrome H-Ras alleles regulate cortical development

Genetic mutations in H-Ras cause Costello syndrome (CS), a complex developmental disorder associated with cortical abnormalities and profound mental retardation. Here, we have asked whether there are perturbations in precursor cell proliferation, differentiation, or survival as a consequence of expressing CS H-Ras alleles that could explain the cognitive deficits seen in this disorder. Two different H-Ras alleles encoding mutations present in CS patients, H-RasG12V and H-RasG12S were expressed in cortical progenitors in culture and in vivo by in utero electroporation and their effects on cortical precursor cell fate examined. Expression of both mutants in cultured precursors inhibited neurogenesis and promoted proliferation and astrogenesis. In vivo, expression of either form of CS H-Ras promoted cell proliferation and inhibited neurogenesis. Moreover, these H-Ras mutants promoted premature gliogenesis, causing formation of astrocytes at a time when normal gliogenesis has not yet begun, ultimately leading to an increase in the number of astrocytes postnatally. Thus, aberrant H-Ras activation enhances neural precursor cell proliferation, and perturbs the normal genesis of neurons and glial cells, effects that likely contribute to the cortical abnormalities and cognitive dysfunction seen in CS.

Coffin-Lowry syndrome: a role for RSK2 in mammalian neurogenesis.

Coffin-Lowry Syndrome (CLS) is an X-linked genetic disorder associated with cognitive and behavioural impairments. CLS patients present with loss-of-function mutations in the RPS6KA3 gene encoding the mitogen-activated protein kinase (MAPK)-activated kinase p90 ribosomal S6 kinase 2 (Rsk2). Although Rsk2 is expressed in the embryonic brain, its function remains largely uncharacterized. To this end, we isolated murine cortical precursors at embryonic day 12 (E12), a timepoint when neuronal differentiation is initiated, and knocked-down Rsk2 expression levels using shRNA. We performed similar experiments in vivo using in utero electroporations to express shRNA against Rsk2. Rsk2 knockdown resulted in a significant decrease in neurogenesis and an increase in the proportion of proliferating Pax6-positive radial precursor cells, indicating that Rsk2 is essential for cortical radial precursors to differentiate into neurons. In contrast, reducing Rsk2 levels in vitro or in vivo had no effect on the generation of astrocytes. Thus, Rsk2 loss-of-function, as seen in CLS, perturbs the differentiation of neural precursors into neurons, and maintains them instead as proliferating radial precursor cells, a defect that may underlie the cognitive dysfunction seen in CLS.

The p53 family member, p63, regulates neural precursor cell survival during cortical development

thinner cortex due to fewer numbers of mature neurons and an increase in gliogenesis. The Erk2 cKO also exhibits a deficit in learning and memory. Detailed behavioural analysis of the Erk2 cKO also revealed that the mice are anosmic. Here we describe a novel mouse model in which both Erk isoforms are inactivated in developing telencephalon. Simultaneous silencing of Erk1 and Erk2 exacerbates the Erk2 cortical phenotype. At E14.5 proliferating NPCs within Erk1/2 DKO animals exhibit decreased BrdU incorporation and almost complete loss of mitotic abventricular intermediate progenitor cells. These changes correlate with a decrease in Map2+ neurons and an increase in uncommitted Nestin+ NPCs in the VZ. Importantly, at E16.5we see an even greater exacerbation of proliferation and differentiation defects during neurogenesis due to a lack of compensation from Erk1. The perturbations of NPC cell cycle dynamics are associated with the disregulation of CyclinD1 and P27 expression; both are downstream effectors of Erk1/2. These data document a critical role of Erk1/2 in regulating neuronal progenitor cells dynamics and cortical development.

A role for activated H-RAS in cortical development and costello syndrome

ATF5 is a bZIP transcription factor that is a member of the ATF/ CREB family. Recent studies have indicated a role for ATF5 in neural, astrocyte and oligodendrocyte progenitor proliferation. Studies done in vivo and in vitro indicate that ATF5 promotes neuroprogenitor cell proliferation and that down-regulation of ATF5 is necessary for such cells to exit the cell cycle and differentiate. Furthermore, expression of ATF5 is high in glioblastomas where its expression appears to be required for survival. Though existing data shed light on biological functions of ATF5, there is little information regarding mechanisms that govern the induction and maintenance of ATF5 expression in proliferating neuroprogenitors and in tumor cells. Because of its well characterized properties, we chose the developing mouse cerebellum as a model system to address these issues. ATF5 protein is highly expressed by cerebellar granule neuron progenitors (GNPs) in the EGL and expression diminishes as GNPs differentiate into granule neurons. Such expression coincides with regions of Sonic Hedgehog (Shh)-driven proliferative activity in the EGL. We therefore assessed in GNP cultures whether ATF5 might be under the control of Shh, a major GNP mitogen. Without added Shh, both ATF5 expression and GNP proliferation rapidly fell. In Shh-treated cultures, in contrast there was a robust increase in the proportion of ATF5 positive cells. Childhood medulloblastomas are thought to arise largely from GNPs and examination of a number of such tumors revealed high expression of ATF5. In summary, our in vivo and in vitro observations are consistent with the idea that ATF5 plays a required role in cerebellar neuroprogenitor cell proliferation and suggest that Shh signaling is involved in ATF5 regulation in this population. Our findings also suggest a potential role for ATF5 in medulloblastomas.