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Neural precursor cell (NPC) dysfunction has been consistently implicated in autism. Induced pluripotent stem cell (iPSC)-derived NPCs from two autism groups (three idiopathic [I-ASD] and two 16p11.2 deletion [16pDel]) were used to investigate if proliferation is commonly disrupted. All five individuals display defects, with all three macrocephalic individuals (two 16pDel, one I-ASD) exhibiting hyperproliferation and the other two I-ASD subjects displaying hypoproliferation. NPCs were challenged with bFGF, and all hyperproliferative NPCs displayed blunted responses, while responses were increased in hypoproliferative cells. mRNA expression studies suggest that different pathways can result in similar proliferation phenotypes. Since 16pDel deletes MAPK3, P-ERK was measured. P-ERK is decreased in hyperproliferative but increased in hypoproliferative NPCs. While these P-ERK changes are not responsible for the phenotypes, P-ERK and bFGF response are inversely correlated with the defects. Finally, we analyzed iPSCs and discovered that 16pDel displays hyperproliferation, while idiopathic iPSCs were normal. These data suggest that NPC proliferation defects are common in ASD.

Abnormalities in genes that regulate early brain development are known risk factors for neurodevelopmental disorders. Engrailed-2 (En2) is a homeodomain transcription factor with established roles in cerebellar patterning. En2 is highly expressed in the developing mid-hindbrain region, and En2 knockout (KO) mice exhibit major deficits in mid-hindbrain structures. However, En2 is also expressed in forebrain regions including the hippocampus, but its function is unknown. Previous studies have shown that the hippocampus of En2-KO mice exhibits reductions in its volume and cell numbers due to aberrant neurogenesis. Aberrant neurogenesis is due, in part, to noncell autonomous effects, specifically, reductions of innervating norepinephrine fibers from the locus coeruleus. In this study, we investigate possible cell autonomous roles of En2 in hippocampal neurogenesis. We examine proliferation, survival, and differentiation using cultures of hippocampal neurospheres of P7 wild-type (WT) and En2-KO hippocampal neural progenitor cells (NPCs). At 7 days, En2-KO neurospheres were larger on average than WT spheres and exhibited 2.5-fold greater proliferation and 2-fold increase in apoptotic cells, similar to in vivo KO phenotype. Further, En2-KO cultures exhibited 40% less cells with neurite projections, suggesting decreased differentiation. Lastly, reestablishing En2 expression in En2-KO NPCs rescued excess proliferation. These results indicate that En2 functions in hippocampal NPCs by inhibiting proliferation and promoting survival and differentiation in a cell autonomous manner. More broadly, this study suggests that En2 impacts brain structure and function in diverse regions outside of the mid-hindbrain.

Adenosine acts as a neuromodulator and metabolic regulator of the brain through receptor dependent and independent mechanisms. In the brain, adenosine is tightly controlled through its metabolic enzyme adenosine kinase, which exists in a cytoplasmic (ADK-S) and nuclear (ADK-L) isoform. We recently discovered that ADK-L contributes to adult hippocampal neurogenesis regulation. Although the cerebellum is a highly plastic brain area with a delayed developmental trajectory, little is known about the role of ADK. Here we investigated the developmental profile of ADK expression in C57BL/6 mice cerebellum and assessed its role in developmental and proliferative processes. We found high levels of ADK-L during cerebellar development, which was maintained into adulthood. This pattern contrasts with that of the cerebrum, in which ADK-L expression is gradually downregulated postnatally and largely restricted to astrocytes in adulthood. Supporting a functional role in cell proliferation, we found that the ADK inhibitor 5-iodotubercidin reduced DNA synthesis of granular neuron precursors in a concentration-dependent manner in vitro. In the developing cerebellum, immunohistochemical studies indicated ADK-L is expressed in immature Purkinje cells and granular neuron precursors, whereas in adulthood, ADK is absent from Purkinje cells, but widely expressed in mature granule neurons and their molecular layer processes. Furthermore, ADK-L is expressed in developing and mature Bergmann glia in the Purkinje cell layer, and in astrocytes in major cerebellar cortical layers. Together, our data demonstrate an association between neuronal ADK expression and developmental processes of the cerebellum, which supports a functional role of ADK-L in the plasticity of the cerebellum. Significance statement The role through which adenosine metabolism functions in the developing and adult cerebellum is poorly understood. Here we investigated the expression and possible function of ADK during cerebellum development. We report that ADK-L expression is associated with cerebellar development and linked to neural progenitor cell proliferation in vitro. In contrast to cerebrum, the adult cerebellum maintained high levels of both isoforms of ADK (L and S).

The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57(Kip2) and p27(Kip1), control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57(Kip2) remain poorly defined. Using in vivo and culture approaches, we show p57(Kip2) overexpression at E14.5-15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57(Kip2)-deficient precursors. Studies at later ages indicate p57(Kip2) overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57(Kip2) overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57(Kip2) was twice as effective as p27(Kip1) in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57(Kip2) alone inhibits in vivo migration. Furthermore, p57(Kip2) effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27(Kip1) studies report cell cycle-independent functions. These observations suggest p57(Kip2) coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27(Kip1).

Although survival-promoting effects of insulin-like growth factor-1 (IGF-1) during neurogenesis are well characterized, mitogenic effects remain less well substantiated. Here, we characterize cell cycle regulators and signaling pathways underlying IGF-1 effects on embryonic cortical precursor proliferationin vitroandin vivo. In vitro, IGF-1 stimulated cell cycle progression and increased cell number without promoting cell survival. IGF-1 induced rapid increases in cyclin D1 and D3 protein levels at 4 h and cyclin E at 8 h. Moreover, p27KIP1and p57KIP2expression were reduced, suggesting downregulation of negative regulators contributes to mitogenesis. Furthermore, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway specifically underlies IGF-1 activity, because blocking this pathway, but not MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase), prevented mitogenesis. To determine whether mechanisms defined in culture relate to corticogenesisin vivo, we performed transuterine intracerebroventricular injections. Whereas blockade of endogenous factor with anti-IGF-1 antibody decreased DNA synthesis, IGF-1 injection stimulated DNA synthesis and increased the number of S-phase cells in the ventricular zone. IGF-1 treatment increased phospho-Akt fourfold at 30 min, cyclins D1 and E by 6 h, and decreased p27KIP1and p57KIP2expression. Moreover, blockade of the PI3K/Akt pathwayin vivodecreased DNA synthesis and cyclin E, increased p27KIP1and p57KIP2expression, and prevented IGF-1-induced cyclin E mRNA upregulation. Finally, IGF-1 injection in embryos increased postnatal day 10 brain DNA content by 28%, suggesting a role for IGF-1 in brain growth control. These results demonstrate a mitogenic role for IGF-1 that tightly controls both positive and negative cell cycle regulators, and indicate that the PI3K/Akt pathway mediates IGF-1 mitogenic signaling during corticogenesis.

While extracellular signals play a major role in brain neurogenesis, little is known about the cell cycle machinery underlying mitogen stimulation of precursor proliferation. Current models suggest that the D cyclins function as primary sensors of extracellular mitogens. Here we define the mechanisms by which basic fibroblast growth factor (bFGF) stimulates cortical precursors, with particular attention to the responses of cell cycle promitogenic and antimitogenic regulators. bFGF produced a 4-fold increase in DNA synthesis and a 3-fold rise in bromodeoxyuridine labeling, suggesting that the factor promotes the G1/S transition. There was also a 3-fold increase in cyclin-dependent kinase 2 (CDK2) kinase activity, which is critical for S phase entry. CDK2 activation was apparently cyclin E dependent, since only its protein and mRNA levels were elevated at 24 h, whereas CDK2, p27KIP1 and p57KIP2 levels were unaltered. Late G1 phase CDK2/cyclin E activity depends on early G1 D cyclin function. Indeed, cyclin D1, but not cyclin D3, was upregulated selectively at 8 h after bFGF treatment, a time when cyclin E was unchanged. The sequential activation of cyclin D1 and cyclin E supports the idea that cyclin E gene transcription is regulated by cyclin-D/CDK4/6-mediated pRb phosphorylation and subsequent E2F transcription factor release. However, in addition to increased D1 cyclin, we unexpectedly detected a 75% reduction in p27KIP1 protein at 8 h, suggesting that both pro- and antimitogenic regulators are targets of extracellular mitogens during brain development.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors that through its neurotrophic tyrosine kinase, receptor, type 2 (TrkB) receptor, increases 5-bromo-2-deoxyuridine incorporation in oligodendrocyte progenitor cells (OPCs) in culture. Roles in vivo are less well understood; however, increases in numbers of OPCs are restricted in BDNF+/− mice following cuprizone-elicited demyelination. Here, we investigate whether these blunted increases in OPCs are associated with changes in proliferation. BDNF+/+ and BDNF+/− mice were fed cuprizone-containing or control feed. To assess effects on OPC numbers, platelet-derived growth factor receptor alpha (PDGFRα)+ or NG2+ cells were counted. To monitor DNA synthesis, 5-ethynyl-2′-deoxyuridine (EdU) was injected intraperitoneally and colocalized with PDGFRα+ cells. Alternatively, proliferating cell nuclear antigen (PCNA) was colocalized with PDGFRα or NG2. Labeling indices were determined in the BDNF+/+ and BDNF+/− animals. After 4 or 5 weeks of control feed, BDNF+/− mice exhibit similar numbers of OPCs compared with BDNF+/+ animals. The labeling indices for EdU and PCNA also were not significantly different, suggesting that neither the DNA synthesis phase (S phase) nor the proliferative pool size was different between genotypes. In contrast, when mice were challenged by cuprizone for 4 or 5 weeks, increases in OPCs observed in BDNF+/+ mice were reduced in the BDNF+/− mice. This difference in elevations in cell number was accompanied by decreases in EdU labeling and PCNA labeling without changes in cell death, indicating a reduction in the DNA synthesis and the proliferative pool. Therefore, levels of BDNF influence the proliferation of OPCs resulting from a demyelinating lesion.

The Eph family of tyrosine kinase receptors and ligands play key roles in cell segregation and axon targeting in the developing nervous system. Interactions between the ligands and receptors cause repulsion or degeneration of receptor-positive axons from several brain regions including the retina, hippocampus, thalamus, and midbrain dopaminergic system. We extend these previous observations by showing that three A-ephrins also negatively regulate the growth of neurites from striatal and olfactory neurons. In addition to negative effects, however, we also report a trophic activity of the A-ephrins: Ephrin-A2 and A5 promote survival and neurite outgrowth of sympathetic neurons. These observations provide support to the notion that ephrins may function as either negative or positive signals in the developing nervous system.

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor (PAC1) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via PAC1 receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via PAC1 receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular Ca2+, the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis.

Once thought only to contribute to cell adhesion as a structural link between a cell's cytoskeleton and its surface, the protein-catenin now appears to be a key regulator of cytoskeletal dynamics and cell proliferation.

Background The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes. Methods To define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted. Results Proliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation. Conclusions In aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis.

Neurons throughout the cen- tral nervous system (CNS) undergo proliferation, migration, and differentiation during their histo- genesis. Although numerous regulatory mol- ecules are expressed in developing neurons, it is unknown whether most of these molecules have the same function throughout the CNS or play different roles in different neuronal popula- tions. Previous studies have shown that Id-1 and NSCL-1 are expressed at high levels in the ven- tricular and subependymal zones, respectively, of the embryonic brain. In the present study, the expression of Id-1 and NSCL-1 was further inves- tigated during postnatal development of the cer- ebellum. By Northern blot hybridization analy- sis, the expression levels of Id-1 and NSCL-1 mRNA were developmentally regulated in the cerebellum, with the highest mRNAlevels coincid- ing with the time of maximal granule cell histo- genesis. By in situ hybridization, NSCL-1 mRNA was found in the premigratory zone of the exter- nal granule layer (EGL), a structure developmen- tally analogous to the subependymal zone of the embryonic brain. In normal mice, Id-1 mRNA was found to be transiently expressed in the upper internal granule layer (IGL), a population of cells that recently completed their migration from the EGL. In the mouse mutant weaver ,I d mRNA was only seen in granule cells that have reached their normal positions in the IGL. No Id-1 hybridization signal was observed in the large numbers of granule cells remaining in the EGL of weaver mice, indicating that Id-1 expres- sion is controlled by spatial cues. The lack of Id-1 expression in ectopic weaver granule cells is compatible with previous suggestions of arrested differentiation. These results support the idea that transcriptional regulators of the helix- loop-helix gene family play important roles in neuronal development, exhibiting region-specific expression and function. Dev Dyn 208:107-114

During nervous system development, the generation of neuronal populations is subject to regulation by extracellular growth factors. Traditional views suggest that growth factors promote proliferation by increasing the fraction of precursors that enter the mitotic cycle and subsequently divide (that is, enhance mitosis). However, recent evidence indicates that dividing precursors may undergo cell death. Consequently, a given molecule may also increase neuroblast proliferation by promoting survival of dividing precursors. In the present work, we developed a new approach to distinguish these two distinct effects of growth factors on dividing neuroblasts. By using a brief 6 h culture paradigm of embryonic day 15.5 sympathetic neuroblasts, we minimized cell death, thereby excluding the survival-promoting (trophic) activity of growth factors. In the absence of trophism, measured increases in [3H]thymidine incorporation reflected growth factor mitogenic activity only. Using a well-characterized sympathetic model, we found that insulin, EGF and vasoactive intestinal peptide (VIP) increased [3H]thymidine incorporation 30%, 20% and 46% respectively, consistent with their previously reported mitogenic activity. In contrast, neurotrophin-3 (NT3) and nerve growth factor (NGF), which serve as trophic signals for the neuroblasts, did not elicit any change in [3H]thymidine incorporation, indicating that the neurotrophins are not mitogenic for sympathetic precursors. This approach may be useful in distinguishing mitogenic and trophic regulation of proliferation in other brain precursor populations.

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57(KIP2), an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57(KIP2) regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27(KIP1) controlled IPC proliferation exclusively. Furthermore, p57(KIP2) deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27(KIP1) increased IPC proliferation at E16.5. Consequently, loss of p57(KIP2) increased primarily layer 5-6 neuron production, whereas loss of p27(KIP1) increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57(KIP2) and p27(KIP1) control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation.

The members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitory proteins (CKIs), including p57KIP2, p27KIP1, and p21CIP1, block the progression of the cell cycle by binding and inhibiting cyclin/CDK complexes of the G1 phase. In addition to this well-characterized function, p57KIP2 and p27KIP1 have been shown to participate in an increasing number of other important cellular processes including cell fate and differentiation, cell motility and migration, and cell death/survival, both in peripheral and central nervous systems. Increasing evidence over the past few years has characterized the functions of the newest CIP/KIP member p57KIP2 in orchestrating cell proliferation, differentiation, and migration during neurogenesis. Here, we focus our discussion on the multiple roles played by p57KIP2 during cortical development, making comparisons to p27KIP1 as well as the INK4 family of CKIs. © 2011 Wiley Periodicals, Inc. Develop Neurobiol 72: 821–842, 2012

While mammalian neurogenesis has been characterized extensively, the molecules involved in regulating neural cell determination and differentiation remain ill-defined. There is accruing evidence that various members of the basic helix-loop-helix (bHLH) protein family critically regulate these biological processes in a number of tissues. Id, a negative regulator of bHLH proteins, was found to exhibit peak gene expression during mouse embryogenesis with a striking pattern in the central nervous system. Id transcripts were specifically localized to undifferentiated neural precursors of the ventricular zone and were not present in their differentiated derivatives. In addition, in the peripheral nervous system, dorsal root ganglia sensory precursors, known to be undifferentiated while dividing, also expressed Id mRNA. However, in the sympathetic nervous system and adrenal medulla, where differentiation and division occur simultaneously in precursors, Id was not expressed. Since Id transcript abundance inversely correlated with differentiation, this protein, similar to its Drosophila homolog, extramacrochaetae, may play a negative regulatory role in neural 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.

As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to “masked” antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.

Hedgehog (HH) is a secreted protein named for the bristle phenotype observed in Drosophila embryos that lack the corresponding gene. Three homologs have been characterized in vertebrates, all which have critical roles in the development of multiple organ systems. Moreover, these proteins regulate stem cell production and activation during tissue repair after injury, and appear to drive proliferation in a variety of type of tumors, including those arising in the brain, foregut, lung, breast, pancreas, stomach, and prostate. Early evidence from Drosophila, and later work in vertebrates established the cAMP/protein kinase A (PKA) pathway as a major pathway which opposes HH signaling, doing so by phosphorylating intracellular signaling mediators and targeting them for degradation. Thus, it seems possible that ligands which activate G protein-coupled receptors (GPCR) may act in some cases to oppose or enhance HH signaling. We studied a possible interaction of pituitary adenylyl cyclase-activating peptide (PACAP) with sonic hedgehog (SHH) in the developing cerebellum, where both PACAP and SHH are know to act. PACAP and the PAC1-specific agonist, maxadilan, were found to completely block the proliferative action of SHH on developing cerebellar granule neurons. It remains to be determined if HH/GPCR antagonistic interactions play additional important roles in development, plasticity, tissue repair, cancer, and other processes.

The developing brain is highly sensitive to methylmercury (MeHg). Still, the initial changes in cell proliferation that may contribute to long-term MeHg effects are largely undefined. Our previous studies with growth factors indicate that acute alterations of the G1/S-phase transition can permanently affect cell numbers and organ size. Therefore, we determined whether an environmental toxicant could also impact brain development with rapid (6–7 h) effects on DNA synthesis and cell cycle machinery in neuronal precursors. In vivo studies in newborn rat hippocampus and cerebellum, two regions of postnatal neurogenesis, were followed by in vitro analysis of two precursor models, cortical and cerebellar cells, focusing on the proteins that regulate the G1/S transition. In postnatal day 7 (P7) pups, a single subcutaneous injection of MeHg (3 μg/g) acutely (7 h) decreased DNA synthesis in the hippocampus by 40% and produced long-term (2 weeks) reductions in total cell number, estimated by DNA quantification. Surprisingly, cerebellar granule cells were resistant to MeHg effects in vivo at comparable tissue concentrations, suggesting region-specific differences in precursor populations. In vitro, MeHg altered proliferation and cell viability, with DNA synthesis selectively inhibited at an early timepoint (6 h) corresponding to our in vivo observations. Considering that G1/S regulators are targets of exogenous signals, we used a well-defined cortical cell model to examine MeHg effects on relevant cyclin-dependent kinases (CDK) and CDK inhibitors. At 6 h, MeHg decreased by 75% levels of cyclin E, a cell cycle regulator with roles in proliferation and apoptosis, without altering p57, p27, or CDK2 nor levels of activated caspase 3. In aggregate, our observations identify the G1/S transition as an early target of MeHg toxicity and raise the possibility that cyclin E degradation contributes to both decreased proliferation and eventual cell death.

Atrial natriuretic peptide (ANP) binding sites have been detected in the embryonic brain, but the specific receptor subtypes and biological functions for ANP family ligands therein remain undefined. We now characterize the patterns of gene expression for the natriuretic peptides [ANP, brain natriuretic peptide (BNP), type-C natriuretic peptide (CNP)] and their receptors (NPR-A, NPR-B, NPR-C) at several early stages in the embryonic mouse nervous system by in situ hybridization, and begin to define the potential developmental actions using cell culture models of peripheral (PNS) and central nervous systems (CNS). In the CNS, gene transcripts for CNP were present at the onset of neurogenesis, embryonic day 10.5 (E10.5), primarily in the dorsal part of the ventricular zone (VZ) throughout the hindbrain and spinal cord. On E14.5, new CNP signals were observed in the ventrolateral spinal cord where motor neurons reside, and in bands of cells surrounding the spinal cord and hindbrain, localized to dura and/or cartilage primordia. ANP and BNP gene transcripts were not detected in embryonic brain, but were highly abundant in the heart. The CNP-specific receptor (NPR-B) gene was expressed in cells just outside the VZ, in regions where post-mitotic neurons are differentiating. Gene expression for NPR-C, which recognizes all natriuretic peptides, was present in the roof plate of the hindbrain and spinal cord and in bilateral stripes just dorsolateral to the floor plate at E12.5. In the PNS, NPR-B and NPR-C transcripts were highly expressed in dorsal root sensory (DRG) and cranial ganglia beginning at E10.5, with NPR-C signal also prominent in adjoining nerves, consistent with Schwann cell localization. In contrast, NPR-A gene expression was undetectable in neural tissues.To define ontogenetic functions, we employed embryonic DRG and hindbrain cell cultures. The natriuretic peptides potently stimulated DNA synthesis in neuron-depleted as well as neuron-containing Schwann cell cultures and differentially inhibited neurite outgrowth in DRG sensory neuron cultures. CNP also exhibited modest survival-promoting effects for sensory neurons. In marked contrast to PNS effects, the peptides inhibited proliferation of neural precursor cells of the E10.5 hindbrain. Moreover, CNP, alone and in combination with sonic hedgehog (Shh), induced the expression of the Shh target gene gli-1 in hindbrain cultures, suggesting that natriuretic peptides may also modify patterning events in the embryonic brain. These studies reveal widespread, but discrete patterns of natriuretic peptide and receptor gene expression in the early embryonic nervous system, and suggest that the peptides play region- and stage-specific roles during the development of the peripheral and central nervous systems.

Developing axons are guided to their appropriate targets by environmental cues through the activation of specific receptors and intracellular signaling pathways. Here we report that gradients of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide widely expressed in the developing nervous system, induce marked attraction ofXenopusgrowth conesin vitro. PACAP exerted its chemoattractive effects through PAC1, a PACAP-selective G-protein-coupled receptor (GPRC) expressed at the growth cone. Furthermore, the attraction depended on localized cAMP signaling because it was completely blocked either by global elevation of intracellular cAMP levels using forskolin or by inhibition of protein kinase A using specific inhibitors. Moreover, local direct elevation of intracellular cAMP by focal photolysis of caged cAMP compounds was sufficient to induce growth cone attraction. On the other hand, blockade of Ca2+, phospholipase C, or phosphatidyl inositol-3 kinase signaling pathways did not affect PACAP-induced growth cone attraction. Finally, PACAP-induced attraction also involved the Rho family of small GTPases and required local protein synthesis. Taken together, our results establish cAMP signaling as an independent pathway capable of mediating growth cone attraction induced by a physiologically relevant peptide acting through GPCRs. Such a direct cAMP pathway could potentially operate in other guidance systems for the accurate wiring of the nervous system.

Generation of distinct cell types and numbers in developing cerebral cortex is subject to regulation by extracellular factors that positively or negatively control precursor proliferation. Although signals stimulating proliferation are well described, factors halting cell cycle progression are less well defined. At the molecular level, production and association of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) regulate cycle progression. We now report that the endogenous peptide, pituitary adenylate cyclase activating polypeptide (PACAP), negatively regulates the cell cycle by inhibiting p57(Kip2)-dependent CDK2 activity in embryonic cortex. Protein levels of CDK2 and members of the CIP/KIP family of CKIs (p27(Kip1), p57(Kip2)) were detected in developing rat cortex from embryonic day 13.5 through postnatal day 2. With advancing development, CDK2 protein levels decreased, whereas CKI expression increased, suggesting that stimulatory and inhibitory cycle proteins control cell cycle exit. Using a well defined, nonsynchronized, 8 hr precursor culture, PACAP decreased the fraction of cells crossing the G(1)/S boundary, inhibiting DNA synthesis by 35%. CDK2 kinase activity was inhibited 75% by PACAP, whereas kinase protein and its regulatory cyclin E subunit were unaffected. Moreover, decreased kinase activity was accompanied by a twofold increase in levels of p57(Kip2) protein, but not p21(Cip1)or p27(Kip1), suggesting that p57(Kip2) mediates PACAP anti-mitogenic effects. Indeed, immunoprecipitation of CDK2 complex revealed increased p57(Kip2) association with the kinase and concomitant reduction in free inhibitor after PACAP exposure, suggesting that p57(Kip2) interactions directly regulate CDK2 activity. These observations establish a mechanism whereby anti-mitogenic signals actively induce cell cycle withdrawal in developing cortex.

The Eph receptor ligands, the ephrins, are membranebound molecules that play important roles in establishing intercellular communication after neurogenesis by regulating cell migration, axon pathfinding, and topographic mapping. In diverse systems, such as embryonic day 17.5 (E17.5) hippocampal and cortical neurons, repulsive/inhibitory mechanisms underlie these cellular effects. However, although ligand/receptor expression occurs far earlier, during brain neurogenesis, little is known about potential ephrin functions in initial process outgrowth. We have examined ligand/receptor expression in E13.5 cortex in vivo and in culture, using alkaline phosphatase (AP)-conjugated reagents and RNase protection assay. B ephrins are highly expressed, including B1, B2, and B3, whereas A ephrins exhibit low expression levels. In contrast, the Eph receptors demonstrate an opposite pattern, exhibiting high levels of Eph A3, A4, and A5 mRNA transcripts and low levels of the B-class receptors. To examine effects on neurite outgrowth, soluble ephrins were incubated with antihuman IgG antibody, producing oligomeric agonist complexes, and dried onto culture dishes. Unexpectedly, both ephrin A and B complexes increased process outgrowth: Seventy to eighty percent of neuronal precursors exhibited long neurites on ephrins, whereas only 5‐10% of cells had neurites on IgG control substrates, indicating that ephrins stimulated neuritogenesis by early cortical neurons. These observations suggest that ephrin ligand/ receptor systems play ontogenetic roles not previously considered, activating mechanisms other than cellular repulsion. Ephrin systems may induce initial process elaboration by early cortical neurons that is restricted at later stages by well-characterized repulsive signaling mechanisms. J. Neurosci. Res. 66:1054 ‐1063, 2001. © 2001 Wiley-Liss, Inc.

Pituitary adenylate cyclase activating peptide (PACAP) may play a role in neurogenesis, nerve injury, and neural tumor growth. A PACAP ligand receptor system functionally coupled to cAMP production was found to be expressed in the embryonic mouse neural tube at the onset of neurogenesis. PACAP was found to inhibit DNA synthesis and antagonize sonic hedgehog signaling in cells isolated from the neural tube, suggesting that PACAP interacts with patterning factors to regulate neurogenesis and phenotypic specification in the developing CNS. PACAP and PACAP receptor (PAC1) mRNA levels were strongly increased and decreased, respectively, in motor neurons in adult rats after facial nerve axotomy, indicating that PACAP may also act in nerve regeneration. Experiments using a neuroblastoma tumor cell line model indicate that PACAP may execute growth-related functions by activating MAP kinase in addition to cAMP-dependent protein kinase A.

Neuronal birth and neurite outgrowth have been regarded as discrete, sequential stages of development. However, we recently found that sympathetic neuroblasts often elaborate axons before mitosis, in culture [E. Wolf, I.B. Black, E. DiCicco-Bloom, Mitotic neuroblasts determine neuritic patterning of progeny, J. Comp. Neurol. 367 (1996) 623–635] and in vivo [E. Wolf, I.B. Black, E. DiCicco-Bloom, Central and peripheral neuroblasts elaborate neurites prior to division in vivo and in vitro, Soc. Neurosci. Abstr. 21 (1995) 785; E. Wolf, I.B. Black, E. DiCicco-Bloom, Mitotic neuroblasts engage in axonal outgrowth and pathfinding in vivo, Soc. Neurosci. Abstr. 22 (1996) 525]. Here, we report that cerebellar granule cells often divide with heritable neurites in vitro. Therefore, mitotic CNS precursors, in addition to peripheral neuroblasts, simultaneously undergo proliferation and process formation. Potentially, neurites on dividing precursors may allow target fields to influence directly the course of neurogenesis.

Neuronal precursor proliferation and axodendritic outgrowth have been regarded as strictly sequential, with process formation presumably beginning after mitotic activity ceases. We now report that sympathetic precursors in vitro often elaborate long neurites before dividing. Of 437 neuroblasts observed in 48 time-lapse recordings, 42 neuroblasts divided. Thirty (71%) of these mitotic neuroblasts had neurites prior to cytokinesis. “Paramitotic” neurites were found to contain microtubules (MTs), indicating that precursors elaborate neuritic cytoskeleton during proliferation. Remarkably, the precise neuritic pattern exhibited by parental neuroblasts was consistently reproduced by daughter cell pairs. Preservation of neuritic morphology occurred through asymmetric division, with individual neurites allocated to specific daughter cells. Paramitotic neurites either remained intact throughout mitosis (12 of 65), or “retracted” into the soma during prophase and then “regrew” within minutes after cytokinesis (53 of 65). “Retraction” and “regrowth” involved resorption of cytoplasm into the soma, then refilling of residual cell membrane, resulting in recapitulation of the parental neurite pattern. Paramitotic neuritogenesis appears to be intrinsically driven, but is responsive to environmental signals. The culture substrate influenced neurite length, but not the response of paramitotic neurites during mitosis or the preservation of neuritic morphology. However, the incidence of neurite-bearing neuroblasts increased from 38 ± 1.3% to 94 ± 1.1% with growth factor treatment. The surprisingly high incidence of paramitotic neurites and the fidelity with which patterning was conserved across cell generations raise the possibility that mitotic precursors engage in pathfinding. Our studies suggest a novel link between neurogenesis and cytoarchitectonic patterning. © 1996 Wiley-Liss, Inc.

Although microglia-mediated cytotoxicity has been extensively investigated, little is known about the potential microglial role in neuronal and glial support. Characterization of trophin elaboration by microglia and identification of responsive populations may define novel functions. We now report that microglia/brain macrophages express neurotrophins of the nerve growth factor (NGF) gene family in vitro and in vivo, suggesting that these cells promote development and normal function of neurons and glia. Moreover, neurotrophins promote microglial proliferation and phagocytic activity in vitro. We found that microglia express neurotrophins in a region-specific manner and that within any region only subpopulations elaborate trophins. Using an antiserum specific for neurotrophin-3 (NT-3) with the microglial/macrophage marker OX-42 on postnatal day 10 in vivo, double- labeled cells were identified in the cerebral cortex, globus pallidus, and medulla; NT-3 was undetectable in OX-42-positive cells in the ependyma, the external capsule, choroid plexus, and meninges. In contrast, ramified microglia in the adult brain did not exhibit NT-3 immunoreactivity, suggesting developmental regulation of microglial NT- 3 expression. In situ hybridization studies on purified microglial cultures confirmed that only subpopulations express the NGF and NT-3 genes, substantiating the existence of microglial heterogeneity. We tentatively conclude that microglial subtypes serve trophic roles in the normal brain, in addition to exerting well documented deleterious actions in illness and injury. Microglia were also responsive to neurotrophins: brain-derived neurotrophic factor (BDNF) and NT-3 increased [3H]thymidine incorporation in vitro, and NT-3 promoted proliferation. Moreover, NT-3 induced phagocytic activity, suggesting that the factor plays a role in processes associated with cellular activation.

The present study examines the role of Ca2+ in the regulation of sympathetic neuroblast mitosis. Employing a fully defined neuroblast culture system, we previously found that insulin growth factors (IGFs), depolarization and vasoactive intestinal peptide (VIP) regulated precursor mitosis. We now report that Ca2+ entry via voltage-sensitive channels was required for depolarization-stimulated mitogenesis. Ca2+ channel blockade with nitrendipine completely inhibited the increase in [3H]thymidine incorporation elicited by depolarizing stimuli including 30 mM KCl and the Na+ channel agonist veratridine. However, Ca2+ channel activity was not involved in the stimulation of DNA synthesis by IGFs or VIP. Thus, neuroblast mitosis may be regulated by multiple intracellular as well as extracellular signals.

ALTHOUGH acute, millisecond-to-millisecond actions of neurotransinitters are well documented, diverse longer-term effects have been discovered only recently1–5. Emerging evidence indicates that these signals regulate a variety of neuronal processes, from phenotypic expression to neurite outgrowth6–19. Here we show that a single putative transmitter, vasoactive intestinal peptide, can exert multiple, long-term effects simultaneously: it stimulates mitosis, promotes neurite outgrowth and enhances survival of sympathetic neuron precursors in culture. As the peptide seems to be a normal presynaptic transmitter in the sympathetic system19–25, synaptic transmission may exert hitherto unexpected effects.

Although positive and negative signals control neurogenesis in the embryo, factors regulating postnatal proliferation are less well characterized. In the vertebrate cerebellum, Sonic Hedgehog (Shh) is an efficacious mitogen for cerebellar granule neuron precursors (GNPs), and mutations activating the Shh pathway are linked to medulloblastoma, a tumor derived from GNPs. Although the mitogenic effects of Shh can be blocked by increasing cAMP or protein kinase A activity, the physiological factors antagonizing this stimulation are undefined. In the embryo, pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1) signaling regulates neural precursor proliferation. We now show that in the developing cerebellum, PAC1 mRNA colocalizes with gene transcripts for Shh receptor Patched 1 and target gene Gli1 in the external germinal layer. We consequently investigated the interactions of PACAP and Shh in proliferation of purified GNPs in culture. Shh exhibited mitogenic activity in both rat and mouse cultures, stimulating DNA synthesis approximately 10-fold after 48 hr of exposure. PACAP markedly inhibited Shh-induced thymidine incorporation by 50 and 85% in rat and mouse GNPs, respectively, but did not significantly affect the stimulation induced by other mitogens. This selective effect was reproduced by the specific PAC1 agonist maxadilan, as well as by the adenylate cyclase activator forskolin, suggesting that PAC1 provides a potent inhibitory signal for Shh-induced proliferation in developing cerebellum. In contrast, in the absence of Shh, PACAP and maxadilan modestly stimulated DNA synthesis, an effect reproduced by activating protein kinase C. These observations suggest that G-protein-coupled receptors, such as PAC1, serve as sensors of environmental cues, coordinating diverse neurogenetic signals.