Engrailed2 modulates cerebellar granule neuron precursor proliferation, differentiation and insulin-like growth factor 1 signaling during postnatal development
© Rossman et al.; licensee BioMed Central Ltd. 2014
Received: 12 April 2013
Accepted: 14 January 2014
Published: 7 February 2014
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.
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.
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.
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.
KeywordsAutism Engrailed2 IGF1 Cerebellum Neurodevelopment Cell cycle Proliferation Phospho-S6 kinase
The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the organization of the midbrain/hindbrain region and the cerebellum, where it exhibits dynamic, prenatal and postnatal expression patterns and complex regional functions [1, 2]. Because of its roles in regulating cerebellar morphogenesis and Purkinje neuron development, and evidence of cerebellar pathology in human disease [3–6], we previously examined ENGRAILED2’s association with human autism spectrum disorder (ASD) and found evidence to support EN2 as an ASD susceptibility gene. These results, initially observed in 167 families, were subsequently replicated in two additional data sets (518 families; P = 0.00000035), and six other groups have demonstrated EN2 association with ASD [7–12].
In the developing mouse brain, En2 restricts the fate of progenitor cells to a midbrain/hindbrain lineage and regulates cerebellar growth, patterning and connectivity. For example, En2 deletion mutants exhibit hypoplastic cerebella with reduced numbers of Purkinje neurons as well as foliation defects and mistargeted spinocerebellar afferents [13–17]. Interestingly, transgenic misexpression of En2 that increases gene expression in postnatal cerebellum also produced similar phenotypes, suggesting that proper levels of En2 expression are required for normal development [18–20]. The fetal expression of En2 in the mouse follows a complex pattern, initially expressed diffusely at the mid-hindbrain junction of the brainstem, but becoming increasingly restricted to the postnatal, developing cerebellum [1, 21, 22]. While the major cerebellar output neurons, the Purkinje cells, are generated prenatally, cerebellar expansion and its adult morphology result from massive proliferation of the granule neuron precursors (GNP) located in the postnatal external germinal layer (EGL) covering the cerebellum [13, 16, 18, 23]. In the EGL, GNPs proliferate in the outer portion, whereas postmitotic precursors start differentiating in the inner layer. Significantly, En2 gene expression is increased in GNP as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may participate in these developmental processes [14, 15, 24]. Because En2 mutants have decreased numbers of Purkinje neurons that provide mitogenic growth factors for GNP proliferation, cerebellar hypoplasia has been considered a consequence of their deficiency. However, as an alternative hypothesis, En2 expression in GNPs themselves may play a cell-autonomous role in regulating proliferation and differentiation. We now explore the function of En2 during GNP development by comparing wild-type (WT) and knockout (KO) GNPs both in vivo and in culture, as well as by using En2 overexpression constructs.
ASD is a highly heritable genetic disorder [25, 26], with interactions between multiple susceptibility genes as well as environmental factors manifesting as diverse clinical presentations . How individual susceptibility genes such as EN2 contribute to disease risk (individually and in aggregate with other genes) remains to be elucidated. Cerebellar granule neurons are the largest population of neurons in the brain, and the only major population to continue neurogenesis postnatally, compared to more limited adult neurogenesis in the forebrain [23, 28, 29]. In humans, this process extends through infancy, the period when ASD symptoms first manifest. Significantly, multiple lines of evidence suggest that cerebellar dysfunction contributes to ASD symptomology . Neuropathological studies demonstrate Purkinje neuron deficits in the majority of brains examined to date, whereas structural MRIs indicate that subsets of individuals have hypoplastic cerebellar vermis and others have enlarged cerebellar hemispheres [5, 30, 31]. Functional brain imaging studies suggest that cerebellar dysfunction contributes to the motor, cognitive and language deficits observed in ASD [4, 32–35]. Thus, a disruption of early postnatal cerebellar development could potentially contribute to ASD pathogenesis. From this perspective, by defining the role of En2 in postnatal GNP neurogenesis, we may identify cellular pathways by which variations in the levels of En2 expression may contribute to disordered cerebellar development, potentially providing insight into ASD pathophysiology. We now define En2 function in postnatal cerebellar development, specifically the period when GNPs transition from proliferation to differentiation concomitantly with their expression of En2[15, 36]. We demonstrate that without En2, GNPs favor proliferation over differentiation, whereas En2 overexpression promotes GNP cell cycle exit and differentiation. Furthermore, we identify previously unrecognized interactions between En2 and IGF1 signaling.
Animals and genotyping
Time-mated Sprague-Dawley rats were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA) and maintained on a 12:12 light:dark cycle with ad libitum Purina rat chow and water. Rats were killed by CO2 gas asphyxiation as approved by RWJMS IACUC. Male and female breeding pairs of C57BL6 J/129S2SV PAS mice heterozygous (HT) for a functional deletion of En2 were obtained from The Jackson Laboratory (no. 002657; Bar Harbor, ME) and maintained. An En2 mutant colony was propagated by HT × HT intercrosses, and genotyping of the progeny was performed as described (jaxmice.jax.org). The mice were initially maintained as heterozygous matings, but, to generate adequate numbers of pups of known genotype for GNP cultures, WT × WT and KO × KO mating pairs were established.
All animal procedures were assessed and approved by the Robert Wood Johnson Medical School Institutional Animal Care and Use Committee. Animals were managed by Robert Wood Johnson Animal Facility, and maintenance, husbandry, transportation, housing and use were in compliance with the Laboratory Animal Welfare Act (PL 89–544; PL-91-579) and National Institutes of Health guidelines (Manual Chapter 4206).
A full-length En2 cDNA was cloned from C57BL6 J/129S2SV PAS adult mouse cerebellum as described previously . To identify transfected cells, the En2 coding sequence was moved to the pCMS-EGFP expression vector (Clontech, Mountain View, CA) by EcoRI digestion and ligation. Orientation of positive clones was determined by Kpn1 digestion.
Mouse and rat cerebellar granule neuron precursor (GNP) culture
Postnatal day (P)7 rat and WT or KO mice were decapitated and cerebellar GNPs isolated as described previously [37, 38]. Briefly, 3–6 cleaned cerebella were incubated 3 min in trypsin-DNase solution (1% trypsin, 0.1% DNase, Worthington, Lakewood, NJ) and dissociated in DNase solution (0.05% in DMEM) by trituration. After pelleting, cells were filtered (30-u m nylon mesh; Tekton, Tarrytown, NY), resuspended and centrifuged at 3,200 rpm on a Percoll (Sigma, St. Louis, MO) 35:60% step gradient [39, 40]. Cells at the 35:60% interface were collected and washed in phosphate buffer, then plated onto a poly-D-lysine (0.1 mg/ml)-coated 60-mm culture dish in defined medium (DM) composed of a 1:1 mixture of F12 and DMEM, 10 ng/ml insulin, 100 u g/ml transferrin, 10 mg/ml bovine serum albumin, 100 u M putrescine, 20 nM progesterone, 30 nM selenium, 6 mg/ml glucose, 50 U/ml penicillin and 50 u g/ml streptomycin. Unless stated otherwise, components were obtained from Sigma. After 1 h of preplating to remove adherent flat cells (<2% of cells), small round (granule) cells were dislodged by gentle pipetting and plated at ~104-5 × 104/cm2 on poly-D-lysine-coated 35-mm Nunc (Thermo Fischer Scientific, Rochester, NY) culture dishes or 24 multiwells. Cultures were maintained in a humidified 5% CO2/air incubator at 37°C.
For thymidine incorporation studies, cells were cultured in DM alone or with additional factors: high-dose insulin (10 u g/ml), 1–100 ng/ml IGF1 (Cell Sciences, Canton, MA), 0.1-3 u g/ml Shh-N (N-terminal fragment, R&D Systems, Minneapolis, MN), 10-12-10-7 M PACAP1-38 in 0.01 N acetic acid vehicle (American Peptide, Sunnyvale, CA), 10 ng/ml FGF2 (gift of Scios, Mountain View, CA), 10–30 ng/ml BDNF (PeproTech, Rocky Hill, NJ), 100 ng/ml EGF, 100–300 ng/ml VEGF or 10-30% conditioned media (CM) harvested from control- or Wnt3a-transfected fibroblasts.
For differentiation, cells were assessed live 24 h post plating by phase microscopy or were fixed in 4% cold paraformaldehyde and processed for immunocytochemistry, then assessed by phase or fluorescence microscopy. To assess neurites, cells bearing processes greater than two-cell soma diameters were assessed at 24 h in the live state, as reported previously , or following fixation and immunostaining against beta-III-tubulin as described below. Cells in two or three 1-cm rows (1.0-1.5% of the culture dish area) bearing neurites >2 cell soma were counted blind in two to four dishes per group, and experiments were performed three or more times.
Assessment of DNA synthesis
Incorporation of 3H-deoxythymidine (3H-dT, 1u Ci/ml) was used to assess DNA synthesis, as described previously [38, 42, 43]. Cells were incubated with 3H-dT during the final 4 h of culture. DNA that had incorporated 3H-dT was collected onto glass fiber filters by a semiautomatic cell harvester (Skatron), and the radioligand was assessed by scintillation spectroscopy. To visualize cells synthesizing DNA, GNPs were exposed to the S-phase marker bromodeoxyuridine (BrdU) (10 u M, Sigma) during the final 4 h of incubation. After fixation, cells were either exposed to 2 N HCl (30 min) or pretreated with PBS/0.3% Triton X-100 and then DNaseI (500 U/ml, Worthington)  and subsequently processed for BrdU immunocytochemistry using monoclonal anti-BrdU (1:200; Dako, Carpinteria, CA) and visualized using a Vectastain avidin-biotin complex kit and Vector SG peroxidase substrate with a 3,3′-diaminobenzidine (DAB) chromogen (Vector Laboratories, Burlingame, CA) as previously described . The labeling index, defined as the proportion of total cells incorporating BrdU into the nucleus, was determined by scoring the cells in five randomly selected, non-overlapping fields in each of the two to four dishes per group per experiment.
cDNA transfection of GNPs
Rat and mouse GNPs were plated 2-4 × 105cells/cm2 onto 12- or 25-mm glass coverslips (VWR International, West Chester, PA) pretreated with 2 N HCl for 30 min, rinsed in dH2O for 30 min, serially washed in 90% and 100% ethanol and fire-polished, then coated with poly-D-lysine (0.1 mg/ml) and fibronectin (1 u g/cm2, Sigma). Transfection media consisted of Neurobasal supplemented with 2% B27 (Invitrogen) containing glutamine (Gln, 2 mM), penicillin (50 U/ml), streptomycin (50 mg/ml), BSA (1 mg/ml), and either FGF2 (10 ng/ml) for rat GNPs or Shh (3 u g/ml) and BDNF (30 ng/ml) for mouse GNPs. For both species, 1 h after plating, cells were gently washed with Neurobasal supplemented with 10 u g/ml Gln, then incubated with transfection reaction media containing Neurobasal supplemented with 2% B27 and 10 u g/ml Gln, and either Lipofectamine Plus or Lipofectamine LTX Plus (Invitrogen) and 1 u g/ml pCMS-EGFP (GFP) or 1.2 u g/ml pCMS-EGFP-En2 (En2). After 5 h, transfection reaction media were replaced with species-specific transfection media (as above) and cells were incubated an additional 24 h, then fixed in cold 4% paraformaldehyde. Successful transfection was determined by GFP autofluorescence, assessed by fluorescence microscopy prior to fixation or other procedures.
Following fixation, cells were incubated 1 h at room temperature (RT) in 33% goat, rabbit or horse serum, then 1 h at RT or overnight at 4°C in one of the following primary antibodies diluted in PBS/0.3% Triton X-100, 2% serum and 0.05% NaN3: TuJ1, monoclonal mouse anti-beta-III-tubulin (1:9,000, Covance Research, Inc., Berkeley, CA), monoclonal mouse anti-PCNA (F2) (1:1,000, Santa Cruz, Inc., Santa Cruz, CA) or polyclonal chicken anti-GFP (1:5,000, Chemicon). Cells were then incubated 1 h with biotinylated horse anti-mouse secondary antibody and visualized with DAB chromogen or, alternatively, Alexafluor 488 (green) or 594 (red) fluorescent goat anti-mouse, goat anti-rabbit, rabbit anti-chicken, rabbit anti-mouse or rabbit anti-goat secondary antibody (1:1,000, Chemicon), followed by 5 min incubation with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 u g/ml, Sigma). Glass coverslips were inverted and mounted onto glass slides with Fluoromount-G (EMS, Hatfield, PA) and assessed by fluorescence microscopy. Cells were counted blind from two randomly selected fields within each of ten predetermined regions covering greater than 80% of the coverslip, from 2–4 coverslips per group per experiment. Images were acquired at 400× with and without background subtraction via an Apotome filter using Axiovision 4.5 software (Carl Zeiss Microimaging, Inc., Thornwood, NY).
Protein collection and western blot analysis
Isolated P7 KO and WT GNPs (2.5 × 106 cells/ml) were incubated in 4 ml DM on poly-D-lysine-coated (0.01 mg/ml) 60-mm dishes. After 4 h incubation, 10 ng/ml IGF1 was administered, followed by 15 min to 8 h incubation, then cells were washed with PBS, lysed by 70 u l lysis buffer containing 20 mM HEPES-KOH, pH 7.5, 5 mM KCl, 0.5 mM MgCl2 with 0.5 mM dithiothretiol (DTT), 2 mM phenylmethylsulfonylfluoride (PMSF), 1 mM leupeptin and 3.5 mg/ml aprotinin, and collected by scraping with rubber policeman. Samples were stored at -80°C. For blotting, defrosted samples were lysed by sonication and centrifuged, and the supernatant was normalized to 0.1 M NaCl. Protein concentrations were determined by Bio-Rad protein assay (Bio Rad Laboratories, Inc., Hercules, CA), and bovine serum albumin (BSA) dilutions served as standard curve. Protein extracts (50 u g per lane) were analyzed by 12% SDS-PAGE and electrically transferred to a polyvinylidenediflouride (PVDF) membrane. The membrane was blocked with 5% milk or 5% BSA and incubated with primary antibody against phospho- or total Akt, phospho- or total ERK1/2, phospho- or total GSK3beta, phospho- or total S6 kinase (1:1,000, Cell Signaling Technology, Danvers, MA) or total actin (1:2500, Chemicon), followed by monoclonal anti-mouse or polyclonal anti-rabbit horseradish peroxidase-conjugated secondary antibody, and visualized with an enhanced chemiluminescence system (Pierce, Rockford, IL), as previously reported [44, 46].
In vivo IGF1 administration and cerebellar 3H-dT incorporation
Groups of 3–5 P7 KO and WT pups were injected with PBS and 0.01 N HCl vehicle or IGF1 (10 u g/g, Cell Sciences) subcutaneously (sc) at time zero, followed at 6 h by 3H-dT (5 u Ci/g, sc), and were killed at 8 h. Cerebella were cleaned of meninges, removed from the brainstem, weighed in pre-tared microcentrifuge tubes and homogenized in dH2O (volume = mass × 5). An aliquot was removed for determination of total isotope uptake into the tissue. In an equal aliquot, DNA was precipitated with 10% trichloroacetic acid, sedimented by centrifugation, and washed by resuspension and resedimentation. The final pellet was dissolved and counted along with the original aliquot in a scintillation spectrophotometer. 3H-dT incorporation was defined as the fraction of total radionuclide taken up by the whole cerebellum that was present in the precipitated DNA and was reported as percent incorporation [47, 48].
In vivo BrdU immunohistochemistry
Two groups of three P7 KO and WT mice were injected sc with BrdU (50 u g/g) and killed at 2 h. Whole brains were removed and drop-fixed in fresh, cold 4% paraformaldehyde. Brains were serially dehydrated in 50, 60, 70, 80, 95 and 100% ethanol, 100% butanol and 100% xylene under vacuum and embedded in paraffin wax. Brains were then serially sectioned at 5 μm parasagittally, and sections were mounted on glass slides, with 5–6 sections/slide collected and numbered consecutively. Every fifth slide from WT and every fourth from KO (due to the smaller cerebellar size) plus two slides from either side of the central-most slide representing the central vermis were deparaffinized and rehydrated for anti-BrdU immunohistochemical processing. BrdU incorporated in DNA was detected by monoclonal antibody (Becton-Dickinson), amplified by biotinylated secondary antibody (Vectastain ABC kits), visualized by peroxidase reaction with DAB and subsequently counterstained with basic fuchsin. BrdU immunoreactive GNPs in the dorsal outer external germinal layer of lobules IV and VIII were counted by camera-lucida microscopy, and the BrdU labeling index quantified as the number of BrdU-positive cells/total cells × 100%. Four to eight sections from the hemispheres and four to eight sections from the vermis were counted for each pup [37, 48].
Data are expressed as mean ± SEM. Statistical comparisons were made by unpaired two-tailed Student’s t test or two-way ANOVA using Excel (Microsoft) or Vassarstats Website for Statistical Computation (http://www.vassarstats.net), respectively.
Proliferation is enhanced in vivo in the absence of En2
Differences in proliferation between En2 KO and WT GNPs defined in vivo are preserved in culture
Differences in GNP proliferation in vivo may reflect the effects of distinct environmental signals such as growth factors derived from Purkinje neurons, which are reduced in the mutants, or alternatively, action of cell autonomous signals, such as En2 expression . Previously, we reported that in the absence of mitogens, specifically Shh, mouse GNPs in culture rapidly exit the cell cycle . Therefore, WT and KO GNPs were isolated from the cerebellum and cultured at low density in defined media without growth factors, and DNA synthesis was assessed at 24 h by BrdU immunocytochemistry. The proportion of GNPs in mitotic S-phase was two-fold higher in En2 KO cells (Figure 1E,F). Further, there were similar numbers of live KO and WT GNPs counted at 24 h (WT = 143 ± 10.4; KO = 120 ± 9.8, total cells/five fields ± SEM; N = 5-6 per genotype; p = 0.14). These in vitro results recapitulate the in vivo finding that En2 KO GNPs fail to exit the cell cycle at the same rates as WT GNPs, an effect not likely due to changes in survival. These data also suggest that En2 participates in cell-autonomous regulation of the GNP cell cycle, independent of extracellular growth factors in the EGL. However, this raises the question, does En2 also function to modulate the effects of extracellular signals on cell cycle regulation?
IGF1 stimulates enhanced proliferation in the absence of En2 in culture and in vivo
Growth factors secreted by underlying Purkinje cells have been well characterized as regulators of GNP proliferation, differentiation and survival [49–52]; therefore, abnormal GNP responses to growth factor signaling in the absence of En2 expression may contribute to the KO phenotype. To address this issue, we isolated En2 KO and WT GNPs and compared proliferation, differentiation and survival in culture without and with developmentally relevant extracellular growth factors.
To determine whether IGF1 signaling is differentially regulated by the absence of En2 in vivo, we administered a single subcutaneous injection of IGF1 (10u g/gbw) to P7 KO and WT mice and measured total cerebellar tissue DNA synthesis using radiolabeled 3H-dT thymidine. As previously described for effects of peripherally injected bFGF on neonatal cerebellum , changes in the magnitude of 3H-dT thymidine incorporation into whole cerebellar homogenates (see , Figure one) closely paralleled changes in the labeling index of isolated GNPs (see , Figure four), supporting the value of this approach. With regard to IGF1, previous studies indicate that peripherally administered growth factor actively passes the blood-brain-barrier via a saturable transport mechanism and penetrates the brain parenchyma [41, 57]. Preliminary studies conducted at 4, 8 and 12 h post-IGF1 injection, with a subcutaneous 3H-dT (5u Ci/g) injection 2 h prior to sacrifice, indicated the greatest increase in DNA synthesis was observed at 8 h. As we detected in culture, IGF1 significantly increased total cerebellar DNA synthesis in both genotypes in vivo. However, in the absence of En2, IGF1 elicited a far greater increase in cerebellar DNA synthesis, increasing 3H-dT incorporation by 28% over saline injection in the KO, whereas the increase was only 9.7% in the WT (Figure 2D). These data recapitulate in vivo that IGF1-induced mitogenesis is differentially regulated in the absence of En2 and suggest En2 negatively regulates IGF1 signaling during postnatal cerebellar development.
Differentiation is diminished in the absence of En2
However, there may be an alternative model for the ability of PACAP plus IGF1 to overcome the neurite outgrowth deficiency observed in KO GNPs. Specifically, the anti-mitogenic effect of PACAP on both WT and KO GNPs, as above (Figure 2C), may allow cells to differentiate that would have otherwise continued to proliferate. Thus, any anti-mitogenic signal would be expected to promote IGF1-induced neuritogenesis. To address this issue, KO and WT GNPs were cultured with FGF2, another anti-mitogenic signal, and effects of IGF1 were examined. FGF2, a strong anti-mitogenic signal in both WT and KO GNPs (Figure 2C), elicited reductions in neurite outgrowth in both genotypes, suggesting it is not a pro-differentiation signal either (Figure 3C). FGF2 significantly attenuated IGF1-induced neurite outgrowth by more than 30% in both genotypes (Figure 3C). Thus, FGF2 represents both an anti-mitogenic and anti-neuritogenic signal that is equally active in both KO and WT GNPs. These findings are consistent with at least one report suggesting FGF2 inhibits neurite outgrowth in cerebellar GNPs . Further, these data suggest KO GNP deficits in neurite outgrowth are not due to failed cell cycle exit, but rather a specific failure or delay in differentiation in the absence of En2.
In addition to regulating the cell cycle and differentiation, both PACAP and IGF1 exert trophic support for developing GNPs in the EGL [38, 52]. Therefore, differences observed in DNA synthesis and neurite outgrowth between WT and En2 KO GNPs could reflect differences in survival. To examine survival, cells were plated at low density (10,000 cells/cm2) to limit cell-cell contact and counted at 24 h in defined media without and with PACAP, IGF1, or PACAP and IGF1 together. Initial counts were taken at 2 h post plating to ensure equal numbers of cells were aliquoted and attached between genotypes. As reported previously, both PACAP and IGF1 increased the number of WT cells, and they exhibited a trend toward additivity when co-administered (Figure 3D). Significantly, En2 KO cells responded identically to WT GNPs, exhibiting similar increases in cell numbers when incubated with PACAP, IGF1 and combined factors (Figure 3D). Thus, the absence of En2 does not appear to be deleterious to GNP survival or to alter the trophic responses to growth factors. Further, these data suggest differences in DNA synthesis and neurite outgrowth observed between KO and WT GNPs are due to differentially regulated mechanisms underlying mitosis and differentiation, and not differences in cell survival or cell death.
IGF1 signaling is altered in the absence of En2
To determine whether IGF1 signaling through PI3K-Akt or MAPK is altered specifically in GNPs in the absence of En2, cells were isolated from each genotype and cultured at cell densities similar to those used in the 3H-dT proliferation assays. To assess IGF1 activity, KO and WT GNPs were cultured in defined media for 2 h without IGF1 and were then pulsed for 30 min with vehicle or 10 ng/ml IGF1. Both KO and WT GNPs exhibited almost no P-Akt activation in vehicle-treated control cultures (Figure 4B). Conversely, IGF1 elicited identical, robust phosphorylation of Akt in both genotypes. Previous studies show that PI3K pathway activation, measured by phosphorylation of Akt, occurs within minutes of growth factor treatment and becomes maximal at 1 h . However, our culture results on mitosis and differentiation were obtained following continuous growth factor exposure for 24 h. Therefore, experiments were repeated at several later time points, including IGF1 treatments of 15 min, 4 h and 8 h. Though IGF1 increased P-Akt early at 15 min, and to a lesser degree at 4 h and 8 h, there were no differences between the genotypes; further, phospho-GSK3-beta protein levels were also no different between genotypes (data not shown). Furthermore, IGF1 pulses did not significantly increase P-ERK at any time point, nor were P-ERK levels different between the genotypes (Figure 4C). This suggests that in our culture models, IGF1 preferentially activates the PI3K-Akt pathway, whereas the MAPK pathway is constitutively activated, similar to previous reports . Genotype-specific differences in IGF1 responses do not appear to be mediated by these dominant upstream pathways.
Given the dominance of the PI3K pathway in our culture models, differences in En2 KO and WT GNP responses to IGF1 were explored further downstream of Akt. The downstream Akt target, mammalian target of rapamycin (mTOR), is known to regulate cell cycle progression as well as protein translation via activation of S6 kinase (S6K) and eukaryotic initiation factor 4B (eIF4B) . In contrast to the upstream signals, IGF1 induced a differential genotype response in phospho-S6 kinase (P-S6K), eliciting a 25-fold increase in P-S6K levels in KO GNPs at 30 min, but inducing no significant increase in WT GNPs (Figure 4D). These data suggest that the differential genotype response of WT and KO GNPs to IGF1 may reflect a previously undefined interaction between En2 and downstream effector molecules of the IGF1-PI3K-Akt-mTOR signaling cascade.
Overexpression of En2 cDNA promotes cell cycle exit and differentiation
Given our findings that the absence of En2 resulted in increased GNP proliferation and decreased differentiation, it follows that overexpression should elicit opposite effects: increased GNP cell cycle exit and differentiation. Previously, we demonstrated that ectopic En2 overexpression in embryonic cortical precursors altered neurogenesis , and additional studies in HEK293 cells confirmed that transfection produces overexpression of En2 cDNA (not shown). Using these same vectors, we overexpressed En2 by lipid transfection of P7 GNPs.
Based on both loss- and gain-of-function approaches, our studies suggest En2 promotes GNP cell cycle exit and differentiation during postnatal cerebellar development. In the absence of En2, GNPs displayed increased proliferation markers in vivo and in culture. Further, GNPs elaborated fewer neuronal processes in response to growth factors but exhibited no differences in survival. Conversely, overexpression of En2 promoted cell cycle exit and stimulated neurite outgrowth, an effect demonstrated in both mouse and rat GNPs. Finally, we identified previously unknown interactions of En2 with IGF1 signaling. In the absence of En2, IGF1 elicited greater stimulation of DNA synthesis in culture and in vivo, effects associated with marked activation of phospho-S6K, while there were no changes in upstream PI3K and ERK signaling. Significantly, regulation of proliferation by other mitogenic signals was unaffected. These studies characterize En2’s roles in postnatal cerebellar GNP neurogenesis and differentiation and interactions with IGF1, which invite further study with respect to the role of En2 in the pathogenesis of development diseases where cerebellar structures are affected, such as ASD and schizophrenia.
En2 promotes cell cycle exit
During postnatal cerebellar development, outer EGL GNPs proliferate robustly, producing a large population of internal granule layer neurons. Underlying Purkinje neurons regulate this process by producing extracellular growth factors, including Shh, IGF1 and PACAP [49, 50, 75]. However, cell intrinsic mechanisms also regulate the GNP transition from proliferation to post-mitotic differentiation . While mechanisms remain undefined, we present evidence suggesting En2 serves to promote GNP cell cycle exit. These findings parallel evidence that outer EGL GNPs that are devoid of En2 remain proliferative while inner EGL GNPs begin expressing En2 as they exit the cycle [15, 24]. Indeed, when isolated in culture, GNPs from the WT mouse were less proliferative than those from the KO. These observations suggest En2 normally inhibits cell cycle progression in a cell-autonomous fashion, a model that is also supported by En2 overexpression that reduces S phase entry and precursor PCNA expression.
En2 modulates IGF1 signaling
Purkinje neurons secrete growth factors, such as Shh, FGF, IGF1, BDNF and PACAP, that support local GNP proliferation, survival and differentiation [37, 46, 49, 52, 65, 77–79]. We find En2 expression modulates GNP responses to IGF1, suggesting that the onset of En2 expression contributes to cell cycle exit and differentiation in the growth factor-rich environment of the EGL. In En2 KO GNPs, IGF1 induced two- to three-fold greater increases in DNA synthesis in culture and three-fold greater stimulation of cerebellar DNA synthesis in vivo. Importantly, En2 did not appear to regulate overall cell cycle machinery or signaling by other growth factors that employ signaling systems similar to IGF1, such as tyrosine-kinase engaging receptors or PI3K/Akt or MAPK pathways. Rather, in the absence of En2, IGF1 markedly upregulated P-S6K, an effect not observed in WT cells. While P-S6K is a well-defined regulator of mitogen-induced cell cycle progression , there were no genotype differences in upstream PI3K-Akt-GSK3beta or MEK/ERK pathways. To our knowledge, this is the first report of interactions between En2 expression and IGF1 signaling.
In addition to altered mitogenesis, En2 KO GNPs grew fewer neurites in response to neuritogenic factors, IGF1 and PACAP, though their combination synergistically overcame differentiation deficits. Decreased IGF1-induced neuritogenesis was not merely due to failed cell cycle exit, a possibility because the PACAP rescue of reduced neurite outgrowth was accompanied by inhibition of proliferation . Rather, treatment with another anti-mitogen, FGF2, did not rescue the differentiation defect, but in fact attenuated IGF1-stimulated neuritogenesis in both genotypes. Significantly, GNP survival was also not compromised in the absence of En2, nor was growth factor-induced trophism. In aggregate, these data suggest that a complex array of mitogenic, trophic and differentiative signals within the EGL act in concert with cell-autonomous gene expression (i.e., En2 expression) to execute GNP maturation.
Previous evidence indicates that multiple interactions of intrinsic and extrinsic signals control GNP proliferation. With regard to extrinsic signals, we previously found that PACAP inhibits Shh-induced GNP mitosis by upregulating adenylate cyclase , whereas others show similar roles for bone morphogenetic proteins via Smad [53, 85]. Conversely, in the current study, we find IGF1 and Shh synergistically stimulate DNA synthesis. Furthermore, interactions between intrinsic signals and growth factors are also known. For example, transcription factor Atoh1/MATH1 expressed in EGL precursors promotes Shh-induced proliferation  by regulating expression of downstream target, Gli1 . Similar promitogenic effects have been defined for Zic family members, including 1, 2 and 4, as well as ATF5 [88–90]. On the other hand, ZNF238/RP58 is expressed as GNPs exit the cell cycle, and overexpression inhibits proliferation [91, 92], findings that parallel our observations with En2. Thus, a number of gene-growth factor interactions likely contribute to GNP development. That no other mitogenic or anti-mitogenic signal elicited genotypic differences in En2 KO and WT GNPs suggests there is a specific yet indirect interaction between En2 and IGF1 signaling (Figure 7), with one feature being altered S6K activation. The expression of En2 by GNPs may facilitate their transition from proliferation to differentiation in an environment rich in mitogenic signals , thereby allowing subpopulations of cells to continue cycling while others exit and differentiate. En2 expression has been reported to be greater in the vermis than the hemispheres [15, 24], which may be one mechanism explaining the delay between vermis and hemisphere GNP proliferation during development .
En2 overexpression promotes cell cycle exit and differentiation
Previously, we found that ectopic En2 overexpression in embryonic cortical precursors increased proliferation . This stimulatory effect, opposite to what we observe here, is consistent with the universal expression of En2 in prenatal hindbrain progenitors and suggests that before birth En2 serves to maintain proliferation. In the current study of postnatal GNPs, En2 overexpression completely abolished BrdU labeling in WT mouse cells and reduced markers of proliferation (PCNA and BrdU) in rat GNPs. Additionally, En2 overexpression more than doubled the proportion of mouse and rat GNPs that exhibited neurite outgrowth. Significantly, neurite outgrowth was stimulated even in KO GNPs, suggesting that acute En2 expression may reverse morphological deficits associated with the absence of En2 for all of development, though additional studies are warranted. Furthermore, despite biological differences in GNPs from mice and rats , En2 overexpression produced similar phenotypes in both species, suggesting En2 function is evolutionarily conserved. The opposite consequences of En2 deletion and overexpression on GNP maturation suggest En2 promotes cell cycle exit and differentiation during postnatal cerebellar development.
One limitation to our current model, however, remains the unexplored role of En2 in prenatal cerebellar neurogenesis. While postnatal granule neurogenesis ultimately dictates final cerebellar morphology and size, prenatal patterning events organize the developing cerebellum and delineate neuronal progenitor cell populations, ultimately producing a cytoarchitectural framework for future axonal pathway elaboration [15, 94–97]. In the early cerebellar anlagen, En2 expression is ubiquitous, as is progenitor cell proliferation; therefore, En2 is unlikely to promote cell cycle exit and differentiation at this time. Previous studies suggest En2 may function during this period to specify numbers as well as types of progenitor cells that will give rise to various cerebellar neuronal populations [14, 16, 50, 95, 96, 98]. Thus, our characterization of postnatal En2 function is likely a time-locked developmental phenomenon that cannot address its prenatal activities. Rather, our demonstration of increased GNP proliferation may potentially suggest why En2 KO mice are capable of producing cerebella at all, despite the prenatal insult.
EN2 and ASD
We reported previously that intronic polymorphisms (A-C haplotype) of human EN2 are associated with ASD [7, 9] and that the presence of these SNPs results in altered transcription factor binding as well as increased levels of gene expression . Furthermore, our recent studies indicate that in human cerebellum, the disease-associated allele produces increased EN2 expression . If the En2 activity defined here in rodent studies is relevant to primates, how then would altered EN2 expression be expected to affect humans? Increased EN2 expression during postnatal cerebellar development, the period when the majority of human granule neurons are generated, would likely produce a granule neuron deficit by eliciting premature cell cycle exit. However, as with the mice, such a simple prediction is unwarranted because the prenatal effects of gene alteration remain undefined. But at the broader level, children with ASD exhibit a range of cerebellar structural abnormalities, including a diminished vermis as well as enlarged hemispheres in which granule neuron numbers are dysregulated [3, 101–103], a phenotype to which EN2 may contribute. Indeed, in mouse models, both gene deletion as well as ectopic overexpression produces cerebellar hypoplasia [13, 18, 20]. In addition, cerebellar abnormalities have been described in human and mouse studies of autistic phenotypes in tuberous sclerosis as well as a number of neuropsychiatric disorders including schizophrenia, attention deficit hyperactivity disorder, and cognitive and language disabilities [35, 104–106].
Another interesting implication of our studies is identification of an interaction between En2 expression and IGF1, the latter being an important pleiotropic growth factor associated with disease as well as a possible therapeutic target. Riikonen et al.  reported reduced IGF1 in cerebrospinal fluid (CSF) of ASD children compared to age-matched controls and that CSF levels correlated with head circumference in ASD, but not control children. Given the En2-IGF1 interaction we describe, one might speculate whether altered EN2 expression may coincide with abnormal IGF1 to contribute to ASD pathogenesis, a question that remains to be investigated. From a therapeutic perspective, the Akt-mTOR-S6K pathway is dysregulated in multiple animal models of monogenic causes of ASD including fragile X mental retardation , Rett syndrome  and tuberous sclerosis , whereas IGF1 ligands may improve neurodevelopmental symptoms in Rett  and the SHANK3 autism-related mouse model of Phelan-McDermid syndrome . It is intriguing that yet another autism-associated gene, in this case EN2, implicates disordered Akt-mTOR-S6K signaling in the disease phenotype . The exact molecular mechanisms mediating En2’s modulation of IGF1 signaling remain to be elucidated.
Our current studies aimed to define the function of one autism-associated gene, EN2, at a specific time during postnatal brain development. Using both a loss of function KO mouse and a gain of function cDNA overexpression vector, we demonstrated that En2 promotes postnatal cerebellar GNP cell cycle exit and differentiation. Further, we characterized a previously unknown interaction between En2 and an important developmental growth factor, IGF1, and demonstrated downstream signaling pathway activation through S6K, a target of the mTOR pathway. These data add to the understanding of postnatal cerebellar development and the complex gene-growth factor interactions that regulate cell biologic processes such as proliferation and differentiation. Further, they provide insight into a possible pathogenetic mechanism by which ASD-associated alleles in human EN2 may alter neurodevelopment during a critical period in susceptible patients. While further investigation remains to define the mediators of the phenomena described here, these observations add further support to a previously recognized signaling pathway as a potential target for therapy.
Autism spectrum disorder
Brain-derived neurotrophic factor
Epidermal growth factor
External germinal layer
Extracellular signal-regulated protein kinase 1/2
Fibroblast growth factor
Eukaryotic initiating factor 4B/E
Granule neuron precursor
Glycogen synthase kinase 3-beta
Insulin-like growth factor 1
Insulin response structure 1
Microtubule-associated protein 1b
Mitogen activated protein kinase
Magnetic resonance imaging
Mammalian target of rapamycin
Pituitary adenylate cyclase activating peptide
Proliferating cell nuclear antigen
3-phosphoinositide-dependent protein kinase 1
Single nucleotide polymorphism
Vascular endothelial growth factor
We would like to thank Xiaofeng Zhou for her invaluable contributions to the execution of these experiments. We greatly appreciate the critical reviews and suggestions of Drs. Karl Herrup and Carol Mason throughout these studies. This research was supported by New Jersey Governor’s Council for Medical Research and Treatment of Autism (E.D.-B., J.H.M.); NIH MH076624 (J.H.M., E.D.-B.); NIH MH070366 (J.H.M., E.D.-B.); NIH ES11256/USEPA R82939101 (E.D.-B.); NINDS NS048649-01 (I.R., E.D.-B.).
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