Evidence That Helix-Loop-Helix Proteins Collaborate with Retinoblastoma Tumor Suppressor Protein to Regulate Cortical Neurogenesis

MATERIALS AND METHODS

Primary cultures of cortical progenitors and neurons.The preparation of cortical progenitors from embryonic day 12 (E12) to E13 mouse embryos has been described in detail previously (Slack et al., 1998; Gloster et al., 1999). Briefly, cortical tissue, obtained from E12–E13 mice, was dissected in ice-cold HBSS (Life Technologies, Burlington, CA) and then transferred into 37°C Neurobasal media (Life Technologies) containing 500 μm glutamine, 1% N2 supplement, 2% B27 supplement, and 1% penicillin–streptomycin (Life Technologies); this media was supplemented with 40 ng/ml basic fibroblast growth factor (bFGF) (Collaborative Research, Bedford, MA). The tissue was triturated with a fire-polished glass Pasteur pipette into small clusters of cells that were plated in multiwell tissue culture dishes (Nunc, Naperville, IL) or chamber slides precoated with laminin and poly-d-lysine (Collaborative Research). Cell density was 100,000 cells per well. Cultures were maintained at 37°C in a 5% CO₂ incubator. Mature postmitotic neurons were prepared from E16 embryos, from which cortices were collected, triturated in culture media (Neurobasal with 0.5 mm glutamine, 1% penicillin–streptomycin, 1% N2, 2% B27 supplements, and 20 ng/ml bFGF), and plated at a density of 50–75,000 cells per well. For analysis of Tα1:nlacZ expression, cultures were prepared from the K6 line of transgenic mice, which has been characterized in detail previously (Gloster et al., 1994, 1999; Bamji and Miller, 1996). These animals are homozygous for the transgene, eliminating the need for genotyping.

Recombinant adenovirus vectors. The adenovirus vectors carrying the Escherichia coli lacZ expression cassette (Ad5CA17lacZ; gift of Dr. Frank Graham, McMaster University, Hamilton, Ontario, Canada) (Slack et al., 1998) and constitutively activated pRb (Chang et al., 1995) have been described previously. The adenoviruses expressing green fluorescent protein (GFP) and myc-tagged human Id2 were both constructed in the Ad5 backbone (Bett et al., 1994), which drives expression from the cytomegalovirus promoter. The GFP-expressing virus was obtained commercially from Quantum Biotechnologies (Montreal, Canada), and that expressing myc-tagged human Id2 was constructed using standard protocols, as we have described previously (Mazzoni et al., 1999).

All recombinant adenoviruses were purified on CsCl gradients, as we have described previously (Aloyz et al., 1998; Mazzoni et al., 1999), and extensively purified. Infectious titer was determined by plaque assay on HEK293 cells (Graham and Prevec, 1991).

Cortical progenitor cells were infected at the time of plating, and 24 hr after infection, half of the media was changed, and cells were fed every 2 d. The multiplicity of infection (MOI) indicates the number of plaque-forming units added per cell.

Immunocytochemistry of cultured cells. For immunocytochemical detection of nestin (1:1000; gift from Dr Ron McKay, National Institute of Neurological Disorders and Stroke, Bethesda, MD), neurofilament M (1:400; Chemicon, Temecula, CA), β-galactosidase (1:1000; ICN Biomedicals, Cleveland, OH), the myc-tag (1:200; PharMingen, San Diego, CA), and the hemagglutinin (HA)-tag [1:500; monoclonal from Boehringer Mannheim (Indianapolis, IN) and polyclonal from Babco (Richmond, CA)], cells were fixed for 20 min with 4% paraformaldehyde. Cultures were then washed with HEPES-buffered saline (HBS), pH 7.4, permeabilized for 5 min in 0.2% NP-40 in HBS, and then blocked for 45 min with buffer containing 6% goat serum and 0.5% bovine serum albumin. Cells were then incubated at 4°C overnight with primary antibodies in HBS containing 3% goat serum. After three washes with HBS, cells were incubated at room temperature for 60 min with indocarbocyanine (Cy3)-conjugated goat anti-mouse (1:400; Jackson ImmunoResearch, West Grove, PA), Cy3-conjugated anti-rabbit (1:400; Jackson ImmunoResearch), FITC-conjugated anti-mouse (1:100; Jackson ImmunoResearch), or FITC-conjugated anti-rabbit (1:100; Jackson ImmunoResearch) secondary antibodies (as necessary) prepared in HBS containing 3% goat serum. Samples were washed three times with HBS and then were counterstained for 2 min with Hoechst 33258 (Sigma, St. Louis, MO) before examination by fluorescence microscopy.

For quantitation, three to six random images of each treatment (per experiment) were captured and processed. Digital image acquisition and analysis was performed with the Northern Eclipse software (Empix Inc.) using a Sony (Tokyo, Japan) XC-75CE CCD video camera.

Cell survival assays and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling.MTT survival assays were performed as described previously (Slack et al., 1998; Vaillant et al., 1999). In brief, 20 μl of MTT reagent was added to the medium in each well of a 96-well plate containing the cultured progenitors or neurons. After a 2.5 hr incubation at 37°C, the medium–MTT mixture was removed, and the cells were lysed with 100 μl of isopropanol containing 2 μl/ml concentrated HCl. The absorbance of the lysate at 570 and 630 nm was determined using a Bio-Tek Instruments (Burlingame, CA) model Elx-800 UV plate reader (Mandel Scientific Inc.). All MTT assays were performed in triplicate.

For the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) experiments, cells were briefly rinsed in HBS, pH 7.4, and fixed for 20 min in 4% paraformaldehyde (Sigma) in HBS. Cells were then permeabilized with 0.4% NP-40 in HBS for 5 min and washed three times with HBS. TUNEL reaction was performed for 1 hr at 37°C. Each 100 μl of TUNEL reaction mixture contained 20 μl of 5× terminal deoxynucleotidyl transferase (TdT) buffer, 1.5 μl of TdT enzyme (both from Promega, Madison, WI), and 1 μl of biotin-16-dUTP (Boehringer Mannheim). After the TUNEL reaction, cells were rinsed three times in HBS and incubated for 45 min at room temperature with Cy3-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:2000 in HBS. In those experiments in which cells were double-labeled, cells were then incubated overnight in the primary antibody, as above, and immunocytochemical analysis was performed as described.

Western blot analysis and coimmunoprecipitations. For biochemistry, cortical progenitors or neurons were plated in 60 mm dishes and were lysed in TBS lysis buffer (Knusel et al., 1994) containing 137 mm NaCl, 20 mm Tris, pH 8.0, 1% (v/v) NP-40, 10% (v/v) glycerol, 1 mm PMSF, 10 μg/ml aprotinin, 0.2 μg/ml leupeptin, 1.5 mm sodium vanadate, and 0.1% SDS. Cells were collected in cold PBS by gentle scraping to detach them, were washed three times with the same buffer, and then were resuspended in 50–100 μl of lysis buffer, followed by rocking for 10 min at 4°C. After a 5 min centrifugation, the lysates were normalized for protein concentration using a BCA Protein Assay Reagent (Pierce, Rockford, IL). Equal amounts of protein (50–100 μg) were then boiled in sample buffer for 5 min and separated by 7.5–15% SDS-PAGE gradient gels. Alternatively, for Id2, samples were separated on tricine–SDS-PAGE gels, which are optimized for small proteins (Schagger and von Jagow, 1987). After electrophoresis, proteins were transferred to 0.2 μm nitrocellulose for 3 hr at 0.75 A, and the membrane was washed three times with TBS. For all antibodies, the membranes were blocked in 5% nonfat milk in TBS plus 0.2% Tween (TBST) (blotto) for 2 hr at room temperature. The membranes were then incubated overnight at 4°C with the primary antibodies in blotto:anti-myc (1:500), anti-HA (1:1000), anti-Id2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-extracellular signal-regulated kinase (ERK) (1:500; Santa Cruz Biotechnology). After incubation with the primary antibodies, membranes were washed four times with TBST over 40 min and incubated with the secondary antibody for 1.5 hr at room temperature. The secondary antibodies (goat anti-mouse or goat anti-rabbit HRP from Boehringer Mannheim) were used at 1:10,000 dilution. After three washes with TBST, detection was performed using the ECL chemiluminescence reagent from Amersham Pharmacia Biotech (Arlington Heights, IL) and XAR x-ray film from Eastman Kodak (Rochester, NY).

For coimmunoprecipitations, HEK293 cells or sympathetic neurons of the superior cervical ganglion, cultured as described previously (Aloyz et al., 1998), were infected with recombinant adenoviruses expressing HA-pRb, myc-Id2, or β-galactosidase, cells were lysed in lysis buffer, and 200 μg of protein from the relevant lysates were mixed and incubated overnight at 4°C in the presence of anti-HA. Protein G-Sepharose (Amersham Pharmacia Biotech) was added to the lysates and incubated for 2 hr at 4°C, and the lysates were then centrifuged to collect the immunoprecipitated protein. Immunoprecipitates were washed twice with lysis buffer, boiled 5 min in 2× sample buffer, and loaded on 7.5–15% SDS-PAGE gradient gels for electrophoresis.

RESULTS

DISCUSSION

In this paper, we have tested the hypothesis that HLH proteins collaborate with pRb to regulate the progenitor-to-neuron transition. Data presented here support four major conclusions. First, these studies support the hypothesis that transcription by positively acting bHLHs is essential for induction of neuronal gene expression in cortical progenitor cells; when bHLH transcriptional activity is inhibited by overexpression of Id2, progenitor cells do not express neuronal genes. Second, these studies demonstrate that perturbation of the progenitor-to-neuron transition by overexpression of Id2 leads to apoptosis. We propose that this phenotype is attributable to inhibitory interactions between Id2 and endogenous pRb, because (1) this apoptotic phenotype is similar to that observed when the pRb family is ablated in these same progenitor cells (Slack et al., 1998), and (2) constitutively activated pRb can rescue the Id2-driven apoptosis. Third, our data indicate that once a progenitor cell has become a postmitotic cortical neuron, then Id2 overexpression does not inhibit neuronal gene expression or cause neuronal apoptosis, suggesting that the bHLH transcription factors and pRb are required for induction of the neuronal phenotype, but not necessarily for its maintenance. Fourth, our data demonstrate that constitutively activated pRb can rescue both the Id2-driven apoptosis and the inhibition of neuronal genes, suggesting that interactions between pRb and HLHs are likely to regulate both terminal mitosis and the induction of neuronal gene expression. Together, these studies indicate that collaborative interactions between the pRb family and HLHs play a key role in regulating cortical neurogenesis and suggest that endogenous Id2 may well be critical in enacting the progenitor-to-neuron decision.

The idea that interactions between the pRb family and HLH proteins may regulate cellular differentiation is a particularly attractive one when considering neurogenesis in the CNS. With a few exceptions, such as the precursors that migrate in the rostral migratory stream from the lateral ventricles to the olfactory bulb (Menezes et al., 1995), when CNS progenitor cells become postmitotic neurons, they coordinately undergo terminal mitosis and induce neuronal gene expression (Gloster et al., 1999). Moreover, when one aspect of this process is perturbed, such as occurs after functional ablation of the pRb family, the end result is cellular apoptosis (Slack et al., 1998). This integral relationship between terminal mitosis and neuronal gene expression suggests that the same molecules may well regulate both events. Data presented here suggest that Id2, which can interact with and inhibit pRb-driven terminal mitosis and bHLH-driven induction of neuronal gene expression, might be one such molecule.

It is likely that Id2 overexpression inhibits the induction of neuronal gene expression by directly binding to the ubiquitous E2A bHLH transcription factors (for review, see Norton et al., 1998), thereby titrating out the requisite binding partners for neurogenic bHLHs, such as Mash-1 (Johnson et al., 1990), neurogenins (Ma et al., 1996; Sommer et al., 1996), or NeuroD (Lee et al., 1995), although Id2 may also directly bind the neurogenic bHLHs themselves (Langlands et al., 1997). A similar mechanism is thought to underlie the ability of Id1 and Id3 to inhibit myogenesis (Jen et al., 1992; Atherton et al., 1996) and adipogenesis (Moldes et al., 1997) in cell lines and of Id1 (Sun, 1994) and Id2 (Morrow et al., 1999) to inhibit B cell and T cell developmentin vivo. Genetic evidence that endogenous Id proteins play a similar inhibitory role in the nervous system derives fromDrosophila (Ellis et al., 1990) (for review, seeCampos-Ortega, 1993; Jan and Jan, 1993) in which the extramachrochaete gene product (the Drosophilaequivalent of Id) antagonizes both Daughterless (DrosophilaE2A) and achaete-scute (Drosophila Mash) bHLH proteins, which are involved in sex determination and neurogenesis. Similarly, Id2 was also suggested to be involved in cell fate decisions in the chick neural crest (Martinsen and Bronner-Fraser, 1998). More recently, the targeted deletion of the Id1 and Id3 genes was shown to cause altered nervous system development accompanied by perturbed angiogenesis in the embryonic brain (Lyden et al., 1999). Deletion of either gene on its own produced no detectable phenotype, presumably because of compensation by different family members. An essential role for Id2 during development is also indicated by the recent report of Id2−/− mice, which display arrested development and lack lymph nodes and Peyer's patches (Yokota et al., 1999). Although no information is yet available regarding the nervous system of these animals and the underlying cellular deficit is unclear, these data do indicate that Id2 is essential for normal mammalian development.

Studies presented here also strongly support the idea that endogenous Id2 interacts with the pRb family to regulate neurogenesis and cell survival. As confirmed here, previous studies demonstrated that Id2 binds to the hypophosphorylated form of pRb and inhibits its ability to mediate growth arrest (Iavarone et al., 1994;Lasorella et al., 1996). Because pRb is required for cortical progenitors to exit the cell cycle and survive (Slack et al., 1998), then this finding predicts that increased Id2 would mimic the phenotype observed when the pRb family is ablated. In fact, this is what we observe; cortical progenitor cells, but not postmitotic neurons, undergo apoptosis when Id2 is overexpressed. Moreover, coexpression of a mutant pRb that is constitutively hypophosphorylated rescues this apoptotic phenotype, presumably by sequestering Id2 and allowing progenitor cells to undergo terminal mitosis. Thus, although our studies do not directly demonstrate that endogenous Id2 regulates the ability of pRb to mediate terminal mitosis in progenitor cells, they suggest that such may be the case.

Why does constitutively activated pRb rescue the deficit in neuronal gene expression induced by Id2 when (1) the hypophosphorylated pRb mutant does not, on its own, induce neuronal gene expression (shown here), and (2) functional ablation of the pRb family has no effect on induction of the same neuron-specific genes (Slack et al., 1998)? We propose that hypophosphorylated pRb, by binding to Id2, inhibits its ability to bind to and inhibit positively acting bHLH transcription factors. In this model, a cycling progenitor cell would have relatively high levels of Id2, and pRb would be primarily present as a hyperphosphorylated protein. At this point in time, Id2 levels would be sufficient to bind and inhibit any positively acting bHLH transcription factors and to bind and inhibit any hypophosphorylated pRb. In response to an as-yet undefined cue to become a postmitotic neuron, Id2 levels would decrease and/or Id2 activity would be altered by phosphorylation (Nagata et al., 1995; Hara et al., 1997), and the ratio of hypophosphorylated to hyperphosphorylated pRb would increase. Id2 would then no longer be able to sequester all of the hypophosphorylated pRb, thereby allowing pRb to “stop” the cell cycle. Moreover, the decrease in Id2 levels–activity, coupled with the increase in hypophosphorylated pRb, would ensure that little or no Id2 was available to bind and sequester E2A bHLHs, which could then form productive transcription complexes with neurogenic bHLHs. Thus, pRb, Id2, and neurogenic bHLHs would all collaborate to enact the neuronal commitment decision, once such a decision had been made. Such a mechanism would ensure (1) the coordinate induction of neuronal gene expression and terminal mitosis, and (2) the apoptosis of any progenitor cell that failed to properly enact this transition.