Skip to main content
  • Research article
  • Open access
  • Published:

The extracellular matrix, p53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors, and reciprocally, Fas-ligand modifies estrogen control of cell-cycle proteins



Apoptosis is important for normal cerebral cortical development. We previously showed that the Fas suicide receptor was expressed within the developing cerebral cortex, and that in vitro Fas activation resulted in caspase-dependent death. Alterations in cell-surface Fas expression may significantly influence cortical development. Therefore, in the following studies, we sought to identify developmentally relevant cell biological processes that regulate cell-surface Fas expression and reciprocal consequences of Fas receptor activation.


Flow-cytometric analyses identified two distinct neural sub-populations that expressed Fas on their cell surface at high (FasHi) or moderate (FasMod) levels. The anti-apoptotic protein FLIP further delineated a subset of Fas-expressing cells with potential apoptosis-resistance. FasMod precursors were mainly in G0, while FasHi precursors were largely apoptotic. However, birth-date analysis indicated that neuroblasts express the highest levels of cell-surface Fas at the end of S-phase, or after their final round of mitosis, suggesting that Fas expression is induced at cell cycle checkpoints or during interkinetic nuclear movements. FasHi expression was associated with loss of cell-matrix adhesion and anoikis. Activation of the transcription factor p53 was associated with induction of Fas expression, while the gonadal hormone estrogen antagonistically suppressed cell-surface Fas expression. Estrogen also induced entry into S-phase and decreased the number of Fas-expressing neuroblasts that were apoptotic. Concurrent exposure to estrogen and to soluble Fas-ligand (sFasL) suppressed p21/waf-1 and PCNA. In contrast, estrogen and sFasL, individually and together, induced cyclin-A expression, suggesting activation of compensatory survival mechanisms.


Embryonic cortical neuronal precursors are intrinsically heterogeneous with respect to Fas suicide-sensitivity. Competing intrinsic (p53, cell cycle, FLIP expression), proximal (extra-cellular matrix) and extrinsic factors (gonadal hormones) collectively regulate Fas suicide-sensitivity either during neurogenesis, or possibly during neuronal migration, and may ultimately determine which neuroblasts successfully contribute neurons to the differentiating cortical plate.


The developing cerebral cortex and other brain regions undergo substantial cell suicide during the period of neurogenesis and early differentiation [110], to generate a mature brain. Mechanisms that control the survival or death of neuroblasts and neurons in the developing cerebral cortex are likely to have profound effects on the organization of cognition, affect and sensori-motor integration in the adult. One generally accepted mechanism that is invoked to explain developmental apoptosis is the competition among neurons for limited supplies of trophic molecules within the environment (reviewed in [11]). According to this model, a neuron's inability to find growth factor support within its environment precedes the initiation of apoptosis. However, the presence of cell-surface suicide receptors, like the Fas/Apo [Apoptosis]-1/CD95 receptor, and their trans-membrane ligands (e.g., FasL) in the developing brain [9] suggests that neural cells may actively communicate apoptosis signals to each other. In addition to competing for a limited supply of trophic factors, developing neural progenitors and differentiating neurons may engage in an active killing process whereby 'killer cells' induce apoptosis in 'suicide-receptive' cells, to limit cell number in the brain. It is therefore important to understand the signaling mechanisms and circumstances that regulate cell-suicide receptor expression in the developing brain.

The Fas cell-suicide receptor plays an important role in limiting cell proliferation in the immune system by apoptosis [12]. Recent evidence suggests that Fas is also an important regulator of cell death in the brain. Fas is expressed by the developing cerebral cortex during the peak period of apoptosis [7, 9, 13], and by other differentiating neural cells [1418]. Fas is also re-expressed in neurological disease conditions including ischemia, multiple sclerosis, Alzheimer's disease, and in neural tumors [15, 1927]. We previously reported that Fas activation leads to unscheduled DNA synthesis, the activation of NF-κB, and caspase-dependent cell death in embryonic cortical neuroblasts [9]. Neuronal cultures obtained from developing gld (Fas-deficient) mice are less sensitive to apoptosis signals than wild-type controls [17]. Finally, haplo-insufficiency of the tyrosine phosphatase Pten/MMAC1 results in the inactivation of Fas [28], resistance to apoptosis in neural progenitors [29], and consequently, an increased incidence of tumors of neural origin [30]. These data indicate that the Fas/Apo-1 suicide system may determine cell number in the brain, both during development, and following injury in the adult. However, we know very little about the types of neural progenitors and neuronal cells that are particularly vulnerable to Fas-induced death. We also know little about the signaling mechanisms in differentiating neural progenitors that promote cell-surface expression of Fas, and consequently, sensitivity to apoptosis.

The transcription factor p53 is important for brain development [31]. In lung and prostate cancer cell lines, Fas activation is dependent on the presence of functional p53 [32]. P53 induces Fas gene transcription [33], the translocation of Fas from Golgi complex to cell surface [34] and activates Fas-mediated apoptosis in un-transformed vascular smooth muscle cells [34] and mammary epithelial tumors [35]. In the adult rat hippocampus, seizure leads to the co-localized expression of p53 and Fas in CA1 neurons [36]. However, it is likely that p53 acts in concert with a coterie of transcription factors to regulate the availability of Fas in the developing brain. For example, we found that estrogen promotes p53 phosphorylation [37], but uncouples p53 from its regulation of Bax, a mitochondrial-associated pro-apoptotic factor. Furthermore, estrogen receptors block the DNA binding activity of NF-kB [38], a transcription factor downstream of members of the TNFr/Fas family [9, 39]. Thus estrogen, and consequently estrogen receptors, may be an important regulator of Fas expression in the developing cerebral cortex.

In the following experiments, we set out to identify cell biological processes that predict cell-surface expression of Fas, and to identify consequences of Fas activation. Our experiments focused predominantly on the pool of Fas receptors that localized to the cell surface, because this population constitutes the bioactive proportion of Fas receptors that contributes to apoptosis sensitivity. We hypothesized that, in embryonic cerebral cortex, cell-surface Fas expression would be associated with exit from cell cycle, and activation of p53. We also hypothesized that estrogen controls neurogenesis and neuron elimination, either by regulating the Fas receptor, or by regulating specific anti-apoptotic cell cycle elements in a Fas-dependent manner.

Our results indicate that embryonic cortical precursors are heterogeneous with respect to cell surface Fas expression and the co-expression of the anti-apoptotic protein FLIP (FLICE/caspase-8 interacting protein). Cell-surface Fas expression in embryonic cortical neurons is associated with recent exit from cell cycle, and with loss of contact with the extra-cellular matrix and anoikis. Fas expression is coincidentally increased following p53 activation. In contrast, estrogen decreases Fas expression, and following the activation of Fas by soluble Fas-ligand (sFasL), estrogen suppresses expression of p21/Waf-1/Cip-1 and the proliferating cell nuclear antigen (PCNA). Estrogen and sFasL separately and together induce expression of cyclin A.


Expression of Fas and related proteins in embryonic cerebral cortical precursors

In the initial experiment, we utilized flow cytometric analysis to examine the localization of Fas to the cell surface of freshly isolated embryonic rat (gestational day (GD)-15) cortical neural cells by immuno-labeling for Fas in the absence of detergent. At this developmental age, the neuroepithelium proliferates rapidly and is the dominant cortical structure, and therefore, neuroepithelial precursors are the most abundant component of this tissue. Furthermore, at this age, the neuroepithelium mainly consists of neuronal (as opposed to glial) precursors (for review, see [40]). Flow cytometric analysis of samples obtained from GD15 rat cortex indicated that 10.69 ± 3.37% (mean ± SEM) of cortical neuronal precursors expressed Fas on their cell surface in vivo on embryonic day 15 (data obtained from 6 independent samples of E15 cortex, Figure 1A). Once cortical precursors are cultured however, immunohistochemical analysis of detergent-permeabilized cultures (a measure of total Fas expression) indicates that a majority of these differentiating cells localize Fas-immunoreactivity within their cytoplasm and proximal processes (Figure 1B vs. control Figure 1C and [9]). In contrast, flow cytometric analyses of non-permeabilized cultured embryonic cortical-derived neuronal precursors (Figure 2) indicate that 23–28% of these cells expressed Fas on their cell surface in vitro, representing ~2-fold induction of cell surface Fas expression in vitro, as compared to in vivo. Collectively, these data indicate that only a fraction (~1/4th) of all Fas-expressing cells actually localize Fas to the cell-surface, in vitro.

Figure 1
figure 1

(A) Flow cytometric analyses of independent samples GD15 cortex show more cells expressing Fas immuno-fluorescence in samples #1–#4, compared to pre-immune serum control. Gate 'M' was set to exclude background fluorescence, or 98% of neuroblasts in the control sample (B & C) immunohistochemical analysis of cultured embryonic cortical neurons indicates Fas-immunoreactivity is localized to the soma and proximal processes (arrow). Immunohistochemical controls (arrow, C) show lack of staining in neurons. (D) Graph (Mean ± SEM) of the percentage of Fas-expressing precursors that also co-localize the pro-apoptotic DISC adapter protein FADD, or the anti-apoptotic inhibitor FLIP.

Figure 2
figure 2

Identification of two unique populations of cell-surface Fas-expressing precursors (A-D). Flow cytometric analysis of the cell-cycle distribution of cultured embryonic cortical precursors (A), based on frequency histogram analysis of propidium iodide (PI) incorporation into DNA in 10,000 cells from each sample, indicates that a majority of cells precursors are in G0. Precursors with DNA content <G0 were identified as apoptotic, while precursors with >G0 DNA content were identified as being in S-G2-M. (B,C) Scatter-plots of two representative independent samples of embryonic cortical-derived neuronal precursors analyzed for combined cell-surface Fas immuno-fluorescence (y-axis) and PI incorporation (cell-cycle stage, x-axis). Based on background immuno-fluorescence patterns in the pre-immune serum controls (D), precursors were characterized as negative Fas-expressing (Fas-ve, expressing <2 × 100 fluorescence units [FUs]), moderate Fas-expressing (FasMod, expressing >2 × 100 and <102 FUs) and high cell surface Fas-expressing (FasHi, >102 FUs). These categories remained consistent across experiments. (E & F) Graphical representation (Mean ± SEM) of the proportion of precursors expressing high (FasHi, E) and moderate (FasMod, F) levels of cell-surface Fas at different stages of cell cycle and apoptosis, expressed as a percentage of control. Asterisks indicate statistically significant differences in cell-stage-specific expression of cell surface Fas, p < 0.05.

Co-localization of Fas with FADD and FLIP

The protein FADD (Fas-Associated Death Domain) is the major intracellular death-inducing signaling complex (DISC) adapter protein that couples Fas to intracellular caspase pathways [4144], while FLIP is the major proximal intracellular protein that blocks Fas/Apo-1-mediated apoptosis [45]. Since FADD and FLIP are antagonistic elements of the DISC-complex and are important intracellular gates of Fas/Apo-1 activation, we examined the extent to which these functionally divergent proteins co-localized with Fas/Apo-1 in differentiating embryonic cortical-derived neuroblasts. Two- parameter FACS analysis (Figure 1D) showed that, among adherent cultures, 72.5 ± 2.1% of Fas-expressing neuroblasts also co-localized FADD. In contrast, only 23.9 ± 3.2% Fas-expressing cells co-localized the anti-apoptotic protein FLIP suggesting that only a portion of Fas/Apo-1-expressing embryonic cortical-derived neuronal precursors may be protected from Fas-induced apoptosis.

Cell-cycle-related expression of Fas in embryonic cortical neuronal precursors

Using flow cytometric analyses, we identified three distinct populations of neural cells in vitro; based on differences in the intensity of cell-surface Fas expression (Figure 2). Approximately 3% of cortical cells expressed Fas on their cell surface at a high intensity (FasHi), while 20% of cultured neural cells expressed moderate levels of Fas on their cell surface (FasMod). In contrast, 77% of cultured cortical neural cells do not express Fas on their cell-surface above background levels (Fas-ve). Cultured cortical cells were counterstained with propidium iodide (PI) to measure cellular DNA content and to stage Fas-expressing cells into apoptosis (defined as cells with less than G0 DNA content), cell cycle arrest (G0/G1) or cell cycle (G2/S/M) phases (Figure 2A,2B,2C,2D). Our results indicate that the largest proportion of neural cells in the FasHi population were apoptotic (Figure 2E). Few FasHi cells were in G0/G1 or S/G2/M stages of cell cycle. In contrast, within the FasMod population, the greatest percentage of Fas-expressing cells was observed in G0/G1 (Figure 2F).

Functional determinants of Fas expression in cortical neuroblasts

The majority of FasHi cells were apoptotic. We therefore examined the extent to which Fas expression was related to specific apoptosis-sensitive cell-biological processes including cell-matrix interactions, cell proliferation, and the activation of cell-cycle inhibitory proteins like p53.

Fas expression is dependent on cell-matrix interactions and 'anoikis'

The loss of adhesion to the extracellular matrix has been reported to induce apoptosis (a process referred to as anoikis), by activating caspases like caspase-2 [46] that are down-stream of suicide receptors like Fas/Apo-1. Such cell-matrix interactions appear to be contextually relevant to the developing brain as well, since deletion of the Matrix Metalloproteinase (MMP)-9 gene (consequently suppressing type-4 collagen degradation) leads to delayed migration of granule cells, and decreased apoptosis in the developing cerebellum [47]. Since post-mitotic neuroblasts migrate to their lamina-specific positions within the cortical plate using guidance cues that are dependent on the integrity of the extra-cellular matrix, we sought to determine if there was a relationship between cell-adhesion and Fas/Apo-1 expression. Non-adherent cells were harvested and analyzed separately from cells that were adherent to the culture dish. Among adherent cells (Figure 3A,3B), a majority of Fas-expressing neural cells belonged to the FasMod group. In contrast, most FasHi neural cells were non-adherent (Figure 3C,3D) and predominantly apoptotic.

Figure 3
figure 3

Relationship between Anoikis (apoptosis due to loss of cell adhesion) and cell surface Fas-expression. (A,C) Sample frequency histograms of PI incorporation (DNA content) in adherent (A) and non-adherent (C) precursors indicates that adherent precursors are mainly in G0, while non-adherent precursors are mainly apoptotic. (B, D) Scatter-plots of combined analysis of PI (x-axis) and cell-surface Fas immunofluorescence (y-axis) indicates that adherent precursors largely express moderate levels of cell-surface Fas (FasMod, B), while non-adherent precursors also express high levels of Fas (FasHi, D). (E) Graph (Mean + SEM) showing that collagenase-A treatment significantly increased the number of cells expressing cell-surface Fas, relative to controls. Asterisk indicates p < 0.05.

Since primary cultured cells of the FasHi group were predominantly non-adherent, we sought to determine if the induction of 'anoikis' could lead to an increase in Fas/Apo-1 expression. We therefore cultured embryonic day 15 cortical-derived neuroblasts on a collagen substrate (rat tail collagen, UBI) and treated adherent cultures with 50 U of Collagenase-A (MMP-1, microbial metalloendopeptidase, EC, Sigma) for 24 hours. Treatment of adherent neuronal precursors with collagenase-A led to a statistically significant, 2.5-fold increase in the number of cells that expressed Fas/Apo-1 on their cell surface (Figure 3E). Because of this relationship between 'anoikis' and Fas/Apo-1 expression, subsequent flow cytometric analyses focused on cells that were adherent to the extracellular matrix deposited onto the culture dish.

Cell surface Fas/Apo-1 expression is associated with cell cycle

We had previously observed that Fas activation was followed by a transient increase in the uptake of 5-Bromo-2'-deoxyuridine (BrdU, [9]) a marker of DNA replication. Though this increase in BrdU incorporation was not related to the induction of cell cycle, we hypothesized that the expression of Fas itself was related to cell proliferation and DNA synthesis. We therefore utilized three-parameter flow cytometric analyses to examine the relationship between cell-surface Fas expression and BrdU incorporation among neuronal precursors that were in cell cycle arrest (G0/G1), or actively in cell cycle (G2/S/M). Cells were first immuno-labeled for Fas, then detergent permeabilized and immuno-labeled for BrdU incorporation. Using propidium iodide (PI) fluorescence intensity as a classification parameter, cortical precursors were classified as belonging to one of three phases, G2/S/M, G0/G1, or apoptosis (sub-G0 DNA content). BrdU incorporation was significantly correlated with the cell-surface expression of Fas. In G0/G1 and S/G2/M conditions, correlation coefficients (r) ranged from 0.98 to 0.99, p < 0.05 (Figure 4A,4B). These data suggest that Fas/Apo-1 is most highly expressed in neuronal precursors that have recently completed their final cycle (for neuroblasts in G0/G1) or recently exited S-phase (for neuroblasts in S/G2/M). Furthermore, among cells in cell cycle (S/G2/M, Figure 4A), a discrete group of FasHi precursors (arrows) also expressed the highest levels of BrdU incorporation. During cell cycle, the highest levels of BrdU incorporation are attained at the completion of chromosome replication (4N DNA content at the end of S-phase) and before the completion of mitosis. Therefore, G2 and early mitosis may represent periods during which some sub-populations of proliferating cortical neuroblasts are particularly vulnerable to receptor-mediated suicide signals. Our data therefore indicate that while Fas activation does not induce or suppress cell cycle [9], the highest expression of Fas is associated with progression past the S-phase of cell cycle or recent exit from cell cycle.

Figure 4
figure 4

Relationship between cell-surface Fas expression and BrdU incorporation at different stages of cell cycle or apoptosis (PI incorporation) in adherent primary precursors. (A,B) Representative frequency histogram of PI incorporation (A) and scatter-plot of combined PI incorporation and Fas immunofluorescence (B) indicates the thresholds for the separate analysis of BrdU content in neuroblasts at S/G2/M (pink), G0 (green), or apoptosis (red). (C-F) Representative scatter-plots of the overall positive relationship between Fas expression and BrdU incorporation (C) and separated by cell-phase, S/G2/M (D), G0 (E) and apoptosis (F). Arrows in S/G2/M condition (D) indicate a group of cells that exhibited the highest levels of BrdU incorporation (suggesting recent completion of S-phase) and cell-surface Fas expression. Insets in figures D,E and F indicate Pearson's product moment correlations (obtained by averaging intensities across 6 independent samples). Asterisks indicate that the correlation was statistically significant with a 2-tailed test. These data indicate that the correlation between BrdU incorporation and cell-surface Fas expression is higher during cell cycle and G0 as compared to apoptosis.

Fas expression in embryonic cortical-derived precursors is associated with p53 activation

The transcription factor p53 induces Fas gene transcription [33], the translocation of Fas from Golgi complex to cell surface [34], and activates Fas-mediated apoptosis in un-transformed vascular smooth muscle cells [34] and mammary epithelial tumors [35]. We had previously shown that p53 activation in cortical progenitor cells was associated with the induction of the mitochondrial death-associated protein Bax [37]. We therefore examined the extent to which p53 status predicted the expression of Fas/Apo-1 on the cell surface of cortical neuronal precursors. We used phosphorylation of p53 at serine 392 as a marker for p53 activation [37]. Cells were first immuno-labeled for Fas, then detergent-permeabilized and immuno-labeled for p(phospho)p53. The intensity of pp53 expression was statistically significantly correlated (p < 0.05, r = 0.73, Figure 5A) with the intensity of cell surface Fas expression in primary cortical neuronal precursors. Thus, cells that exhibit high levels of p53 phosphorylation also express high cell-surface levels of Fas.

Figure 5
figure 5

Fas expression is associated with p53 activation. (A) Sample flow cytometric scatter-plot showing a strong positive association between the intensity of p53 phosphorylation (pp53) and cell surface Fas expression in primary cortical precursors. Asterisk indicates statistical significance of Pearson's product moment correlation (inset). (B,C) Sample western blot (B) and quantitative densitometric analysis of Fas expression in conditionally immortalized CHB50 cerebral cortical neuroblasts under -p53 conditions (+tsTA/-pp53) and under +p53 conditions (-tsTA/+pp53) for 24 or 48 hours.

As a follow-up experiment, to determine whether p53 activation is required for the expression of Fas/Apo-1, we utilized a rodent cerebral cortical cell line (CHB50), conditionally immortalized with a temperature sensitive mutation of the SV40 large T antigen (tsTA), that we had previously developed and characterized [37]. In the +tsTA condition (incubation at 33°C), neuroblasts proliferate indefinitely and p53 is inactive as indicated by the absence of phosphorylated p53 (-pp53). However, cessation of large T antigen expression (-tsTA, 39°C) is accompanied by p53 phosphorylation (+pp53) and the induction of p53-dependent proteins like Bax [37]. CHB50 neuroblasts were therefore cultured under +tsTA and -tsTA conditions. Western immunoblot analysis indicated that CHB50 neuroblasts expressed very low-to-undetectable levels of Fas in the -pp53 (+tsTA) condition. However, following the activation of p53, [+pp53 (-tsTA) condition], there was a statistically significant 12-fold increase in the expression of Fas, 48 hours following activation of p53 (Figure 5B,5C).

Estrogen regulation of Fas expression is cell-cycle dependent

We had previously observed that estrogen prevented apoptosis following p53 activation and decreased the expression of the mitochondrial-associated pro-apoptotic protein Bax in differentiating cerebral cortical neuroblasts [37]. Furthermore, in the context of p53 activation, estrogen led to an increase in neuroblast proliferation. We therefore examined the extent to which cell-surface Fas expression was estrogen-dependent.

Flow cytometric analysis of non-synchronized GD15 cortical neuronal precursors (Figure 6A) indicated that 24 hours exposure to estradiol-17β (E2) at 2 nM led to a statistically significant decrease in the total number of cortical cells in G0 (Figure 6B) and a corresponding significant increase in the number of neuroblasts in the DNA synthesis (S)-phase (Figure 6C). At 24 hours there was not a statistically significant change in the numbers of cells progressing through G2/M (Figure 6D), suggesting that estrogen-stimulated cells may have not yet completed cell cycle. There was also a non-statistically-significant trend towards a decrease in the total number of neuronal precursors that were apoptotic (Figure 6E). Our previous data also showed that estrogen decreased the expression of another member of the Fas family, the pan-neurotrophin receptor p75NTR [48, 49]. We therefore also examined estrogen regulation of cell-surface Fas expression at different stages of cell cycle.

Figure 6
figure 6

Estrogen promotes cell cycle in adherent primary cortical precursors: (A) Sample frequency (y-axis) distributions of PI intensity (x-axis) along with the best-fit distributions of cells in apoptosis, G0/G1, S-phase or G2/M. The area under the curve delineated by diagonal lines (S-phase) is greater in estrogen (E2)-treated cultures compared to controls. (B-E) Quantitative analysis of control and E2 treated cultures showing the mean % of cells ± SEM in G0 (B), S-phase (C), G2/M (D) or apoptosis (E). Asterisks indicate statistical significance at p < 0.05.

As observed earlier, in adherent embryonic cortical cultures, most Fas expressing cells belong to the FasMod category and very few FasHi-type cells can be observed (Figure 7A, control). Twenty-four hours of exposure to 2 nM E2 led to a statistically significant, 1.5-fold decrease in the percentage of cells in G0/G1 that expressed Fas (Figure 7A:E2 and 7B). In contrast, the numbers of Fas expressing cells in cell cycle, S/G2/M, were not altered by estrogen exposure (Figure 7C), though estrogen led to a statistically significant 2-fold decrease in the intensity of cell-surface Fas expression (on a per-cell basis, Figure 7D). Additionally, estrogen led to a statistically significant, 2-fold decrease in the numbers of Fas-expressing cells that were apoptotic, suggesting that estrogen suppresses apoptosis in Fas-expressing cells (Figure 7E).

Figure 7
figure 7

Estrogen suppresses Fas expression in adherent primary cortical precursors: (A) Sample flow cytometric scatter plots from two control and two E2 treated cultures, showing Fas intensity (y-axis) plotted against cell-cycle stage (PI intensity, x-axis). Scatter plots depict a general suppression in cell surface Fas intensity in E2 treated cultures compared to controls. (B,C,E) Quantitative analysis (mean ± SEM) of the number of cells expressing Fas (FasMod + FasHi) in control and E2 treated cultures, normalized to controls. Since these analyses were performed on adherent cells, the FasHi population was insignificant. (D) Quantitative analysis of the mean cell-surface intensity of Fas expression in control and E2 treated cultures. Asterisks indicate statistical significance at p < 0.05.

Since Fas expression was also associated with p53 activation, we hypothesized that estrogen would suppress Fas expression in the context of p53 activation as well. We therefore examined the expression of Fas in tsTA-CHB50 cortical neuroblasts cultured for 24 and 72 hours under +pp53 (-tsTA) conditions, and treated for those durations with either estradiol-17β (2 nM) alone or estradiol-17β with the antagonist 4-hydroxytamoxifen (at 1 uM, Sigma). Our previous western immunoblot analyses indicated that in control CHB50 cortical cultures, Fas expression was statistically significantly induced 72 hours following p53 activation (Figure 5B,5C). In the context of p53 activation, estrogen led to a significant decrease in the expression of Fas at 72 hours but not at 24 hours (Figure 8). Surprisingly (see discussion), concurrent exposure to the estrogenic antagonist 4-hydroxytamoxifen did not prevent the estrogen-induced reduction in Fas.

Figure 8
figure 8

Densitometric analyses of western immunoblots of samples obtained from conditionally immortalized CHB50 cortical neuroblasts cultured for 24 or 72 hours following p53 induction (-tsTA/+pp53 condition). During this duration, cultures were maintained either under control conditions or were exposed to E2 alone or E2 with tamoxifen. E2 induced a significant decrease in Fas that was not reversed by concurrent exposure to tamoxifen. Asterisks indicate statistical significance at p < 0.05.

Estrogen and Fas regulation of the cell cycle-associated proteins

Flow cytometric analysis of primary cortical cultures indicated that estrogen led to an increase in the number of cells in S-phase (Figure 6A,6C), consistent with our previously published observations that estrogen promotes neurogenesis in cortical neuroblasts [37]. Furthermore, estrogen prevented apoptosis in cortical neuroblasts. We therefore hypothesized that in the context of Fas activation, estrogen would alter the expression of specific cell-cycle related proteins such as Cyclin-A [50], the Proliferating Cell Nuclear Antigen (PCNA, [51, 52]) and p21/waf-1 [53], that also serve anti-apoptotic and DNA repair functions in addition to their roles in cell cycle. For example, PCNA and p21/Waf-1 bind to each other to prevent apoptosis induction [51], and to promote repair of double strand breaks in DNA, a characteristic of apoptosis. We therefore examined if Fas activation and estrogen could interact to regulate the expression of cyclin A, PCNA and p21/waf-1 and compared the expression of these anti-apoptotic cell cycle proteins with other cell-cycle proteins. Dissociated embryonic cortical cells were exposed to soluble Fas ligand (sFasL, 5 ng/ml, Alexius Corp) or estradiol-17β (2 nM) either alone or concurrently, for 12 hours. Western immunoblot analysis indicated that neither estrogen nor FasL alone led to a change in the expression of PCNA. However, concurrent exposure to both estrogen and FasL did lead to a significant decrease in PCNA expression (Figure 9). Since p21/Waf-1 may prevent apoptosis by interacting with PCNA [51], we examined the expression of p21/Waf-1 in dissociated embryonic cortical cells that were exposed to sFasL, estrogen or both sFasL and estrogen for 12 hours. Neither estrogen, nor sFasL alone induced an alteration in p21/Waf1 expression, though sFasL-treated cultures exhibited a non-statistically significant trend towards decreased p21/waf-1 expression. However, the concurrent administration of estrogen and sFasL led to a significant decrease in the expression of p21/Waf-1 (Figure 9).

Figure 9
figure 9

Estrogen (E2) and sFasL cooperate to regulate cell-cycle proteins. Densitometric analyses of western immunoblots of primary embryonic precursors treated with sFasL or E2 alone or sFasL concurrently with E2. Concurrent exposure sFasL and E2 suppress PCNA (A) and p21/Waf-1 (B) expression at 12 hours. sFasL and E2 separately and together induce cyclin-A (C). sFasL and E2 either alone or together do not regulate the expression of cyclin-E, CDK1/CDC2 or CDK2 (D). (E) Sample western immunoblots showing regulation of the expression of cell-cycle proteins following treatment with sFasL or E2 alone or together. p21Waf-1, cyclin-E and CDK-2 images were each cropped from one single immunoblot to eliminate non-relevant treatment conditions. Asterisks indicate statistical significance at p < 0.05.

Cyclin-A prevents apoptosis by disrupting the formation of a complex between the E2F1 and p53 transcription factors [50]. Therefore, we examined the expression of cyclin-A following exposure to E2 and/or FasL. Both estrogen and sFasL alone led to a statistically significant increase in the expression of cyclin-A compared with controls at 12 hours post-treatment. Concurrent exposure to sFasL and estrogen was not different from exposure to either agent alone (Figure 9).

Estrogen and Fas regulation of other cell cycle-related proteins

Neither estradiol-17β nor sFasL alone or together altered the expression of cdk2 or cyclin E compared to control cultures (Figure 9). Similarly, neither estrogen nor sFasL significantly altered the expression of Cyclin-dependent Kinase-1 (cdk1/cdc2) relative to control cultures, suggesting that that interactions between estrogen and Fas activation were not specifically related to cell cycle.


The perinatal cerebral growth spurt is maintained by several competing biological processes including neurogenesis, neuronal migration, differentiation and cell death, and hence, represents a critical period of vulnerability to signals that disrupt cortical development. Cues that alter the expression of suicide receptors like Fas/Apo-1 are likely to elicit profound and enduring alterations in the organization of the developing brain. Our previous data indicate that Fas-mediated, caspase-dependent death mechanisms are present during this critical period of cortical development [9]. We therefore set out to ask the following two questions: (1) 'what developmentally relevant cell biological processes predict cell-surface expression of Fas?' and (2): 'what are the consequences of Fas activation for some of these processes?' The data reported here indicate that there are at least two subpopulations of cortical neuronal precursors that express Fas receptors, a small population that expresses extremely high levels of receptor on their cell surface (the FasHi group) and the majority of Fas-expressing cells that express moderate levels of Fas on their cell surface (the FasMod group). Since ventricular-zone cortical neuroblasts also express the Fas ligand [9], FasHi neuronal precursors may be particularly sensitive to local suicide signals, and it is not surprising that, at any given time, a majority of cells in the FasHi group are apoptotic. In culture, the FasHi and FasMod groups together account for approximately one third of all cells.

An analysis of our data shows that a majority (though not all) of Fas-expressing precursors express the adapter protein FADD, which enables the activation of caspase-8-dependent death [42, 43]. Presumably, the non-FADD expressing neuroblasts (~27% of Fas-expressing cells) couple Fas to other death mechanisms, such as RIP [9] or DAXX [54]. Furthermore, only a small proportion of Fas-expressing cells also express the protein FLIP that is capable of blocking Fas activation [45]. We do not as yet know whether FLIP expression defines a phenotypically distinct sub-population of neural precursors. However, it is likely that this subpopulation is at least functionally distinct, since neural precursors that are resistant to suicide receptor-induced death may contribute disproportionately to the overall number of neurons in the cortical plate.

Our data indicate that cell-surface Fas/Apo-1 expression is responsive to stage of cell cycle. Though a majority of FasMod precursors were in G0, cortical cells express higher levels of Fas (determined by immunofluorescence intensity) in cell cycle, than in G0. Concurrent-analysis of BrdU incorporation indicates that Fas-expression is particularly high in cells that have recently completed S-phase or their final round of mitosis. One possible interpretation of these data is that Fas expression, and consequently, vulnerability to Fas-induced cell death, is tied to cell cycle checkpoints. Thus, the Fas-suicide system may play a regulatory role in eliminating cortical progenitor cells that exhibit signs of genomic instability, cell senescence, errors in chromosome replication, or DNA damage. Our observation of a positive relationship between the activation of p53 (p53 phosphorylation) and Fas expression is particularly germane to the issue of cell cycle control, since p53 regulates progression through cell-cycle checkpoints [55, 56]. Presumably, a p53-associated increase in cell-surface Fas expression would allow 'sentinel' Fas-ligand expressing cells in the local ventricular zone environment to eliminate damaged neuroblasts from the pool of progenitors, thereby limiting the likelihood that aberrant neurons will be added to the cortical plate.

An interesting phenomenon related to our observation that cell-surface Fas expression is highest at the end of S-phase, is the association between cell cycle and oscillatory, interkinetic movements of progenitor cells within the ventricular zone (see Figure 10, ventricular zone model). As progenitor cells transition from S-phase to mitosis, their nuclei migrate from the outer margin of the neuroepithelium to the ventricular surface [57]. Our data suggest that during this ventricular-fugal nuclear migration (post-S-phase), cortical progenitors would exhibit the highest sensitivity to Fas-induced apoptosis. This scenario is likely to be biologically important, because genetic defects in proteins such as lissencephaly-1 (LIS1) that control interkinetic nuclear movements, lead to enhanced cell death of ventricular-zone neuroblasts [58]. LIS1 regulates interkinetic movements by interacting with the microtubule-dynein motor system (reviewed in [59]). Interestingly, de-polymerization of microtubules protects hepatocytes from Fas-induced apoptosis [60]. Together, these data suggest a hypothesis that Fas expression and consequently, suicide-sensitivity, is linked to the interkinetic movements that accompany cell cycle in the cortical neuroepithelium.

Figure 10
figure 10

Models of Fas-mediated suicide sensitivity of precursors during cell cycle, and following disruption of cell-matrix interactions. Ventricular zone (VZ), interkinetic nuclear movement model: Our data shows that during cell cycle, cell-surface Fas expression is highest in neuroblasts that also exhibit the highest level of BrdU incorporation. Such a relationship would occur at the end of S-phase, perhaps reflecting DNA replication errors. Therefore, Fas expression (indicated in the cartoon by a green peri-cellular halo), and hence suicide-sensitivity would be highest during the ventricular-fugal interkinetic movement of nuclei transitioning through G2. Resident Fas-ligand expressing cells (indicated by pacman figures) could eliminate defective Fas-expressing neuroblasts. Cortical plate (CP), 'anoikis' model: Cortical neuroblasts utilize integrin-mediated signals to migrate along radial glia and into the laminae of the cortical plate [98]. Collagenase-A disrupts integrin-collagen interactions, and our data shows that collagenase-A leads to increased cell-surface Fas expression. Therefore, the induction of the Fas receptor may underlie the process of 'anoikis'. 'Anoikis' in turn, may protect the developing cerebral cortex from migration errors. Abbreviations: V = ventricular zone, VZ = ventricular zone, CP = cortical plate.

Motility is also an important feature of the post-mitotic cortical neuroblast. Differentiating cortical neuroblasts exit the ventricular zone and populate the cortical plate in an inside-out gradient, with the younger neuroblasts populating successively superficial laminae [61]. Migration into the cortical plate is mediated by the interactions of receptors like the integrins with the extracellular matrix (reviewed in [59]). Our initial observation was that non-adherent neural cells predominantly segregated to the FasHi category of cell-surface Fas-expressing cells. Therefore, in a subsequent experiment, we manipulated the interactions of neural cells with the extra-cellular matrix. Since type-1 collagen engages integrins, we used Collagenase-A to disrupt neuroblast interactions with the extra-cellular collagen matrix. Our results showed that loss of contact with the collagen matrix resulted in a significant increase in Fas expression, and was associated with the phenomenon of 'anoikis'. These data are consistent with a recent report showing that integrin-mediated T-lymphocyte adhesion to extracellular matrix resulted in the suppression of Fas expression [62]. Anoikis may be relevant to brain development as well, since suppression of matrix reorganization by deletion of the MMP-9 gene, leads to delayed migration of cerebellar granule cells and decreased apoptosis [47]. Interestingly, in the context of our data on estrogen suppression of Fas expression, homozygous estrogen receptor-beta knockout animals exhibit defects in the organization of radial glia and increased apoptosis in cortical ventricular zone neuroblasts [63]. These data suggest that migration and apoptosis are contextually linked to each other in vivo. Perhaps, differentiating neurons that migrate to inappropriate positions within the cortical plate are eliminated by receptor-mediated suicide mechanisms (see Figure 10, cortical plate model), analogous to the mechanism that is used to eliminate self-antigen recognizing lymphocytes in the immune system [12].

The developing brain's hormonal environment has an important impact on cell fate determination and the hormone estrogen is an important regulator of cell cycle and cell suicide in cerebral cortical neuroblasts [37]. Both identified estrogen receptor subtypes, ERα and ERβ, are ligand-activated transcription factors [6468]. Nuclear estrogen receptors and ERα mRNA are transiently expressed, at high levels in the developing cerebral cortex during the peak of neurogenesis and apoptosis [6971]. ERβ is also expressed in the cerebral cortex [68, 72, 73].

Estrogen has divergent, region-specific actions on the survival of neural tissues. For example, estrogen metabolites induce apoptosis in neuroblastoma cells [74], while estrogen promotes survival in hypothalamic cell lines [75] and in the bed nucleus of Stria Terminalis [10]. Estrogen also inhibits apoptosis in the sexually dimorphic nucleus of the preoptic area while inducing apoptosis in the anteroventral periventricular nucleus of the preoptic area [76]. Furthermore, ERα and ERβ may also have opposing anti- and pro-apoptotic roles that are perhaps Fas-ligand dependent, as supported by evidence from hypothalamic cell lines [77]. In cerebral cortical neuroblasts, we have previously shown that estrogen promotes activation of the transcription factor p53, but uncouples p53 from down-stream cell suicide signals to promote neuronal survival [37]. We therefore reasoned that estrogen suppresses apoptosis in the developing cerebral cortex, in part, by decreasing the cell-surface expression of the Fas receptor. This hypothesis was verified by our experiments. The numbers of neuroblasts in G0 expressing the Fas receptor declined significantly, following exposure to estradiol-17beta. Though estrogen did not alter the numbers of cells in cell cycle (S/G2/M) that expressed Fas, estrogen did lead to a significant decrease in the average intensity of Fas expression at the cell-surface. The estrogen-mediated reduction in numbers of Fas-expressing neuroblasts in G0 is open to several interpretations. The most parsimonious explanation for the data is that estrogen directly decreased Fas expression. Alternatively, since estrogen also promotes neuroblast proliferation [37], estrogen could have led to the expansion of a progenitor pool that did not express Fas on the cell surface.

We had previously observed that estrogen prevented apoptosis in T-antigen-synchronized cultures, following p53 activation (as indicated by p53 phosphorylation at serine 392, and activation of prototypic p53-responsive genes like MDM2, p21/Waf-1 and Bax) [37]. In the current study on non-synchronized primary cortical neuroblasts, we observed a non-statistically significant trend towards an estrogen-mediated decrease in apoptosis as reflected by the presence of cells with less than G0 DNA content. However, when we restricted the analysis to the Fas population, we found that estrogen induced a statistically significant decrease in the numbers of Fas-expressing cells that were apoptotic. Since Fas expression was positively associated with p53 phosphorylation, it is likely that estrogen protection against apoptosis is contextual, i.e., dependent on the activation of p53. Thus, following p53 activation in our CHB50 cortical progenitors, we found that estrogen also led to a significant decrease in Fas expression. Interestingly, the estrogen receptor antagonist, 4-hydroxytamoxifen, did not block estrogen suppression of Fas expression. We previously observed a similar tamoxifen-independent effect of estrogen on the regulation of p53 itself [37], suggesting that estrogen may regulate cell-surface Fas expression by non-classical mechanisms including transcriptional activation mediated by AP-1 [78] or activation of protein kinase pathways [79, 80].

One issue that remains to be addressed is whether estrogen suppresses cell-surface Fas expression by acting as a non-classical transcription repressor, or by suppressing translocation of Fas from intracellular compartments to the cell-surface in a manner that is antagonistic to that observed for p53 [34]. The timeframe of estrogen's actions is certainly more consistent with transcription repression, and perhaps, an antagonism of p53-mediated transcription. Both the mouse and human Fas genes contain a p53 response element (p53RE) within intron #1 that drives transcription of the Fas gene [33]. In silico analysis of the murine Fas gene (GenBank Accession #AF282865) also indicates a potential estrogen response element (ERE) in intron#1. This presumptive ERE consists of a perfect palindrome whose halves are separated by an integer multiple of 3 random base-pairs GGTCA(NNN)83TGACC (where the canonical ERE is GGTCA(NNN)TGACC [81]). Four additional hemi-palindromic sequences, identical to half a canonical ERE are dispersed throughout intron#1, and may also contribute to estrogen repression of Fas. These possibilities are supported by our previous research showing that intronic EREs (e.g., in the Brain-derived neurotrophic factor or BDNF gene) are functional and can contain integer multiples of the canonical triplet base-pair spacer, between two adjacent hemi-palindromes [82]. Furthermore, repressor functions have been ascribed to estrogen receptors acting at the promoters for IGF-1 and IGF-1 receptor in aortic smooth muscle cells [83] and specific residues on ERα recruit co-repressor protein complexes [84]. One hypothesis that remains to be tested, is that estrogen receptors and p53 may be mutually repressive. While our data indicates that estrogen suppresses the expression of p53-induced proteins like Bax [37] and Fas, other data indicates that p53 can suppress estrogen receptor activation [85], by preventing ER-alpha binding to EREs.

Since estrogen induces neuroblasts to enter S-phase of cell cycle, while suppressing the cell-surface expression of Fas, we decided to examine the extent to which estrogen regulates cell cycle proteins concurrent with Fas activation. We were particularly interested in the regulation of dual-purpose proteins like cyclin-A, p21/waf-1 and PCNA that not only regulate progression through cell cycle, but also participate in DNA repair and blockage of apoptosis. For example PCNA and p21/waf-1 initiate repair of double-strand DNA breaks (a hallmark of apoptosis) [51] while cyclin A prevents apoptosis by disrupting p53 signaling [50]. Unexpectedly, concurrent exposure to both estrogen and to sFasL led to a decrease in the expression of both PCNA and p21/waf1. Since these proteins play antagonistic roles during cell cycle, it is likely that this co-regulation will have little net effect on cell cycle per se. On the other hand, it is likely that capacity to repair double stranded DNA breaks is curtailed. It is unclear at this time why the coordinate activation of a suicide receptor and a hormone receptor would decrease both PCNA and p21/waf1, potentially compromising DNA repair capacity. Perhaps, once cell suicide is initiated, and caspase-activated DNAses create double strand breaks, DNA repair would be an inefficient mechanism to reverse apoptosis. In this context, the role of estrogen may be to cleanly facilitate suicide. This hypothesis is consistent with observations from other laboratories [76, 77] that estrogen can induce apoptosis in specific brain regions. Estrogen's anti-apoptotic role may be to prevent the initiation of apoptosis in the first place, by suppressing expression of p53-activated proteins like Fas (as reported above) and Bax [37] or alternatively, antagonizing p53 more directly. The latter hypothesis is supported by our observation that estrogen and Fas-ligand both separately and together, significantly increased Cyclin-A. Cyclin-A is a G1/S-phase cyclin, so it is not surprising that estrogen would up-regulate this cyclin as a consequence of increasing the number of neuroblasts that enter S-phase. However, cyclin-A can also antagonize p53-dependent apoptosis [50] by preventing the association between p53 and E2F, and therefore, may represent the preferred intermediary for estrogen suppression of apoptosis. Interestingly, cyclin-E, which does not compete with E2F for binding to p53 [50], was not induced by estrogen in our experiments. Additionally, cyclin-A complexes with CDK2 to induce phosphorylation of ER-alpha on serines 104 and 106, and consequent transactivation [86]. It is likely therefore, that the induction of cyclin-A increases activation of associated kinases, even though CDK2 levels were not altered, leading to feedback activation of the estrogen receptor, further suppression of Fas expression, and neuro-protection. If cyclin-A represents a means for limiting apoptosis, then the induction of cyclin A by sFasL, suggests that neuronal precursors may initiate adaptive anti-apoptotic mechanisms (aside from FLIP expression), in response to the activation of a suicide receptor.


Overall, our results indicate that cell-surface Fas expression identifies two distinct sub-populations of cortical neuroblasts, the FasHi and FasMod populations, with FLIP co-expressing cells comprising perhaps a third population. Developmentally, cell-surface Fas expression is modulated by a variety of competing factors including cell-cycle stage, extracellular matrix interactions, p53 phosphorylation and hormone availability. Dynamic changes in suicide Fas-mediated sensitivity during development are likely to play a significant role in the genesis of a variety of developmental neurodegenerative diseases including the Fetal Alcohol Syndrome [87], neonatal cerebral ischemia [15], neonatal posthemorrhagic hydrocephalus [88], neonatal brain trauma [89], and genetic diseases resulting from the expansion of polyglutamine repeats [16].



Timed-pregnant rats (Sprague Dawley) were purchased from Harlan (TX). Gestational Day (GD) zero was defined as the day on which dams were sperm positive. GD-15 rat fetuses were obtained under aseptic conditions, from pregnant dams that were anesthetized with Phenobarbital (50 mg). Fetal brains were dissected out aseptically and, in all cases, care was taken to minimize any pain and discomfort to the animals.

Dissociated primary embryonic cortical cultures

The cerebral cortical mantle of GD-15 rat fetuses was dissected out under sterile conditions, and separated from overlying meningeal tissue under a microscope. We selected GD15 because this age represents the peak period for proliferation within the cortical neuroepithelium, to generate neurons of the cortical plate. At GD15, the neuroepithelium is the dominant cortical structure, though a smaller number of differentiated neurons (the cortical preplate) and radial glia are also present. Furthermore, at this age, the neuroepithelium is mainly comprised of proliferating neuronal (rather than glial) precursors (for review see [40]). The earliest developing layer of the cortical plate (layer VI) does not form until one day later (GD16-17). Morphologically, these cortical cultures are immature and continue to proliferate in vitro, but over a period of 24 hours, cultured neuroblasts become increasingly polarized and exhibit growth cones and process characteristic of early differentiating neurons. Cultures were established according to our previously published protocols [9]. Briefly, neuroblasts were dispersed by trituration in 0.5% trypsin and 6.84 mM EDTA (Sigma) and were plated on collagen-coated 24-well plates in sterile culture medium (89% Dulbecco's modified eagle media [DMEM], 10% gelding serum and 1% penicillin-streptomycin). Cultures were maintained at 37°C for 24 hours and the culture medium was replaced. For initial experiments, whole cultures were assayed 24 hours later by FACS (Fluorescence-assisted cell sorting) analysis. For subsequent experiments, only adherent cells were harvested at 24 hours and fixed for FACS analysis. For some experiments, soluble protein was extracted for western immunoblot analysis. Some experimental groups were exposed to soluble Fas Ligand (sFasL 5 ng/ml, Alexis Corp.), estradiol-17β (2 nM, Sigma) or sFasL together with estradiol-17β. Control and sFasL-treated cultures were maintained at 37°C in normal culture medium for 24 hours and then exposed to the appropriate treatments. Estrogen-treated cultures were pre-treated with estradiol-17β for 24 hours and then re-fed with estrogen alone or estrogen concurrently with sFasL for 12 hours.

Immortalized cerebral cortical culture model

To examine the regulation of Fas by p53 and estrogen, we also utilized a rodent cerebral cortical cell line (CHB50) conditionally immortalized with a temperature sensitive mutation of the SV40 large T antigen (tsTA) that we had previously developed and characterized [37]. In the +tsTA condition (incubation at 33°C), neuroblasts proliferate indefinitely and p53 is inactive. However, cessation of large T antigen expression (-tsTA, 39°C) is accompanied by induction of the p53 phosphorylation and the induction of p53-dependent proteins, p21/Waf1, Bax and MDM2 that lead to cell cycle-arrest, suicide, and p53 inhibition/cell cycle re-entry respectively. We had previously observed that CHB50 neuroblasts express the estrogen receptor ER-α and are estrogen-responsive. Estrogen prevents apoptosis following p53 activation and induces both neurogenesis and neuronal differentiation in this cell line. CHB50 cortical neuroblasts were therefore cultured under the +tsTA and -tsTA conditions as published previously [37] to examine the p53-related expression of Fas, and under the -tsTA conditions to examine the estrogen regulation of Fas during neuroblast differentiation (in the context of p53 activation).

Flow cytometric analysis

Triple parameter flow cytometric analysis was used to measure immunolabeled cell surface Fas protein, BrdU incorporation (a measure of DNA incorporation) and DNA content [propidium iodide (PI) dye intercalation] as a measure of cell cycle distribution. Dissociated embryonic cortical cultures were administered a single BrdU pulse (24 hours) at the time the cultures were established. Non-adherent and adherent [harvested by administration of collagenase (Sigma, 30 units)] cells were obtained after the initial 24-hour BrdU pulse and centrifuged at 300 × g to sediment the cells. The pellet was washed with Dulbecco's Phosphate buffered saline (PBS) and fixed in 1% paraformaldehyde and re-suspended in PBS. Cells were sedimented by centrifugation between subsequent immunohistochemical steps. To detect cell-surface Fas/Apo-1, immuno-histofluorescence chemistry was performed in the absence of detergent. Following exposure to a blocking solution (in Tris-buffered saline, (TBS) pH 7.4, with 0.1%Bovine Serum Albumin, and 2% Normal Goat Serum), cell-surface Fas/Apo-1 expression was detected with a rabbit polyclonal anti-Fas antibody (Santa Cruz, 1:500), followed by biotinylated, secondary donkey anti-rabbit (Jackson Immunochemicals, 1:1000) conjugated to streptavidin-FITC. Following Fas/Apo-1 immunofluorescence, cells were permeabilized with detergent (0.3% Trition-X100) in blocking solution. BrdU incorporation was detected with a primary anti-BrdU antibody (Sigma, 1:500) followed by biotinylated, secondary horse anti-mouse (Vector, 1:1000), conjugated to streptavidin-allophycocyanin (APC, Molecular Probes, 1:500). The cells were then washed with PBS and exposed to a PI solution (see above) for 10 minutes at room temperature. BrdU-allophycocyanin, Fas-FITC and PI fluorescence was analyzed from 104 cells with excitation at 488 nm (argon laser) and detection of FITC fluorescence at approximately 530 nm, PI fluorescence at 585 nm and allophycocyanin at 650 nm.

In studies examining the relationship between cell surface Fas and Fas-associated protein expression, binding of the primary antibody, mouse monoclonal antibody to Fas (Transduction Laboratories, 1:500), was detected using a rat-absorbed, biotinylated horse-anti-mouse secondary antibody (Vector, 1:1000), conjugated to streptavidin-APC or rabbit polyclonal anti-Fas antibody (Santa Cruz, 1:500), followed by biotinylated, secondary donkey anti-rabbit (Jackson Immunochemicals, 1:1000) conjugated to streptavidin-FITC. Subsequently, FLIP or phosphorylated P53 was detected using rabbit polyclonal anti-FLIP (gift from Drs. Sato and Walsh, Tufts University School of Medicine; 1:500) or phospho-p53 at serine 392 (New England BioLabs, 1:500) followed by a secondary donkey anti-rabbit (Jackson Immunochemicals, 1:1000) conjugated to streptavidin-FITC. FADD was detected using a monoclonal anti-FADD antibody (Transduction Laboratories, 1:500) followed by a rat-absorbed, biotinylated horse-anti-mouse secondary antibody (Vector, 1:1000). Phosphorylation of the casein kinase II-sensitive site at serine 392 was used as a marker for activated p53. Phosphorylation at this site promotes tetramerization of p53 [90] and specificity of p53 binding to DNA [91], while regulating re-annealing of double-stranded DNA [92]. Cells were then washed with PBS and exposed to a PI solution [PBS, 0.1% triton (Sigma), 0.5 mmol EDTA, 0.05 mg/ml RNAse A and 50 mg/ml PI] for 10 minutes at room temperature. Fas-FITC and PI fluorescence was analyzed from 10,000 cells using a flow cytometer (Becton Dickinson) with excitation at 488 nm (argon laser) and detection of FITC fluorescence at approximately 530 nm and PI fluorescence at 585 nm. The specificity of all antibodies used for flow cytometric analysis was previously verified by western immunoblot analysis and/or immunohistochemical analysis [9, 37]. Immunological controls were exposed to pre-immune serum in place of the appropriate primary antibody and were used to determine background immunofluorescence.

Western immunoblot analysis

The expression of cell cycle elements that regulate S-phase [proliferating cell nuclear antigen (PCNA)], the G1/S transition [cyclin dependent kinase 2 (cdk2) and cyclin E], the G2/M transition [cyclin dependent kinase (cdk1) and cyclin A], and a cell cycle arrest factor [p21/wild-type p53-activated fragment (Waf1)/Cip1 (cdk-interacting protein-1)], and Fas were verified by western immunoblot analysis of E15 cortical neuroblasts, according to our previously published protocols [9, 37, 93, 94]. Detergent (1% SDS) soluble protein was isolated using the Trizol reagent (Invitrogen). Protein samples were size fractionated on an 8% SDS-polyacrylamide gel, and blotted onto supported nitrocellulose (Hibond-C-super, Amersham). Blots were blocked (5% milk, TBS (1.4 M NaCl, 0.2 M Tris)-0.1% Tween 20), exposed to primary antibody [mouse monoclonal anti-PCNA antibody (Calbiochem, 1:66); rabbit polyclonal anti-cyclin E (Calbiochem, 1:250); mouse monoclonal anti-cyclin A (Calbiochem, 1:100), mouse monoclonal anti-cdk2 (Transduction Laboratories, 1:500), mouse monoclonal anti-cdk1 (Calbiochem, 1:100) or mouse monoclonal anti-Fas (Transduction Labs)], washed and exposed to horse-anti-mouse secondary antibody (Vector, 1:1000) or donkey anti-rabbit (Jackson Laboratories, 1:5000), washed again and exposed to streptavidin horseradish-peroxidase conjugate (Amersham). Immunoreactive bands were detected using enzyme-linked chemiluminescense (NEN).

Data analysis

Data analyses for the experiments reported here were based on 5–9 independent samples. For FACS analysis, the incorporation of PI into cellular DNA was used to categorize cells in G0/G1 (diploid DNA content), S/G2/M (hyper-diploid DNA content) and apoptosis (sub-diploid DNA content) [9597]. Cells were categorized, and intensities calculated using standard cell sorting software (Cell Quest, Becton Dickinson). Western immunoblots were analyzed using standard densitometric software (Molecular Analyst, Bio-Rad). Sample size for western blot analyses was 6. Statistical differences were calculated using ANOVAs followed by post-hoc tests (Student-Newman-Keuls, p < 0.05). Correlational analyses were calculated by combining mean intensity values from 6–8 replications using Pearson's product moment correlation (r) followed by a two-tailed test for the significance of the correlation coefficient. Levels of statistical significance were set at p < 0.05. Data were expressed in terms of mean ± standard error of the mean (SEM).






CDK1 = CDC2:

Cyclin-dependent kinase 1


Cyclin-dependent kinase 2


Cortical Plate


Death Inducing Signaling Complex




Estrogen response element


Fas-associated death domain containing protein


Fas/Apo [apoptosis]-1/CD95 suicide receptor


High intensity of cell surface Fas immuno-fluorescence


Moderate intensity of cell surface Fas immuno-fluorescence


Undetectable cell surface Fas immuno-fluorescence (not different from background)


Fluorescein isothiocyanate


FLICE (caspase-8)–inhibitory protein


Fluorescence Units


Wild-type p53-activated fragment 1 or cyclin-dependent kinase inhibitor 1A


p53 response element


Proliferating cell nuclear antigen


Propidium iodide


Phosphorylated p53 (on serine 392)


Standard error of the mean


soluble Fas-ligand


temperature-sensitive SV40 T antigen




Ventricular Zone


  1. Ferrer I, Bernet E, Soriano E, DelRio T, Fonseca M: Naturally occurring cell death in the cerebral cortex of the rat and removal of dead cells by transitory phagocytes. Neuroscience. 1990, 39: 451-458. 10.1016/0306-4522(90)90281-8.

    Article  CAS  PubMed  Google Scholar 

  2. Ferrer I, Soriano E, DelRio J, Alcantara S, Auladell C: Cell death and removal in the cerebral cortex during development. Prog.Neurobiol. 1992, 39: 1-43. 10.1016/0301-0082(92)90029-E.

    Article  CAS  PubMed  Google Scholar 

  3. Finlay B, Slattery M: Local differences in the amount of early cell death in neocortex predict adult local specializations. Science. 1983, 219: 1349-1351.

    Article  CAS  PubMed  Google Scholar 

  4. Spreafico R, Frassoni C, Arcelli P, Selvaggio M, De Biasi S: In situ labeling of apoptotic cell death in the cerebral cortex and thalamus of rats during development. J Comp Neurol. 1995, 363: 281-295. 10.1002/cne.903630209.

    Article  CAS  PubMed  Google Scholar 

  5. Rabinowicz T, De Courten-Myers GM, Petetot JM-S, Xi G, De Los Reyes E: Human cortex development: Estimates of neuronal numbers indicate major loss late during gestation. Journal of Neuropathology and Experimental Neurology. 1996, 55: 320-328.

    Article  CAS  PubMed  Google Scholar 

  6. Thomaidou D, Mione MC, Cavanagh JF, Parnavelas JG: Apoptosis and its relation to cell cycle in the developing cerebral cortex. Journal of Neuroscience. 1997, 17: 1075-1085.

    CAS  PubMed  Google Scholar 

  7. Verney C, Takahashi T, Bhide PG, Nowakowski RS, Caviness VS: Independent controls for neocortical neuron production and histogenic cell death. Dev Neurosci. 2000, 22: 125-138. 10.1159/000017434.

    Article  CAS  PubMed  Google Scholar 

  8. Blaschke AJ, Weiner JA, Chun J: Programmed cell death is a universal feature of embryonic and postnatal neuroproliferative regions throughout the central nervous system. Journal of Comparitive Neurology. 1998, 396: 39-50. 10.1002/(SICI)1096-9861(19980622)396:1<39::AID-CNE4>3.0.CO;2-J.

    Article  CAS  Google Scholar 

  9. Cheema ZF, Wade SB, Sata M, Walsh K, Sohrabji F, Miranda RC: Fas/Apo[Apoptosis]-1 and associated proteins in the differentiating cerebral cortex: Induction of caspase dependent cell death and activation of NF-kappaB. Journal of Neuroscience. 1999, 19: 1754-1770.

    CAS  PubMed  Google Scholar 

  10. Chung WC, Swaab DF, Vries GJ: Apoptosis during sexual differentiation of the bed nucleus of the stria terminalis in the rat brain. J Neurobiol. 2000, 43: 234-243. 10.1002/(SICI)1097-4695(20000605)43:3<234::AID-NEU2>3.3.CO;2-V.

    Article  CAS  PubMed  Google Scholar 

  11. Burek MJ, Oppenheim RW: Programmed cell death in the developing nervous system. Brain Pathol. 1996, 6: 427-446.

    Article  CAS  PubMed  Google Scholar 

  12. Nagata S, Goldstein P: The Fas death factor. Science. 1995, 267: 1449-1456.

    Article  CAS  PubMed  Google Scholar 

  13. Park C, Sakamaki K, Tachibana O, Tetsumori Y, Junkoh Y, Yonehara S: Expression of Fas antigen in the normal mouse brain. Biochemical and Biophysical Research Communications. 1998, 252: 623-628. 10.1006/bbrc.1998.9572.

    Article  CAS  PubMed  Google Scholar 

  14. van Landeghem FK, Felderhoff-Mueser U, Moysich A, Stadelmann C, Obladen M, Bruck W, Buhrer C: Fas (CD95/Apo-1)/Fas ligand expression in neonates with pontosubicular neuron necrosis. Pediatr Res. 2002, 51: 129-135.

    Article  CAS  PubMed  Google Scholar 

  15. Felderhoff-Mueser U,, Taylor DL, Greenwood K, Kozma M, Stibenz D, Joashi UC, Edwards AD, Mehmet H: Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain. Brain Pathol. 2000, 10: 17-29.

    Article  CAS  PubMed  Google Scholar 

  16. Sanchez I, Xu CJ, Juo P, Blenis Y, Yuan J: Caspase-8 is required for cell death induced by expanded polyglutamine repeats. Neuron. 1999, 22: 623-633. 10.1016/S0896-6273(00)80716-3.

    Article  CAS  PubMed  Google Scholar 

  17. Le-Niculescu H, Bonfoco E, Kasuya Y, Claret F-X, Green DR, Karin M: Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas Ligand induction and cell death. Molecular and Cellular Biology. 1999, 19: 751-763.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Raoul C, Henderson CE, Pettmann B: Programmed cell death of embryonic motoneurons triggered through the Fas death receptor. Journal of Cell Biology. 1999, 147: 1049-1061. 10.1083/jcb.147.5.1049.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Bonetti B, Raine CS: Multiple scelerosis: oligodendrocytes display cell death-related molecules in situ but do not undergo apoptosis. Annals of Neurology. 1997, 42: 74-84. 10.1002/ana.410420113.

    Article  CAS  PubMed  Google Scholar 

  20. D'Souza SD, Bonetti B, Balasingam V, Cashman NR, Barker PA, Troutt AB, Raine CS, Antel JP: Multiple sclerosis: Fas signaling in oligodendrocyte cell death. Journal of Experimental Medicine. 1996, 184: 2361-2370. 10.1084/jem.184.6.2361.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Harrison DC, Roberts J, Campbell CA, Crook B, Davis R, Deen K, Meakin J, Michalovich D, Price J, Stammers M, Maycox PR: TR3 death receptor expression in the normal and ischaemic brain. Neuroscience. 2000, 96: 147-160. 10.1016/S0306-4522(99)00502-3.

    Article  CAS  PubMed  Google Scholar 

  22. Matsuyama T, Hata R, Tagaya M, Yamamoto Y, Nakajima T, Furuyama J, Wanaka A, Sugita M: Fas antigen mRNA induction in postichemic murine brain. Brain Research. 1994, 657: 342-346. 10.1016/0006-8993(94)90989-X.

    Article  CAS  PubMed  Google Scholar 

  23. Matsuyama T, Hata R, Yamamoto Y, Tagaya M, Akita H, Uno H, Wanaka A, Furuyama J, Sugita M: Localization of Fas antigen mRNA induces in postischemic murine forebrain by in situ hybridization. Molecular Brain Research. 1995, 34: 166-172. 10.1016/0169-328X(95)00162-L.

    Article  CAS  PubMed  Google Scholar 

  24. Nishimura T, Akiyama H, Yonehara S, Kondo H, Ikeda K, Kato M, Iseki E, Kenji K: Fas antigen espression in brains of patients with Alzheimers-type dementia. Brain Research. 1995, 695: 137-145. 10.1016/0006-8993(95)00699-Q.

    Article  CAS  PubMed  Google Scholar 

  25. Sakurai M, Hayashi T, Abe K, Sadahiro M, Tabayashi K: Delayed selective motor neuron death and Fas antigen induction after spinal cord ischemia in rabbits. Brain Research. 1998, 797: 23-28. 10.1016/S0006-8993(98)00290-X.

    Article  CAS  PubMed  Google Scholar 

  26. Tachibana O, Nakazawa H, Lampe J, Watanabe K, Kleihues P, Ohgaki H: Expression of Fas/Apo-1 during the progression of astrocytomas. Cancer Research. 1995, 55: 5528-5530.

    CAS  PubMed  Google Scholar 

  27. Weller M, Kleihues P, Dichgans J, Ohgaki H: CD95 ligand - lethal weapon against malignant gliomas. Brain Pathology. 1998, 8: 285-293.

    Article  CAS  PubMed  Google Scholar 

  28. Di Cristofano A, Kotsi P, Peng YF, Cordon-Cardo C, Elkon KB, Pandolfi PP: Impaired Fas Response and Autoimmunity in Pten +/- Mice. Science. 1999, 285: 2122-2125. 10.1126/science.285.5436.2122.

    Article  CAS  PubMed  Google Scholar 

  29. Li L, Liu F, Salmonsen RA, Turner TK, Litofsky NS, Di Cristofano A, Pandolfi PP, Jones SN, Recht LD, Ross AH: PTEN in neural precursor cells: regulation of migration, apoptosis, and proliferation. Mol Cell Neurosci. 2002, 20: 21-29. 10.1006/mcne.2002.1115.

    Article  CAS  PubMed  Google Scholar 

  30. Rasheed BK, Stenzel TT, McLendon RE, Parsons R, Friedman AH, Friedman HS, Bigner DD, Bigner SH: PTEN gene mutations are seen in high-grade but not in low-grade gliomas. Cancer Res. 1997, 57: 4187-4190.

    CAS  PubMed  Google Scholar 

  31. Amson R, Lassalle JM, Halley H, Prieur S, Lethrosne F, Roperch JP, Israeli D, Gendron MC, Duyckaerts C, Checler F, Dausset J, Cohen D, Oren M, Telerman A: Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice. PNAS. 2000, 97: 5346-5350. 10.1073/pnas.97.10.5346.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Li Y, Raffo AJ, Drew L, Mao Y, Tran A, Petrylak DP, Fine RL: Fas-mediated apoptosis is dependent on wild-type p53 status in human cancer cells expressing a temperature-sensitive p53 mutant alanine-143. Cancer Res. 2003, 63: 1527-1533.

    CAS  PubMed  Google Scholar 

  33. Munsch D, Watanabe-Fukunaga R, Bourdon J-C, Nagata S, May E, Yonish-Rouach E, Reisdorf P: Human and Mouse Fas (APO-1/CD95) Death Receptor Genes Each Contain a p53-responsive Element That Is Activated by p53 Mutants Unable to Induce Apoptosis. J Biol Chem. 2000, 275: 3867-3872. 10.1074/jbc.275.6.3867.

    Article  CAS  PubMed  Google Scholar 

  34. Bennet M, Macdonald K, Chan S-W, Luzio JP, Simari R, Weissberg P: Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science. 1998, 282: 290-293. 10.1126/science.282.5387.290.

    Article  Google Scholar 

  35. Sheard MA, Vojtesek B, Janakova L, Kovarik J, Zaloudik J: Up-regulation of Fas (CD95) in human p53wild-type cancer cells treated with ionizing radiation. Int.J.Cancer. 1997, 73: 757-762. 10.1002/(SICI)1097-0215(19971127)73:5<757::AID-IJC24>3.0.CO;2-1.

    Article  CAS  PubMed  Google Scholar 

  36. Tan Z, Sankar R, Tu W, Shin D, Liu H, Wasterlain CG, Schreiber SS: Immunohistochemical study of p53-associated proteins in rat brain following lithium-pilocarpine status epilepticus. Brain Res. 2002, 929: 129-138. 10.1016/S0006-8993(01)03360-1.

    Article  CAS  PubMed  Google Scholar 

  37. Wade SB, Oommen P, Conner WC, Earnest DJ, Miranda RC: Overlapping and divergent actions of estrogen and the neurotrophins on cell fate and p53-dependent signal transduction in conditionally immortalized cerebral cortical neuroblasts. J Neurosci. 1999, 19: 6994-7006.

    CAS  PubMed  Google Scholar 

  38. McKay LI, Cidlowski JA: Cross-talk between nuclear factor-kappa B and the steroid hormone receptor: mechanisms of mutual antagonism. Molecular Endocrinology. 1998, 12: 45-56. 10.1210/me.12.1.45.

    Article  CAS  PubMed  Google Scholar 

  39. Beg AA, Baltimore D: An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science. 1996, 274: 782-784. 10.1126/science.274.5288.782.

    Article  CAS  PubMed  Google Scholar 

  40. Bayer SA, Altman J: Principles of Neurogenesis, Neuronal Migration, and Neural Circuit Formation. The Rat Nervous System. Edited by: Paxinos G. 1995, San Diego, Academic Press, Inc., 1079-1098. 2

    Google Scholar 

  41. Hsu H, Shu H-B, Pan M-P, Goeddel DV: TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor-1 signal transduction pathways. Cell. 1996, 84: 299-308. 10.1016/S0092-8674(00)80984-8.

    Article  CAS  PubMed  Google Scholar 

  42. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM: FADD, a novel death domain-containing protein interacts with the death domain of Fas and initiates apoptosis. Cell. 1995, 81: 505-512. 10.1016/0092-8674(95)90071-3.

    Article  CAS  PubMed  Google Scholar 

  43. Chinnaiyan AM, Tepper CG, Seldin MF, O'Rourke K, Krischel FC, Hellbardt S, Krammer PH, Dixit VM: FADD/MORT is a common mediator of CD95 (Fas/Apo-1) and TNF-receptor-induced apoptosis. Journal of Biological Chemistry. 1996, 271: 4961-4965. 10.1074/jbc.271.9.4961.

    Article  CAS  PubMed  Google Scholar 

  44. Boldin MP, Goncharov TM, Golstev YV, Wallach D: Involvement of MACH, a novel MORT1/FADD-interacting protease in FAS/APO-1- and TNF receptor-induced cell death. Cell. 1996, 85: 803-815. 10.1016/S0092-8674(00)81265-9.

    Article  CAS  PubMed  Google Scholar 

  45. Srinivasula SM, Ahmad M, Ottilie S, Bullrich F, Banks S, Wang Y, Fernandes-Alnemri T, Croce C, Litwack G, Tomaselli KJ, Armstrong RC, Alnemri ES: FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis. Journal of Biological Chemistry. 1997, 272: 18542-18545. 10.1074/jbc.272.30.18542.

    Article  CAS  PubMed  Google Scholar 

  46. Grossmann J, Walther K, Artinger M, Kiessling S, Scholmerich J: Apoptotic signaling during initiation of detachment-induced apoptosis ("anoikis") of primary human intestinal epithelial cells. Cell Growth & Differentiation. 2001, 12: 147-155.

    CAS  Google Scholar 

  47. Vaillant C, Meissirel C, Mutin M, Belin M-F, Lund LR, Thomasseta N: MMP-9 deficiency affects axonal outgrowth, migration, and apoptosis in the developing cerebellum. Molecular and Cellular Neuroscience. 2003, 24: 395-408. 10.1016/S1044-7431(03)00196-9.

    Article  CAS  PubMed  Google Scholar 

  48. Sohrabji F, Greene L, Miranda R, Toran-Allerand CD: Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells. J Neurobiol. 1994, 25: 974-988. 10.1002/neu.480250807.

    Article  CAS  PubMed  Google Scholar 

  49. Sohrabji F, Miranda RC, Toran-Allerand CD: Estrogen differentially regulates estrogen and nerve growth factor receptor mRNAs in adult sensory neurons. J Neurosci. 1994, 14: 459-471.

    CAS  PubMed  Google Scholar 

  50. Hsieh JK, Yap D, O'Connor DJ, Fogal V, Fallis L, Chan F, Zhong S, Lu X: Novel function of the cyclin A binding site of E2F in regulating p53-induced apoptosis in response to DNA damage. Mol Cell Biol. 2002, 22: 78-93. 10.1128/MCB.22.1.78-93.2002.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Scott M,, Bonnefin P,, Vieyra D,, Boisvert F-M,, Young D,, Bazett-Jones D,, Riabowol K,: UV-induced binding of ING1 to PCNA regulates the induction of apoptosis. J Cell Science. 2001, 114: 3455-3462.

    CAS  PubMed  Google Scholar 

  52. Maga G,, Hübscher U,: Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Science. 2003, 116: 3051-3060. 10.1242/jcs.00653.

    Article  CAS  PubMed  Google Scholar 

  53. Asada M,, Yamada T,, Ichijo H,, Delia D,, Miyazono K,, Fukumuro K,, Mizutani S,: Apoptosis inhibitory activity of cytoplasmic p21Cip1/WAF1 in monocytic differentiation. EMBO J. 1999, 18: 1223-1234. 10.1093/emboj/18.5.1223.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. Xiaolu Y, Khosravi-Far R, Chang HY, Baltimore D: Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell. 1997, 89: 1067-1076. 10.1016/S0092-8674(00)80294-9.

    Article  Google Scholar 

  55. Cross SM, Sanchez CA, Morgan CA, Schimke MK, Ramel S, Idverda RL, Rasking WH, Reid BJ: A p53-dependent mouse spindle checkpoint. Science. 1995, 267: 1353-1356.

    Article  CAS  PubMed  Google Scholar 

  56. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B: Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science. 1998, 282: 1497-1501. 10.1126/science.282.5393.1497.

    Article  CAS  PubMed  Google Scholar 

  57. Takahashi T, Nowakowski RS, Caviness VS: Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall. Journal of Neuroscience. 1996, 16: 5762-5776.

    CAS  PubMed  Google Scholar 

  58. Gambello MJ, Darling DL, Yingling J, Tanaka T, Gleeson JG, Wynshaw-Boris A: Multiple Dose-Dependent effects of Lis1 on cerebral cortical development. Journal of Neuroscience. 2003, 23: 1719-1729.

    CAS  PubMed  Google Scholar 

  59. Gupta A, Tsai L-H, Wynshaw-Boris A: Life is a journey: A genetic look at neocortical development. Nature Reviews: Genetics. 2002, 3: 342-355. 10.1038/nrg799.

    Article  CAS  PubMed  Google Scholar 

  60. Feng G, Kaplowitz N: Colchicine protects mice from the lethal effect of an agonistic anti-Fas antibody. J Clin Invest. 2000, 105: 329-339.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  61. Angevine JB, Sidman RL: Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature. 1961, 192: 766-768.

    Article  PubMed  Google Scholar 

  62. Gendron S, Couture J, Aoudjit F: Integrin alpha2 beta1 inhibits Fas-mediated apoptosis in T lymphocytes by protein phosphatase 2A-dependent activation of the MAPK/ERK pathway. J Biol Chem. 2003, [Epub ahead of print].

    Google Scholar 

  63. Wang L, Andersson S, Warner M, Gustafsson J-A: Estrogen receptor (ER)beta knockout mice reveal a role for ER beta in migration of cortical neurons in the developing brain. Proc Nat Acad Sci USA. 2002, 100: 703-708. 10.1073/pnas.242735799.

    Article  Google Scholar 

  64. Beato M: Gene regulation by steroid hormones. Cell. 1989, 56: 335-344. 10.1016/0092-8674(89)90237-7.

    Article  CAS  PubMed  Google Scholar 

  65. Evans RM: The steroid and thyroid hormone receptor superfamily. Science. 1988, 240: 889-895.

    Article  CAS  PubMed  Google Scholar 

  66. O'Malley B: The steroid receptor superfamily: more excitement predicted for the future. Molecular Endocrinology. 1990, 4: 363-369.

    Article  PubMed  Google Scholar 

  67. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson G, Gustafsson JA: Cloning of a novel estrogen receptor expressed in rat prostrate and ovary. Proc Nat Acad Sci USA. 1996, 93: 5925-5930. 10.1073/pnas.93.12.5925.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  68. Mosselman S, Polman J, Dijkema R: ER·B: identification and characterization of a novel human estrogen receptor. FEBS. 1996, 392: 49-53. 10.1016/0014-5793(96)00782-X.

    Article  CAS  Google Scholar 

  69. Friedman WJ, McEwen BS, Toran-Allerand CD, Gerlach JL: Perinatal development of hypothalamic and cortical estrogen receptors in mouse brain: Methodological aspects. Brain Research, Development Brain Research. 1983, 11: 19-27. 10.1016/0165-3806(83)90198-0.

    Article  CAS  Google Scholar 

  70. Gerlach JL, McEwen BS, Toran-Allerand CD, Friedman WJ: Perinatal development of estrogen receptors in mouse brain assessed by radiautography, nuclear isolation and receptor assay. Brain Research, Development Brain Research. 1983, 11: 7-18. 10.1016/0165-3806(83)90197-9.

    Article  CAS  Google Scholar 

  71. Miranda R, Toran-Allerand CD: Developmental expression of estrogen receptor mRNA in the rat cerebral cortex: a nonisotopic in situ hybridization histochemistry study. Cerebral Cortex. 1992, 2: 1-15.

    Article  CAS  PubMed  Google Scholar 

  72. Couse JF, Lindzey J, Grandien K, Gustafsson J, Korach KS: Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology. 1997, 138: 4613-4621. 10.1210/en.138.11.4613.

    CAS  PubMed  Google Scholar 

  73. Perez SE, Chen EY, Mufson EJ: Distribution of estrogen receptor alpha and beta immunoreactive profiles in the postnatal rat brain. Brain Res Dev Brain Res. 2003, 145: 117-139. 10.1016/S0165-3806(03)00223-2.

    Article  CAS  PubMed  Google Scholar 

  74. Nakagawa-Yagi Y, Ogane N, Inoki Y, Kitoh N: The endogenous estrogen metabolite 2-methoxyestradiol induces apoptotic neuronal cell death in vitro. Life Sciences. 1996, 58: 1461-1467. 10.1016/0024-3205(96)00116-6.

    Article  CAS  PubMed  Google Scholar 

  75. Rasmussen JE, Torres-Aleman I, MacLusky NJ, Naftolin F, Robbins RJ: The effects of estradiol on the growth patterns of estrogen receptor-positive hypothalamic cell lines. Endocrinology. 1990, 126: 235-240.

    Article  CAS  PubMed  Google Scholar 

  76. Arai Y, Sekine Y, Murakami S: Estrogen and apoptosis in the developing sexually dimorphic preoptic area in female rats. Neuroscience Research. 1996, 25: 403-407. 10.1016/0168-0102(96)01070-X.

    Article  CAS  PubMed  Google Scholar 

  77. Nilsen J, Mor G, Naftolin F: Estrogen-regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system. J Neurobiol. 2000, 43: 64-78. 10.1002/(SICI)1097-4695(200004)43:1<64::AID-NEU6>3.0.CO;2-7.

    Article  CAS  PubMed  Google Scholar 

  78. Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-A, Kushner PJ, Scanlan TS: Differential ligand activation of estrogen receptors ER-alpha and ER·beta at AP1 sites. Science. 1997, 277: 1508-1510. 10.1126/science.277.5331.1508.

    Article  CAS  PubMed  Google Scholar 

  79. Singh M, Setalo G, Guan X, Frail DE, Toran-Allerand CD: Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-alpha knock-out mice. Journal of Neuroscience. 2000, 20: 1694-1700.

    CAS  PubMed  Google Scholar 

  80. Singh M, Setalo G, Guan X, Warren M, Toran-Allerand CD: Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. Journal of Neuroscience. 1999, 19: 1179-1188.

    CAS  PubMed  Google Scholar 

  81. Klein-Hitpass L, Schorpp M, Wagner U, Ryffel G: An estrogen-responsive element derived from the 5' flanking region of xenopus vitellogenin A2 gene functions in transfected human cells. Cell. 1986, 46: 1053-1061. 10.1016/0092-8674(86)90705-1.

    Article  CAS  PubMed  Google Scholar 

  82. Sohrabji F, Miranda R, Toran-Allerand D: Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor. Proc Natl Acad Sci. 1995, 92: 11110-11114.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  83. Scheidegger KJ, Cenni B, Picard D, Delafontaine P: Estradiol decreases IGF-1 and IGF-1 receptor expression in rat aortic smooth muscle cells. Mechanisms for its atheroprotective effects. J Biol Chem. 2000, 275: 38921-38928. 10.1074/jbc.M004691200.

    Article  CAS  PubMed  Google Scholar 

  84. Huang HJ, Norris JD, McDonnell DP: Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists. Mol Endocrinol. 2002, 16: 1778-1792. 10.1210/me.2002-0089.

    Article  CAS  PubMed  Google Scholar 

  85. Liu G, Schwartz JA, Brooks SC: p53 down-regulates ER-responsive genes by interfering with the binding of ER to ERE. Biochem Biophys Res Commun. 1999, 264: 359-364. 10.1006/bbrc.1999.1525.

    Article  CAS  PubMed  Google Scholar 

  86. Rogatsky I, Trowbridge JM, Garabedian MJ: Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex. J Biol Chem. 1999, 274: 22296-22302. 10.1074/jbc.274.32.22296.

    Article  CAS  PubMed  Google Scholar 

  87. Cheema ZF, West JR, Miranda RC: Ethanol induces Fas/Apo [apoptosis]-1 mRNA and cell suicide in the developing cerebral cortex. Alcohol Clin Exp Res. 2000, 24: 535-543. 10.1097/00000374-200004000-00029.

    Article  CAS  PubMed  Google Scholar 

  88. Felderhoff-Mueser U, Buhrer C, Groneck P, Obladen M, Bartmann P, Heep A: Soluble Fas (CD95/Apo-1), soluble Fas ligand, and activated caspase 3 in the cerebrospinal fluid of infants with posthemorrhagic and nonhemorrhagic hydrocephalus. Pediatr Res. 2003, 54: 659-664. 10.1203/01.PDR.0000084114.83724.65.

    Article  CAS  PubMed  Google Scholar 

  89. Felderhoff-Mueser U, Sifringer M, Pesditschek S, Kuckuck H, Moysich A, Bittigau P, Ikonomidou C: Pathways leading to apoptotic neurodegeneration following trauma to the developing rat brain. Neurobiol Dis. 2002, 11: 231-245. 10.1006/nbdi.2002.0521.

    Article  CAS  PubMed  Google Scholar 

  90. Sakaguchi K, Sakamoto H, Lewis M, Anderson C, Erickson J, Appella E, Xie D: Phosphorylation of serine 392 stabilizes the tetramer formation of tumor supressor protein p53. Biochemistry. 1997, 36: 10117-10124. 10.1021/bi970759w.

    Article  CAS  PubMed  Google Scholar 

  91. Hoffman R, Craik D, Pierens G, Bolger R, Otvos LJ: Phosphorylation of the C-terminal site of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides. Biochemistry. 1998, 37: 13755-13764. 10.1021/bi980760a.

    Article  Google Scholar 

  92. Filhol O, Baudier J, Chambaz EM, Cochet C: Casein kinase 2 inhibits the renaturation of complementary DNA strands mediated by p53 protein. Biochem J. 1996, 316: 331-335.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  93. Miranda R, Sohrabji F, Singh M, Toran-Allerand CD: Nerve growth factor (NGF) regulation of estrogen receptors in explant cultures of the developing forebrain. J Neurobiol. 1996, 31: 77-87. 10.1002/(SICI)1097-4695(199609)31:1<77::AID-NEU7>3.0.CO;2-C.

    Article  CAS  PubMed  Google Scholar 

  94. Donovan M, Miranda R, Kraemer R, McCaffrey T, Tessarollo L, Mahadeo D, Kaplan D, Tsoulfas P, Parada L, Toran-Allerand D, Hajjar D, Hempstead B: Neurotrophin and neurotrophin receptors in vascular smooth muscle cells: Regulation of expression in response to injury. Am.J.Path. 1995, 147: 309-324.

    PubMed Central  CAS  PubMed  Google Scholar 

  95. Daryzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F: Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry. 1997, 27: 1-20. 10.1002/(SICI)1097-0320(19970101)27:1<1::AID-CYTO2>3.3.CO;2-X.

    Article  Google Scholar 

  96. Urdiales JL, Becker E, Andrieu M, Thomas A, Jullien J, van Grunsven LA, Menut S, Evan GI, Martin-Zanca D, Rudkin BB: Cell cycle phase-specific surface expression of nerve growth factor receptors TrkA and p75NTR. Journal of Neuroscience. 1998, 18: 6767-6775.

    CAS  PubMed  Google Scholar 

  97. Zong W-X, Farrell M, Bash J, Gelinas C: v-Rel prevents apoptosis in transformed lymphoid cells and blocks TNF-alpha-induced cell death. Oncogene. 1997, 15: 971-980. 10.1038/sj.onc.1201266.

    Article  CAS  PubMed  Google Scholar 

  98. Forster E, Tielsch A, Saum B, Weiss KH, Johanssen C, Graus-Porta D, Muller U, Frotscher M: Reelin, Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus. Proc Nat Acad Sci USA. 2002, 99: 13178-13183. 10.1073/pnas.202035899.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references


The authors would like to thank Jane Miller for technical assistance, and Dr. Kenneth Walsh (Tufts University School of Medicine) for the gift of the anti-FLIP antibody. This study was funded by grants from NIH (AA13440 and MH55724) to RCM.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Rajesh C Miranda.

Additional information

Authors' contributions

ZFC, DRS, JMN & SBW contributed to the conduct of the experiments. ZFC & RCM contributed to the data analysis, ZFC and RCM conceived the experiments and the experimental design, ZFC, DRS and RCM wrote the manuscript.

Authors’ original submitted files for images

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheema, Z.F., Santillano, D.R., Wade, S.B. et al. The extracellular matrix, p53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors, and reciprocally, Fas-ligand modifies estrogen control of cell-cycle proteins. BMC Neurosci 5, 11 (2004).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: