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. 2011 Feb 17;21(9):2177–2186. doi: 10.1093/cercor/bhr006

Fezf2 Regulates Telencephalic Precursor Differentiation from Mouse Embryonic Stem Cells

Zhi-Bo Wang 1, Erin Boisvert 1,2, Xiaoqing Zhang 3,4,5, Min Guo 1,6, Adedayo Fashoyin 4,5, Zhong-Wei Du 3,4,5, Su-Chun Zhang 3,4,5, Xue-Jun Li 1,7,✉
PMCID: PMC3155607  PMID: 21330470

Abstract

The mechanisms by which transcription factors control stepwise lineage restriction during the specification of cortical neurons remain largely unknown. Here, we investigated the role of forebrain embryonic zinc finger like (Fezf2) in this process by generating Fezf2 knockdown and tetracycline-inducible Fezf2 overexpression mouse embryonic stem cell (mESC) lines. The overexpression of Fezf2 at early time points significantly increased the generation of rostral forebrain progenitors (Foxg1+, Six3+) and inhibited the expression of transcription factors which are expressed by the midbrain and caudal diencephalon (En1+, Irx+). This effect was partially achieved by the regulation of Wnt signaling during this critical early time window. The role of Fezf2 in regulating the rostrocaudal patterning was further confirmed by the significant decrease in the expression of Foxg1 and Six3 and the increase in the expression of En1 when Fezf2 was knocked down. In addition, Fezf2 overexpression at later time points had little effect on the expression of Foxg1 and Six3. Instead, Fezf2 promotes the generation of dorsal telencephalic progenitors and deep-layer cortical neurons at later stages. Collectively, our data suggest that Fezf2 controls the specification of telencephalic progenitors from mESCs through differentially regulating the expression of rostrocaudal and dorsoventral patterning genes.

Keywords: cortical neuron, embryonic stem cells, Fezf2, neural patterning, transcription factor

Introduction

The development of diverse neuronal subtypes in the forebrain begins with the specification of forebrain precursors in the most anterior part of the neural tube (Rubenstein and Beachy 1998; Wilson and Houart 2004). Subsequently, these forebrain precursors are patterned along the rostrocaudal (or anterior–posterior, A–P) and dorsal–ventral (D–V) axes to form regional progenitors. The anterior part of the forebrain, the telencephalon, comprises the cortex in the dorsal regions and the basal ganglion in the ventral area (Sussel et al. 1999; Campbell 2003). In the cortex, 6 layers of cells develop from dorsal telencephalic progenitors (cortical progenitors), and each layer is composed of different types of neurons that have unique axonal connections and molecular properties (Arlotta et al. 2005; Molyneaux et al. 2007). The diencephalon, the caudal part of the forebrain, is further divided into rostral and caudal parts by the zona limitans intrathalamica (Lim and Golden 2007). Although the exact molecular mechanisms that underlie forebrain A–P and D–V patterning, especially the subsequent lamination of different cortical neuronal subtypes are unknown, gene expression studies have verified that this stepwise specification and lineage restriction is marked by unique transcription factor expression patterns (Molyneaux et al. 2007; Wigle and Eisenstat 2008).

The putative transcription factor forebrain embryonic zinc finger like (Fezf2, Fezl, or Zfp312) is evolutionarily conserved from Drosophila to humans (Hashimoto, Yabe, et al. 2000; Shimizu and Hibi 2009). In mice, Fezf2 is expressed in the prospective forebrain at E8.5 and continues to be expressed as deep-layer neurons are generated and specified (Hirata et al. 2004; Chen, Rasin, et al. 2005), after which Fezf2 becomes restricted to neurons within the deep layers of the neocortex. Consistent with the expression pattern of Fezf2, the corticospinal tract is absent in Fezf2-null mice (Chen, Schaevitz, et al. 2005; Molyneaux et al. 2005). In addition, the misexpression of Fezf2 in layers II and III induces the expression of the subcerebral projection neuron marker and the formation of ectopic subcortical axonal projections (Molyneaux et al. 2005), demonstrating the salient role of Fezf2 in the specification of subcerebral projection neurons, especially of cortical–spinal motoneurons (CSMNs). In zebra fish, Fezf2 has been identified as an anterior neuroectoderm–specific gene, and it is one of the earliest forebrain-specific genes (Hashimoto, Itoh, et al. 2000; Hashimoto, Yabe, et al. 2000). The downregulation of Fezf2 in zebra fish shrinks the rostral diencephalon and expands the caudal diencephalon (Jeong et al. 2007). Conversely, Fezf2 overexpression in zebra fish induces the expansion of the rostral diencephalon and telencephalon. These data suggest that Fezf2 is vital for rostrocaudal patterning of the forebrain in zebra fish (Jeong et al. 2007). Using Fezf2 and Fezf1 doubly deficient (Fezf2−/−Fezf1−/−) mice, Hirata et al. (2006) showed that the rostrocaudal patterning of diencephalon is altered and the cortex is reduced in these mice. However, it is still largely unclear whether and how Fezf2 regulates the early A–P and D–V patterning of the forebrain in mice.

Mouse embryonic stem cells (mESCs), isolated from the inner cell mass of the embryo blastocyst, have the capacity to become all the cell types in the body including neurons (Martin 1981; Ying et al. 2003). Culture systems have been established for differentiating mESCs to telencephalic progenitors and neurons using a serum-free suspension culture (Watanabe et al. 2005; Eiraku et al. 2008). These forebrain neural precursors can be patterned further into ventral or dorsal progenitors and then into different layers of cortical neurons, similar to what has been demonstrated in vivo (Gaspard et al. 2008). Thus, ESCs are a very useful tool to study early forebrain development and explore the transcriptional networks of this complex process. Here, by establishing Fezf2 knockdown and Fezf2 overexpression mESC lines and analyzing these cells at critical time points during neural differentiation, we have shown that Fezf2 temporally regulates the expression of A–P and D–V patterning genes and subsequent cortical layer–specific genes.

Materials and Methods

mESC Cultures

The mESCs (D3 line) were cultured on 6-well plates, which were preplated with a layer of irradiated mouse embryonic fibroblast cells (MEFs). The mESC medium consisted of 50% Buffalo rat liver cell conditioned medium, 32% Dulbecco's modified Eagle's medium (DMEM; Invitrogen), 15% knockout serum replacement (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 mM L-glutamine (Invitrogen), and 1 mM pyruvate (Invitrogen).

Establishment of Fezf2 Overexpression mESC Lines

The tetracycline-inducible mESC line was kindly gifted by Dr Kyba at University of Minnesota (Kyba et al. 2002). In this system, the rtTA was integrated into the ROSA26 locus for the constitutive expression of Fezf2 (ATCC). The tetracycline-inducible cassette was integrated into the HPRT locus. Cre-mediated recombination of targeting vectors was used to restore the resistance to G418 (Neomycin), which helped in the efficient isolation of clones. We inserted Fezf2 into the LoxP vector and established Fezf2-inducible mESC lines. When doxycycline (Dox; MP Biomedicals United States) was added to the cells, the expression of Fezf2 increased dramatically. Various techniques such as western blotting and quantitative polymerase chain reaction (qPCR) were then utilized to test the Fezf2 expression levels (see Results).

Neural Differentiation from mESCs

mESCs were dissociated and suspended on 10-cm Petri dishes at a density of 50 000/mL with 10 mL of neural medium consisting of DMEM/F12 (Invitrogen), neural basal medium (Invitrogen), N2 supplement (Invitrogen), 2-mercaptoethanol, L-glutamine, 1× chemically defined lipid concentrate (Invitrogen), N-acetyl-cysteine (Sigma-Aldrich), and leukemia inhibitory factor (LIF; 2 ng/mL, Millipore Corporate). After 2 days of incubation, the cells aggregated to form embryoid bodies (EBs). The EBs were then collected and cultured in suspension in new 6-cm Petri dishes using the same medium without LIF for the next 4–6 days. The EBs were then dissociated and plated onto polyornithine (Sigma-Aldrich)/laminin (Invitrogen)–coated coverslips and were cultured in neural basal medium supplemented with B27 (Invitrogen), cAMP (Sigma-Aldrich), and neurotrophic factors (brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and insulin-like growth factor 1; 10 ng/mL, PeproTech Inc.).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Quantitative Polymerase Chain Reaction (qPCR)

Total RNA was extracted from the cells after differentiation at multiple stages using TRIzol (Invitrogen). The SuperScript kit (Invitrogen) was utilized to synthesize cDNA. Regular PCR was used to assess gene expression with primers for specific genes (Supplementary Table 1). qPCR was then performed with the appropriate SYBR Green gene expression assay in a 20-μL mixture containing cDNA, primers, and 1× iQ SYBR Green Supermix (Bio-Rad). Standard curves were measured by each set of primers to confirm that only one amplicon was generated at the same efficiency as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a house-keeping gene. The messenger RNA (mRNA) expression levels were calculated using the comparative CT method. The primers for qPCR are listed in Supplementary Table 2.

Immunocytochemistry and Quantification

The coverslips were fixed using 4% paraformaldehyde (Sigma-Aldrich) (Li et al. 2005). Next, they were rinsed with phosphate-buffered saline (PBS; Invitrogen) and were incubated with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min and 10% normal serum (Jackson ImmunoResearch Laboratories, Inc.) for 1 h before overnight incubation with primary antibodies in PBS containing 5% serum at 4 °C. Antigen–antibody reactions were developed by the appropriate fluorescein-conjugated secondary antibodies (Invitrogen and Jackson ImmunoResearch Laboratories, Inc.). Nuclei were stained with Hoechst (Invitrogen). Primary antibodies used in this study included anti-Sox1 (1:500, goat IgG; R&D Systems, Inc.), anti-Ctip2 (1:2000, Abcam), anti-Otx2 (1:2000, goat IgG; Millipore Corporate), anti-Nestin (1:200, mIgG; Santa Cruz Biotechnology, Inc.), anti-Nkx2.1 (1:200, mouse IgG; Millipore Corporate), anti-Tbr1 (1:2000, rabbit IgG; Millipore Corporate). Monoclonal antibodies against Pax6 (1:5000), Otx1 (1:200), Isl 1 (1:1000), and βIII-tubulin (1:1000) were purchased from Developmental Studies Hybridoma Bank (University of Iowa).

The population of Pax6-, Nkx2.1-, and Otx1-positive cells among total differentiated cells (Hoeschst labeled) was counted as previously described (Li et al. 2009). In brief, a Zeiss fluorescence microscope (Carl Zeiss Inc.) was used to capture images. Then at least 5 fields of each coverslip were chosen and counted using the ImageJ software program (National Institute of Mental Health) by an observer blinded to the experimental conditions. In each group, 3–4 coverslips were counted. Data were expressed as mean ± standard deviation (SD).

Western Blots

The cell pellets were suspended in lysis buffer (1% Nonidet P-40, 50 mm Tris–HCl, pH 8.0, 0.5% sodium deoxycholate, 150 mm NaCl, 5 mm ethylenediaminetetraacetic acid, 10 mm NaF with protease inhibitor cocktail; all from Sigma-Aldrich), passed through a 28.5 gauge needle, and were lysed overnight at 4 °C. The particulate fraction was removed by centrifugation. Protein was resolved by using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western blotting analysis with the Fezf2 antibody (1:200, rabbit IgG, a kind gift of Dr Nenad Sestan).

Lentiviral Transfection to Generate Fezf2 Knockdown mESC Lines

Two short hairpin RNA sequences targeting mouse Fezf2 was chosen and inserted into the PLKO.1 lentiviral vector (Addgene Inc.). The lentivirus was produced by HEK293-FT cells by transfecting the HEK cells via the calcium phosphate method with 7.5 μg of psPAX2 (Addgene), 10 μg of the lentiviral transfection vector, and 5 μg of the VSV-G (Addgene) envelope protein pMD2.0G (Addgene). Sixty hours after transfection, the cell culture medium containing the viral particles was collected and filtered through a 0.45-μm filter (Millipore). The viral particles were further concentrated by ultracentrifugation (SW 28 rotor; Beckman Coulter, Inc.) at 50 000 × g for 2 h at 16 °C. The pellets were resuspended in mESC medium. During the transduction of mESCs, the mESCs were passaged using standard technique and pelleted via centrifugation. The cell pellets were then incubated with 100 μL of concentrated virus (106 transducing units/mL) at 37 °C for 30 min. During the incubation period, the cells were gently mixed every 10 min. The cells and the virus were then transferred to a 6-well plate containing standard mESC medium and a mouse embryonic fibroblast feeder layer overnight. The medium was then changed the next day. Incremental drug selection Blasticidin S (BSD; InvivoGen) was utilized to eliminate any mESCs, which did not contain the virus. Individual clones were then picked based on their green fluorescent protein (GFP) intensity, replated, and expanded to produce pure cultures.

Statistics

The Dunnett's test and Tukey's studentized range test were used to compare the mean values among multiple sample groups after one-way analysis of variance (Fig. 2). The mean value between Dox-treated group and control group was compared by Student's t-test. The significance level was defined as 2-sided P < 0.05, and statistical analysis was conducted using SAS 9.1 (SAS Institute).

Figure 2.

Figure 2.

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Fezf2 regulates the A–P patterning of the forebrain. (A) Differences in the expression levels of Fezf2 and other patterning markers at day 6 between control and Dox-treated groups. (B) qPCR indicating the changes in the expression of Fezf2 and rostrocaudal genes at different time points after being treated with Dox during days 4–6. (C) Analysis of the expression of the rostrocaudal genes at day 10 showed that there was an increase in the expression of Foxg1 and Six3 and a decrease in En1 expression when Dox was added and that these changes were time dependent. Data are presented as Mean ± SD. *P < 0.05 versus Control (No Dox) group, n = 3.

Results

Fezf2 Overexpression Does Not Alter Neural Induction

To investigate the function of Fezf2, we first established mESC lines with inducible Fezf2 overexpression by modifying the tetracycline-inducible system (Kyba et al. 2002). Two clones were established to exhibit inducible expression of Fezf2. qPCR showed that treatment of Dox induced a high expression of Fezf2 gene in both clone 1 and clone 2 (Fig. 1A) and there was more than a 20-fold increase in the Fezf2 mRNA expression when Dox was added (Fig. 1B). Western blot analysis also showed the induction of Fezf2 expression at the protein level after Dox treatment (Fig. 1C).

Figure 1.

Figure 1.

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Neural differentiation from Fezf2 overexpression mESCs. (A) The treatment of Dox induced high Fezf2 mRNA expression in the Fezf2 overexpression ESC clones. (B) qPCR data indicating that when Dox was added, there was more than a 20-fold increase in the Fezf2 expression. (C) The induction of Fezf2 protein was observed in the Fezf2-inducible ESC line after Dox treatment using western blotting. (D) A schematic procedure for neural differentiation from mESCs. (E) qPCR indicating the gene expression profiles at different time points during neural differentiation from mESCs. (F) At day 8 after differentiation, immunostaining showed that the addition of Dox yielded no significant changes in the expression of NESTIN and SOX1. Bar = 50 μm. The overexpression of Fezf2 also did not affect the expression of Nestin and Sox1 mRNA as shown by regular PCR (day 6, G) and qPCR (day 6, H). Mean ± SD, n = 3. *P < 0.05 versus Control (No Dox) by 2-sided t-test.

The transgenic mESCs were differentiated toward the neural lineage according to the method established by Dr Sasai with some modifications as described in the Materials and Methods section (Fig. 1D) (Watanabe et al. 2005). RT-qPCR analysis revealed a significant increase in the mRNA expression of neuroepithelial (NE) genes, Nestin and Sox1 between days 6 and 8 in the absence of Dox (Fig. 1E). To examine the expression of these markers at the protein level, mESC agregates were dissociated and plated on polyornithine/laminin–coated coverslips at day 6, which were then fixed for immunohistochemistry analysis. A large population of cells expressed Nestin and Sox1 at day 8 suggesting that the NE cells are specified at days 6–8 after differentiation (Fig. 1F). These cells also organized to form rosette-like structures (Fig. 1F).

To examine whether or not the overexpression of Fezf2 has an effect on neural induction, Dox was supplied during neural differentiation. The expression of endogenous Fezf2 remained at a low level at the ESC stage and increased significantly at day 6 after neural differentiation (Fig. 1E). The overexpression of Fezf2 during neural induction via the addition of Dox (days 2–6 or days 4–6) did not alter the expression of Nestin or Sox1 mRNA at day 6 as shown by RT-PCR (Fig. 1G) and qPCR (Fig. 1H). Similarly, at day 8, the expression of Nestin and Sox1 protein was very similar between the cells regardless of whether or not they were treated with Dox (Fig. 1F). Moreover, the overexpression of Fezf2 at an earlier time point (days 0–2) showed no significant differences in the expression of Nestin as compared with the control group (Supplementary Fig. 1), suggesting that the overexpression of Fezf2 does not alter neural induction.

Fezf2 Affects Early A–P Patterning of the Forebrain

To determine the role of Fezf2 on the A–P patterning of neural cells, we first examined the expression of rostrocaudal transcriptional factors, including Foxg1 (telencephalic marker), Six3 (rostral forebrain marker), Irx1 and Irx3 (caudal forebrain markers), and En1 (midbrain marker). The treatment of Dox after day 2 of differentiation increased the expression of Foxg1 and Six3 mRNA but decreased the expression of Irx1, Irx3, and En1 at day 6 as shown by RT-PCR (Fig. 2A). The expression of Hoxb4, a transcription factor expressed by cells in hindbrain and spinal cord, remained at a low level (Fig. 2C). Since the treatment of Dox for 2 days (days 4–6) or 4 days (2–6) showed a similar effect on the expression of these genes, Dox treatment from days 4 to 6 was selected for subsequent analysis. After further differentiation (at day 10), there was a significant increase in the population of FOXG1+ cells in the Dox 4–6-treated group compared with that in control (Supplementary Fig. 2).

To investigate the temporal change of gene expression after Dox treatment, Dox was added during days 4–6 and samples were collected at days 4, 5, 6, and 8. The overexpression of Fezf2 induced by Dox peaked at 1 day post-Dox treatment (day 5) and decreased on day 6 (Fig. 2B). qPCR analysis showed a significant increase in the expression of Six3 and Foxg1, during days 5–8 (over 10-fold increase) after Dox treatment (Fig. 2B). Correspondingly, Fezf2 overexpression resulted in a dramatic decrease in the expression of En1. The expression of caudal diencephalon markers, Irx1 and Irx3, were also decreased in the Dox-treated group (Fig. 2B, Supplementary Fig. 3). These data suggest that the overexpression of Fezf2 affects the A–P patterning by promoting the generation of telencephalon and rostral diencephalon and repressing caudal diencephalon and midbrain identities. Interestingly, when we tested the endogenous expression of Fezf2, we found that at day 8 its expression increased significantly and remained high at day 10. This suggests that Fezf2 may promote its own expression (Fig. 2B).

As we have shown in human ESC-derived neural differentiation, cells are more responsive to patterning signals at an early stage of development before the expression of Sox1 (Li et al. 2005). We hypothesized that the overexpression of Fezf2 during an early time window would be important for its function on forebrain specification. To test this hypothesis, we applied Dox at different time windows and tested the expression of rostrocaudal genes at day 10 (Fig. 2C). Although the treatment of Dox during days 4–6 significantly increased the expression of Foxg1 and Six3 at day 10, the change in Foxg1 and Six3 expression as well as the decrease in the En1 expression was much smaller when Dox was added on days 6–8 (Fig. 2Ci). Notably, the treatment of Dox during days 8–10 had little effect on increasing the expression of Foxg1/Six3 or decreasing En1 expression (Fig. 2Ci). This suggests that after day 8, the overexpression of Fezf2 does not affect the expression of Foxg1, Six3, or En1 genes. We then asked whether the treatment of Dox for an extended period will further increase the expression of Foxg1. The treatment of Dox during days 4–8 did not further increase the expression of Foxg1/Six3 or decrease the expression of En1 as compared with treatment during days 4–6 (Fig. 2Cii). Treatment of Dox during days 6–10 moderately decreased the En1 expression and had a smaller effect on the expression of Foxg1 and Six3 (Fig. 2Cii). Together, these data suggest that the overexpression of Fezf2 at an early stage (days 4–6) is critical for Fezf2's role in promoting the generation of the rostral forebrain and the repression of midbrain identities.

Fezf2 Affects Early A–P Patterning of Forebrain Through Regulating Wnt Signaling

Wnts including Wnt1, Wnt3a, and Wnt8b are key regulators in the caudalization of progenitor cells to form the diencephalon and midbrain (Sasai and De Robertis 1997; Kim et al. 2002; Lagutin et al. 2003). Since Fezf2 is essential for regulating the A–P patterning, we examined whether the role of Fezf2 is achieved through the regulation of Wnts. We first tested the temporal change in the expression of Wnt1, Wnt3a, and Wnt8b during neural differentiation. We also examined the expression of Wnt4, 6, and 11, which are not involved in the A–P patterning. Our data showed that the expression of Wnt1, Wnt3a, and Wnt8b increased significantly at day 6, while the expression of other Wnts especially Wnt 6 and Wnt11 did not change much during neural differentiation (Fig. 3A). Since our previous results showed that the addition of Dox during days 4–6 lead to a significant change in the rostrocaudal patterning, Dox was added to the cells at day 4 to induce Fezf2 expression. This resulted in a significant decrease in the expression of Wnt1, Wnt 3a, and Wnt8b at day 6 (Fig. 3B). Interestingly, the expression of Wnt4, Wnt6, and Wnt11 remained unchanged when Dox was added, indicating a specific effect of Fezf2 on the expression of Wnts related to A–P patterning. Moreover, the decrease of Wnts (Wnt1, Wnt3a, and Wnt8b) at day 6 was correlated to the increased expression of the rostral forebrain markers Foxg1 and Six3 and a decrease in the expression of the caudal diencephalon and midbrain markers Irx and En1 during this specific time window. Thus, these data suggest that Fezf2 regulates the rostrocaudal patterning through, at least in part, inhibiting the Wnt signaling.

Figure 3.

Figure 3.

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Fezf2 regulates Wnt expression. (A) qPCR showing the expression of Wnt1, Wnt3a, Wnt8b, Wnt4, Wnt6, and Wnt11 mRNA at different time points after neural differentiation from mESCs. (B) Dox was added to the Fezf2 overexpression line at day 4 after neural differentiation. After 2-day (total Day 6) treatment, the expression of Wnt genes was examined using qPCR in both Dox-treated and non–Dox-treated (control) cells. The mRNA level at day 4 was considered as 1. Mean ± SD, n = 4. *P < 0.05 versus Control by 2-sided t-test.

Fezf2 Knockdown Inhibits the Rostral Forebrain Differentiation

To examine whether Fezf2 is necessary for the generation of the rostral forebrain, we established Fezf2 knockdown mESC lines by infecting mESCs with lentivirus containing shRNA targeting either Fezf2 or luciferase (as a control) (Fig. 4A). The knockdown of Fezf2 had no affect on the morphology or the self-renewal of the ESCs (Fig. 4B). A GFP tag was added to the vector so that viral integration into the cells could be tracked. The GFP expression was present in the mESCs, EBs, and NE cells, indicating that the expression of the Fezf2 knockdown viral vector was present during neural differentiation (Fig. 4B). The Fezf2 expression was significantly decreased in the knockdown cell lines (Fig. 4C). When Fezf2 was knocked down, there was a significant decrease in the mRNA expression of Foxg1 and Six3 and a significant increase in the mRNA expression of En1 at day 10 (Fig. 4D). No significant changes were observed in the expression of Irx1 gene when Fezf2 was knocked down. Meanwhile, the expression of the neural epithelial genes (Sox1 and Nestin) at day 6 was comparable with that in control group (Fig. 4E). Thus, these results suggest that Fezf2 is necessary for the generation of rostral forebrain cells. To further confirm the role of Fezf2 in regulating the expression of A–P patterning genes, we have used a second shRNA, which targets a different area of the Fezf2 gene. When Fezf2 was knocked down using this shRNA, it yielded similar changes to the first shRNA including a reduced expression of Foxg1 and Six3 genes and an increase in the expression of En1 at day 10 after differentiation (Supplementary Fig. 4).

Figure 4.

Figure 4.

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The establishment and differentiation of Fezf2 mESC knockdown mESC lines. (A) Fezf2 and luciferase RNAi sequences were inserted into the PLKO.1 vector and lentivirus was produced. (B) mESCs were transduced with the lentivirus. The RNAi-expressing cells, as indicated by the expression of GFP, were then differentiated into EBs and NE cells. (C) The expression of Fezf2 was significantly decreased in the Fezf2 knockdown mESC lines. The Fezf2 expression remained very low when the cells were differentiated for 10 days. (D) The Fezf2 knockdown cell line showed a decrease in Foxg1 and Six3 mRNA expression and an increase in En1 expression relative to the luciferase RNAi cell line at day 10. (E) RT-PCR showed that the expression of Nestin and Sox1 mRNA was similar between control and Dox-treated cells at day 6 after differentiation. Mean ± SD, n = 4. *P < 0.05 versus Control by 2-sided t-test. Bar = 50 μm.

Fezf2 Overexpression at an Early Time Point Results in the Increase of Both Dorsal and Ventral Forebrain Neurons

Fezf2, which is expressed by prospective forebrain cells at E8.5, is then enriched in the dorsal telencephalon at E10.5 (Hirata et al. 2004). We then further examined whether Fezf2 differentially affects the expression of dorsal versus ventral forebrain progenitors when overexpressed at an early time point. qPCR analysis showed that both dorsal forebrain (Pax6, Emx1) and ventral forebrain (Dlx2, Nkx2.1) genes were increased in the Dox-treated group (Fig. 5A). To exclude the nonspecific effect of Dox treatment, we induced neural differentiation using a control tetracycline-inducible line (without recombination) and added Dox to these cells during days 4–6. After 4 more days (day 10), the cells were collected to analyze the expression of A–P and D–V patterning genes. The addition of Dox to the control line did not alter the expression of either A–P or D–V genes (Supplementary Fig. 5A), suggesting that the patterning changes observed after dox-induced Fezf2 overexpression are specific. At the protein level, immunocytochemical analysis confirmed the significant increase of PAX6- and NKX2.1-positive cells when Dox was added during days 4–6 (Fig. 5B,C) as compared with the control group (Fig. 5B,C). Moreover, NKX2.1-positive cells did not double stain with PAX6 (Supplementary Fig. 6A). Together, our data suggest that Fezf2 promotes the differentiation of both dorsal and ventral forebrain cells.

Figure 5.

Figure 5.

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Changes in neural differentiation after Fezf2 overexpression at an early time point. (A,B) The overexpression of Fezf2 on days 4–6 lead to an increase in the expression of both dorsal (Pax6, Emx1) and ventral (Nkx2.1 and Dlx2) forebrain transcriptional factors at both the mRNA (A) and the protein (B) levels at day 10 after differentiation. (C) Statistical analysis showed that there was a significant increase in the population of NKX2.1+ and PAX6+ cells after Dox treatment. (D) Upon further differentiation, Dox treatment resulted in an increased expression of Islet1, Tbr1, and BCL11B (Ctip2) proteins. Glutamatergic neurons which stained positive for Tbr1 were also positive for the neuronal markers, βIII-tubulin (D) and MAP2 (E). Some cells were positive for GABA (E). Blue indicated Hoechst-stained nuclei. Mean ± SD, n = 4. *P < 0.05 versus Control by 2-sided t-test. Bar = 50 μm.

Dorsal and ventral forebrain progenitors develop into glutamatergic and γ-aminobutyric acid (GABA)ergic neurons, respectively (Olsson et al. 1997; Marin and Rubenstein 2001). To investigate the ability of our forebrain progenitor cells to differentiate into glutamatergic and GABAergic neurons, the neural precursors were dissociated and plated on polyornithine/laminin–coated coverslips. Two weeks after differentiation, we examined the expression of Isl1, a transcription factor expressed by ventral telencephalic progenitors and neurons, Tbr1, a marker for glutamatergic neurons (Hevner et al. 2006), and B-cell CLL/lymphoma 11B (Bcl11b or Ctip2), a transcriptional factor expressed by subcerebral neurons and also ventral striatum neurons (Arlotta et al. 2008) (Fig. 5D). The expression of all these forebrain-related markers remained at very low levels within the control group, while Dox treatment significantly increased the population of ISL1+, TBR1+, and BCL11B + forebrain neurons (Fig. 5D). After another week in culture, the TBR1-positive neurons also double stained with MAP2, a marker for mature neurons. GABA-positive neurons were observed in Dox-treated cells (Fig. 5E), suggesting that the overexpression of Fezf2 via Dox treatment promotes the differentiation of both glutamatergic and GABAergic neurons.

Fezf2 Overexpression Confers Dorsal and Cortical Deep-Layer Differentiation at a Later Time Point

Since the overexpression of Fezf2 during an early time window promotes the generation of both ventral and dorsal (cortical) neurons in the forebrain, next, we investigated whether Fezf2 preferentially promotes the formation of cortical and subcerebral neurons at later stages. To achieve this, Dox was added to the Fezf2-inducible overexpression cells at later time points (days 12–14) when the forebrain is already specified. Fezf2 overexpression ESC-derived neural cells were exposed to Dox at either an early time point (days 4–6) or at both an early time point (days 4–6) and a later time point (days 12–14) (Fig. 6A). qPCR analysis indicated that when Dox was added at both the early and the later stages, there was an increase in the expression of the dorsal forebrain markers (Emx1 and Pax6), while the expression of ventral forebrain genes (Dlx2 and Nkx2.1) was decreased at day 15 (Fig. 6B). Immunostaining analysis further confirmed that the population of PAX6+ cells were much higher in the groups treated with Dox at both early and late stages (over 50%) compared with those that were only treated with Dox at the early stage (around 30%) (Fig. 6C,D). In contrast, the population of NKX2.1+ cells showed the opposite trend (Fig. 6C,D). Thus, while the overexpression of Fezf2 during an early time window yields both ventral and dorsal (cortical) progenitors, when Fezf2 is overexpressed at a later time point after the telencephalic precursors are specified, Fezf2 preferentially promotes the generation of dorsal telencephalic progenitors.

Figure 6.

Figure 6.

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The time-dependent effect of Fezf2. (A) A schematic demonstrating the time points during which cells were exposed to Dox. (B) The expression levels of different D–V markers were evaluated at day 15 via qPCR. When the Fezf2-inducible overexpression cell lines were exposed to Dox during both the early and the late stages of differentiation, there was an increase in the expression of both dorsal telencephalic progenitor and deep-layer cortical projection neuron markers. Moreover, the expression of ventral forebrain and upper cortical layer markers were decreased when Fezf2 was overexpressed at both early and late stages. (C,D) Immunostaining of the Fezf2-inducible overexpression cells was evaluated and it showed that there was a similar trend in the expression of dorsal forebrain progenitor (PAX6+ cells), ventral forebrain progenitors (NKX2.1+ cells), and layer V projection neurons (OTX1+ cells). Mean ± SD, n = 3. *P < 0.05 versus No Dox Control by 2-sided t-test. Blue indicated Hoechst-stained nuclei. Bar = 50 μm.

Cortical progenitors develop into neurons constituting 6-layer cortex in an inside-out manner. Subcerebral projection neurons are mainly located in layers V and VI (deep layers). We further compared the expression of subcerebral and deep layer–related markers between 2 groups that were treated with Dox at an early time point (days 4–6) or at both early and later time points. The expression of BCL11B and ETS variant gene 1 (Etv1, also known as Er81), transcription factors which are expressed by subcerebral and layer V projection neurons (Hevner et al. 2003; Arlotta et al. 2005), was increased after being treated with Dox at both early and late stages as shown by qPCR analysis in cells at day 15 (Fig. 6B). In contrast, the expression of upper layer marker such as CUT-like 1 (Cutl1, or Cux1) was decreased. The addition of Dox to a control tetracycline-inducible cell line at a later stage yielded no significant changes in the expression of these transcription factors including Pax6, Emx1, Nkx2.1, Bcl11b, Etv1, and Cutl1 (Supplementary Fig. 5B). After another week in culture (total 3 weeks), few cells (less than 3%) further differentiated to OTX1+ (a marker for neurons in layer V) (Weimann et al. 1999) cells in cultures that were treated with Dox at an early time point. Notably, there was a dramatic increase in the expression of OTX1+ cells (around 10% of total cells) after being treated with Dox at both stages (Fig. 6C). As expected, OTX1+ cells were negative for CUTL2 (or Cux2), an upper layer marker (Supplementary Fig. 6B). Together, these data revealed that Fezf2 preferentially promotes the generation of cortical and subcerebral neurons when it is overexpressed at a later time point, suggesting that the Fezf2 function is temporally regulated during cortical differentiation.

Discussion

By establishing Fezf2 knockdown and overexpression mESC lines and analyzing them at critical time points during neural differentiation, we have demonstrated that Fezf2 plays an important role in controlling the rostrocaudal patterning during neural differentiation from mESC. Specifically, Fezf2 promotes the generation of the telencephalon and rostral diencephalon cells and inhibits the generation of caudal diencephalon and midbrain cells from mESCs. This effect occurs when Fezf2 is overexpressed at an early but not late stage during neural differentiation from mESCs, which is achieved at least in part by the regulation of Wnt signaling. At a later time point, Fezf2 plays an essential role in cortical neuron differentiation by promoting the specification of dorsal telencephalic progenitors and subcerebral projection neurons. Thus, Fezf2 can possess distinct roles during cortical differentiation along both the anterior/posterior and the dorsal/ventral axes, and these intrinsic roles of Fezf2 are dependent on the temporal cell context.

The neocortex is the most developed structure in mammals and is very important for our motor, sensory, and cognitive functions. The deep layers of the cortex (layers 5 and 6) contain neurons, which project to distant parts of the body and help to control movement. Fezf2 is critical for the specification of deep-layer cortical neurons, which have been extensively studied in mice especially with Fezf2−/− mice (Chen, Schaevitz, et al. 2005; Molyneaux et al. 2005). Fezf2−/− mice experience multiple defects in the development of projection neurons in the cortex, including abnormal development of subplate neurons and thalamocortical axons and impairment of subcerebral projection neurons, especially CSMNs (Hirata et al. 2004; Chen, Rasin, et al. 2005; Molyneaux et al. 2005). Our data using mESCs also indicate that Fezf2 promotes the expression of deep layer/subcerebral neurons, supporting the in vivo evidence. The generation of dorsal progenitors is also promoted when Fezf2 is overexpressed. Notably, we have shown that the Fezf2 function modulates over time. At early time points during the specification of neuroectodermal cells, Fezf2 plays a novel role in the specification of telencephalic precursors from mESCs, which results in an increase in the generation of both dorsal and ventral progenitors and neurons when Fezf2 is overexpressed. At this early stage, overexpression of Fezf2 also affects the rostrocaudal patterning of the diencephalon in mESCs by promoting the rostral diencephalon and inhibiting the caudal diencephalon, which is consistent with the findings in zebra fish and mice (Hirata et al. 2006; Jeong et al. 2007).

The Fez2-null mice did not show any obvious changes in the A–P or D–V patterning of the forebrain (Hirata et al. 2004; Chen, Schaevitz, et al. 2005). However, Fezf1/Fezf2 double knockout (Fezf1−/−/Fezf2−/−) mice exhibited an expansion of their caudal diencephalon and a reduction of their rostral diencephalon (Hirata et al. 2006). Double knockout mice also have a smaller cortex, however, the detailed changes in the telencephalon have not been studied. Using Fezf2 knockdown mESCs, we have observed similarities (the decrease in the expression of Six3/Foxg1) and differences (the change in En1 expression) as compared with the Fezf2-deficient zebra fish or the Fezf1−/−/Fezf2−/−mice. One explanation regarding the differences between in vitro differentiated cells and in vivo development is that during in vitro differentiation, NE cells are cultured in a simplified environment. In this way, the defects on forebrain cell development in Fezf2 knockdown cells more reflect a cell autonomous or intrinsic role of Fezf2. Previous studies have shown that neural cells derived from mESCs are more likely to have midbrain/hindbrain identities and thus it had been difficult to obtain forebrain neurons from mESCs (Du and Zhang 2004). Overexpression of Fezf2 promotes the generation of Foxg1+ and Six3+ forebrain cells, and thus provides a powerful tool to enrich forebrain neurons for both basic and translational research. This culture system, which is much more simplified as compared with in vivo environment, also has advantages in studying cell autonomous versus nonautonomous effects as well as analyzing the time-dependent requirement.

The mechanisms by which Fezf2 regulates the cortical cell development at distinct stages remain to be elucidated. Our data suggest that the inhibition of the critical caudalizing factors (Wnt1, Wnt3a, and Wnt8b) may contribute to the effect that Fezf2 has on rostrocaudal patterning at early time points. As a transcriptional repressor, Fezf2 may also directly interact with certain transcription factors to control the rostrocaudal patterning. After the telencephalic progenitors were specified (later time points), we found that Fezf2 specifically promotes dorsal (cortical) progenitors and Bcl11b and Otx1, transcription factors which are expressed by subcerebral and layer V cortical neurons, during neural differentiation from mESCs. It has been reported that Fezf2 may interact with Bcl11B (also known as Ctip2) and Sox5 during corticogenesis (Chen et al. 2008; Kwan et al. 2008). A recent study revealed that Fezf2 binds directly to the Hes5 promoter, which results in the inhibition of Hes5 and the subsequent initiation of forebrain neurogenesis (Shimizu et al. 2010). The differential regulation of distinct sets of transcription factors might contribute to the temporally regulated functions of Fezf2 during forebrain development. Further identification of potential targets of Fezf2 using ESCs will also broaden our understanding of the complex process of cortical development.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/

Funding

National Institutes of Health R21 NS055261 (to X.J.L.) and RO1 NS045926 (to S.C.Z.). Amyotrophic Lateral Sclerosis Association and University of Connecticut Health Center start-up funds to X.J.L.

Supplementary Material

Supplementary Data
supp_21_9_2177__index.html (1.8KB, html)

Acknowledgments

We thank Drs Nenad Sestan, Yoshiki Sasai, and Alain Nepveu for generously providing the Fezf2, Foxg1, and Cutl2 antibodies. We would also like to thank Dr Michael Kyba for kindly providing the tetracycline-inducible mESC line. Conflict of Interest : None declared.

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