FoxP2在胚胎時期的神經新生中所扮演的角色

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(1)國立臺灣師範大學生命科學系碩士論文. FoxP2 在胚胎時期的神經新生中所扮演的角色 The Role of FoxP2 in Embryonic Neurogenesis. 研究生：邱怡綺 Yi-Chi Chiu 指導教授：王慈蔚博士 Tsu-Wei Wang. 中華民國一百零一年六月.

(2) Contents. Chinese abstract…………………………………………………..……..3. English abstract………………………………………………………….5. 1. Introduction………………………………………………...………...7 1.1. Embryonic neurogenesis 1.2. Neurol stem cell 1.3. Forebrain 1.4. FoxP2 1.5. Nkx2-1 1.6. Sonic hedgehog pathway 1.7. PDGFRα. 2. Materials and Methods………………………………………………15 2.1. Primary cell cultures 2.2. Plasmids 2.3. Electroporation 2.4. P19 cell culture and transfection 2.5. Immunostaining analysis 2.6. Western blotting 2.7. Image analysis and statistical analysis. 3. Results………………………………………………….……………21 1.

(3) 3.1. Overexpression and knockdown of FoxP2 in primary and P19 cell cultures 3.2. FoxP2 positively regulates neuronal differentiation in the embryonic telencephalon 3.3. FoxP2 has no effects on cell proliferation and cell survival 3.4. FoxP2 increases the expression of PDGFRα and the neurognic effect of FoxP2 is PDGFRα-dependent 3.5. FoxP2 does not regulate oligodendrocyte differentiation in the embryonic telencephalon 3.6. FoxP2 regulates the specification of neuronal subtypes. 4. Discussion..……………………………….…………………………30 4.1. FoxP2 regulates neuronal differentiation in the embryonic forebrain 4.2. The role of FoxP2 and PDGFRα in neuronal differentiation 4.3. FoxP2 acts as a regulator in neuronal subtype specification. 5. References………………………………………………………......37. 6. Figures……………………………………………………….……...50. 2.

(4) 摘要目前已知 FoxP2 為一轉錄因子，在人類中如產生突變，會影響語言的能力。此外，在斑胸草雀有一個學習唱歌的神經迴路 Area X，降低 Area X 的 FoxP2 的表現量，會使之無法學會該物種鳴叫的旋律和降低中度棘神經元的發育。以小鼠為例，剔除 FoxP2 會使小鼠的動作學習產生缺失。這些都暗示著 FoxP2 可能會調控神經元新生，但 FoxP2 影響神經元新生的機制尚未明確。因此本論文要探討在神經系統的發育過程中，FoxP2 所扮演的角色為何。將胚胎鼠前腦的細胞進行初級細胞培養，我發現 FoxP2 會增加神經元分化和突觸的長度而不會去影響細胞增生及細胞死亡。另外，我也發現 FoxP2 會增加 PDGFRα的表現量且 FoxP2 所影響的神經元分化是透過 PDGFRα。在前腦腹側神經元特化的部分，我發現 FoxP2 對於中度棘神經元的分化是必須的，且 FoxP2 會抑制中間神經元的分化。根據日前研究指出 FoxP2 和另一已知調控前腦中間神經元的轉錄因子 Nkx2-1 有交互作用。我也發現維持 Nkx2-1 表現的 Sonic hedgehog pathway 會影響 FoxP2 調控神經元類型的特化。由以上結果可知 FoxP2 在調控胚胎時期前腦的神經元特化過程中扮演重要的角色。. 關鍵字: 胚胎時期神經新生、神經幹細胞、FoxP2、Nkx2-1、Sonic 3.

(5) hedgehog pathway、PDGFRα. 4.

(6) Abstract In humans, mutations of FoxP2, a transcription factor, cause a severe speech and language disorder. In addition, reduction of FoxP2 expression in the basal ganglia song nucleus Area X in zebra finches causes deficits in song learning and the development of medium spiny neurons. In mouse models, loss-of-function of FoxP2 causes defects in motor learning. These suggest that FoxP2 plays important roles during neural development. However, the mechanism is still unclear. Therefore, the present study is aimed to investigate effects of FoxP2 during the development of the nervous system. By using primary cell cultures from the embryonic telencephalon, I observed that FoxP2 increased neuronal differentiation and neurite length without affecting cell proliferation and cell survival. I also found that FoxP2 increased the expression of platelet-derived growth factor receptor α (PDGFRα) and the neurognic effect of FoxP2 was PDGFRα-dependent. In terms of neuronal subtype specification, I found that FoxP2 positively regulated the differentiation of lateral ganglionic eminence (LGE)-derived medium spiny neurons and negatively regulated the formation of medial ganglionic eminence (MGE)-derived interneurons. Since previous studies showed that there was interaction between FoxP2 and Nkx2-1, a transcription factor known for the development of the forebrain interneurons, I found that the effect of FoxP2 on the differentiation of forebrain interneurons was mediated by the Sonic hedgehog pathway, which maintains Nkx2-1 expression and the specification of interneurons. Taken together, my study suggests that FoxP2 regulates the development of the embryonic forebrain. 5.

(7) Key word: Embryonic neurogenesis、Neurol stem cell、FoxP2、Nkx2-1、 Sonic hedgehog pathway、PDGFRα. 6.

(8) 1. Introduction 1.1 Embryonic neurogenesis The development of the nervous system is a complex process. It occurs in all regions of the developing brain and produces all types of cells of the nervous system, such as neurons, astrocytes and oligodendrocytes. New neurons are derived from neural stem cells. Neural stem cells first produce progenitor cells that then give rise to neurons and glia (Johansson et al., 1999). In the cerebral cortex, new neurons migrate to outer layers along radial glia fibers and establish cortical layers (Nadarajah et al., 2001). However, the detail mechanism is still unknown. Previous studies found that peptide growth factors play critical roles in regulating neurogenesis within the developing and adult central nervous systems. They function as regulators of proliferation, differentiation, migration, maturation, or survival of cell populations (Lee et al., 2009; Sanders et al., 2008). For example, basic fibroblast growth factor (bFGF) and transforming growth factor-α (TGF-α), both act as mitogens for a wide variety of neural precursors and may be responsible for inducing proliferation in the developing brain (Liboi et al., 1987; Romo et al., 2011). Also, several peptide growth factors, such as platelet-derived growth factor (PDGF) and ciliary neurotrophic factor (CNTF), appear to direct the fate choice of cells (Hirschi et al., 1998; Johe et al., 1996). In addition, many transcription factors have been found to regulate neurogenesis. For instance, Neurogenin2 (Ngn2), a basic helix-loop-helix (bHLH) transcription factor, is expressed in the neuronal progenitor cells and strongly induces the production of glutamatergic neurons (Ma et al., 7.

(9) 1999). NeuroD is another bHLH transcription factor expressed in the neocortex and hippocampus and it is necessary for proliferation and postnatal differentiation of dentate gyrus (DG) neurons (Hallam et al 2000; Liu et al., 2000).. 1.2 Neural stem cell In mammals, neural stem cells (NSC) appear early in the development. Stem cells have two properties: self-renewal and multi-potency (Taupin et al., 2002). First, stem cells divide asymmetrically to give rise to a stem cell and a progenitor cell. There are two types of progenitors: neuronal progenitors and glia progenitors. Neuronal progenitors produce neurons and glia progenitors generate astrocytes and oligodendrocytes. This process is both controlled by extrinsic and intrinsic signals (Qian et al., 2000). The major group of extrinsic factors is growth factors/morphogens such as FGFs, bone morphogenetic proteins (BMPs), Sonic hedgehog (Shh), Wnt and retinoic acid (RA) (Przybyla et al., 2012, Dhruv et al., 2011, Wang et al., 2005). They have been shown to regulate cell cycle, cell survival or differentiation during the brain development. Besides, intrinsic signals provided by transcription factors have been shown to play roles in progenitor cell proliferation and differentiation (Dessaud et al., 2007, Maden et al., 2002). It is possible that other unknown transcription factors may also influence various aspects of neurogenesis (Laub et al., 2001).. 1.3 Forebrain 8.

(10) The embryonic forebrain is the most anterior neurol plate derivative that will give rise to brain centers controlling preceptions, memory, emotions voluntary movements and endocrine functions. The vertebrate forebrain is a complex neural network consisting of a wide variety of highly specified neurons and glia. During forebrain development, the embryonic telencephalon is subdivided into the pallium and subpallium. At this stage, neural progenitors have acquired their spatial identity defined by distinct combinations of transcription factors (Shimamura et al., 1997). Several secreted molecules including Shh, FGFs, Wnts and BMPs, as well as RA have been implicated in early forebrain regionalization (Wilson et al., 2004). In the dorsal telencephalon, the pallium develops into the cerebral cortex, whereas the subpallium becomes the basal ganglia in the ventral telencephalon. There are several transcription factors that define and maintain the identity of the ventral and dorsal telencephalon, such as Emx2, Pax6 and Nkx2.1 (Sussel et al., 1999; Stoykova et al., 2000; Muzio et al., 2002; Fuccillo et al., 2004). These transcription factors are required for correct specification of the telencephalon and regulate expression of region-specific genes (Fode et al., 2000). The basal ganglia can be divided into the pallidum and the striatum. They are derived from the medial (MGE) and lateral ganglionic eminence (LGE), respectively (Eriksson et al., 2003). The majority of telencephalic GABAergic neurons arise from either LGE or MGE where neural progenitor cells give rise to projection neurons or interneurons. Projection neurons from LGE are medium spiny neurons. GABAergic interneurons can be further subdivided by the expression of calcium 9.

(11) binding proteins or neural peptides, such as parvalbumin (PV), somatostatin (SST) or calretinin (Calr). Different types of interneurons are from different origins (Vitalis et al., 2011). For example, the PV+ and SST+ subgroups originate from the MGE, whereas the Calr+ subgroups originate from the LGE (Butt et al., 2005, Xu et al., 2004). Over the past decade, there were many researches showing that the neuronal subtype specification could be directly related to their embryonic spatial and temporal origins. For example, Nkx2-1, which is a member of the Nkx2 class of homeodomain-containing transcription factor, acts as a molecular switch to induce MGE-specific patterns of gene expression but repress LGE-specific genetic programs (Butt et al., 2008, Elias et al., 2008, Corbin et al., 2003). However, there might be unknown molecular mechanisms to specify neuronal subtypes in the mammalian forebrain.. 1.4 FoxP2 FoxP2, a transcriptional factor of the forkhead box family, has a winged-helix DNA binding domain. FoxP2 consists of approximately 110 amino acids and is highly conserved in vertebrates. It is expressed in various tissues including the lung and the brain. Currently, FoxP2 is the only gene linked to the development of speech and language (Spiteri et al., 2007, Enard et al., 2002). Recent studies show that mutations of FoxP2 causes language disorders and suggest that it correlates to speech development (Lai et al., 2003). In human, FoxP2 is mapped to chromosome 7a31. A missense mutation in the FoxP2 forkhead (R553H) is found in a group of patients who is called the KE family (Kaminen et 10.

(12) al., 2003). Mutated FoxP2 (R553H) reduces DNA binding and affects nuclear localization. Members of the KE family have severe abnormalities in speech and language (Lai et al., 2001). They have verbal dyspraxia, dysphasia and difficulties in performing complex movements of the mouth and face. However, why mutated FoxP2 leads to language disorder is still unknown. In non-human species such as Zebra finches which are one kind of songbirds (White et al., 2006), FoxP2 is expressed at high levels in the basal ganglia song neuclues Area X, a brain area related to the striatum in mammals, and is required for song learning (Miller et al., 2008). When Zebra finches are learning songs, the expression of FoxP2 is increased in Area X, whereas FoxP2 is decreased after the song is learned (Rochefort et al., 2007). Knockdown of FoxP2 in Area X not only decreases neuron spine density but also affects song learning (Schulz et al., 2010). Therefore, FoxP2 is required for vocal learning in Zebra finches. In mammals, human FoxP2 only differs with its homolog in mouse in three amino acids (Enard et al., 2009). In embryonic mice, FoxP2 is expressed in the LGE. Transgenic mice that lack FoxP2 or have mutations in FoxP2 show defects in motor learning, ultrasonic pup vocalization and synaptic plasticity in the striatum (Fujita et al., 2008). However, how FoxP2 contributes to song/language learning is still unknown.. 1.5 Nkx2-1 Nkx genes and their homologs are known regulators of neuronal cell fates. Nkx2-1 is a member of the Nkx2 class of homeodomain-containing 11.

(13) transcription factor. It is also known as thyroid transcription factor 1 (TTF-1). Nkx2-1 is expressed in the ventral forebrain during mouse embryonic development. During embryonic forebrain neurogenesis, Nkx2-1 is expressed in all of the MGE progenitors and is entirely excluded from the LGE. Nkx2-1 is required for MGE development (Butt et al., 2005, Sussel et al., 1999). Moreover, Nkx2-1 is required for the production of MGE-derived cortical interneuron subtypes such as SST+ and PV+ interneurons (Butt et al., 2008). Previous studies show that both FoxP2 and Nkx2-1 are expressed in alveolar epithelial cells and there are interactions between FoxP2 and Nkx2-1. Furthermore, the direct interaction between FoxP2 and Nkx2-1 inhibits DNA-binding and transcriptional activity of Nkx2-1 (Zhou et al., 2008). It is interesting to see whether FoxP2 interacts with Nkx2-1 to regulate forebrain development in embryonic mice.. 1.6 Sonic hedgehog pathway Sonic hedgehog (Shh), a member of the hedgehog family, is known to plays critical roles in multiple aspects of embryogenesis (Porter et al., 1996). Binding of Shh to the transmember receptor Patched (Ptc) relieves constitutive inhibition of a second transmember receptor, Smoothened (Smo) and initiates a cascade of events leading to induction of Gli transcription (Fuccillo et al., 2006). Shh signaling is involved in the determination of cell fate and patterening during early forebrain development. It serves a permissive role in ventral patterning (Rallu et al., 2002). One of the Shh signaling downstream effector is Nkx2-1. Previous 12.

(14) studies have indicated that Shh signaling during embryonic neurogenesis maintains the identity of Nkx2-1 expressing progenitors in the MGE (Xu et al.,2005) and suggest that Nkx2-1 level in MGE progenitors are dependent on Shh signaling. Therefore, it is possible that Shh signaling might interact with FoxP2.. 1.7 PDGFRα Platelet-derived growth factor receptor (PDGFR) is a receptor tyrosine kinase which is located on the cell membrane and can be activated by platelet-derived growth factors (PDGF). PDGF has four types of monomers: PDGF-A, B, C and D. They can form functional homodimers and heterdimers. PDGFR also has two kinds of monomer, PDGFRα and β. They can form functional dimmers: PDGFR -αα and -αβ. PDGF-AA,-AB, -BB,-CC,-DD can activate PDGFR-αα, whereas PDGF-AB,-BB,-CC can activate PDGFR-αβ (Wiens et al., 2010). PDGF signaling regulates cell growth, cell proliferation, embryonic development, angiogenesis and neurogenesis (Xie et al., 2001, Erlandsson et al., 2001). During mouse development, PDGFR-α is expressed by neuroepithelial cells as early as embryonic day 8.5 (E8.5). NSC in the subventricular zone have high level expression of PDGFR-α. PDGF can promote NSC growth and self-renewal (Jackson et al., 2006). Besides, treatment of PDGF in stem cells from central nervous system can induce their differentiation into neurons (Johe et al., 1996). Previous studies found that PDGFR-α was one of the downstream target gene of FoxP2 by a microarray analysis (Konopka et al., 2009). An important unanswered question is whether the 13.

(15) effect of FoxP2 during neural development is mediated by PDGFR-α.. Previous studies showed that FoxP2 was related to language and also regulated neuron spine density. However, how FoxP2 contributes to these is still unknown. I reason that FoxP2 may affect neurogenesis. Therefore, the aims of this study are to investigate the role of FoxP2 and related mechanisms in embryonic neurogenesis.. 14.

(16) 2. Materials and Methods 2.1 Primary cell cultures Embryonic day 13.5 (E13.5) embryos were removed from pregnant female mice and their forebrains were separated and transferred into ice-cold HBSS. The forebrains were then dissected into two parts: the cerebral cortex and GE. Tissues were then triturated using a fire-polished Pasteur pipette and resuspended in L15 medium (N2/B27/glucose/penn-strep; GIBCO, Sigma) containing basic FGF (1:5000; R&D system). 1×106 cells were plated in each well of 6-well plates containing coverslips (36 mm2; Corning) that were previously coated with polylysine (10 μg/ml) and laminin (5 μg/ml). 0.9×106 donor cells per well were added as feeder layers 30 minutes previously. Cultures were maintained at 37°C in 5% CO2 and ambient oxygen. On the next day (1 d.i.v.), half of the medium was replaced with neurobasal medium containing 10 ng/ml of basic FGF. Medium was changed every other day for the following time of culture. This approach promotes the initial proliferation of progenitors that grossly mimics what occurs in the early embryonic forebrain in vivo. Cells were cultured for 2–12 d.i.v.. 2.2 Plasmids For FoxP2 overexpression experiment, US2-FoxP2 and US2-GFP were used and US2-GFP was used as control. For FoxP2 knockdown experiment, UI4-shFoxP2860-GFP, shFoxP2-5, shFoxP2-6 and 15.

(17) shFoxP2-7 were used. UI4-GFP was used as control. For Nkx2-1 knockdown experiment, shNkx2-1-4, shNkx2-1-6, shNkx2-1-7 and US2-GFP were used and shLacZ and US2-GFP were used as control. Plasmids were electroporated into the cells. US2-FoxP2, US2-GFP, UI4-shFoxP2860-GFP and UI4-GFP were constructed by Jenn-Yah Yu’s lab and UI4-shFoxP2860-GFP was designed to target FoxP2 mRNA sequence 860-882. shFoxP2-5, shFoxP2-6, shFoxP2-7, shNkx2-1-4, shNkx2-1-6, shNkx2-1-7 and shLacZ were obtained from the National RNAi Core Facility and the target sequences are the following: ShFoxP2-5: 5’-CCACACATACATTCAATCCAT-3’; shFoxP2-6: 5’-GCAACAGTTCAATGAATCA AA-3’; ShFoxP2-7:5’-GCGACATTCAGA CAAATACAA-3’; ShNkx2-1-4: 5’GCGACGTTTCAAGCAACAGAA-3’; ShNkx2-1-6: 5’-CGCCATGTCTTGTT CTACCTT-3’; ShNkx2-1-7: 5’-GTT CTC AGTGTCTGACATCTT-3’; shLacZ: 5’-CGCTAAATACTGGCAGGCGT T-3’; shGli2: 5’-CACCAACCCTTCAGACTATTA.. 2.3 Electroporation The primary cells from the embryonic forebrain were prepared for nuclofection and mixed with 100 μL of the nucleofection solution (Lonza) and 10 μg of plasmid per transfection. Cells and the nucleofection mixture were added into the cuvette (Lonza) and electroporated with the Nucleofector (Lonza), program A-033 (Mouse NSC) according to the 16.

(18) instructions of the manufacturer. Cells were then incubated at 37°C in 5% CO2 and ambient oxygen.. 2.4 P19 cell culture and transfection The mouse embryonic carcinoma cell line P19 was cultured in α-Minimal Essential Medium supplemented with 2.5% FBS, 7.5% newborn calf serum and 1% Penicillin/Streptomycin. To introduce various constructs into P19 cells, cells were transfected by Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. The medium was changed 24 hours after transfection. For the FoxP2 knockdown experiments (in one well of a twelve-well culture plate), P19 cells were transfected with US2-GFP, FoxP2 and three shFoxP2 including shFoxP2-5, -6, -7. Cells were cultured for three days. For the Nkx2-1 knockdown experiments, P19 cells were transfected with US2-GFP, Nkx2-1 and three shNkx2-1 including shNkx2-1-4, -6, -7. Cells were cultured for one day. 24 h after transfection, the culture medium was changed to MEMα serum-free medium (Segal et al., 1992).. 2.5 Immunostaining analysis Transfected cells grown on coverslips were rinsed once with PBS and then fixed in 4% paraformaldehyde for 15 min. After washed with 1X PBT, cells were incubated with goat serum blocking buffer for 1 hour. 17.

(19) Samples were then incubated with the appropriate primary (16~18 hours at 4℃) and secondary antibodies and finally stained with 4, 6-diamidino-2-phenylindole (DAPI) (5 μg/ml) for 30 min. The primary antibodies used included Calretinin (mouse; 1:5000; Millipore Co.), Caspase-3 (rabbit; 1:200; Cell Signaling Co.), DARPP32 (rabbit; 1:5000; Cell Signaling Co.), FoxP2 (rabbit; 1:5000; Abcam Co.), GFP (rabbit; 1:2000; Molecular probes Co.), Nkx2-1 (mouse; 1:500; Thermo Co.), O4 (mouse IgM; 1:200; Millipore Co.), PDGFRα (Goat; 1:100; R&D Co.), Tuj1(mouse; 1:1000; Covance Co.), GAD67 (mouse; Chemicon; 1:1000) and Somatostatin (rat;1:200; Molecular probes Co.). Then, cells were washed and incubated with secondary antibody such as goat anti-rat 488, 549, goat anti-mouse 549, goat anti-rabbit 488, 549 (Jackson Immuno-Research Laboratory, USA; 1:1000). For BrdU staining, cells were treated with 2N hydrochloride for 30 mins at 37°C and 0.1 M sodium borate for 10 mins at room temperature. After washed with 1X PBT, cells were incubated with goat serum blocking buffer for 1 hour. BrdU (rat; 1:500; ACCURATE CHEMICAL & SCIENTIFIC Co.) were used as primary antibody. Then, cells were washed and incubated with secondary antibody (goat anti-rat 549 (Jackson Immuno-Research Laboratory, USA; 1:1000). Samples were examined using a fluorescence microscope (DMI 4000; Leica).. 2.6 Western blotting. 18.

(20) Transfected P19 cells were washed once with PBS and lysed in lysis buffer (20 mM Tris-Base, pH7.9, 20 mM NaCl, 20 mm β-glycerol phosphate, 1 mM EDTA, 1mM PMSF, 25 mM Calyculm A, 0.5% Triton and protease inhibitor cocktail (Roche)). Cell lysates were centrifuged at 13,000 × g for 10 min, and the supernatants were collected for Western blot analysis. The protein concentration was determined using a BCA kit (Bio-Rad). For Western blot analysis, equal amounts (10 μg) of cell lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% skimmed milk and incubated with the primary antibody at 4°C overnight. Primary antibody used for Western blotting were FoxP2 (rabbit; 1:5000; Abcam Co.), Nkx2-1 (mouse; 1:500; Thermo Co.) and β-tubulin (mouse; 1:1000; Sigma). After the membrane was washed and incubated with Goat anti rabbit HRP-conjugated secondary antibodies (1:1000; Jackson Immuno-Research Laboratory, USA) and Goat anti mouse HRP-conjugated secondary antibodies (1:1000; Jackson Immuno-Research Laboratory, USA) at room temperature for 1 h, it was then developed with an ECL kit (Thermo). Chemiluminescent was detected by Luminescent image analyzer Las4000. The intensity of protein band was quantified by Image J software.. 2.7 Image analysis and statistical analysis Cell counting was performed under a fluorescence microscope (DMI 4000; Leica) at 200 X magnification. In cell culture experiments, cells in ramdomly selectted five to ten flields were counted in each coverslip, 19.

(21) about 8-20 cells/field and 50-130 cells/coverslip were counted. All Data were shown as mean ± SEM. We used unpaired two-tailed Sudent t test, one-way ANOVA or two-way ANOVA followed by Tukey post test of group means were used for two-group or multiple comparisons. Significance level was p＜0.05. Asterisks indicate the following p values: *p ＜0.05, **p ＜0.01, ***p ＜0.001.. 20.

(22) 3. Results 3.1 Overexpression and knockdown of FoxP2 in primary and P19 cell cultures To examine whether FoxP2 affects embryonic neurogenesis, loss-of-function and gain-of-function experiments were designed. It has been known that FoxP2 is expressed in the LGE but not in the cortex (Ferland et al., 2003). Therefore, I decided to overexpress FoxP2 in the cortex and knock down FoxP2 in the GE by electroporation. Electroporation permits examination of targeted cells in a background of normal cells, which is essential to test for cell-autonomous effects. I selected E13.5, the peak stage of neurogenesis for primary cell culture. I electroporated FoxP2 into the cortical progenitor cells and cultured them for 2 days. As expected, the expression of FoxP2 was greatly increased (Control: 16.60 ± 0.04%; FoxP2: 88.33 ± 0.01%; p < 0.01; Fig. 1A, 1B). For the knockdown FoxP2 experiments, we used P19 cells which is a multipotent embryonic carcinoma cell line that can differentiate into neurons, glial cells, cardiac cells and muscle cells (McBurney et al., 1982) as a model to test the knockdown efficiency of shFoxP2. We found that shFoxP2860 could efficiently block FoxP2 expression (data not shown). In the following experiments, I used shFoxP2860 as my knockdown construct. I suppressed endogenous FoxP2 expression in GE progenitor cells by electroporating shRNA against FoxP2. Since FoxP2 is expressed in postmitotic neurons in LGE, I cultured these cells for 5 days. I found that only 3.35 ± 0.01% of the GFP-expressing cells in the knockdown 21.

(23) group expressed FoxP2 compared with 86.67 ± 0.08% in the control group (Control: 86.67 ± 0.08%; shFoxP2: 3.35 ± 0.01%; p < 0.01; Fig. 1A, 1C). Besides, I used three other shRNA expression constructs (shFoxP2-5, shFoxP2-6 or shFoxP2-7) which were obtained from the National RNAi Core Facility targeting different sequences of FoxP2 to silence FoxP2 expression. The immunostaining (Fig. 2A) and Western blot (Fig. 2C) result show that the knockdown efficiency of shFoxP2-6 was the highest (Immunostaining: Control: 88.53 ± 0.01%; shFoxP2-5: 63.27 ± 0.09%; shFoxP2-6: 4.23 ± 0.02%, p < 0.01; shFoxP2-7: 47.07 ± 0.06%, p < 0.05; Fig. 2B, Western blot: Control: 100%; shFoxP2-5: 97.82 ± 0.13%; shFoxP2-6: 16.87 ± 0.04%, p < 0.01; shFoxP2-7: 42.58 ± 0.05%, p < 0.01; Fig. 2D ). Based on this, I also used shFoxP2-6 for the knockdown experiment.. 3.2 FoxP2 positively regulates neuronal differentiation in the embryonic telencephalon First, I investigated whether FoxP2 regulated neuronal differentiation. I overexpressed FoxP2 in E13.5 cortical progenitor cells and cultured them for 2 or 5 days to observe the short term and long term effect of FoxP2. I found that more GFP-positive cells in the FoxP2 group also expressed Tuj1, a neuron marker (2 d.i.v: Control: 84.27 ± 0.01%; FoxP2: 90.00 ± 0.01%; p < 0.05; 5 d.i.v: Control: 70.43 ± 0.02%; FoxP2: 91.10 ± 0.01%; p < 0.01; Fig. 3A, 3B). This result confirms that FoxP2 positively regulates neuronal differentiation. In the control group, Tuj1-positive cells in the 5-d culture appeared to be less than the 2-d one. This could be 22.

(24) due to that other cell types such as astrocytes or oligodendrocytes are generated at this stage, whereas only neurons were produced at the earlier time. For the loss-of-function experiments, I electroporated UI4 plasmid and shFoxP2-860 into GE progenitor cells as the control and knockdown group, respectively (Fig. 3A) and cultured them for 5 days. When compared to the control group, there were less GFP-Tuj1 double positive cells in the shFoxP2-860 group (Control: 98.43 ± 0.03%; shFoxP2: 82.38 ± 0.04%; p < 0.01; Fig. 3C). Therefore, FoxP2 is sufficient and necessary for neuronal differentiation in the embryonic telencephalon. To futher comfirm this finding, I also used shFoxP2-6 to knockdown FoxP2 and found that the result was consistent to that of shFoxP2-860 (Control: 96.10 ± 0.01%; shFoxP2: 80.78 ± 0.02%; p < 0.01; Fig. 4A, 4B).. A previous study reported that deficits in vocal learning and spiny density occur in zebra finches after reduction of FoxP2 in Area X, a striatal nucleus involved in song acquisition (Schulz et al., 2010). Therefore, I were interested in whether FoxP2 affected neuronal morphology in mammals. I choose neurite number and length as two indices of neuronal differentiation. I found that the length of the longest neurite of GFP-positive neurons was significantly increased when overexpressed FoxP2 in cortical progenitor cells (2 d.i.v: Control: 43.8 ± 23.94 µm; 100%; FoxP2: 50.57 ± 23.28 µm; 122%; 5 d.i.v: Control: 81.21 ± 6.78 µm; 100%, FoxP2: 134.56 ± 13.06 µm; 166%; p < 0.01; Fig. 5A, 5B, 5E) whereas it was decreased when knocked down FoxP2 in GE progenitor cells (Control: 80.19 ± 15.37 µm; 100%, shFoxP2: 60.38 ± 23.

(25) 8.53 µm; 76%; p < 0.01; Fig. 5C, 5D, 5F). In 2 d and 5 d culture, I observed that there was no difference of neurite number between control group and FoxP2 overexpression group or control group and shFoxP2 group (Fig 6). Taken together, FoxP2 increases neurite length without affecting neurite number.. 3.3 FoxP2 has no effects on cell proliferation and cell survival The neurogenic effect of FoxP2 I observed may be due to increased cell proliferation or decreased cell death. Therefore, I examined cell cycle or cell death markers in my cultures. When neural progenitor cells differentiate into neurons, they exit cell cycle. For cell proliferation experiments, I cultured cortical progenitor cells for two days and added BrdU, the thymidine analog, two hours before fixation to label proliferating cells (Fig. 7A). The percentage of BrdU labeling in GFP-positive cells in the control and FoxP2 overexpression group were similar (Control: 2.23 ± 0.01%; FoxP2: 2.43 ± 0.004%; Fig. 7B). Furthermore, I cultured cortical progenitor cells for five days to observe whether FoxP2 affected cell survival. I found that just a few cultured cells expressed caspase-3, the apoptotic cell marker, in both groups (Fig. 8A). The percentage of caspase-3 labeling in GFP-positive cells in the FoxP2 overexpression group was similar to that of the control group (Control: 3.9 ± 0.01%; FoxP2: 5.37 ± 0.01%; Fig. 8B). Therefore, these data suggest that FoxP2 promotes neuronal differentiation without affecting cell proliferation or cell survival. 24.

(26) 3.4 FoxP2 increases the expression of PDGFRα and the neurognic effect of FoxP2 is PDGFRα-dependent The effect of FoxP2 on neuronal differentiation provides an opportunity to identify downstream effectors. A lead candidate is PDGFRα, a member of the receptor tyrosine kinase related to neural development and can be activated by PDGF. By a microarray analysis, PDGFRα is likely to be one of FoxP2 downstream target genes (Konopka et al., 2009). First, I cultured primary cell cultures for five days to examine whether FoxP2 regulated PDGFRα expression (Fig. 9A). PDGFRα expression was significantly higher in FoxP2 group than in the control group (Control: 21.24 ± 0.04%; FoxP2: 43.89 ± 0.03%; p < 0.05; Fig. 9B), whereas it was significantly lower in the shFoxP2 group than in the control group (Control: 46.13 ± 0.01%; shFoxP2: 24.79 ± 0.03%; p < 0.05; Fig. 9C ).To further explore the possibility that PDGFRα acts as a FoxP2 downstream target to induce neuronal differentiation, I overexpressed FoxP2 in cortical progenitor cells and added the functional blocking PDGFRα antibody (PDGFRα ab) during the five-day culture. There were four groups: Ctrl-PBS, FoxP2-PBS, Ctrl-PDGFRα ab and FoxP2-PDGFRα ab (Fig. 10A). As expected, overexpression of FoxP2 increased neuronal differentiation identified by Tuj1 expression (US2-PBS: 66.01 ± 0.06%; FoxP2-PBS: 90.11 ± 0.01%; p < 0.05; Fig. 10B). PDGFRα ab inhibited neuronal differentiation in FoxP2 overexpression group to the control level (FoxP2-PBS: 90.11 ± 0.01%; FoxP2- PDGFRα: 68 ± 0.01%; p < 0.01; Fig. 10B). Since there was no endogenous FoxP2 expression in cortical progenitor cells, PDGFRα ab 25.

(27) treatment alone did not affect the number of GFP-, Tuj1-double positive cells (US2-PBS: 66.01 ± 0.06%; US2-PDGFRα: 65 ± 0.02%; Fig. 10B). These results support my hypothesis that FoxP2 increases the expression of PDGFRα and the neurognic effect of FoxP2 is PDGFRα-dependent.. 3.5 FoxP2 does not regulate oligodendrocyte differentiation in the embryonic telencephalon Neural progenitor cells can differentiate into three different cell types: neurons, oligodendrocytes and astrocytes. I tested whether FoxP2 also regulated the differentiation of these other cell types. I used O4, an oligodendrocyte marker to see whether FoxP2 affects oligodendrocyte differentiation. I overexpressed FoxP2 in the cortical progenitor cells and cultured them for five days. I found that there was no difference in oligogendrocyte differentiation between the two groups (Control: 21.49 ± 0.03%; FoxP2: 24.06 ± 0.06%; Fig. 11A, 11B). Therefore, FoxP2 does not affect oligodendrocyte differentiation in the embryonic telencephalon. However, I cannot obtain GFAP-positive astrocytes in any of my culture conditions. The effect of FoxP2 on astrocyte differentiation is still unknown. Nevertheless, my data suggests that FoxP2 positively regulate neuronal but not glial differentiation.. 3.6 FoxP2 regulates the specification of neuronal subtypes Next, I investigated whether FoxP2 regulates the specification of neuronal subtypes. Since FoxP2 is expressed in the LGE, I focused on 26.

(28) neurons originated from this area. The majority of neurons from GE are GABAergic. First, I examined whether FoxP2 regulated the generation of these neurons. I electroporated shFoxP2 in GE to knock down FoxP2 expression and cultured these cells for twelve days (Fig. 12A). I found that knockdown of FoxP2 did not change the percentage of GABAergic neurons in my cultures (Control: 44.93 ± 0.05%; shFoxP2: 45.45 ± 0.03%; Fig. 12B).. Since GABAergic neurons in the ventral telencephalon include DARPP-32-positive medium spiny projection neurons, Calr-positive, SST-positive and PV-positive interneurons, I decided to examine whether FoxP2 affectted the production of these subtypes of neurons. DARPP-32-positive medium spiny neurons and calretinin-positive interneurons originate from FoxP2-positive LGE (Farries et al., 2000). Also, previously studies showed that FoxP2 was exclusively expressed in medium spiny neurons (Miller et al., 2008). Therefore, FoxP2 might regulate the generation of medium spiny neurons. It has been previously reported that retinoic acid (RA) is a key regulator of the development of medium spiny neurons (Toresson et al., 1999). To examine whether FoxP2 is also required for the differentiation of these neurons, I knocked down FoxP2 with shFoxP2-860 construct and added RA into the culture to create an environment permissive for the differentiation of medium spiny neurons. There were just a few DARPP-32-positive cells in both the control and shFoxP2 groups when adding DMSO instead of RA (Control-DMSO: 2.10 ± 0.01%; shFoxP2-DMSO: 4.1 ± 0.002%; Fig. 13). 27.

(29) As expected, RA induced the differentiation of DARPP-32-positive medium spiny neurons (Fig 13). More importantly, shFoxP2 blocked the production of DARPP-32-positive neurons in RA-treated group (Control-RA: 19.06 ± 0.005%; shFoxP2-RA: 11.86 ± 0.02%; Fig. 13B).. I also examined whether FoxP2 affected the production of Calr-positive interneurons. I knocked down FoxP2 in GE progenitor cells and cultured them for twelve days. However, there were just a few GFP-positive cells expressing Calr (Fig. 14A) and there was no difference between control and shFoxP2 group (Control 6.93 ± 0.01%; shFoxP2: 6 ± 0.01%; Fig. 14B). Together, these data suggest that FoxP2 is required for the development of LGE-derived medium spiny neurons.. I was interested in whether FoxP2 can switch the neuronal subtypes. FoxP2 can increase LGE-derived neurons so I expected it might decrease MGE-derived neurons. In the control group, I observed that numerous GFP-positive cells were SST-positive cells, which represent MGE-derived interneurons (Fig. 15A). I found that the neumber of GFPand SST-positive neurons was significantly increased in shFoxP2 group (Control: 30.85 ± 0.01%; shFoxP2: 43.57 ± 0.02%; p < 0.01; Fig. 15B). Furthermore, previous studies indicate that there are interactions between FoxP2 and Nkx2-1 (Zhou et al., 2008), a transcription factor known for the development of the ventral forebrain. I was interested in whether FoxP2 and Nkx2-1 together regulate the development of neuronal subtypes here. Nkx2-1 is maintained by Shh signal and expressed in 28.

(30) postmitotic neurons derived from the MGE (Sussel et al., 1999), such as SST-positive and PV-positive interneurons. However, it is entirely excluded from the LGE where FoxP2 is expressed (Butt et al., 2008, Du et al., 2008). Besides, FoxP2 also inhibits Nkx2-1-mediated transcription of surfactant protein C during lung development (Zhou et al., 2008). I hypothesize that FoxP2 may inhibit Nkx2.1 function to regulate the specification of neuronal fates in the ventral forebrain. Previous studies also show that Nkx2-1 is required for the production of MGE-derived cortical interneuron subtypes such as SST+ and PV+ interneurons (Butt et al., 2008). To test this hypothesis, I knockdowned Gli2, a transcription activators which is downstream of Shh pathway and upstream of Nkx2-1. I found that the neumber of GFP- and SST-positive neurons was significantly decreased in shGli2 group (Control: 30.85 ± 0.01%; shGli2: 16.30 ± 0.02%; p < 0.01; Fig. 15B). Based on this, I could further investigate whether FoxP2 and Shh-Nkx2-1 pathway together control the number of the SST-positive interneurons. I found that double knockdown of FoxP2 and Gli2 could rescue the number of SST-positive interneurons (shFoxP2: 43.57 ± 0.02%; shFoxP2+shGli2: 23.17 ± 0.03%; p < 0.01; Fig. 15B). Taken together, FoxP2 positively regulates the differentiation of LGE-derived medium spiny neurons and inhibits the production of MGE-derived interneurons.. 29.

(31) 4. Discussion First, I found that FoxP2 increased neuronal differentiation and neurite length without affecting cell proliferation and cell survival. Second, FoxP2 increased the expression of PDGFRα and the neurognic effect of FoxP2 was PDGFRα-dependent. Finally, FoxP2 regulated the specification of neuronal subtype in GE (Fig. 16). Taken together, these findings provide new insights that FoxP2 controls neurogenesis in the embryonic forebrain.. 4.1 FoxP2 regulates neuronal differentiation in the embyonic forebrain In the past years, accumulating evidence showed that FoxP2 was expressed in multiple species, such as humans, songbirds and mice (Teramitsu et al., 2004; White et al., 2006). In human, FoxP2 mutation causing a speech-language disorder was found in the KE family (Lai et al., 2001). The distribution of FoxP2 is changed in songbirds during vocal learning (Schulz et al., 2010). FoxP2 knockout mice show abnormal ultrasonic vocalizations (Fujita et al., 2008). However, the FoxP2-mediated molecular mechanism is still unclear. Previous studies showed that FoxP2 was expressed in medium spiny neurons and these cells were subject to postnatal recruitment, especially during song learning in zebra finches (Rochefort et al., 2007). Besides, reducing the expression of FoxP2 in an area of striatum called Area X leads to incomplete and inaccurate imitation of tutor songs (Haesler et al., 2007). 30.

(32) Futhermore, it has been proposed that two amino acid substitutions in the transcription factor FoxP2 have been positively selected during human evolution due to effects on aspects of speech and language (Enard et al., 2009). Transgenic mice with humanized FoxP2 had qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain. Besides, dendrite lengths and synaptic plasticity in striatal medium spiny neurons in these mice were increased (Reimers-kipping et al., 2011). Because vocal learning depends on neural plasticity in a number of brain nuclei in zebra finches and mice carrying nonfunctional FoxP2 showed abnormal in neuronal development and behavior, I hypothesize that FoxP2 affects song learning and language development by regulating neurogenesis. There are four contributing parts during neurogenesis: stem/progenitor cell proliferation, neuronal differentiation, cell survival, and migration. Several factors have been implicated to be involved in the process of neural stem cells into mature neurons, such as the specification of neuronal subtype identities, neuronal differentiation and migration (Bertrand et al., 2002; Schuurmans et al., 2004). Previous studies show that the basic-helix-loop-helix (bHLH) transcription factors encoded by proneural genes play a major and evolutionary conserved role in neurogenesis (Bertrand et al., 2002; Ross et al., 2003). For example, Mash1, Neurogenin1 and Neurogenin2 can promote the differentiation of neural progenitors into neurons, thus demonstrating that they are sufficient to activate neuronal differentiation program (Farah et al., 2000; Britz et al., 2006). Moreover, members of the NeuroD family and the T-box proteins Tbr1 can also affect neuronal 31.

(33) differentiation (Englund et al., 2005). NeuroD and the related gene Math2/Nex have been implicated in the terminal differentiation of neurons in the hippocampal dentate gyrus (Huang et al., 2000; Schwab et al., 2000) and neurogenesis in the adult hippocampus (Deisseroth et al., 2004). Tbr1 is required for the differentiation of certain populations of cortical neurons (Hevner et al., 2001). Although a large number of transcription factors have been implicated in neuronal development and differentiation, the detailed mechanism of the transcription factors that promote the differentiation of basal ganglia neurons is still not clear. In this study, I analyzed the function of FoxP2 in the mouse forebrain during embryonic neurogenesis. Using primary cell culture system, I overexpressed FoxP2 in the cortex and knocked down FoxP2 in the GE by electroporation. Indeed, I found that FoxP2 positively regulated neuronal differentiation. One study showed that reduced FoxP2 levels in the new spiny neurons in the Area X of zebra finches led to lower spine density (Schulz et al., 2010). I found that the length of the longest neurite of electroporated neurons was significantly increased when overexpressed FoxP2 in cortical progenitor cells, whereas it was decreased when knocked down FoxP2 in GE progenitor cells. The changes in neurite length could be important for the firing patterns of neurons or affect their connectivity. Depending on neuronal morphology, cells extending long processes are considered to be more mature neurons. Therefore, I suggest that FoxP2 increases neuronal maturation. This idea is consistent with a previous in vivo study that FoxP2 increased the neuron spine density in the Area X of zebra finches (Schulz et al., 2010). 32.

(34) In this study, I found that FoxP2 positively regulated neuronal differentiation, but not proliferation and survival. However, I did not examine whether FoxP2 was involved in cell migration. For the speed of migration, I can observe the transfected cells by time-lapse video microscopy to answer this question. However, it is difficult to use primary cell culture to answer whether FoxP2 affects neuronal migration. FoxP2 mutant mice will be useful by labeling migrating cells in mouse embryos with a tracer to investigate their migratory pattern.. 4.2 The role of FoxP2 and PDGFRα in neuronal differentiation PDGFRα is predicted as one of the FoxP2 target genes from a microarray study (Konopka et al., 2009). The PDGF network supports a variety of cell process including oligodendrogenesis, vascular development and angiogenesis (Hu et al., 2012; Sun et al., 2005). Furthermore, two previous reports indicate that PDGF signal can enhance neuronal differentiation (Johe et al., 1996; Williams et al., 1997). In my study, I found that FoxP2 increased the expression of PDGFRα and the neurognic effect of FoxP2 was PDGFRα-dependent. It raises the question of whether FoxP2 directly activates PDGFRα expression, I can use chromatin immunoprecipitation to answer this question.. Multipotent neural stem cells give rise to neurons, astrocytes, and oligodendrocytes. Moreover, external signals are thought to play an 33.

(35) important role for the regulation of neural stem/progenitor cell (NSPC) fate (Ruhnke et al., 2003). In this study, I found that FoxP2 increased neuronal differentiation without affecting oligodendrocyte differentiation. However, my experimental environment wasn’t suitable for astrocytes formation. Since previous studies showed that CNTF directed NSPC to an astrocytic cell fate (Erlandsson et al., 2006), I could add CNTF into my cell culture medium to induce astrocytes and examine whether FoxP2 affects astrocyte differentiation.. 4.3 FoxP2 acts as a regulator in neuronal subtype specification During development, neurons are instructed to migrate and send projections to specific regions in the brain. A fundamental question is how cells differentiate into specific neuronal subtypes. The subdivision of the GE can give rise to different subtypes of neurons by the expression of transcription factors. The LGE gives rise to not only cortical GABAergic interneurons but also striatal projection neurons and the MGE gives rise to many cortical GABAergic interneurons (Sussel et al., 1999). Several recent studies have shown that the GE regional identities are maintained through cross-regulatory interactions by transcription factors (Tucker et al., 2008) For example, homeobox transcription factors Dlx1 and Dlx2 are expressed by progenitors within the ventral telencephalon, regulate the fate and migration of ventral precursors, and promote GABAergic interneuron differentiation (Maqdelena et al., 2007). Another transcription factor Mash1 has a role in the specification of neuronal 34.

(36) identity in different regions of the nervous system and can induce expression of markers of GABAergic neurons in the cerebral cortex (Fode et al. 2000). Moreover, Shh signaling is centrally involved in the patterning of the nervous system. It has been shown that expression of Shh is required for the specification of neuronal identity (Ericson et al., 1996) and GABAergic populations such as interneurons derived from the MGE also depend on Shh-signaling (Nery et al., 2001). Many Shh-dependent homeodomain transcription factors are involved in ventral patterning in GE (Corbin et al., 2003). Previous study has demonstrated that Gli family of transcriptional regulators – Gli1, Gli2 are collectively necessary and sufficient for all aspects of intracellular Shh-responses involved in ventral forebrain patterning (Matise et al., 1998). Another study shows that the Shh-mediated induction of Nkx2-1 expression initiates MGE development (Xu et al., 2005). However, the mechanisms controlling the segregation of LGE and MGE neuronal populations remain poorly understood. Previous reports have shown that transcription factors are thought to play a major role in regulating the specification of neuronal subtype. For example, one previous study showed that FoxP2 was expressed by LGE-derived neurons and absent from MGE-derived interneurons (Takahashi et al., 2009). Therefore, FoxP2 may be important to specify GE-derived neuronal subtypes. Using loss-of-function approaches, my experiments demonstrate that loss of FoxP2 expression results in a reduction of LGE-derived medium spiny neurons. In contrast, MGE-derived interneurons are increased. These results suggest that in the absence of FoxP2, LGE progenitors are specified to a more MGE fate. 35.

(37) This transformation leads to a dramatic change in the number of GABAergic cortical interneurons, striatal interneurons and projection neurons derived from the MGE and LGE, respectively (Sussel et al., 1999). Furthermore, other factors may be involved in transcriptional regulation of neuronal subtype specification. I believe that evolution has selected distinct regulatory networks of gene expression for the cell types derived from each of the two GE. For example, Nkx2-1, unlike FoxP2, is expressed in the MGE and is critical for the development of the mammalian forebrain (Gulacsi et al., 2006). Also, previous studies have revealed that Nkx2-1 regulates the specification of MGE-derived cells during early patterning of the telencephalon (Corbin et al., 2003). Interestingly, FoxP2 inhibits Nkx2-1 transcription activity in alveolar cells (Zhou et al., 2008). It is likely that FoxP2 interacts with other transcription factors to regulate neuronal subtype specification. Taken together, it implies that these two transcription factors FoxP2and Nkx2-1 may regulate the specification of neuronal fates in the ventral forebrain.. Overall, in this study, I investigate the effects and mechanisms of FoxP2 in embryonic neurogenesis. Therefore, the next step is to investigate whether FoxP2 and Shh/Nkx2-1 together control the specification of GABAergic neurons and potential interactions between FoxP2 and Shh/Nkx2-1 in the embryonic brain.. 36.

(38) 5. References Bertrand N, Castro DS, Guillemot F (2002) Proneural genes and the specification of neural cell types. Nat Rev Neurosci 3:517-530. Besnard V, Xu Y, Whitsett JA (2007) Sterol response element binding protein and thyroid transcription factor-1 (Nkx2.1) regulate Abca3 gene expression. Am J Physiol Lung Cell Mol Physiol 293:L1395-1405. Britz O, Mattar P, Nguyen L, Langevin LM, Zimmer C, Alam S, Guillemot F, Schuurmans C (2006) A role for proneural genes in the maturation of cortical progenitor cells. Cereb Cortex 16 Suppl 1:i138-151. Butt SJ, Fuccillo M, Nery S, Noctor S, Kriegstein A, Corbin JG, Fishell G (2005) The temporal and spatial origins of cortical interneurons predict their physiological subtype. Neuron 48:591-604. Butt SJ, Sousa VH, Fuccillo MV, Hjerling-Leffler J, Miyoshi G, Kimura S, Fishell G (2008) The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes. Neuron 59:722-732. Corbin JG, Rutlin M, Gaiano N, Fishell G (2003) Combinatorial function of the homeodomain proteins Nkx2.1 and Gsh2 in ventral telencephalic patterning. Development 130:4895-4906. Deisseroth K, Singla S, Toda H, Monje M, Palmer TD, Malenka RC (2004) Excitation-neurogenesis coupling in adult neural stem/progenitor cells. Neuron 42:535-552. Dessaud E, Yang LL, Hill K, Cox B, Ulloa F, Ribeiro A, Mynett A, Novitch BG, Briscoe J (2007) Interpretation of the sonic hedgehog 37.

(39) morphogen gradient by a temporal adaptation mechanism. Nature 450:717-720. Dhruv NT, Tailby C, Sokol SH, Lennie P (2011) Multiple adaptable mechanisms early in the primate visual pathway. J Neurosci 31:15016-15025. Du T, Xu Q, Ocbina PJ, Anderson SA (2008) NKX2.1 specifies cortical interneuron fate by activating Lhx6. Development 135:1559-1567. Elias LA, Potter GB, Kriegstein AR (2008) A time and a place for nkx2-1 in interneuron specification and migration. Neuron 59:679-682. Enard W, Gehre S, Hammerschmidt K, Holter SM, Blass T, Somel M, Bruckner MK, Schreiweis C, Winter C, Sohr R, Becker L, Wiebe V, Nickel B, Giger T, Muller U, Groszer M, Adler T, Aguilar A, Bolle I, Calzada-Wack J, Dalke C, Ehrhardt N, Favor J, Fuchs H, Gailus-Durner V, Hans W, Holzlwimmer G, Javaheri A, Kalaydjiev S, Kallnik M, Kling E, Kunder S, Mossbrugger I, Naton B, Racz I, Rathkolb B, Rozman J, Schrewe A, Busch DH, Graw J, Ivandic B, Klingenspor M, Klopstock T, Ollert M, Quintanilla-Martinez L, Schulz H, Wolf E, Wurst W, Zimmer A, Fisher SE, Morgenstern R, Arendt T, de Angelis MH, Fischer J, Schwarz J, Paabo S (2009) A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell 137:961-971. Enard W, Przeworski M, Fisher SE, Lai CS, Wiebe V, Kitano T, Monaco AP, Paabo S (2002) Molecular evolution of FOXP2, a gene involved in speech and language. Nature 418:869-872. Englund C, Fink A, Lau C, Pham D, Daza RA, Bulfone A, Kowalczyk T, 38.

(40) Hevner RF (2005) Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. J Neurosci 25:247-251. Eriksson C, Wictorin K (2003) Neuronal and glial differentiation within expanded glial cultures derived from the lateral and medial ganglionic eminences. Exp Neurol 184:1058-1063. Ericson J, Morton S, Kawakami A, Roelink H, Jessell TM (1996) Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 1996-87:661–673. Erlandsson A, Brannvall K, Gustafsdottir S, Westermark B, Forsberg-Nilsson K (2006) Autocrine/paracrine platelet-derived growth factor regulates proliferation of neural progenitor cells. Cancer Res 66:8042-8048. Erlandsson A, Enarsson M, Forsberg-Nilsson K (2001) Immature neurons from CNS stem cells proliferate in response to platelet-derived growth factor. J Neurosci 21:3483-3491. Farah MH, Olson JM, Sucic HB, Hume RI, Tapscott SJ, Turner DL (2000) Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127:693-702. Farries MA, Perkel DJ (2000) Electrophysiological properties of avian basal ganglia neurons recorded in vitro. J Neurophysiol 84:2502-2513. Ferland RJ, Cherry TJ, Preware PO, Morrisey EE, Walsh CA (2003) Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J Comp Neurol 460:266-279. 39.

(41) Fode C, Ma Q, Casarosa S, Ang SL, Anderson DJ, Guillemot F (2000) A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. Genes Dev 14:67-80. Fuccillo M, Rallu M, McMahon AP, Fishell G (2004) Temporal requirement for hedgehog signaling in ventral telencephalic patterning. Development 131:5031-5040. Fuccillo M, Joyner AL, Fishell G (2006) Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development. Nat Rev Neurosci 7:772-783. Fujita E, Tanabe Y, Shiota A, Ueda M, Suwa K, Momoi MY, Momoi T (2008) Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells. Proc Natl Acad Sci U S A 105:3117-3122. Gulacsi A, Anderson SA (2006) Shh maintains Nkx2.1 in the MGE by a Gli3-independent mechanism. Cereb Cortex 16 Suppl 1:i89-95. Hallam S, Singer E, Waring D, Jin Y (2000) The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification. Development 127:4239-4252. Haesler S, Rochefort C, Georgi B, Licznerski P, Osten P, Scharff C (2007) Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X. PLoS Biol 5:e321. Hevner RF, Shi L, Justice N, Hsueh Y, Sheng M, Smiga S, Bulfone A, Goffinet AM, Campagnoni AT, Rubenstein JL (2001) Tbr1 regulates differentiation of the preplate and layer 6. Neuron 29:353-366. 40.

(42) Hirschi KK, Rohovsky SA, D'Amore PA (1998) PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 141:805-814. Hu JG, Wang YX, Wang HJ, Bao MS, Wang ZH, Ge X, Wang FC, Zhou JS, Lu HZ (2012) PDGF-AA mediates B104CM-induced oligodendrocyte precursor cell differentiation of embryonic neural stem cells through Erk, PI3K, and p38 signaling. J Mol Neurosci 46:644-653. Huang HP, Liu M, El-Hodiri HM, Chu K, Jamrich M, Tsai MJ (2000) Regulation of the pancreatic islet-specific gene BETA2 (neuroD) by neurogenin 3. Mol Cell Biol 20:3292-3307. Jackson EL, Garcia-Verdugo JM, Gil-Perotin S, Roy M, Quinones-Hinojosa A, VandenBerg S, Alvarez-Buylla A (2006) PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling. Neuron 51:187-199. Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96:25-34. Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD (1996) Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 10:3129-3140. Kaminen N, Hannula-Jouppi K, Kestila M, Lahermo P, Muller K, 41.

(43) Kaaranen M, Myllyluoma B, Voutilainen A, Lyytinen H, Nopola-Hemmi J, Kere J (2003) A genome scan for developmental dyslexia confirms linkage to chromosome 2p11 and suggests a new locus on 7q32. J Med Genet 40:340-345. Konopka G, Bomar JM, Winden K, Coppola G, Jonsson ZO, Gao F, Peng S, Preuss TM, Wohlschlegel JA, Geschwind DH (2009) Human-specific transcriptional regulation of CNS development genes by FOXP2. Nature 462:213-217. Lai CS, Fisher SE, Hurst JA, Vargha-Khadem F, Monaco AP (2001) A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 413:519-523. Lai CS, Gerrelli D, Monaco AP, Fisher SE, Copp AJ (2003) FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder. Brain 126:2455-2462. Laub F, Aldabe R, Friedrich V, Jr., Ohnishi S, Yoshida T, Ramirez F (2001) Developmental expression of mouse Kruppel-like transcription factor KLF7 suggests a potential role in neurogenesis. Dev Biol 233:305-318. Lee JS, Wagoner-Johnson A, Murphy WL (2009) Modular peptide growth factors for substrate-mediated stem cell differentiation. Angew Chem Int Ed Engl 48:6266-6269. Liboi E, Pelosi E, Di Francesco P, Gallinari P, Petrini M, Sposi NM, Testa U, Rossi GB, Peschle C (1987) The EL2 rat fibroblasts line: differential effects of growth factors (EGF, PDGF, FGF, TPA and 42.

(44) TGF beta) on cell proliferation and c-fos expression. Ann N Y Acad Sci 511:318-328. Liu M, Pleasure SJ, Collins AE, Noebels JL, Naya FJ, Tsai MJ, Lowenstein DH (2000) Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy. Proc Natl Acad Sci U S A 97:865-870. Ma Q, Fode C, Guillemot F, Anderson DJ (1999) Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev 13:1717-1728. Maden M (2002) Retinoid signalling in the development of the central nervous system. Nat Rev Neurosci 3:843-853. Maqdelena AP, Gregory BP, David HR, John LR (2007) Dlx1 and Dlx2 control neuronal versus oligodendroglial cell fate acquisition in the developing forebrain. Neuron 55(3):417-433. Matise MP, Epstein DJ, Park HL, Platt KA, Joyner AL (1998) Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development 125:2759–2770. McBurney MW, Jones-Villeneuve EM, Edwards MK, Anderson PJ (1982) Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature 299:165-167. Miller JE, Spiteri E, Condro MC, Dosumu-Johnson RT, Geschwind DH, White SA (2008) Birdsong decreases protein levels of FoxP2, a molecule required for human speech. J Neurophysiol 100:2015-2025. 43.

(45) Muzio L, Di Benedetto B, Stoykova A, Boncinelli E, Gruss P, Mallamaci A (2002) Emx2 and Pax6 control regionalization of the pre-neuronogenic cortical primordium. Cereb Cortex 12:129-139. Nadarajah B, Brunstrom JE, Grutzendler J, Wong RO, Pearlman AL (2001) Two modes of radial migration in early development of the cerebral cortex. Nat Neurosci 4:143-150. Nery S, Wichterle H, Fishell G (2001) Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain. Development 128:527–540. Porter JA, Young KE, Beachy PA (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science 274:255-259. Przybyla LM, Voldman J (2012) Attenuation of extrinsic signaling reveals the importance of matrix remodeling on maintenance of embryonic stem cell self-renewal. Proc Natl Acad Sci U S A. Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, Temple S (2000) Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 28:69-80. Rallu M, Machold R, Gaiano N, Corbin JG, McMahon AP, Fishell G (2002) Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling. Development 129:4963-4974. Reimers-Kipping S, Hevers W, Paabo S, Enard W (2011) Humanized Foxp2 specifically affects cortico-basal ganglia circuits. Neuroscience 175:75-84. Riccio O, Murthy S, Szabo G, Vutskits L, Kiss JZ, Vitalis T, Lebrand C, Dayer AG (2012) New pool of cortical interneuron precursors in 44.

(46) the early postnatal dorsal white matter. Cereb Cortex 22:86-98. Rochefort C, He X, Scotto-Lomassese S, Scharff C (2007) Recruitment of FoxP2-expressing neurons to area X varies during song development. DevNeurobiol 67: 809–817. Romo P, Madigan MC, Provis JM, Cullen KM (2011) Differential effects of TGF-beta and FGF-2 on in vitro proliferation and migration of primate retinal endothelial and Muller cells. Acta Ophthalmol 89:e263-268. Ross SE, Greenberg ME, Stiles CD (2003) Basic helix-loop-helix factors in cortical development. Neuron 39:13-25. Ruhnke M, Ungefroren H, Zehle G, Bader M, Kremer B, Fandrich F (2003) Long-term culture and differentiation of rat embryonic stem cell-like cells into neuronal, glial, endothelial, and hepatic lineages. Stem Cells 21:428-436. Sanders EJ, Harvey S (2008) Peptide hormones as developmental growth and differentiation factors. Dev Dyn 237:1537-1552. Schulz SB, Haesler S, Scharff C, Rochefort C (2010) Knockdown of FoxP2 alters spine density in Area X of the zebra finch. Genes Brain Behav 9:732-740. Schuurmans C, Armant O, Nieto M, Stenman JM, Britz O, Klenin N, Brown C, Langevin LM, Seibt J, Tang H, Cunningham JM, Dyck R, Walsh C, Campbell K, Polleux F, Guillemot F (2004) Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways. EMBO J 23:2892-2902. 45.

(47) Schwab MH, Bartholomae A, Heimrich B, Feldmeyer D, Druffel-Augustin S, Goebbels S, Naya FJ, Zhao S, Frotscher M, Tsai MJ, Nave KA (2000) Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. J Neurosci 20:3714-3724. Segal RA, Takahashi H, McKay RD (1992) Changes in neurotrophin responsiveness during the development of cerebellar granule neurons. Neuron 9:1041-1052. Shimamura K, Martinez S, Puelles L, Rubenstein JL (1997) Patterns of gene expression in the neural plate and neural tube subdivide the embryonic forebrain into transverse and longitudinal domains. Dev Neurosci 19:88-96. Spiteri E, Konopka G, Coppola G, Bomar J, Oldham M, Ou J, Vernes SC, Fisher SE, Ren B, Geschwind DH (2007) Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain. Am J Hum Genet 81:1144-1157. Stoykova A, Treichel D, Hallonet M, Gruss P (2000) Pax6 modulates the dorsoventral patterning of the mammalian telencephalon. J Neurosci 20:8042-8050. Sun J, Wang DA, Jain RK, Carie A, Paquette S, Ennis E, Blaskovich MA, Baldini L, Coppola D, Hamilton AD, Sebti SM (2005) Inhibiting angiogenesis and tumorigenesis by a synthetic molecule that blocks binding of both VEGF and PDGF to their receptors. Oncogene 24:4701-4709. 46.

(48) Sussel L, Marin O, Kimura S, Rubenstein JL (1999) Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126:3359-3370. Takahashi H, Takahashi K, Liu FC (2009) FOXP genes, neural development, speech and language disorders. Adv Exp Med Biol 665:117-129. Taupin P, Gage FH (2002) Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 69:745-749. Teramitsu I, Kudo LC, London SE, Geschwind DH, White SA (2004) Parallel FoxP1 and FoxP2 expression in songbird and human brain predicts functional interaction. J Neurosci 24:3152-3163. Teramitsu I, White SA (2006) FoxP2 regulation during undirected singing in adult songbirds. J Neurosci 26:7390-7394. Toresson H, Mata de Urquiza A, Fagerstrom C, Perlmann T, Campbell K (1999) Retinoids are produced by glia in the lateral ganglionic eminence and regulate striatal neuron differentiation. Development 126:1317-1326. Tucker ES, Segall S, Gopalakrishna D, Wu Y, Vernon M, Polleux F, Lamantia AS (2008) Molecular specification and patterning of progenitor cells in the lateral and medial ganglionic eminences. J Neurosci 28:9504-9518. Vitalis T, Rossier J (2011) New insights into cortical interneurons 47.

(49) development and classification: contribution of developmental studies. Dev Neurobiol 71:34-44. Wang TW, Zhang H, Parent JM (2005) Retinoic acid regulates postnatal neurogenesis in the murine subventricular zone-olfactory bulb pathway. Development 132:2721-2732. White SA, Fisher SE, Geschwind DH, Scharff C, Holy TE (2006) Singing mice, songbirds, and more: models for FOXP2 function and dysfunction in human speech and language. J Neurosci 26:10376-10379. Wiens KM, Lee HL, Shimada H, Metcalf AE, Chao MY, Lien CL (2010) Platelet-derived growth factor receptor beta is critical for zebrafish intersegmental vessel formation. PLoS One 5:e11324. Williams BP, Park JK, Alberta JA, Muhlebach SG, Hwang GY, Roberts TM, Stiles CD (1997) A PDGF-regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells. Neuron 18:553-562. Wilson SW, Houart C (2004) Early steps in the development of the forebrain. Dev Cell 6:167-181. Xie J, Aszterbaum M, Zhang X, Bonifas JM, Zachary C, Epstein E, McCormick F (2001) A role of PDGFRalpha in basal cell carcinoma proliferation. Proc Natl Acad Sci U S A 98:9255-9259. Xu Q, Cobos I, De La Cruz E, Rubenstein JL, Anderson SA (2004) Origins of cortical interneuron subtypes. J Neurosci 24:2612-2622. Xu Q, Guo L, Moore H, Waclaw RR, Campbell K, Anderson SA (2005) Sonic hedgehog signaling confers ventral telencephalic progenitors 48.

(50) with distinct cortical interneuron fates. Neuron 65:328–340. Zhou B, Zhong Q, Minoo P, Li C, Ann DK, Frenkel B, Morrisey EE, Crandall ED, Borok Z (2008) Foxp2 inhibits Nkx2.1-mediated transcription of SP-C via interactions with the Nkx2.1 homeodomain. Am J Respir Cell Mol Biol 38:750-758.. 49.

(51) 50.

(52) Figure 1: Overexpression and knockdown of FoxP2 in primary cell culture by electroporation. To set up gain-of-function experiments, I overexpressed FoxP2 and GFP in E13.5 cortical progenitor cells and cultured them for 2 days. Transfected cells were identified by GFP (green) staining and FoxP2 expressing cells were identified by FoxP2 (red) staining. Arrows indicate GFP- and FoxP2-double positive cells; arrowheads indicate GFP-positive but FoxP2-negative cells. The expression of FoxP2 in FoxP2 overexpression group was significantly increased (A, left two columns). For the loss-of-function experiments, I knocked down FoxP2 with shRNA against FoxP2 in GE progenitor cells and cultured them for 5 days. The expression of FoxP2 in the knockdown group was significantly decreased than in the control group (A, right two columns). For quantification anaylysis of US2 and FoxP2 groups, numbers of FoxP2 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧360 GFP-positive cells were counted in each group (B). For quantification anaylysis of UI4 and shFoxP2 groups, n=3, ≧320 GFP-positive cells were counted in each group (C). Data are presented as means ± SEM. ** compared to the control group, p < 0.01 (t-test); scale bar: 50μm.. 51.

(53) 52.

(54) Figure 2: Test of knockdown efficiency of shFoxP2 constructs. To test the knockdown efficiency of three shFoxP2 constructs (shFoxP2-5, shFoxP2-6, shFoxP2-7), P19 cells were transfected with control vector, shFoxP2-5, shFoxP2-6, shFoxP2-7 along with GFP- and FoxP2-expressing constructs and fixed 3 days after transfection (A). Transfected cells were identified by GFP (green) staining and FoxP2 expressing cells were identified by FoxP2 (red) staining. Arrows indicate GFP- and FoxP2-double positive cells; arrowheads indicate GFP-positive but FoxP2-negative cells. More GFP-positive cells were FoxP2-negative in shFoxP2-6 group than in the control one (A). For quantification anaylysis of (A), numbers of FoxP2 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧197 GFP-positive cells were counted in each group (B). Western blot analysis 53.

(55) of shFoxP2 knockdown efficiency in P19 cells (C). Cells were lysed 3 days after transfection. Expression levels of β-tubulin (55 kDa) were used as loading controls. Quantification of FoxP2 protein levels was determined as relative intensity of FoxP2 over β-tubulin. FoxP2 expression was reduced significantly in P19 cells transfected with two shRNA expression constructs (shFoxP2-6 or shFoxP2-7) (D). Data are presented as means ± SEM. * compared to the control group. **: p < 0.01; *: p < 0.05 (t-test); scale bar: 50 μm.. 54.

(56) 55.

(57) Figure 3: FoxP2 is sufficient and necessary for neuronal differentiation in the embryonic telencephalon. To investigate whether FoxP2 regulats neuronal differentiation, I overexpressed FoxP2 and GFP in the cortical progenitor cells and cultured them for 2 or 5 days. Transfected cells were identified by GFP (green) staining and neurons were identified by Tuj1 (red) staining. Arrows indicate GFP- and Tuj1-double positive cells; arrowheads indicate GFP-positive but Tuj1-negative cells. FoxP2 positively regulates neuronal differentiation (A; left two columns). For quantification anaylysis of US2 and FoxP2 groups, numbers of Tuj1 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧273 GFP-positive cells were counted in each group (B). For the loss-of-function experiment, I knocked down FoxP2 expression in GE progenitor cells and cultured them for 5 days. The number of neurons in the knockdown group was significantly decreased than in the control group (A; right two columns). For quantification anaylysis of UI4 and shFoxP2 groups. n=3, ≧307 GFP-positive cells were counted in each group (C). Data are presented as means ± SEM. * compared to control group, p < 0.05 (t-test); scale bar: 50 μm.. 56.

(58) 57.

(59) Figure 4: The number of neurons is also decreased in the shFoxP2-6 group. I knocked down FoxP2 expression in GE progenitor cells and cultured them for 5 days. The number of neurons in the shFoxP2-6 group was significantly decreased than in the shLacZ group (A). Transfected cells were identified by GFP (green) staining and neurons were identified by Tuj1 (red) staining. Arrows indicate GFP- and Tuj1-double positive cells; arrowheads indicate GFP-positive but Tuj1-negative cells. For quantification anaylysis of (A), numbers of Tuj1 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧ 241 GFP-positive cells were counted in each group (B). Data are presented as means ± SEM. * compared to control group, p < 0.05 (t-test); scale bar: 50 μm.. 58.

(60) 59.

(61) Figure 5: FoxP2 increases neurite length. I overexpressed FoxP2 and GFP in E13.5 cortical progenitor cells and cultured them for 2 or 5 days. The length of the longest neurite of GFP-positive neurons was measured and the distance between two arrows represents the neurite length. Control group (A), FoxP2 overexpression group (B). The length of the longest neurite of GFP-positive neurons was significantly increased when overexpressed FoxP2 in cortical progenitor cells (E). For the loss-of-function experiment, I knocked down FoxP2 in GE progenitor cells and cultured them for 5 days. Control group (C), shFoxP2 group (D). The length of the longest neurite of GFP-positive neurons in the knockdown group was significantly decreased than in the control group (F). Data are presented as means ± SEM. * compared to control group, p < 0.05 (t-test); scale bar: 10 μm.. 60.

(62) 61.

(63) Figure 6: FoxP2 doesn’t augment neurite number. The morphology of represented cortical neurons in the control and FoxP2 overexpression group (A). There was no difference in neurite number between control group and FoxP2 overexpression group (B). The morphology of represented GE neurons in the control group and shFoxP2 group (C). There was no difference in neurite number between control group and shFoxP2 group (D). Data are presented as means ± SEM; scale bar: 10 μm.. 62.

(64) 63.

(65) Figure 7: FoxP2 does not affect cell proliferation. To investigate whether FoxP2 regulates cell proliferation, I overexpressed FoxP2 and GFP in E13.5 cortical progenitor cells and cultured them for 2 days. BrdU was added two hours before fixation. Transfected cells were identified by GFP (green) staining and proliferating cells were identified by BrdU (red) staining. Arrowheads indicate GFP-positive but BrdU-negative cells. There was no difference in GFP- and BrdU-double positive cells between control and FoxP2 group (A). For quantification anaylysis of (A), numbers of BrdU and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧307 GFP-positive cells were counted in each group (B). Data are presented as means ± SEM; scale bar: 50 μm.. 64.

(66) 65.

(67) Figure 8: FoxP2 does not affect cell survival. To investigate whether FoxP2 regulates cell survival, I overexpressed FoxP2 and GFP in E13.5 cortical progenitor cells and cultured them for 5 days. Transfected cells were identified by GFP (green) staining and apoptotic cells were identified by Caspase3 (red) staining. Arrowheads indicate GFP-positive but Caspase3-negative cells. The survival rate was similar between control and FoxP2 group (A). For quantification anaylysis of (A), numbers of Caspase3 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧200 GFP-positive cells were counted in each group (B). Data are presented as means ± SEM; scale bar: 50 μm.. 66.

(68) 67.