Y.J. Lai and M.Y. Li contributed equally to this work.
Patterns & Phenotypes
TRIP6 regulates neural stem cell maintenance in the postnatal mammalian subventricular zone
Version of Record online: 20 JUL 2014
Copyright © 2014 Wiley Periodicals, Inc.
Volume 243, Issue 9, pages 1130–1142, September 2014
How to Cite
Lai, Y.-J., Li, M.-Y., Yang, C.-Y., Huang, K.-H., Tsai, J.-C. and Wang, T.-W. (2014), TRIP6 regulates neural stem cell maintenance in the postnatal mammalian subventricular zone. Dev. Dyn., 243: 1130–1142. doi: 10.1002/dvdy.24161
- Issue online: 20 AUG 2014
- Version of Record online: 20 JUL 2014
- Accepted manuscript online: 5 JUL 2014 04:08AM EST
- Manuscript Revised: 25 JUN 2014
- Manuscript Accepted: 25 JUN 2014
- Manuscript Received: 22 FEB 2014
- National Science Council. Grant Numbers: NSC-100-2320-B-003-001-MY2, NSC-101-2320-B-003-001
- National Taiwan Normal University. Grant Numbers: NTNU-99091023, NTNU-100-D-02, NTNU-103T3040B03
- neural stem cell;
Background: Postnatal neurogenesis persists throughout life in the subventricular zone (SVZ)-olfactory bulb pathway in mammals. Extrinsic or intrinsic factors have been revealed to regulate neural stem cell (NSC) properties and neurogenesis. Thyroid hormone receptor interacting protein 6 (TRIP6) belongs to zyxin family of LIM proteins, which have been shown to interact with various proteins to mediate cellular functions. However, the role of TRIP6 in NSCs is still unknown. Results: By performing double immunofluorescence staining, we found that TRIP6 was expressed by Sox2-positive NSCs in embryonic and postnatal mouse forebrains. To study the function of TRIP6 in NSCs, we performed overexpression and knockdown experiments with neurospheres derived from postnatal day 7 SVZ. We found that TRIP6 was necessary and sufficient for self-renewal and proliferation of NSCs, but inhibited their differentiation. To further investigate the mechanism of TRIP6 in NSCs, we performed Luciferase reporter assay and found that TRIP6 activated Notch signaling, a pathway required for NSC self-renewal. Conclusions: Our data suggest that TRIP6 regulates NSC maintenance and it may be a new marker for NSCs. Developmental Dynamics 243:1130–1142, 2014. © 2014 Wiley Periodicals, Inc.
Neurogenesis is a tightly regulated process from the embryonic stage through adulthood (Gotz and Huttner, 2005). During the developmental stage of the mammalian nervous system, neural stem cells (NSCs) present at the ventricular zone (VZ) of the neural tube generate neurons and glia through symmetric and asymmetric cell division (Gotz and Huttner, 2005). Postnatally, NSCs are restricted at the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) (Corotto et al., 1993; Luskin, 1993; Seki and Arai, 1993). During embryonic and postnatal neurogenesis, newly generated cells exit cell cycle and undergo guided migration to reach their final destination and differentiate into neurons (Gotz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009). In the postnatal forebrains, glial fibrillary acidic protein (GFAP)- and Sox2-expressing neural stem cells in the SVZ generate transit-amplifying cells, which give rise to doublecortin (DCX)-positive neuroblasts. Neuroblasts migrate tangentially along the rostral migratory stream (RMS) to the olfactory bulb (OB) and differentiate into interneurons, whereas neuroblasts generated in the SGZ migrate radially into the nearby DG and differentiate into granule cells (Ming and Song, 2011).
In these neural stem cell niches, morphogens and transcription factors play important roles in regulating self-renewal and multi-potency, two properties of NSCs (Wang et al., 2005, 2006, 2011; Gong et al., 2007; Lin et al., 2012). The major morphogens, including wingless-type MMTV integration site family (Wnt), bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), sonic hedgehog (Shh), and Notch, mediate the activation of different transcription factors to regulate self-renewal and differentiation of NSCs (Liu and Zhang, 2011; Yao et al., 2012). Many transcription factors have also been shown to regulate embryonic neural development and postnatal neurogenesis (Liu and Zhang, 2011). For example, Sox2 promotes stem cell proliferation and maintenance (Ferri et al., 2004), whereas Pax6 and Tbr1 induce neuronal differentiation (Hevner et al., 2001; Englund et al., 2005). Whether there are other factors controlling NSC properties remains to be investigated.
Thyroid hormone receptor interacting protein-6 (TRIP6) is first identified as a thyroid hormone receptor β1 interacting protein using a yeast two-hybrid screening system (Lee et al., 1995). It contains a proline-rich region in its amino-terminus and three LIM domains (named for homeodomain proteins Lin-11, Isl-1, and Mec-3) in the carboxy-terminus (Yi and Beckerle, 1998; Murthy et al., 1999). With these features, TRIP6 and other focal adhesion proteins, such as zyxin (Beckerle, 1997), lipoma preferred partner (LPP) (Petit et al., 1996), Ajuba (Kanungo et al., 2000), and LIMD1 (Kiss et al., 1999) all belong to the zyxin family proteins. Most zyxin family members localize at sites of focal adhesions, but may also shuttle between the plasma membrane and nucleus to mediate different signaling events (Wang and Gilmore, 2001). Through the three LIM domains, the PDZ-binding motif, the Crk SH2-binding motif, and/or other protein-interacting domains, TRIP6 serves as a platform for the recruitment of a number of molecules involved in actin assembly, cell motility, survival, and transcriptional control (Xu et al., 2004; Lai et al., 2005, 2007, 2010; Li et al., 2005; Solaz-Fuster et al., 2006; Bai et al., 2007; Chastre et al., 2009; Hadjipanayis and Van Meir, 2009). Besides, TRIP6 is also involved in telomere protection (Sheppard and Loayza, 2010) and host-pathogen interactions (Williams et al., 1998; Worley et al., 2006). Interestingly, the expression of TRIP6 mRNA is reported to be elevated in different lineages of stem cells, including neural stem cells (Ramalho-Santos et al., 2002). This study implicates that TRIP6 may play roles in regulating properties of NSCs.
To understand the function of TRIP6 in NSCs, we examined the expression pattern of TRIP6 in stem cell niches of embryonic and adult mouse forebrains. We found that TRIP6 was majorly expressed by Sox2-positive NSCs. Besides, TRIP6 promoted the self-renewal and proliferation of postnatal NSCs and inhibited their differentiation. Furthermore, TRIP6 may regulate stem cell maintenance through the Notch signaling pathway.
TRIP6 Is Expressed by NSCs in the Embryonic and Adult Mouse Brain
To study the role of TRIP6 in the nervous system, we asked whether it was present in the brains of embryonic and adult mice. For this, we first examined the specificity of the TRIP6 antibody, which has been reported to be able to detect endogenous TRIP6 by immunobloting (Xu et al., 2004) and immunostaining (Takizawa et al., 2006). In 3T3 cells, this antibody detected endogenous TRIP6 at focal adhesions in the scrambled shRNA-expressing cell, but not in the cell expressing shTRIP6 (Fig. 1A). Our TRIP6 antibody could not detect TRIP6 signal in shTRIP6-transfected 3T3 cells (Fig. 1B). These results confirm the specificity of our TRIP6 antibody.
From the whole brain extract, we found that TRIP6 mRNA was expressed in embryonic day 15.5–16.5 (E15.5–16.5) mouse brains, but the level was low or undetectable in adult ones (Fig. 1C). Consistent with the mRNA result, TRIP6 protein was only detected in embryonic, but not in adult brains (Fig. 1D). These results implicate that the role of TRIP6 in the nervous system may be mainly in neural development. After the nervous system is developed, TRIP6 is down-regulated.
Since we found that TRIP6 was abundant in the embryonic brain (Fig. 1C, D) and TRIP6 has been reported to be enriched in NSCs (Ramalho-Santos et al., 2002), we carried out immunofluorescence of TRIP6 with E16.5 forebrain sections to investigate whether embryonic NSCs express TRIP6. We found that there was a ventricle-to-mantle gradient of TRIP6 expression in the dorsal and ventral forebrain, where the primordium of cerebral cortex and striatum reside, respectively (Fig. 2A). Since embryonic NSCs are located at the VZ, we double labeled forebrain sections with antibodies against TRIP6 and the neural stem cell marker Sox2. We found that most of the TRIP6-expressing cells were also Sox2-positive in the dorsal and ventral VZ (Fig. 2B, C). The percentage of TRIP6-positive cells that also express Sox2 was 90.2 ± 2.7% (Fig. 2E). During the embryonic stage, NSCs are actively dividing. To examine whether TRIP6-positive cells are proliferative, E16.5 forebrain sections were double-labeled with antibodies against TRIP6 and the cell cycle marker Ki67 (Fig. 2D). We found that 34.1 ± 2.1% of TRIP6-positive cells were labeled with Ki67 (Fig. 2E). These results suggest that TRIP6 is mainly expressed by NSCs in the embryonic forebrain.
The central nervous system is mostly developed during the embryonic stage, but neurogenesis persists in the SVZ-olfactory bulb pathway and the DG of adult brains. Since we found that embryonic NSCs expressed TRIP6, it is possible that TRIP6 may also be expressed by postnatal NSCs. Although we did not find TRIP6 expression in the adult brain by Western blot analysis, its mRNA was detected in some of the analyzed adult brains (Fig. 1C, D). Due to relatively fewer NSCs present in the adult nervous system, it is difficult to detect TRIP6 signal from whole brain extracts. Therefore, immunofluorescence can provide a better resolution of TRIP6-expressing cells in the adult brain. Indeed, we found that TRIP6 was expressed in the SVZ (Fig. 3A, C–H), but not in the SGZ (Fig. 3B). Consistent with the finding from embryonic brains, most TRIP6-expressing cells in the adult SVZ were also Sox2-positive (62.7 ±1.6%; Fig. 3C, D, D', I). However, only 8.0 ± 0.3% of TRIP6-positive cells were labeled with Ki67 (Fig. 3E, F, I). Since adult NSCs are mostly quiescent, it is reasonable that there are fewer TRIP6 and Ki67 double-positive cells in the adult SVZ. It is also proposed that adult NSCs are ependymal cells lining the lateral ventricle (Spassky et al., 2005). We also double labeled adult SVZ sections with antibodies against TRIP6 and the ependymal cell marker S100β, and found that 44.4 ± 1.5% of TRIP6-expressing cells were also S100β-positive (Fig. 3G, H, I). These results suggest that TRIP6 is mainly expressed by adult NSCs in the SVZ.
To examine whether the progeny of adult NSCs also expresses TRIP6, we double-labeled adult SVZ sections with antibodies against TRIP6 and the neuroblast marker, DCX, the neuronal maker, microtubule-associated protein 2 (MAP2), or the astrocyte marker, GFAP. We found that only 6.7 ± 1.8% of TRIP6-expressing cells were DCX-positive (Fig. 4A). However, MAP2-positive neurons do not express TRIP6 (Fig. 4B). Interestingly, TRIP6 was not expressed by GFAP-positive astrocytes in the striatum and the corpus callosum (Fig. 4C and data not shown). To further examine whether TRIP6 is expressed by microglia, we double-labeled SVZ sections with antibodies against TRIP6 and the microglia marker, ionized calcium-binding adapter molecule 1 (Iba1, Fig. 4D). We found that TRIP6 was not expressed by Iba1-positive microglia, either. Taken together, TRIP6 is mainly expressed by NSCs, ependymal cells, and some of the neuroblasts in the adult SVZ, but not in more differentiated cells including neurons, astrocytes, and microglia. These results implicate that TRIP6 may regulate NSC properties.
TRIP6 Maintains Self-Renewal and Proliferation of NSCs
Postnatal NSCs from the SVZ can be cultured in vitro in suspension condition supplied with mitogenic growth factors to form primary neurospheres (1' NS). 1' NS can be dissociated and cultured in the same condition to form secondary NS (2' NS). After growth factor removal, NS can differentiate into neurons, astrocytes, and oligodendrocytes. Therefore, the rate 2' NS formation and neural differentiation are indices of self-renewal and multi-potency of NSCs, respectively. To investigate whether TRIP6 is required for the self-renewal ability of NSCs, 1' NS derived frompostnatal day 7 (P7) SVZ were dissociated, transfected with scrambled shRNA (Ctrl) or shTRIP6 together with GFP plasmids, and cultured to form 2' NS for 5 days. Scrambled control transfected cells formed 2' NS (Fig. 5A). However, shTRIP6 ones did not (Ctrl: 55 ± 3.6 spheres/106 cells, TRIP6: 0.33 ± 0.3 spheres/106 cells, P < 0.01; Fig. 5B, C). To further confirm this result, we also performed gain-of-function experiment by overexpression of TRIP6. 1' NS were dissociated, transfected with control (Ctrl) or TRIP6 together with GFP vectors, and cultured to form 2' NS. Transfected cells formed 2' NS in both the control and TRIP6 groups (Fig. 5D, E). The number of 2' NS was not increased by TRIP6 overexpression (TRIP6 = 41 ± 4.93 spheres/106 cells). This could be due to the abundance of endogenous TRIP6 in neural stem cells. Therefore, we measured the sphere diameter as another index of self-renewal. For the control of the knockdown experiment, the size of the 2' NS tended to be slightly larger than that in the control of the overexpression experiment, which could be due to different vectors transfected. Still, TRIP6 shifted the distribution toward larger spheres where TRIP6 significantly decreased the number of small-sized spheres (100–150 µm; P < 0.05) and increased the number of large-sized ones (250–300 µm; P < 0.05; Fig. 5F). Together, these results suggest that TRIP6 is necessary and sufficient for self-renewal of NSCs.
Since TRIP6 increases the neurosphere size, it is possible that it positively regulates cell proliferation of NSCs. To test this hypothesis, 1' NS were dissociated, transfected with scrambled shRNA (Ctrl) or shTRIP6, and cultured in differentiation condition for 1 day when cells were still actively dividing. BrdU, the thymidine analog, was added 2 hr before fixation. We found that BrdU-positive cells more significantly decreased in the shTRIP6 group than those in the control group (Ctrl: 100%, shTRIP6: 77.3 ± 5.8%, P < 0.05; Fig. 6A–C). We also overexpressed TRIP6 in dissociated 1' NS and cultured them in differentiation condition for 2 days when cells started to exit cell cycle and underwent differentiation. BrdU was again added 2 hr before fixation. We found that BrdU-positive cells significantly increased in the TRIP6 group (Ctrl: 100%, TRIP6: 132.7 ± 5.4%, P < 0.05; Fig. 6D–F). These results together suggest that TRIP6 positively regulates NSC/neural progenitor cell proliferation.
TRIP6 Inhibits Differentiation of Postnatal NSCs
Since TRIP6 positively regulates self-renewal of postnatal NSCs (Fig. 5), TRIP6 may also be involved in NSC differentiation. To test whether TRIP6 modulates NSC differentiation, we started with the loss-of-function approach by knockdown of TRIP6. In our culture condition, NSCs mostly differentiate into astrocytes and neurons labeled by GFAP and class III β-tubulin (Tuj1) antibodies, respectively (Fig. 7), and a few differentiate into oligodendrocytes (data not shown). Therefore, we examined neuronal and astroglial differentiation after manipulation of TRIP6 expression. 1' NS derived from P7 SVZ were dissociated, co-transfected with GFP and shTRIP6 constructs, and cultured in differentiation condition for 3 days. We found that knockdown of TRIP6 significantly enhanced neuronal differentiation of NSCs (Ctrl: 100%, shTRIP6: 208.0 ± 20.8%, P < 0.05; Fig. 7A, B, and E) at the expense of glial differentiation (Ctrl: 100%, shTRIP6: 66.5 ± 3.3%, P < 0.01; Fig. 7C, D, and E), suggesting that TRIP6 negatively regulates neuronal differentiation. To further confirm the role of TRIP6 in NSC differentiation, we transfected 1' NS with control (Ctrl) or TRIP6 constructs and cultured them in differentiation condition for 3 days. Overexpression of TRIP6 decreased neuronal differentiation (Ctrl: 100%, TRIP6: 50.8 ± 3.7%; P < 0.01; Fig. 7F, G, and J) and reduced glial differentiation (Ctrl: 100%, TRIP6: 71.2 ± 6.9%, P < 0.05; Fig. 7H–J), suggesting that TRIP6 inhibits differentiation. Taken together, our results demonstrate that TRIP6 mainly maintains NSC identity and negatively regulates NSC differentiation.
TRIP6 Activates Notch Signaling Pathway
Notch, Shh, and Wnt pathways have been reported to be involved in NSC maintenance (Ming and Song, 2011). Since we found that TRIP6 is necessary and sufficient to regulate self-renewal and negatively regulates differentiation of postnatal NSCs (Figs. 5, 7), it is plausible that these signaling pathways mediate the function of TRIP6. To examine these possibilities, we tested whether TRIP6 regulates Notch, Shh, or Wnt activity by Luciferase reporter assays in P19 mouse embryonic carcinoma cell line. P19 cells are pluripotent cells, which can be induced to differentiate into neurons by retinoic acid (RA) treatment or expression of neural basic-helix-loop-helix (bHLH) transcription factors (Jones-Villeneuve et al., 1982, 1983; Farah et al., 2000). First, we tested whether TRIP6 interacted with Notch pathway. Upon ligand binding, Notch intracellular domain (NICD) is cleaved from the cell membrane and translocates into the nucleus to increase the transcription of genes containing CBF binding sites (Hsieh et al., 1996). We transfected P19 cells with Luciferase reporter constructs containing wild-type (WT) or mutant (MT) CBF binding sites and found that TRIP6 significantly promoted the Luciferase activity with WT CBF binding sites up to 2-fold (Ctrl: 100 ± 0%, TRIP6: 192.5 ± 26.2%, P < 0.05; Fig. 8A). However, TRIP6 did not promote Luciferase activities with Gli or TCF binding sites, which are consensus binding sites for Shh and Wnt pathways, respectively (data not shown). To rule out the possibility that this is a P19 cell-specific effect, we repeated the same experiment with the SH-SY5Y neuroblastoma cell line, which can be induced to differentiate into neurons with RA (Biedler et al., 1978). Consistent with the result from P19 cells, TRIP6 also significantly increased Notch activity in SH-SY5Y cells (Ctrl: 100 ± 0%, TRIP6: 260.41 ± 32.6%, P < 0.05; Fig. 8B). Taken together, our results suggest that TRIP6 may activate Notch pathway to maintain NSC identity.
As a focal adhesion molecule, studies of TRIP6 have been focused on its role in regulating cell migration, including cancer metastasis and pathogen invasiveness in the body. Its functions in progression and survival of cancer cells have also been reported (Lai et al., 2010; Lin et al., 2013). Since a microarray analysis of different lineages of stem cells reveals that TRIP6 may serve as a stemness gene (Ramalho-Santos et al., 2002), here we investigate its role in NSCs. We found that TRIP6 was expressed by NSCs in the embryonic and adult forebrain. It was required for their self-renewal and proliferation and inhibited differentiation. In addition, TRIP6 activated the Notch signaling pathway.
Sox1, 2, and 3 belonging to SoxB1 transcription factors are expressed by NSCs (Zappone et al., 2000; Wang et al., 2006). SoxB1 transcription factors and the Notch pathway are well known for their roles in maintaining NSCs in an undifferentiated state (Bylund et al., 2003; Ross et al., 2003; Ferri et al., 2004). Moreover, the Notch pathway has been shown to activate Sox2 expression and inhibit neural differentiation (Ehm et al., 2010). It has also been reported that the Notch signaling is downregulated in RA-induced differentiation of glioblastoma stem cells, further suggesting that the Notch pathway is required to maintain stem cells in an undifferentiated state (Ying et al., 2011). Here, we found that TRIP6 was expressed by Sox-2-positive NSCs in the embryonic and adult forebrain (Figs. 2, 3). TRIP6 maintained self-renewal ability and inhibited differentiation of postnatal NSCs (Figs. 5, 7). More importantly, TRIP6 increased the Notch activity (Fig. 8). In addition, Notch signaling is reported to inhibit the neuronal fate while inducing astrocyte differentiation in embryonic and postnatal neural stem cells (Tanigaki et al., 2001; Grandbarbe et al., 2003). Moreover, the Notch pathway has been shown to maintain adherens junctions of neural stem cells (Hatakeyama et al., 2014). Loss of adherens junctions promotes detachments of neural progenitor cells and induces precocious neurogenesis (Rousso et al., 2012; Hatakeyama et al., 2014). Here, we found that knockdown of TRIP6 increased neuronal differentiation at the expense of glial differentiation (Fig. 7). This result further supports that TRIP6 may act upstream of the Notch-Sox2 signaling pathway to maintain NSC identity and regulate their differentiation.
During neurogenesis, adhesion status of NSCs regulates the balance of self-renewal and differentiation. NSCs form a self-supporting niche by adhering to the luminal surface of ventricle and neighboring progenitor cells (Meng and Takeichi, 2009; Zhang et al., 2010). Loss of adhesion status correlates with differentiation, while secured adhesion promotes the self-renewal of NSCs (Rousso et al., 2012; Malaguti et al., 2013). These reports mainly concentrate on adherens junctions and cadherin proteins. However, studies of focal adhesion molecules in neurogenesis are limited and most of them focus on cell migration. Interestingly, the dynamics of focal adhesions has been shown to correlate with differentiation of NSCs (Lyu et al., 2013) and deficiency of a focal adhesion molecule, vinculin, disrupts neural tube formation (Xu et al., 1998). Here, we provide evidence that TRIP6 is involved in regulating of NSC self-renewal and differentiation. Taken together, this finding suggests that focal adhesion molecules also play a role in neurogenesis.
Previously, we found that TRIP6 not only regulates cell motility, but also cell proliferation and survival in cancer cells (Lai et al., 2005, 2010; Lin et al., 2013). It activates ERK, Akt, and NF-κB pathways to promote cell motility, proliferation, and survival in ovarian cancer and glioblastoma cells (Lai et al., 2005, 2010; Lin et al., 2013). During neurogenesis, ERK, Akt, and NF-κB signaling pathways are also important regulators for proliferation and survival of NSCs (Widera et al., 2006; Zhou and Miller, 2006; Shioda et al., 2009). Therefore, TRIP6 may also regulate properties of NSCs through these pathways.
In the adult SVZ, TRIP6 is also expressed by ependymal cells (Fig. 3). Ependymal cells are one kind of glial cells that remain in the VZ during neural development (Spassky et al., 2005). Johansson et al. have reported that ependymal cells serve as NSCs that give rise to new neurons in the adult olfactory bulb (Johansson et al., 1999). Ependymal cells can also respond to spinal cord injury and generate migratory astrocytes to the injured sites (Johansson et al., 1999). Our results show that TRIP6 is expressed in these cells and Sox2-positive cells, suggesting that TRIP6 is majorly expressed in cells with stem cell properties and may maintain the stemness features of these cells.
Ependymoma is a glioma transformed from ependymal cells. According to the microarray data from the Neale Multi-cancer data set, TRIP6 mRNA expression is also higher in ependymoma and anaplastic ependymoma. In addition, we have studied the role of TRIP6 in glioma tumorigenesis and found that TRIP6 is highly expressed in glioblastoma compared to control tissues (Lai et al., 2010). Moreover, TRIP6 enhances the anti-apoptotic ability of glioblastoma cells and promotes tumor growth in vivo (Lai et al., 2010; Lin et al., 2013). Consistent with this, the expression level of TRIP6 is reversely correlated with the overall survival of patients; poor prognosis of patients usually has a higher level of TRIP6 (Lin et al., 2013). These studies suggest that TRIP6 may be a key factor in various gliomas. Since our results showed that mature astrocytes do not express TRIP6 (Fig. 4C) but NSCs do (Figs. 2, 3), it is possible that the ectopic expression of TRIP6 in differentiated glial cells may transform them into cancer cells and make them tumorigenic. Therefore, TRIP6 may not only serve as a biomarker, but also be a therapeutic target for malignant brain diseases.
Handling of mice was according to university guidelines and the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Normal University (Approval Number 101026). Adult and pregnant CD1 mice at gestation day 15.5 and 16.5 (E15.5, E16.5) were exposed to photoperiod (12L: 12D) in IVC system with unlimited food and water. The sample size of each group of experiments was at least three.
For the TRIP6-expressing construct, cDNA sequence of human TRIP6 was inserted into the pEGFP-C1 expressing vector with CMV promoter (Clontech, Palo Alto). Short hairpin TRIP6 RNA (shTRIP6) and scrambled shRNA were inserted in pSUPER and pLVTHM vector (Lai et al., 2005, 2007; Lin et al., 2013). Enhanced green fluorescence protein (GFP), renilla Luciferase, and the human Notch intracellular domain (NICD) were inserted into the US2 vector with human Ubiquitin C promoter. Luciferase reporter constructs CBF1-WT and CBF1-MT (4xwtCBFLuc and 4xmtCBFLuc), Gli-WT and Gli-MT (8x3'Gli-BS and 8xm3'Gli-BS), TCF/LEF binding site and mutant sites (8xTOP and 8xFOP), and the human NICD (US2-NICD), Gli2 expression construct (pcDNA3.1-HisB-Gli2), and β-catenin expression construct (CAG-EYFP-CAG-ctnnb1) were described previously (Lin et al., 2012).
Fixation and Sectioning
Brain fixation and sectioning was described previously (Wu et al., 2013). In brief, adult mice were deeply anesthetized with Avertin and perfused with saline and 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO). Brains were then postfixed with 4% PFA, cryoprotected, and frozenly cut into 40-μm coronal sections with a microtome (Leica). For embryonic brains, mother mice were anesthetized and perfused as described above. Embryonic brains were frozen and cut into 30-μm coronal sections with a cryostat (Leica, Exton, PA). Three embryonic forebrain sections (120 µm apart/section) from each animal were selected for staining. Six equivalent SVZ sections (160 µm apart/section) from each adult animal throughout the anterior to posterior part were selected after the corpus callosum and before the anterior commissure were connected from both sides of the brain.
Brain tissues of female pregnant mice and their fetuses at E16.5 were subjected to RNA extraction using Trizol® (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Two micrograms of total RNA were reverse transcribed by GoScript™ kit (Promega, Madison, WI) and then 2 µl of cDNA was used for PCR reaction by GoTaq®Green Master Mix (Promega). Mouse TRIP6 forward primer: 5′-GAAGCCCAGTGGAGGTGCTG-3′. Reverse primer: 5′-ACTCCAGAAGGTCCCTCCGG-3′.
NIH3T3 Cell Culture
NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) with 10% calf serum (Invitrogen). Two micrograms of pLVTHM-scamble shRNA or pLVTHM-shTRIP6 were transfected into 60 to 80% confluent 3T3 cells in 6-wells using Lipofectamine® 2000 (Invitrogen) according to the manufacturer's manual. Sixteen hours after transfection, cells were starved by 0.1% BSA containing medium overnight and focal adhesions were induced by 10% FBS DMEM for 15 min (Xu et al., 2004). Cells were then fixed by 3% formaldehyde (Bionovas, Bremerton, WA) for immunofluorescence.
Brain tissues of female pregnant mice and their fetuses at E15.5 were lysed with 1xSDS lysis buffer (10% SDS, 60 mM Tris-HCl pH 6.8) and sonicated for 20 sec. Sixty micrograms of lysates were then applied to SDS-PAGE analysis and TRIP6 (1:2,000, Bethyl Laboratories) and GAPDH (1:5,000, GeneTex) were detected by their specific antibodies.
Immunoblotting for 3T3 cells, 4 µg plasmids were used for transfection. Two days after transfection, cells were lysed by 1xSDS lysis buffer and 40 µg of cell lysates were then used for SDS-PAGE analysis. TRIP6 (1:2,000, Clone 16, BD Bioscience, Franklin Lakes, NJ) and β-actin (1:2,000, Sigma-Aldrich) were detected by their specific antibodies.
Immunofluorescence for 3T3 cells was described previously (Xu et al., 2004). In brief, fixed cells were permeablized with 0.2% Triton X-100 and blocked with phosphate-buffered saline containing 2% BSA. TRIP6 was detected by its specific antibody (1:250, Clone 16, BD Bioscience) and DyLight-549 goat anti-mouse antibody (1:500, Jackson ImmunoResearch, West Grove, PA). Cell nuclei were stained by DAPI (Invitrogen). Cells were photographed by the inverted fluorescence microscope (Leica DMI3000, Exton, PA).
The immunostaining procedure on brain slices was described in Wu et al. (2013). Brain sections were incubated in blocking buffer (10% goat serum in TBS buffer) for 1 hr followed by incubation with primary antibodies overnight at 4°C. After washing, sections were incubated with fluorescence-conjugated secondary antibodies for 2 hr and with DAPI (Invitrogen) for 30 min. Stained sections were then mounted with anti-fade solution (Invitrogen) and photographed by a confocal microscope (TCS SP2, Leica) in the Image Core at NTNU. Antibodies used: mouse anti-TRIP6 (1:250; BD Bioscience), rabbit anti-Sox2 (1:1,000; Millipore), rabbit anti-S100β (1:1,000; Millipore), rabbit anti-Ki67 (1:250; Leica), guinea pig anti-DCX (1:5,000; Millipore), rabbit anti-GFAP (1:350; Sigma), rabbit anti-MAP2 (1:1,000; Millipore), rabbit anti-Iba1 (1:1,000; Wako), DyLight™ 488 goat-anti-mouse, DyLight™549 goat-anti-rabbit (1:500; Thermo Scientific), and DyLight™549 donkey-anti-guinea pig (Jackson ImmunoResearch). Cells in SVZ area were counted in 2-µm confocal sections.
Differentiated NSCs were fixed in 4% PFA for 15 min. After wash and blocking, cells were incubated overnight at 4°C with the following primary antibodies: mouse anti-neuronal class III β-tubulin (Tuj1, 1:1,000, Covance, Princeton, NJ), mouse anti- GFAP (1:1,000, Millipore, Billerica, MA), and rabbit anti-GFP (1:1,000, Invitrogen). Labeling was visualized with DyLight-550 goat anti-mouse and DyLight-488 goat anti-rabbit secondary antibody (1:1,000, Abcam, Cambridge, MA). Cell nuclei were stained by DAPI (Invitrogen). Cells were photographed by an inverted fluorescence microscope in the Image Core at NTNU.
Neurosphere Culture and Electroporation
Neurosphere (NS) cultures were prepared as previously described (Wang et al., 2005) with modifications. P7 CD1 mice were sacrificed by cervical dislocation. SVZ tissue dissected from 2-mm-thick coronal brain sections was minced and dissociated with trypsin (Sigma), hyaluronidase (Sigma), and Kynurenic acid (Sigma). SVZ cells were cultured in a 24-well dish (1.5 mice per well) with Dulbecco's modified Eagle's medium (DMEM/F12) (Invitrogen) with 1% N2 (Invitrogen), 10 ng/ml bFGF (Sigma), 20 ng/ml EGF (Sigma), 2 µg/ml Heparin (Sigma), and 1 % Penicillin-Streptomycin-Gluatmax (Invitrogen) at 37°C, 5 % CO2 incubator for 5 days. Half of the medium were replaced every 2 days. For each experiment, SVZ explants from two litters of P7 mice were combined and cultured to form primary (1') NS first. These 1' NS were then dissociated and transfected with different constructs for either secondary (2') NS formation, proliferation or differentiation experiments. Electroporation was performed with 1x106 NSCs dissociated from 1' NS using Nucleofector (LONZA Amaxa) with Program A-033. In TRIP6 loss-of-function experiments, 4 µg of GFP plasmid was co-electroporated with 6 µg of shTRIP6 or scrambled shRNA plasmid. In TRIP6 gain-of-function experiments, 4 µg of GFP plasmid was co-electroporated with 6 µg of pEGFP-TRIP6 or pEGFP-C1 control plasmid. Post-electroporated NSCs were cultured at 2.5x105 cells per 6-well to form 2' NS.
Transfection and Differentiation of NSCs
1x105 NSCs dissociated from 1' NS were plated in 24-well dishes and cultured in DMEM/F12 with 1% N2 and 1% FBS without antibiotics for 1–2 hr before transfection. In TRIP6 loss-of-function experiments, cells were co-transfected with 0.25 µg of US2-GFP and 0.35 µg of shTRIP6 or scrambled shRNA-harboring plasmid. In TRIP6 gain-of-function experiments, cells were co-transfected with 0.25 µg of US2-GFP and 0.35 µg of GFP-TRIP6 or GFP-expressing vector. Lipofectamine® 2000 (Invitrogen) was used for transfection according to the manufacturer's instruction. The medium was replaced by DMEM/F12 with 1% N2, 1% FBS, and 1% antibiotics 6 hr after transfection. For neuronal differentiation, NSCs were cultured for 3 days on cover slips coated with poly-L-lysine (Sigma) and laminin (Invitrogen).
P19 cells were maintained in MEMα medium with 7.5% calf serum, 2.5% FBS, and 1% antibiotics (Lai et al., 2007). For transfection, cells were plated in 12-well dishes at 80–90% confluence in MEMα with 10% serum without antibiotics. Lipofectamine® 2000 (Invitrogen) was used for transfection according to the manufacturer's instruction. P19 cells were co-transfected with 0.05 µg of US2-renilla Luciferase, 0.5 µg of pEGFP-C1, pGFP-TRIP6, US2-NICD, Gli2, or ctnnb1, and 0.7 µg of Firefly Luciferase reporter constructs with either wild type (WT) or mutated (MT) transcription factor binding sites. Six hours after transfection, medium was replaced with Opti-MEM with 1% FBS and antibiotics. SH-SY5Y cells were maintained in DMEM/F12 medium with 10% FBS, and 1% antibiotics. For transfection, 5x105 SH-SY5Y cells were plated in 12-well dishes and cultured in DMEM/F12 with 10% FBS without antibiotics for one hour before transfection. The transfection protocol was the same as P19 cells. Six hours after transfection, medium was replaced with DMEM/F12 with 1% FBS and antibiotics. Reporter activity was measured 24 hr after transfection using Dual-Luciferase Assay System (Promega). For normalization, firefly Luciferase activity was first normalized to renilla Luciferase activity of each group. The obtained value from the wild-type binding sites (WT CBF1) was then normalized to that of the MT CBF1. Finally, the normalized value from the control vector group was set as 100% and the value from the TRIP6 group was shown as the percentage of the control group. Samples of each experiment were plated in duplicates and each experiment was repeated for three times (n=3).
Two-group comparisons were analyzed by two-tailed Student's t-test. Difference of two distributions was analyzed by Wilcoxons rank-sum test. All data were presented as mean ± standard error of the mean (SEM). Significant level is P < 0.05.
We thank Dr. Fang-Tsyr Lin for providing the GFP-TRIP6, pSUPER-scramble, pSUPER-shTRIP6, pLVTHM-scramble, and pLVTHM-shTRIP6 plasmids, and Dr. Jenn-Yah Yu and Dr. Hsiu-Mei Hsieh for providing experimental materials. We also thank Dr Li-Yih Lin, Dr. Chung-Hsin Wu, and Dr. Ja-An A. Ho for supporting the confocal microscope usage. Images in this article were generated in the Molecular Image Core of National Taiwan Normal University under the auspices of the National Science Council. The authors declare that they have no conflict of interest.
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