Interaction between Oc-1 and Lmx1a promotes ventral midbrain dopamine neural stem cells differentiation into dopamine neurons

brain research 1608 (2015) 40–50

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Research Report

Interaction between Oc-1 and Lmx1a promotes ventral midbrain dopamine neural stem cells differentiation into dopamine neurons Jian Yuana,n,1, Zhi-nian Leib,1, Xi Wangc, Yong-Jian Dengd,n, Dong-Bo Chena a

Department of Pathology and Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, 510006 Guangzhou, China b Department of Human Anatomy and Neuroscience, Medical School of Southeast University, Dingjiaqiao 87, Nanjing, Jiangsu 210009, China c Department of Pathology and Laboratory Medicine, University of Rochester Medical School, 601 Elmwood Ave, Box 626, Rochester, NY 14642, USA d Department of Pathology, School of Basic Medical Science, Southern Medical University, 510515 Guangzhou, China

ar t ic l e in f o

abs tra ct

Article history:

Recent studies have shown that Onecut (Oc) transcription factors may be involved in the

Accepted 24 February 2015

early development of midbrain dopaminergic neurons (mdDA). The expression profile of

Available online 5 March 2015

Oc factors matches that of Lmx1a, an important intrinsic transcription factor in the

Keywords:

development of mDA neuron. Moreover, the Wnt1-Lmx1a pathway controls the mdDA

Oc-1

differentiation. However, their expression dynamics and molecular mechanisms remain to

Lmx1a

be determined. To address these issues, we hypothesize that cross-talk between Oc-1 and

Wnt1

Lmx1a regulates the mdDA specification and differentiation through the canonical Wnt-β-

mdDA

catenin pathway. We found that Oc-1 and Lmx1a displayed a very similar expression

Parkinson's disease

profile from embryonic to adult ventral midbrain (VM) tissues. Oc-1 regulated the proliferation and differentiation of ventral midbrain neural stem cells (vmNSCs). Downregulation of Oc-1 decreased both transcript and protein level of Lmx1a. Oc-1 interacted with lmx1a in vmNSCs in vitro and in VM tissues in vivo. Knockdown of Lmx1a reduced the expression of Oc-1 and Wnt1 in vmNSCs. Inhibiting Wnt1 signaling in vmNSCs provoked similar responses. Our data suggested that Oc-1 interacts with Lmx1a to promote vmNSCs differentiation into dopamine neuron through Wnt1-Lmx1a pathway. & 2015 Elsevier B.V. All rights reserved.

n

Corresponding authors. Fax: þ86 20 39352186. E-mail address: [email protected] (J. Yuan). 1 Jian Yuan and Zhi-nian Lei equally contributed to this work.

http://dx.doi.org/10.1016/j.brainres.2015.02.046 0006-8993/& 2015 Elsevier B.V. All rights reserved.

brain research 1608 (2015) 40–50

1.

Introduction

Ventral midbrain dopamine neural stem cells (vmNSCs) have a potential to be used as a cell source to generate dopamine (DA) neurons for cell replacement therapy in Parkinson's disease (PD). An intensive research in recent years has focused on identifying the molecular mechanisms that regulate midbrain dopaminergic neurons (mdDA) development. The development of mdDA is a complex, multi-step process. A number of molecular pathways have been shown to play key roles in this process (Abeliovich and Hammond, 2007; Flames and Hobert, 2011; Hegarty et al., 2013; Kiecker and Lumsden, 2012). Recent studies showed that the floor plate cells in the murine ventral midbrain (VM) become neurogenic and subsequently give rise to DA neurons(Ono et al., 2007) and vmNSCs in the floor plate demonstrated radial glial characteristics (Bonilla et al., 2008; Hebsgaard et al., 2009). The differentiation of vmNSCs into DA neurons is a tightly controlled and highly dynamic process requiring concerted action of both extrinsic and intrinsic signals such as sonic hedgehog (Shh), fibroblast growth factor 8 (FGF8) and Wnt1, followed by activation of a series of transcription factors including Lmx1a/b, Foxa1/2, En1/2, Nurr1 and Pitx3 during different stages of development (Chakrabarty et al., 2012; Joksimovic and Awatramani, 2014). More interestingly, other transcription factors, including Onecut (Oc) factors, are also involved in the generation of the VM DA neuronal field. The Oc transcription factors are conserved in animals and have been found in Caenorhabditis elegans, Drosophila, sea urchin, frog, and mammals (Roy et al., 2012). There are three members of Oc in mammals: Oc-1 (or HNF-6), Oc-2 and Oc-3 (Audouard et al., 2013). All of them are characterized by a bipartite DNA-binding domain constituted by a single Cut domain and a divergent homeodomain (Wu et al., 2012). Oc1–3 is detected during the development in several endodermal derivatives including liver and pancreas, where they control different aspects of cell fate decision and morphogenetic processes. Furthermore, they have been detected both in the peripheral nervous system (PNS) and in the central nervous system (CNS). In the PNS, Oc1–2 is present in the trigeminal ganglion wherein Oc-2 contributes to proper central projection of the sensory neurons (Hodge et al., 2007). In the CNS, Oc-1 and Oc-2 regulate the generation, maintenance or the projections of different encephalic structures and spinal interneuron (Espana and Clotman, 2012a, 2012b). Recent research further showed that Oc-1 signaling, which is most prominent at E12.5, regulates the early phases of mdDA development, while Oc-3 might be mainly involved in late differentiation events of mdDA neurons (Chakrabarty et al., 2012). Oc-1 and Oc-2 are also expressed in the retina. However, how they integrate into the transcriptional circuits underlying the development of mdDA is still unknown (Wu et al., 2012). Lmx1a belongs to the Lmx group of LIM homeodomain transcription factors. The mesencephalic floor plate cells express Lmx1a (Hebsgaard et al., 2009). Several loss- and gain-of-function studies, mainly in chick and embryonic stem cells (ESCs) have demonstrated that Lmx1a plays an essential role in specifying the mdDA neuronal phenotype and, more specifically, in regulating homeodomain factor Msx1, which

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then either induces neurogenesis by activating the proneural gene Ngn2, or suppresses alternative cell fates by suppressing the Nkx6.1 gene (Yan et al., 2011). Furthermore, Lmx1a overexpression directly binds and promotes the expression of Nurr1 and Pitx3 (Friling et al., 2009). Moreover, Lmx1a can regulate the dopamine transporter (DAT) expression during ESCs differentiation by directly binding to conserved element of DAT's promoter region. The forced expression of Lmx1a enhanced the production of mdDA neurons in embryonic stem cells (Friling et al., 2009). In addition, analysis of the dreher mouse, which contains a mutation in the Lmx1a locus, showed that the loss of Lmx1a leads to a significant decrease in DAT expression. These studies suggest that Lmx1a is an early mdDA differentiation marker. It induces a regulatory cascade involving additional downstream transcription factors, which promote the proliferation, differentiation and maturation of developing mdDA neurons (Hoekstra et al., 2013). However, its precise role is still not clarified. Previously published studies have shown that Oc1–3 displays similar expression profiles to that of Lmx1a in the developing VM, and their loss resulted in the diminished generation of VM DA neurons (Chakrabarty et al., 2012). However, it is not clear if Oc1–3 is regulated by Lmx1a, or if they act in parallel to regulate neuronal differentiation in the VM. In the present study, our results suggest that Oc-1 plays important roles in vmNSC proliferation and differentiation by interacting with Lmx1a.

2.

Results

2.1. Oc-1 directly stimulates the expression of Lmx1a and promotes the proliferation of vmNSCs Oc-1 regulates the expression of Lmx1a in the proliferation and differentiation of vmNSCs and involves in the early phases of development in ventral/lateral domain mdDA neuron. Lmx1a plays an important role in the development of mesodiencephalic dopaminergic neurons by regulating proliferation, cell cycle exit, and differentiation of mDA progenitors. Because the mesencephalic floor plate (FP) gives rise to mDA neurons, we hypothesized that the interaction between the transcription factor Oc-1 and Lmx1a promotes the generation and differentiation of mDA neurons in the FP. To address this idea, we first probed their endogenous expression in both embryonic and adult VM. Both were expressed from Embryonic Day 10 (E10) to adult. Moreover, their expression pattern paralleled with each other (Fig. 1A–C). The highest expression time window was at E12 and E14. Furthermore, Oc-1 colocalized with Lmx1a in E14 rat substantia nigra (Fig. 1J). Then, to analyze the possible effect of Oc-1 on Lmx1a, we aimed to over-express Oc-1 protein in vmNSCs in an in vitro culture model system, which is amenable to various manipulations, not easily feasible in the in vivo system. Oc-1 protein levels in cells transfected with an Oc-1 overexpression construct, or with control construct, were compared on Western blot, revealing a clear overexpression of Oc-1 protein in the experiment group (Fig. 1D and E). Oc-1 overexpression significantly increased the expression of Lmx1a in vmNSCs at DIV7 (Fig. 1D and F). Subsequently, vmNSCs were allowed to proliferate. At day 7 of proliferation, the cells

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Fig. 1 – Oc-1 overexpression promoted the proliferation of vmNSCs. (A–C). Western blot showed that both Oc-1 and Lmx1a were expressed in embryonic and adult Ventral midbrain tissue. The highest expression time window was at E12 and E14 respectively. (D–F) showed that Oc-1 overexpresion enhanced the expression of Lmx1a in vmNSCs at 7 days in vitro (DIV). (G) Phase-contrast photomicrographs of representative spheres of vmNSCs induced by Oc-1 overexpression at DIV7. (H and I) Oc-1 overexpression increased cell sphere number and diameter. (K and J) Oc-1 overexpression increased the number of DATTH double-labeled positive cells. (L) A representative coronal section of the ventral midbrain substantia nigra from E14 rat embryos stained to detect Oc-1þ and Lmx1aþ cells. Data are reported as mean7SEM (n¼ 5 experiments; 15–20 microscopic fields were randomly selected for counting in each experiment). npo0.05; nnpo0.01 versus control group by unpaired 2-tailed Student t-test. Scale bars¼(G) 30 μm; (J) 100 μm; (L) 20 μm.

exhibited neurosphere morphology which is in agreement with our previous results (Lei et al., 2011). Oc-1 overexpression increased not only the neurosphere number (Fig. 1G and H), neurosphere diameter (Fig. 1G and I), but also the THþ–DATþ cell number in cultured vmNSCs (Fig. 1J and K).

2.2.

Oc-1 induces key gene underlying generation of mdDA

To determine whether the key genes involved in the neurogenesis of mdDA neurons play a role in the differentiation of vmNSCs. We investigated the gene expression profile of the cultured vmNSCs treated with Oc-1 overexpression construct during a 3 weeks proliferation and differentiation period by

qRT-PCR (Fig. 2A–G). Oc-1 transcripts level was increased since the initiation of proliferation and sustained the high level throughout the assayed time period. Similarly to the Oc-1, the level of Lmx1a was increased with time, indicating that Lmx1a transcripts were accumulated during the in vitro proliferation and differentiation. Lmx1a acts as a determinant of mdDA neurons and an effector of Wnt1. Wnt1 patterns both the dorsal and ventral mesencephalon. Wnt1 regulates Lmx1a expression through the canonical β-catenin pathway. Conversely, Lmx1a binds directly to the promoter of Wnt1 during mdDA neuron differentiation (Chung et al., 2009). Our data showed that Wnt1 was expressed at all time points. Its level peaked at the 1st week. Nurr1 expression

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Fig. 2 – Effects of Oc-1 overexpression on generation of mdDA neurons in vmNSCs. qRT-PCR and Western blotting were used to follow the expression level of selected genes and their corresponding protein prior to virus infection (0) and during the next 3 weeks. (A–G) qRT-PCR was used to assay the transcript level of selected genes in vmNSCs induced by Oc-1 overexpression. Lmx1a, Wnt1, Nurr1, Pitx3, and TH were elevated at all time points by Oc-1 overexpression, while actin levels remained unchanged. The experiment was repeated at least three times, normalized against to GAPDH. (H) vmNSCs treated with Oc-1 overexpression and whole-cell lysates were prepared and subjected to immunoblotting with anti-Oc-1, anti-Lmx1a, anti-Wnt1, anti-Nurr1, anti-Pitx3, anti-TH, or anti-β-actin antibodies. (I–N) The corresponding bar graph represented the Oc-1, Lmx1a, Wnt1, Nurr1, Pitx3, and TH protein levels in 3 or 4 independent experiments, normalized against β-actin. Data are reported as mean7SEM. npo0.05; nnpo0.01, one-way analysis of variance with Tukey post hoc test followed by unpaired 2-tailed Student t-test.

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in vivo indicates the beginning of mdDA postmitotic differentiation. In our model, Nurr1 levels peaked at the 1st week then displayed a dynamics similar to that of Wnt1. Pitx3 was suggested to define specification and differentiation of mdDA progenitors. Pitx3 transcript level was increased by Oc-1 overexpression as the proliferation and differentiation progressed. The quantity of TH transcripts was gradually increased from the 1st week to the 3rd week, suggesting that the cells matured with time. However, the level of actin control remained stable at all time points. Moreover, our western blot showed that their protein levels displayed a similar trend (Fig. 2H–N). Overall, all markers tested at mRNA

and protein levels have indicated that vmNSCs treated with Oc-1 overexpression have acquired and maintained a DA fate.

2.3. Oc-1 functions cooperatively with Lmx1a to promote the proliferation and differentiation of vmNSCs To test the effect of Oc-1 on the endogenous levels of Lmx1a, we attenuated the expression of Oc-1 in vmNSCs by a short hairpin RNA (shRNA) containing lentiviral vector. Oc-1 shRNA significantly decreased the expression of Oc-1 (Fig. 3A and B). Knockdown of Oc-1 gene reduced the protein level of Lmx1a by 85%, as compared with the group treated with scramble

Fig. 3 – Oc-1-knockdown reduced the transcript level of Lmx1a in vmNSCs. (A and B) vmNSCs were stably transfected with Oc-1 shRNA. (C) Oc-1-knockdown decreased the protein level of Lmx1a in vmNSCs. (D) qRT-PCR analysis showed that Oc-1 knockdown led to significant downregulation of Lmx1a (more than 90%) and its targets Nurr1, Pitx3, and TH. Importantly, the level of Wnt1 was also decreased by Oc-1-knockdown. This assay was repeated three times (n¼ 9). The transcript level of each gene represented the ratio between its expression in the Oc-1 knockdown against the expression in the scramble shRNA treated cells. (E–H) Downregulation of Oc-1 decreased the number of Nurr1-TH and Pitx3-TH double positive cells in cultured vmNSCs (DIV14, n ¼ 5 experiments; 15–20 microscopic fields were randomly selected for counting in each experiment). Data are reported as mean7SEM. npo0.05; nnpo0.01, one-way analysis of variance with Tukey post hoc test followed by unpaired 2-tailed Student t-test.

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shRNA (Fig. 3A and C). Furthermore, we found that the mRNA level of Oc-1 was reduced by 92% in the Oc-1-knockdown vmNSCs (Fig. 3D). Indeed, the transcript levels of Lmx1a, Wnt1, Nurr1, Pitx3, and TH were reduced upon Oc-1 knockdown by 90, 87, 56, 50 and 26%, respectively. Oc-1 knockdown also reduced the Nurr1þ–THþ and Pitx3þ–THþ cell number (Fig. 3E–H). Taken together, during the vmNSCs proliferation and differentiation, Oc-1 may regulate the development of mdDA neurons through activation of Lmx1a. We then further

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investigated the relationship between Oc-1 and Lmx1a, regarding how Lmx1a knockdown will affect the expression of Oc-1 and the key genes involved in the neurogenesis of mdDA neurons. vmNSCs were stably transfected with Lmx1a shRNA. Lmx1a shRNA significantly decreased the expression of Lmx1a during the proliferation and differentiation of vmNSCs (Fig. 4A and B). Lmx1a knockdown significantly reduced the expression of Oc-1, Wnt1 and its downstream targets Nurr1, Pitx3, and TH (Fig. 4C). Furthermore, their

Fig. 4 – Lmx1a regulated the expression of Oc-1 in the proliferation and differentiation of vmNSCs. (A and B) vmNSCs were stably transfected with Lmx1a shRNA, Lmx1a shRNA decreased the protein level of Lmx1a in vmNSCs. (C) qRT-PCR assay indicated that Lmx1a-knockdown induced significant downregulation of Oc-1, Wnt1, Nurr1, Pitx3, and TH. This assay was repeated three times (n ¼9). The transcript level of each gene represented the ratio between its expression in the Lmx1a knockdown against the expression in the scramble shRNA treated cells. (D) vmNSCs treated with Lmx1a and whole-cell lysates were prepared and subjected to immunoblotting with anti-Oc-1, anti-Wnt1, anti-Nurr1, anti-Pitx3, anti-TH, or anti-βactin antibodies. (E–I) The corresponding bar graph represented the Oc-1, Wnt1, Nurr1, Pitx3, and TH protein levels in 3 or 4 independent experiments, normalized against β-actin. (J–L) Downregulation of Lmx1a decreased the number of Nurr1-TH and Pitx3-TH double positive cells in cultured vmNSCs (DIV14, n ¼5 experiments; 15–20 microscopic fields were randomly selected for counting in each experiment). Data are reported as mean7SEM. npo0.05; nnpo0.01, one-way analysis of variance with Tukey post hoc test followed by unpaired 2-tailed Student t-test. Scale bars¼ (J) 100 μm.

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protein levels were down-regulated by Lmx1a knockdown (Fig. 4D–I). More importantly, Lmx1a knockdown decreased the Nurr1þ–THþ and Pitx3þ–THþ cell number (Fig. 4J–L). We then examined if Lmx1a could bind the Oc-1 promoter in the vmNSCs in vivo by ChIP. Chromatin was prepared from differentiated vmNSCs, when both lmx1a and Oc-1 are expressed, and the chromatin within the Oc-1 promoter would be accessible for Lmx1a binding. Our results suggested that the promoter region of Oc-1 was enriched by anti-Lmx1a antibody immunoprecipitation (Fig. 5A). Moreover, compared with the control cells, the level of ChIPed Oc-1 and TH promoter sequences were low in Lmx1a- knockdown cells. Furthermore, our co-immunopreciptation experiments from in vivo VM tissues clearly showed that there was a direct interaction between Oc-1 and Lmx1a (Fig. 5B).

2.4. Inhibition of Wnt/β-catenin pathway downregulates Oc-1 and Lmx1a transcription Our data showed that Oc-1 was a direct target gene of Lmx1a. Therefore, we hypothesized that Oc-1 is a possible downstream effector of the Wnt1 signaling pathway. Wnt1-Lmx1a pathway directly regulates Nurr1 and Pitx3 to control the development of mdDA. Therefore, we used Dickkopf-1 (DKK-1) to antagonize the Wnt1 pathway to observe its effect on Oc-1 transcript levels. Dkk-1 inhibits Wnt1 signaling by preventing Fz-LRP6 complex formation induced by Wnt1 (L'Episcopo et al., 2011). vmNSCs were treated with Dkk-1 (100 ng/ml) during 1st week of differentiation. Consequently, the transcript levels of Oc-1 (Fig. 6A), Lmx1a (Fig. 6B), and Wnt1 signaling component β-catenin (Fig. 6C) were significantly decreased, while the levels of actin control remained the same, measured by qRT-PCR (Fig. 6D). Furthermore, DKK-1 significantly reduced the protein expression level of Lmx1a, Oc-1, and β-catenin (Fig. 6E–H).

3.

Discussion

Although enormous effort has been made to identify the molecular mechanisms that are crucial for mdDA development,

our understanding of transcriptional programs leading to mdDA neuron genesis is still in its infancy. Here, using various molecular tools we have demonstrated that Oc-1 is a novel target of Wnt1 signaling, which controls the mDA proliferation and differentiation. Not only does Oc-1 act directly as downstream molecule of Lmx1a, it also feeds back and modifies the expression level of Lmx1a, Wnt1 and Nurr1. Moreover, our results showed how combinatorial action of Oc-1 and Lmx1a might induce dopaminergic neuron specification (Fig. 5). Taken together, Oc-1 is a novel member of the Wnt1-Lmxla transcriptional network underlying the development of mdDA neurons. Oc family factors regulate neuronal identity, migration, maintenance and projections in different regions of the CNS (Audouard et al., 2013). Oc-1 is present in different structures of the CNS including the trigeminal ganglion (Hodge et al., 2007), the dopaminergic A13 nucleus (Espana and Clotman, 2012b), the Locus Coeruleus and the midbrain trigeminal nucleus (MTN) (Espana and Clotman, 2012a), and numerous spinal populations (Francius and Clotman, 2010). Oc-1 is necessary for the maintenance of A13 dopaminergic neurons and for the differentiation of MTN cells that in turn may be required for maintenance of the Locus Coeruleus (Espana and Clotman, 2012a, 2012b). Recent studies showed that Oc-1 influenced the formation of the mdDA neurons in the brain (Chakrabarty et al., 2012). In our current study, we firstly analyzed the expression of Oc-1 and Lmx1a at the time points of embryo, early postnatal and adulthood and found both Oc1 and Lmx1a were robustly expressed in developing mDA neurons, indicating that Oc-1 and Lmx1a are expressed in embryonic, immature and mature neurons during embryogenesis. Moreover, Oc-1 colocalized with Lmx1a in E14 rat substantia nigra region, the overexpression of Oc-1 promoted the expression of Lmx1a in vmNSCs, increased both the diameter and number of neuroshperes, and increased the TH-DAT double labeled positive cell number (Fig. 1). Lmx1a plays a role in the neurogenesis, proliferation, and part of the differentiation of developing mdDA neurons (Hoekstra et al., 2013). Thus, it is plausible to expect the Oc-1 regulating signaling pathways to be involved in mdDA neuron development and differentiation. Together with previous reports

Fig. 5 – Lmx1a combined with the Oc-1 promoter in vivo and interacted with Oc-1. (A) ChIP assay was performed on control and Lmx1a-knockdown vmNSCs. The genomic locus of TH, serving as positive control, were increased following immunoprecipitation with anti-Lmx1a antibody in conrol but not in lmx1a-knockdown group. The α-actin locus, used as a negative control, was not increased at any condition tested. (B) Whole-cell lysates of VM tissues were subjected to immunoprecipitation using a Lmx1a- and Oc-1-specific antibody and IgG as control and were subjected to SDS-PAGE followed by western blot analysis using the indicated antibodies. Data are presented as the mean7SEM of three independent experiments. n po0.05; nnpo0.01, one-way analysis of variance with Tukey post hoc test followed by unpaired 2-tailed Student t-test.

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Fig. 6 – Inhibition of Wnt1 signaling reduced the transcript and protein expression level of Oc-1. vmNSCs were treated with Dkk-1 (100 ng/ml), a Wnt1 signaling pathway inhibitor at DIV7. (A–D) qRT-PCR analysis showed that inhibition of Wnt1 pathway resulted in a decrease in the transcript level of Oc-1 (B) and Wnt1 signaling member β-catenin (C) and Lmx1a (A). DKK-1 decreased the protein expression level of Lmx1a, Oc-1, and β-catenin (E–H). Data are shown from 4 independent experiments, normalized to GAPDH or β-actin. npo0.05; nnpo0.01, one-way analysis of variance with Tukey post hoc test followed by unpaired 2-tailed Student t-test.

showing that the Oc-1 expression profile matched that of Lmx1a, our data demonstrated that Oc-1 was the direct transcription activator in the proliferation and differentiation of vmNSCs. Furthermore, Oc-1 may promote vmNSC1 differentiation into mdDA neurons by regulating the expression of Lmx1a (Fig. 2). Subsequently, we investigated if the inhibition of endogenous transcription factors (downregulation of Oc-1) could inactivate the role of Lmx1a in mdDA neurons development. Our data showed that Oc-1 knockdown not only reduced the expression of Lmx1a and impaired the generation of DA neurons, but also decreased the mRNA levels of Wnt1, Nurr1,

Pitx3, and TH in vmNSCs cultures (Fig. 3), which is fully in agreement with the idea that Oc-1 promotes the transition of mdDA stem cells into mature DA neurons. Together, our results suggested that Oc-1 played an important role in the differentiation of vmNSCs through Lmx1a and was a key modulator in the development of mdDA neurons. Lmx1a acts as an early intrinsic determinant of mdDA neurons (Andersson et al., 2006). We examined the effect of Lmx1a on the expression of Oc-1. Our results showed that both protein and mRNA levels of Oc-1 were reduced by Lmx1a knockdown. Lmx1a knockdown also reduced the transcript level of Wnt1 and the number of double positive Nurr1-TH

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and Pitx3-TH cells. Our findings indicate that Lmx1a may directly activate Oc-1 transcription as a positive feedback loop and therefore inhibit the differentiation of VmNSCs (Fig. 4). Our ChIP and IP results further showed that Oc-1 directly interacts with Lmx1a to regulate the transcription of Nurr1 and Pitx3 (Fig. 5). Previous studies have shown that the Wnt1-Lmx1a pathway works in a cooperative (Chung et al., 2009) but antagonistic (Joksimovic et al., 2009) fashion with Shh-Foxa2 to promote mDA differentiation. Wnt1 signaling patterned mdDA stem cells and promoted Lmx1a expression through the canonical β-catenin pathway. Conversely, Lmx1a binds directly to the promoter of Wnt1 during mDA neuron differentiation. Furthermore, Wnt1 is expressed more widely than and prior to that of Lmx1a in the ventral midbrain (Brown et al., 2011). The cell cycle progression and neurogenesis of mdDA neurons are largely mediated by Wnt1 via canonical signaling. We investigated the relationship between Oc-1 and Wnt1-Lmx1a pathway. Our data suggested that Oc-1 could in turn regulate Wnt1, directly or indirectly via Lmx1a, thereby forming a feedback loop (Fig. 6). Up to now, little is known on the direct upstream regulators of Nurr1 expression in the ventral midbrain (Prakash and Wurst, 2006). Both Lmx1a and Nurr1 genes are required for TH expression. Furthermore, Nurr1 and Pitx3 activated in vivo a series of genes essential for mdDA development, such as TH, AADC, VMAT2, and DAT (Burbach and Smidt, 2006). Pitx3 acts as a potentiator of Nurr1 in the terminal differentiation of mdDA neuron. Our data suggested that the crosstalk between Oc-1 and Lmx1a affected the expression of Nurr1 and Pitx3 in the differentiation of vmNSCs. Moreover, TH was also affected by manipulating the level of Oc-1. Although Oc-1 may pattern the subtype identity of mdDA Neuron, our results did not directly address whether Oc-1 is a direct regulator of Nurr1 and Pitx3 in adult substantia nigra. Further studies will be needed in this regard. Our findings indicate that Oc-1 can interact with Lmx1a to promote vmNSCs differentiation into DA neuron, which in the long run will be promising for the cell replacement therapy for Parkinson's disease.

4.

Experimental procedures

4.1.

Animals and tissue collection

Female pregnant C57 mice were housed in cage with ad libitum access to food and water in a room 25 1C under 12 h light/dark conditions. Animal care and handling were carried out according to the guidelines of the Animal Care and Use Committee at Guangdong Pharmaceutical University. All efforts were made to minimize animal suffering and reduce the number of animals used. Ventral mesencephalon was obtained from fetuses of the same inbred strain.

4.2.

Preparation of vmNSCs Cultures

C57 mice ED10 VM neural stem cells were prepared as previously described (Cajanek et al., 2013; Lei et al., 2011). In brief, VM tissue was isolated and mechanically triturated

through a Nitex filter to a quasi-single-cell suspension. Viable cells were counted using the Trypan Blue exclusion method. Cell suspensions were plated in 24-well tissue culture plates at a density of 1.25  105 cells/cm2 in 0.4 ml of expansion medium. The expansion medium consisted of Dulbecco Modified Eagle Medium/F12 (GIBCO, Beijing, China) containing 1% N2 (Invitrogen, Beijing, China), 2% B27 (Invitrogen), 20 ng/mL epidermal growth factor (R&D Systems, Shanghai, China), and 20 ng/mL basic fibroblast growth factor (R&D Systems). Cells were cultured in an incubator with 372% CO2 at 37 1C. These culturing methods resulted in the formation of mNSC neurospheres. After 3 days, the cultures were supplemented with fresh medium. At day 7, the cell spheres were passaged using 700 μg/mL collagenase/dispase (Roche Applied Science, Indianapolis, IN) with agitation on an orbital mixer incubator (80 rpm, 20 min). Cells were again plated at a density of 1.25  105 cells/cm2. These cultures were referred to as passage 1. For the differentiation assay, the second passage of vmNSCs spheres were collected, resuspended in differentiation medium, and added 0.5% (vol/vol) fetal bovine serum(GIBCO), then plated onto poly-D-lysine and laminincoated plates (10 μg/mL each), and left to differentiate for 21 days. All differentiation experiments were performed in quadruplicate. For passage 2 cultures, passage 1 spheres were split again after an additional 7 days and treated as described previously mentioned.

4.3.

RT-PCR and quantitative PCR assay

Total RNA was extracted and quantified as previously described (Bani-Yaghoub et al., 2006). In brief, RNA was extracted from tissues (n¼ 3 per group) using Tri-reagent (Invitrogen) treated with the RNase-free DNase and used for complementary DNA (cDNA) synthesis. Oc-1, Lmx1a, Wnt1, β-catenin, Pitx3, Nurr1, TH, Actin and glyceraldehyde-3phosphate dehydrogenase (GAPDH; control) cDNA fragments were amplified, using the following primers: Oc-1 (forward 5'TTCCAGCGCATGTCGGCGCTC-3', reverse 5'-GGTACTAGTCCGTGGTTCTTC-3'), Lmx1a (forward, 5'-CACGGGAAGCTAGACTCAAC-3', reverse 5-CCCTTCACACAGTATGGTTG-3'), Wnt1 (forward 5'-ACAGCAACCACAGTCGTCAG-3', reverse 5'-TTCGTGGAGGAGGCTATGTT-3'), Pitx3 (forward 5'-CCCGTTCGCCTTCAACTCG-3', reverse 5'-CGAGGCGTAAGGGCAGGACAC-3'), Nurr1 (forward 5'-CTGTCGGTTTCAGAAGTGC-3', reverse 5'-TGGACCTGTATGCTAAGCGTA-3'), TH (forward 5'TGTCACGTCCCCAAGGTTCAT-3', reverse 5'-GGGCAGGCCGGGTCTCTAAGT-3'), Actin (Forward 5'-TGTTACCAACTGGGACGACA-3', reverse 5'-TGAGGTAGTCCGTCAGGTCC-3'), and GAPDH (forward 5'- ACCACAGTCCATGCCATCAC-3', reverse 5'-TCCACCACCCTGTTGCTGTA-3'). qRT-PCR was performed with SYBR Green (Applied Biosystems) in MicroAmp 96-well reaction plates. For each primer set, we used a reaction mix of nuclease-free water (7.5 ml), Primer 1 (2.0 ml), Primer 2 (2.0 ml), SYBR Green (12.5 ml). Note that all primers were tested to ensure that they yielded a single amplicon of appropriate size (typically  200 nucleotides). All samples were processed in triplicate. Data were normalized to GAPDH and the relative quantification of gene expression was analyzed using the 2  ΔΔ G method (Livak and Schmittgen, 2001).

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4.4. Construction of the Oc-1-expressing vector and production of lentiviral shRNA particles Oc-1 cDNA was subcloned into the pLenti/CMV/Oc-1 using ViraPower™ Promoterless Lentiviral Gateways Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. pLenti/ CMV/AcGFP acted as control. Expression clones were sequenced to confirm both the CMV promoter and the expression gene presence (Oc-1 or AcGFP1). Then, Virus was produced using ViraPowerLentiviral Expression System (Invitrogen) according to the manufacturer's instructions. Viral titers (transducing units/ mL) were determined by transduction of HeLa cells with serial dilutions of the viral supernatant and colony counting after blasticidin selection (4ng/mL, Invitrogen) with Crystal Violet staining (Sigma, St. Louis, MO, USA). Viral titers ranged from 1  106 to 1  107 transducing units/mL. Lentiviral vectors encoding either scramble shRNA or Lmx1a shRNA (sc-72344-V) and Oc1 shRNA (sc-37937-V) were obtained from Santa Cruz Biotechnology. The viral particles, vesicular stomatitis virus glycoprotein (VSVG) pseudotyped, were produced using the four-plasmid system as previously described (Cajanek et al., 2013). Fortyeight hours later, after transfection of HEK293T cells, the supernatant was collected and filtered. High-titer stocks were obtained by ultracentrifugation (50,000g for 3 h), and the pellets were resuspended in PBS with 1% BSA and stored at  80 1C until further use. The titers used for primary cell transduction were in the range of 1  107–1.5  107 transducing units per ml, and the multiplicity of infection was 1–2. Both Oc-1-expressing vector and lentiviral vectors were added to vmNSCs culture medium at DIV1 for 24 h. The transduction media was then fully replaced with fresh expansion medium.

4.5. Chromatin immunoprecipitation, Immunoprecipitation (IP) and western blotting Chromatin immunoprecipitation was performed on vmNSCs (DIV7) according to the procedure used previously (Stott et al., 2013). The immunoprecipitated DNA was analyzed by quantitative PCR. For IP, VM from E10 embryos were homogenized in lysis buffer and subjected to IP and western blot analysis as previously described (Veenvliet et al., 2013). Blots were incubated with SuperSignal and exposed to ECL films (Pierce). Antibodies used were: goat anti-Oc-1 (Santa Cruz Biotechnology); rabbit anti-Lmx1a (Genway Biotech, San Diego, CA, USA); rabbit antiWnt1 (Abcam); rabbit anti-Nurr1 (Santa Cruz Biotechnology); rabbit anti-Pitx3 (Invitrogen); Rabbit anti-DAT (Sigma); mouse anti-TH (Sigma); mouse anti-β-actin (Sigma). Promoter-specific primers were designed to amplify approximately 100 bp amplicons. The following ChIP primers were used for quantitative PCR: Oc-1 Forward 50 -TTCCAGCGCATGTCGGCGCTC-30 and Reverse 50 -GGTACTAGTCCGTGGTTCTTC-30 ; TH Forward 50 TGAAGACATCCAAAAAGCTAGTGAGA-30 and Reverse 50 -CAAGGGTTCATGTTAGGAAGGCTATA-30 ; α-Actin Forward 50 -ACGGACGTAAGCCTCACTTC-30 and Reverse 50 -TACCTGCTGCTCTGACTCTG-30 .

4.6.

Antagonism of Wnt1 signaling by Dickkopf-1

For antagonism of Wnt1 studies, vmNSCs were cultured as described previously mentioned (Andersson et al., 2013; Lei

49

et al., 2011). At DIV7, cell culture medium was replaced by differentiation medium supplemented with 100 ng/ml Dickkopf-1 (Dkk-1, R&D Systems). After three days of differentiation, half of the medium was replaced by differentiation medium without Dkk-1 and fresh Dkk-1 was added. vmNSCs were allowed to further differentiate under these conditions until DIV14, when RNA and protein were extracted for qRTPCR and immunoblotting respectively.

4.7.

Fluorescence immunolabeling and confocal microscopy

Fluorescence immunostaining combined with confocal laser scanning microscopic analysis was used to determine colocalization of two different signals within the same cell by z-series consecutive scans. Cells were fixed with 4% paraformaldehyde for 10 min at RT. For blocking of the background staining cells were incubated with 10% normal goat serum (NGS) in 0.1% TritonX in PBS for 1 h at RT. Primary antibodies were added for the cells in 10% NGS-0.1% TritonX in PBS for overnight staining in 4 1C. After primary staining cells were washed with 0.1% TritonX in PBS and secondary antibodies were added to the cells in 10% NGS-0.1% TritonX in PBS for 1 h RT. For nuclear detection cells were stained with hoechst (Invitrogen) and coverslips were mounted with mowiol (Sigma). For brain sections, the embryos were fixed by immersion overnight in 4% PFA in PBS at 4 1C. Thereafter, the tissues were cryoprotected overnight in 30% (v/v) sucrose, embedded in Tissue Freezing Medium (Tissue-Tek, Zakura Finetek) and frozen. Coronal sections (20 μm) were rehydrated in PBS. Then, tissue sections were permeabilized and blocked for 1 h at RT with 0.3% Triton X-100 and 10% NGS in PBS. Slides were incubated overnight at 4 1C with the primary antibodies diluted in PBS containing 10% NGS. Fluorescent signals were detected at excitation 535 nm and emission 565 nm (Rhodamine), 490 nm and 525 nm (FITC) by confocal laser scanning microscopy (TCS SP2, Leica, Germany). The number of double-labeled THþ–DATþ, Nurr1þ–THþ, and Pitx3þ–THþ cells were counted under confocal laser scanning microscope in a double-blinded fashion. Every double-labeled cell was confirmed in x–y crosssection, as well as in x–z and y– z cross-sections produced by orthogonal reconstructions from z-series.

4.8.

Statistical analysis

Statistical analysis was performed using statistical software (Microsoft excel and SPSS). All data were expressed as mean7S.E.M. Differences between groups were analyzed by ANOVA or Student's t test, and with a Po0.05 considered statistically significant.

Acknowledgments This work was supported by Science and Technology Planning Project of Guangdong Province (Nos. 2010B031500005 and No. 2013B021800087) and the National Natural Science Foundation of China (Grants 81172381, 81372584). The authors indicate no potential conflicts of interest.

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