Smoothened Agonist – Her2 Signal

Smoothened agonist augments proliferation and survival of neural cells
Olga Braginaa,b, Svetlana Sergejevaa, Martin Sergc, Tamara Zˇarkovsky d, Alla Maloverjanb,
Priit Kogermana,b,e,∗, Aleksandr Zˇarkovsky d
a Institute of Clinical Medicine, Tallinn University of Technology, Estonia
b Department of Gene Technology, Tallinn University of Technology, Estonia
c Department of Cardiology, Centre of Excellence for Translational Medicine, University of Tartu, Estonia
d Department of Pharmacology, Centre of Excellence for Translational Medicine, University of Tartu, Estonia
e Competence Centre for Cancer Research, Tallinn, Estonia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 10 December 2009
Received in revised form 22 June 2010 Accepted 23 June 2010

Keywords: Sonic hedgehog SAG
Gli1 Neurogenesis Differentiation

Sonic hedgehog signaling pathway is important in developmental processes like dorsoventral neural tube patterning, neural stem cell proliferation and neuronal and glial cell survival. Shh is also implicated in the regulation of the adult hippocampal neurogenesis. Recently, nonpeptidyl Smoothened activa- tors of the Shh pathway have been identiﬁed. The aim of this study was to investigate the effects of chlorobenzothiophene-containing molecule, Smo agonist (SAG), which has been shown to activate Shh signaling pathway, in neurogenesis and neuronal survival in in vitro and in vivo models. Our in vitro experiments showed that SAG induces increased expression of Gli1 mRNA, transcriptional target and mediator of Shh signal. In vitro experiments also demonstrated that SAG in low-nanomolar concentra- tions induces proliferation of neuronal and glial precursors without affecting the differentiation pattern of newly produced cells. In contrast to Shh, SAG did not induce neurotoxicity in neuronal cultures. The SAG and Shh treatment also promoted the survival of newly generated neural cells in the dentate gyrus after their intracerebroventricular administration to adult rats. We propose that SAG and similar com- pounds represent attractive molecules to be developed for treatment of disorders where stimulation of the generation and survival of new neural cells would be beneﬁcial.
© 2010 Elsevier Ireland Ltd. All rights reserved.

Sonic hedgehog (Shh) is a member of the Hedgehog (Hh) fam- ily of secreted signaling proteins having diverse functions during vertebrate development [16]. Shh is essential for development of organizing structures at the ventral midline and the speci- ﬁcation of neurons and glia [7]. Shh signaling is launched by binding of the secreted Shh peptide to the 12-span transmem- brane protein Patched (Ptch), resulting in loss of Ptch activity and consequent phosphorylation and posttranscriptional stabilization of 7-span transmembrane protein Smoothened (Smo) [16]. As a result, expression of Hh target genes is initialized through post- translational activation of the Gli family of zinc-ﬁnger transcription factors [11]. Activation of Shh signaling pathway is involved in the regulation of the proliferation of the cerebellar granule neuron pre- cursors [2,25], regulation of a number of embryonic and postnatal cells in neonatal cortex [20], and controls cell proliferation in the adult ventral tegmentum [17]. Recent studies have demonstrated that Shh is required for cell proliferation in the subventricular zone, tuberculum olfactorium, and in the dentate gyrus (DG) of the hip-

∗ Corresponding author. Tel.: +372 6204337; fax: +372 6204401.
E-mail address: [email protected] (P. Kogerman).

pocampal formation in adult animals [3,18]. The role of Shh in the regulation of adult neurogenesis was also conﬁrmed by the expres- sion pattern of the Shh receptor Patched in the hippocampal tissues within the hilar region, in the pyramidal cell layer and in the neu- rogenic niche of subgranular zone (SGZ) [12,22]. Also, Smo mRNA was found in the granule cells of the DG [23].
Recent studies have demonstrated neuroprotective properties of Shh. It was shown that survival of neural precursors is dependent on Shh [19] and intrastriatal injection of Shh reduces behavioral impairment in a rat model of Parkinson’s disease [24]. Moreover, Shh rescues cranial neural crest from cell death induced by ethanol exposure [1]. Contrary, several studies have demonstrated that Shh at certain concentrations can promote neuronal death of various neuronal populations via induction of apoptosis [8,15] and that early postmitotic neurons are the most sensitive to the neurotoxic effects of Shh [8].
Recently, several small molecule activators of the Shh pathway have been identiﬁed [6]. These molecules feature properties that make them attractive as potential therapeutic agents including low-nanomolar potencies and favorable pharmacokinetic pro- ﬁles in targeted tissues. Thus, nonpeptidyl Smo agonists have been shown to have therapeutic potential for the treatment of

Parkinson’s disease and peripheral nerve damage [24]. Importantly, the Hh-pathway agonists can activate Shh signaling pathway in a variety of in vitro and in vivo assays [6,9]. One of these compounds, a chlorobenzothiophene-containing molecule, Smo agonist (SAG) was recently synthesized and was shown to be able to bind directly to Smo and activate Shh-dependent pathway [6].
The aim of the current study was to evaluate the ability of Smo agonist SAG to activate Shh signaling pathway, modulate neuroge- nesis and promote neural cells’ survival.
For both in vivo and in vitro assays, SAG (synthesized at Karolin- ska Institutet, Department of Biosciences and Nutrition, Novum) was dissolved in 100% dimethyl sulfoxide (DMSO), recombinant ShhC24II protein (Shh) [13] was dissolved in a phosphate buffered saline (PBS, 0.1 M, pH 7.4).
To assess the effects of SAG on Gli1 mRNA levels in the cor- tical/hippocampal culture, reverse transcription PCR (RT-PCR) was performed. Brieﬂy, primary cells were treated with DMSO or SAG at concentrations 0.5, 1 or 5 nM for 48 h. Cells were collected and total RNA extracted with RNeasy Micro Kit (Qiagen). The extracted RNA was subsequently used to generate cDNA using SuperScriptTM First Strand Synthesis System for RT-PCR (Invit- rogen). Data were normalized using expression of housekeeping
gene HPRT. The sequences of the primers were: Gli1 primers 5∗-
ACGTTTGAAGGCTGTCGGAA-3∗, 5∗-CACACGTATGGCTTCTCATT-3∗ and HPRT primers 5∗-CAGTCCCAGCGTCGTGATTA-3∗, 5∗- AGCAAGTCTTTCAGTCCTGTC-3∗.
For the luciferase assay the NIH-3T3 cell line clone Shh-LIGHT2 (Shh-L2) [21] was used. Shh-L2 cells are stably expressing a Gli- dependent luciferase reporter and the Renilla luciferase. The latter was used for the normalization of the data. The cells were cultured according to the method described previously [13]. SAG at concen- trations 0.5, 1 or 5 nM was added 24 h after plating and 48 h later cells were subjected to luciferase assay using Dual Renilla/Fireﬂy Luciferase kit (Promega) and Ascent FL Fluoroscan (Thermo Elec- tron Corporation) according to the manufacturers’ instructions.
The possible toxic effects of SAG and Shh were studied in the primary culture of the cerebellar granule cells. This culture was prepared from the cerebella obtained from 8-day-old rat pups according to the method described previously [10]. Shh or SAG were added 2 h after plating and 8 days later the neuronal death was measured using trypan blue assay [10].
Primary culture of the neurons from the cortical and hippocam- pal tissues isolated from the brains of newborn rat pups at P0 was prepared as previously described [14]. The cells were plated on coverslips coated with poly-L-lysine placed in 24-well dishes containing neurobasal A medium supplemented with Bx27, L-
glutamine, 1% penicillin–streptomycin without ﬁbroblast growth factor-2 and were kept at 37 ◦C in CO2–air (5–95%) atmosphere. This culture contains postmitotic neurons and neuronal and glial
precursors. For the proliferation assay, a proliferation marker bromodioxyuridine (BrdU) was added at ﬁnal concentration of 50 µg/ml 3 h after plating. SAG or vehicle was added to the cul- tures together with BrdU and the cells were incubated for 48 h. Next, cells were ﬁxed with 4% paraformaldehyde and the number of BrdU-labeled cells and their phenotype was determined. To detect BrdU-labeled cells, rat-anti-BrdU antibody (1:300; Accurate Chem- icals) was used. To determine phenotypes of the BrdU-labeled cells a mouse-anti-TUJ1 (IgG, 1:800, neuronal class III β-tubulin, marker for neurons, Covance) or rabbit-anti-GFAP (IgG, 1:800; glial ﬁbril- lary acidic protein, marker for astrocytes, Dako) were used. The cells were incubated for 1.5 h at room temperature with antibodies. Sec- ondary antibodies were: goat-anti-rat conjugated with Texas Red (1:1000, Molecular Probes), goat-anti-mouse Alexa-488 IgG, and goat-anti-rabbit Alexa-488 IgG (1:2000; Molecular Probes). Fluo- rescent signals were detected using inverted light microscope Zeiss Axiovert 200 M (600×). The number of the BrdU-labeled cells as

well as the percentage of these cells expressing neuronal or glial markers per ﬁeld were determined. To determine the percentage of positive cells, the labeled cells were counted in each of 4 ran- domly chosen ﬁeld and the data were averaged for each coverslip. For each treatment group, the number of coverslips was 3 and the data were obtained from two independent experiments.
All experiments with animals were conducted in accordance with the guidelines established in the Principles of Laboratory Ani- mal Care (directive 86/609/EEC). 2-Months old female Wistar rats, weighing 250–350 g at the time of surgery, were obtained from the Scanbur National Animal Centre (Sollentuna, Sweden). All rats had free access to food and water.
Animals were deeply anaesthetized with chloral hydrate (350 mg/kg, intraperitonial (i.p.)). Drugs were stereotaxically injected into the ventricles (anteroposterior axis, 1; mediolateral axis, 1.5; dorsoventral axis, 3.4 from skull, with nose bar at 2 mm up) via minipump (2 µl/5 min). Animals received 5 µl of either Shh (n = 3) or PBS (n = 3) solution as control; or 2 µl of either SAG (n =4 for each concentration) or 100% DMSO (n = 3) solution. The cavity was packed with surgical foam and the animals were allowed to recover by warming.
BrdU was used to label newly produced cells. All ani- mals received i.p. injection of BrdU (50 mg/kg; Roche Molecular Biochemicals) every 12 h for the next 3 days after intrac- erebroventricular injections. Three weeks after the last BrdU injection, animals were deeply anaesthetized with chloral hydrate (400 mg/kg) and transcardially perfused with normal saline, and then with 4% paraformaldehyde/PBS; the brains were removed and post-ﬁxed for an additional 24 h in a paraformaldehyde solution. After the post-ﬁxation period (24–48 h), the brains were cut into 40 µm thick coronal sections on a vibratome (Leica VT1000S).
The sections, after antigen retrieval in citrate buffer (Dako) at 90 ◦C for 20 min, were incubated with a mixture of rat-anti-BrdU monoclonal antibody (1:300) and with either mouse-anti-TUJ1
(1:100) or rabbit-anti-GFAP (1:800). After rinsing, the sections were incubated with secondary antibodies (see above) for 1 h. Double- labeled cells for BrdU and TUJ1 or GFAP were visualized and counted with a light microscope (Olympus BX61) using a 60 oil objective. The representative photographs were made using laser confocal microscope Bio-Rad 1024. Z-axis analysis was used for pheno- typic characterization. Sections were optically sliced in the Z-plane by using 0.5–1.0 µm intervals. The data are expressed as a per- centage of BrdU-labeled cells expressing the phenotype marker. In total, 50 BrdU-labeled cells for each animal were analyzed for co-localization.
Data are presented as mean + SEM. Statistical analysis was per- formed using one-way ANOVA followed by Bonferroni post hoc test. P < 0.05 was considered statistically signiﬁcant. To test whether SAG is able to activate Gli-dependent luciferase activity in Shh-L2 cells, we applied SAG at different concentrations into cells. The obtained data were normalized with renilla luciferase values. The experiment was repeated 3 times, and results are shown in Fig. 1A. Application of SAG signiﬁcantly increased the luciferase activity, whereas this effect was the most prominent at the lowest concentration of SAG (0.5 nM). To test the SAG ability to activate Shh pathway in primary cells, we applied SAG at different concentrations onto corti- cal/hippocampal cells and assessed its’ effect on the expression of Gli1 mRNA using Real-Time RT-PCR. The obtained data were normalized with the expression of housekeeping gene HPRT. The experiment was repeated 3 times and the data are shown in Fig. 1B. Application of SAG induced an increase in Gli1 mRNA levels, but in this experiment the effects of SAG were clearly concentration- dependent with maximum effect at concentration 5 nM (Fig. 1B). To determine whether SAG has a direct role in cell proliferation, cultured cortical/hippocampal cells were incubated with BrdU and Fig. 1. Effects of SAG on Gli-dependent reporter activity and on the Gli1 mRNA expression. (A) SAG-induced expression of Gli1 mRNA in luciferase-based reporter assay in NIH-3T3 cell line clone Shh-LIGHT2; (B) SAG-induced expression of Gli1 mRNA in the cortical/hippocampal cell culture. The data are mean + SEM (n = 3). **P < 0.001, *P < 0.01 (Bonferroni test). different concentrations of SAG (0.5, 1 or 5 nM) for 48 h. Analysis of the number of BrdU-labeled cells revealed bell-shaped stimulatory effect of SAG on cell proliferation. The maximum effect of SAG was observed with a concentration of 1 nM (Fig. 2A). The higher con- centration of SAG (5 nM) did not affect signiﬁcantly the number of BrdU-labeled cells (Fig. 2A). To determine whether SAG affects differentiation pattern of the cortical/hippocampal progenitors, we performed co-localization studies using anti-BrdU antibodies and antibodies for the glial (GFAP) and neuronal (TUJ1) markers (Suppl. Fig. 1). No effect of SAG was observed on the differentiation pattern of the precursors (Fig. 2B and C). Next we tested whether SAG and Shh were neurotoxic in the pri- mary culture of the cerebellar granule cells. The data are depicted in Fig. 3. One-way ANOVA revealed that application of Shh induced neuronal death (F2,12 = 8.6, P < 0.01). Bonferroni test indicated sig- niﬁcant effect of Shh at the concentration of 50 nM. In contrast, SAG used in the same concentrations did not demonstrate neurotoxic effect (Fig. 3). The data obtained in in vitro experiments, were extended by our data in vivo. In in vivo experiments, Shh or SAG was admin- istered intracerebroventricularly to adult rats and the number and differentiation pattern of the BrdU-positive cells in the DG was assessed 3 weeks later. The intracerebroventricular admin- Fig. 2. Effect of SAG administration on the number and differentiation of BrdU-labeled cells in the cortical/hippocampal cell culture. Cells were incubated with BrdU and SAG at indicated concentrations for 48 h. The cells were immunostained for BrdU or double-stained with anti-BrdU and anti-GFAP or anti-TUJ1 antibodies. The number of BrdU-labeled cells (A) and a percentage of GFAP+/BrdU+ (B) or TUJ1+/BrdU+ (C) cells from total number of BrdU+cells was determined. The data are expressed as mean + SEM (n = 3). *P < 0.01 (Bonferroni test). Fig. 3. Effects of Shh and SAG on the survival of the cerebellar granule neurons in primary culture. The cells were treated with indicated concentrations of Shh or SAG and their viability was assessed by Trypan Blue exclusion assay. The data are calculated as a percentage of dead cells from total number of cells and are given as mean + SEM (n = 4–5). *P < 0.01 (Bonferroni test). istration of SAG signiﬁcantly increased the number of newly generated cells in the adult rat hippocampus (Fig. 4B). This effect of SAG was evident at both 2.5 nmol and 2.5 µmol doses. BrdU-labeled cells, often found in clusters, were distributed in the inner layer of the granular cell layer and the hilus of the DG. Similar effect on the number of BrdU-labeled was observed after intracerebral administration of Shh in a dose of 0.01 nmol (Fig. 4A). It should be noted that the assessment of the effects of Shh and SAG on the proliferation of the neural stem cells immediately after BrdU administration was complicated because brain tissue damage due to the intracerebroventricular injection of the drugs produced robust increase in proliferation (data not shown), and this stimu- lation of the proliferative activity due to trauma masked a possible stimulatory effects of the drugs. Therefore, the observed increase in the number of survived BrdU-positive cells represents a net result of both proliferation and survival. Next, we tested whether Shh or SAG administration was able to affect the differentiation pattern of newly born cells in DG (Fig. 4C–F). The phenotype of de novo produced, was determined by double immunohistochemistry with antibodies against BrdU and the glial marker, GFAP, or the neuronal marker, TUJ1 (Suppl. Fig. 2). As could be seen in Fig. 4 neither Shh nor SAG affected the pro- portion of cells differentiated into either TUJ1-positive neurons or GFAP-positive glia cells. Our study showed that synthetic molecule SAG is able to acti- vate the expression of Shh-dependent transcription factor Gli1. The enlarged expression of Gli1 was found in different experimental settings using both Shh-L2 cell line and primary culture of the cor- tical/hippocampal cells and conﬁrms the ability of SAG to activate and act via Shh-dependent intracellular pathway. This effect of SAG Fig. 4. Effects of Shh and SAG on the differentiation and survival of newly born cells in the adult rat hippocampus. Shh (A, C, and E) or SAG (B, D, and F) were injected into the cerebral ventricles of adult rats followed by injection of BrdU during the following 3 days. Three weeks later the animals were sacriﬁced and the amount of newly produced cells determined. The fate of newly produced cells was determined by double immunostaining with anti-BrdU and anti-GFAP (C and D) or anti-TUJ1 (E and F) antibodies. The data are given as a number of BrdU-labeled cells/section or as a percentage of TUJ1+/BrdU+ or GFAP+/BrdU+cells from total number of BrdU+cells and expressed as mean + SEM (n = 4). **P < 0.05. was accompanied by an increased cell proliferation in the primary cortical/hippocampal culture as evidenced by an increase in the total number of BrdU-labeled cells. No effect of SAG on the differ- entiation pattern of BrdU-labeled precursors added to the neuronal cultures was found. Our study also demonstrated that Shh in high concentrations was able to induce neuronal death in postmitotic cerebellar granule neurons. These data conﬁrm those previously obtained by others where neurotoxicity of Shh was demonstrated either in vitro in the primary culture of dorsal root ganglion neurons or in vivo after fetal administration of Shh [8,16]. In contrast, SAG did not affect survival of the cerebellar granule neurons. In addition to in vitro experiments, we also demonstrated the effects of Shh and SAG on the survival of newly born cells in the DG of adult rats. Although in the adult rat DG, neural progenitor cells are continuously generated, many newly born cells die shortly after birth, and only some of them survive and differentiate [4]. To deter- mine whether SAG and Shh affect the survival and differentiation of de novo produced cells in the DG, animals were sacriﬁced 3 weeks after BrdU injection. Under these conditions, the number of BrdU- labeled cells represent a net result of the effects of the drugs on the proliferation and survival. Increased number of BrdU-labeled cells in hippocampus of treated rats showed that SAG increases pool of de novo proliferated cells in the DG. In agreement with our in vitro data, the percentage of newly born neurons and glial cells did not differ between Shh, SAG or vehicle-treated groups, indicating that differentiation of new cells in the DG was not inﬂuenced by applied treatments. The lack of the Shh effect on the differentiation into cells with neuronal or glial phenotype is supported by others [12]. Although we failed to ﬁnd any changes in the differentiation pattern of the newly born cells, it is still not excluded that SAG can promote generation of oligodendroglia, since previous studies have demon- strated Shh-induced increased oligodendroglial differentiation [5]. In the present study, we have demonstrated that small molecule SAG promotes neurogenesis and survival of neural cells in vitro and vivo, respectively. Thus, in vitro SAG, similarly to Shh, promoted proliferation of the cortical/hippocampal cells without signiﬁcant effect on their differentiation pattern. In vivo, SAG, as well as Shh, extended survival of hippocampal cells. However, in contrast to Shh, SAG in even in high concentrations did not induce neuronal death. Based on this evidence, it may be possible to develop safe and effective small molecule drugs that can be used to promote expan- sion and survival of endogenous precursors aiming for replacement of dead or damaged neural cells. Identiﬁcation of a small molecule compound that can induce the birth of new cells in the adult brain without disrupting the normal differentiation process can be viewed as a promising ﬁrst step in this direction. Acknowledgments This study was supported by EU FP7 grant HEALTH-F2-2008- 223077 “Neuropro”, Estonian Science Foundation Grants 7955, 7242 and 8116, and European Regional Development Fund. We would like to acknowledge Prof. Jan Bergman and Dr. Robert Engqvist from Karolinska Institutet for synthesizing SAG and Priit Pruunsild for the help with the culture experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.06.068. References [1] S.C. Ahlgren, V. Thakur, M. Bronner-Fraser, Sonic hedgehog rescues cranial neu- ral crest from cell death induced by ethanol exposure, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 10476–10481. [2] O.J. Becher, D. Hambardzumyan, E.I. Fomchenko, H. Momota, L. Mainwaring, A.M. Bleau, A.M. Katz, M. Edgar, A.M. Kenney, C. Cordon-Cardo, R.G. Blasberg, E.C. Holland, Gli activity correlates with tumor grade in platelet-derived growth factor-induced gliomas, Cancer Res. 68 (2008) 2241–2249. [3] S. Blaess, J.D. Corrales, A.L. Joyner, Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region, Development 133 (2006) 1799–1809. [4] H.A. Cameron, C.S. Woolley, B.S. McEwen, E. Gould, Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat, Neuroscience 56 (1993) 337–344. [5] C. Danesin, E. Agius, N. Escalas, X. Ai, C. Emerson, P. Cochard, C. Soula, Ven- tral neural progenitors switch toward an oligodendroglial fate in response to increased Sonic hedgehog (Shh) activity: involvement of Sulfatase 1 in modu- lating Shh signaling in the ventral spinal cord, J. Neurosci. 26 (2006) 5037–5048. [6] M. Frank-Kamenetsky, X.M. Zhang, S. Bottega, O. Guicherit, H. Wichterle, H. Dudek, D. Bumcrot, F.Y. Wang, S. Jones, J. Shulok, L.L. Rubin, J.A. Porter, Small-molecule modulators of Hedgehog signaling: identiﬁcation and char- acterization of Smoothened agonists and antagonists, J. Biol. 1 (2002) 10.
[7] L.V. Goodrich, M.P. Scott, Hedgehog and patched in neural development and disease, Neuron 21 (1998) 1243–1257.
[8] W. Guan, G. Wang, S.A. Scott, M.L. Condic, Shh inﬂuences cell number and the distribution of neuronal subtypes in dorsal root ganglia, Dev. Biol. 314 (2008) 317–328.
[9] J.M. Harper, C. Krishnan, J.S. Darman, D.M. Deshpande, S. Peck, I. Shats, S. Back- ovic, J.D. Rothstein, D.A. Kerr, Axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 7123–7128.
[10] A. Kalda, A. Zharkovsky, Metabotropic glutamate receptor agonists protect from oxygen–glucose deprivation- and colchicine-induced apoptosis in primary cul- tures of cerebellar granule cells, Neuroscience 92 (1999) 7–14.
[11] K.W. Kinzler, J.M. Ruppert, S.H. Bigner, B. Vogelstein, The GLI gene is a member of the Kruppel family of zinc ﬁnger proteins, Nature 332 (1988) 371–374.
[12] K. Lai, B.K. Kaspar, F.H. Gage, D.V. Schaffer, Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo, Nat. Neurosci. 6 (2003) 21–27.
[13] A. Maloveryan, C. Finta, T. Osterlund, P. Kogerman, A possible role of mouse Fused (STK36) in Hedgehog signaling and Gli transcription factor regulation, J. Cell Commun. Signal. 1 (2007) 165–173.
[14] S. Murphy, M.L. Simmons, L. Agullo, A. Garcia, D.L. Feinstein, E. Galea, D.J. Reis, D. Minc-Golomb, J.P. Schwartz, Synthesis of nitric oxide in CNS glial cells, Trends Neurosci. 16 (1993) 323–328.
[15] R.W. Oppenheim, S. Homma, E. Marti, D. Prevette, S. Wang, H. Yaginuma, A.P. McMahon, Modulation of early but not later stages of programmed cell death in embryonic avian spinal cord by sonic hedgehog, Mol. Cell. Neurosci. 13 (1999) 348–361.
[16] T. Osterlund, P. Kogerman, Hedgehog signalling: how to get from Smo to Ci and Gli, Trends Cell Biol. 16 (2006) 176–180.
[17] V. Palma, D.A. Lim, N. Dahmane, P. Sanchez, T.C. Brionne, C.D. Herzberg, Y. Git- ton, A. Carleton, A. Alvarez-Buylla, A. Ruiz i Altaba, Sonic hedgehog controls stem cell behavior in the postnatal and adult brain, Development 132 (2005) 335–344.
[18] V. Palma, A. Ruiz i Altaba, Hedgehog-GLI signaling regulates the behavior of cells with stem cell properties in the developing neocortex, Development 131 (2004) 337–345.
[19] V.F. Rafuse, P. Soundararajan, C. Leopold, H.A. Robertson, Neuroprotective prop- erties of cultured neural progenitor cells are associated with the production of sonic hedgehog, Neuroscience 131 (2005) 899–916.
[20] A. Ruiz i Altaba, V. Nguyen, V. Palma, The emergent design of the neural tube: prepattern, SHH morphogen and GLI code, Curr. Opin. Genet. Dev. 13 (2003) 513–521.
[21] J. Taipale, J.K. Chen, M.K. Cooper, B. Wang, R.K. Mann, L. Milenkovic, M.P. Scott,
P.A. Beachy, Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine, Nature 406 (2000) 1005–1009.
[22] E. Traiffort, D. Charytoniuk, L. Watroba, H. Faure, N. Sales, M. Ruat, Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system, Eur. J. Neurosci. 11 (1999) 3199–3214.
[23] E. Traiffort, D.A. Charytoniuk, H. Faure, M. Ruat, Regional distribution of Sonic Hedgehog, patched, and smoothened mRNA in the adult rat brain, J. Neu- rochem. 70 (1998) 1327–1330.
[24] K. Tsuboi, C.W. Shults, Intrastriatal injection of sonic hedgehog reduces behav- ioral impairment in a rat model of Parkinson’s disease, Exp. Neurol. 173 (2002) 95–104.
[25] C. Vaillant, D. Monard, SHH pathway and cerebellar development, Cerebellum 8 (2009) 291–301.