Hedgehog: Multiple Paths for Multiple Roles in Shaping the Brain and Spinal Cord.

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Hedgehog: Multiple Paths for Multiple Roles in Shaping the Brain and Spinal Cord Julien Ferent and Elisabeth Traiffort Neuroscientist published online 17 April 2014 DOI: 10.1177/1073858414531457 The online version of this article can be found at: http://nro.sagepub.com/content/early/2014/04/17/1073858414531457 A more recent version of this article was published on - May 8, 2014

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NROXXX10.1177/1073858414531457The NeuroscientistFerent and Traiffort

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Hedgehog: Multiple Paths for Multiple Roles in Shaping the Brain and Spinal Cord

The Neuroscientist 1–16 © The Author(s) 2014 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1073858414531457 nro.sagepub.com

Julien Ferent1 and Elisabeth Traiffort2

Abstract Since the discovery of the segment polarity gene Hedgehog in Drosophila three decades ago, our knowledge of Hedgehog signaling pathway has considerably improved and paved the way to a wide field of investigations in the developing and adult central nervous system. Its peculiar transduction mechanism together with its implication in tissue patterning, neural stem cell biology, and neural tissue homeostasis make Hedgehog pathway of interest in a high number of normal or pathological contexts. Consistent with its role during brain development, misregulation of Hedgehog signaling is associated with congenital diseases and tumorigenic processes while its recruitment in damaged neural tissue may be part of the repairing process. This review focuses on the most recent data regarding the Hedgehog pathway in the developing and adult central nervous system and also its relevance as a therapeutic target in brain and spinal cord diseases. Keywords neural stem cells, axonal guidance, Smoothened, Boc, congenital diseases, primary cilium The Hedgehog (Hh) field emerged from studies regarding segmentation of the Drosophila embryo, where the gene was first characterized as necessary for segment organization (Nusslein-Volhard and Wieschaus 1980). Hh is known to regulate growth and patterning of most tissues including the nervous system in both invertebrates and vertebrates, but also to be the source of dysregulations that lead to several human diseases. This review highlights recent advances in the current understanding of Hh signaling within the CNS, from the canonical to the noncanonical mechanisms of action and from cellular to developmental and maintenance functions.

Hedgehog Signaling Pathway Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) peptides form a small family of secreted proteins with key roles in embryonic tissue induction and patterning. Among them, Shh is the protein implicated in the development and maintenance of the CNS. Synthesized as a large precursor of 47 kDa, Shh has first aroused interest because of its unclassical posttranslational processing primarily described in Drosophila (Box 1) and mostly conserved in vertebrates (Fig. 1). The biogenesis of Shh involves the removal of the signal sequence and an autoproteolytic cleavage of the precursor, which generates the biologically active N-terminal

fragment (Shh-N) covalently linked to cholesterol at its C terminus (Porter and others 1996) and to a palmitoyl group transferred by a dedicated acyltransferase at its N-terminus (Chamoun and others 2001). Cholesterol addition is considered as a rare protein modification. It increases Shh lipophilicity and promotes its tethering into the plasma membrane, but is also required for Shh-N potency and ability to signal at long distance, even though the synergic presence of non-sterol-modified Shh is necessary for the full activation of the pathway (Palm and others 2013). A complex trafficking likely prepares Shh-N for release from the synthesizing cells and then adherence to target cells (Fig. 1). As described in recent reviews (Briscoe and Thérond 2013; Eaton 2008), several proteins such as the 12 pass-transmembrane protein Dispatched and the extracellular matrix proteins heparan sulfate proteoglycans (HSPG) are involved in the 1

IRCM, Molecular Biology of Neural Development, Montreal, Quebec, Canada 2 INSERM-Université Paris Sud, Neuroprotection and Neuroregeneration: Small Neuroactive Molecules UMR 788, Le Kremlin-Bicêtre, France Corresponding Author: Elisabeth Traiffort, INSERM-Université Paris Sud, Neuroprotection and Neuroregeneration: Small Neuroactive Molecules UMR 788, 80 Rue du Général Leclerc, F-94276, Le Kremlin-Bicêtre, France. Email: [email protected]


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Figure 1. Biosynthesis, release, and reception of Shh protein. Shh is synthesized as a precursor autocatalytically cleaved into amino- (N-term) and carboxy- (C-term) terminal peptides. C-term leaves the endothelial reticulum (ER) and is targeted to the proteosomal system. N-term is modified by the addition of two lipid groups, cholesterol and palmitic acid which promote the tethering of the biologically active Shh peptide (ShhN) at the cell membrane. Active mechanisms required for ShhN secretion include the proteins Dispatched and Scube. Alternatively, ShhN may either bind to lipoprotein particles in the presence of extracellular matrix proteins called heparin sulfate proteoglycans (HSPG), or form multimers or be targeted to the surface of exovesicles derived from the plasma membrane. Specialized filopodia (not yet reported in the CNS) might also direct long range transport of ShhN and allow its interaction with filopodia present on the responding cells. The most well described reception mechanism of ShhN takes place in the primary cilium. In the presence of the co-receptors Cdon/Boc/Gas1, ShhN binds Ptc located in the cilium. The binding relieves the inhibition exerted by Ptc on Smo. After phosphorylation, Smo enters the cilium, the Gli/Su(Fu) complex is dissociated and leads to the transport of Gli activator forms toward the nucleus. This pathway called “canonical” results in the transcriptional activation of target genes mainly implicated in cell proliferation and differentiation. The non-canonical pathways independent of Gli transcription include (a) type I, which involves Ptc (but not Smo) and leads to cell apoptosis and cell cycle regulation and (b) type II, which involves Smo and is composed of the type IIa leading to calcium release in a cilium-dependent manner and type IIb implicated in cilium-independent chemotaxis via cytoskeleton remodeling.


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Ferent and Traiffort mechanism of Shh secretion. Several secreted forms with distinct signaling activities may account for the diversity of Shh-mediated responses. Indeed, Shh may bind to lipoprotein particles by tethering its lipid moieties into the outer phospholipid monolayer of these particles, form multimers as evidenced in vivo in the mouse neural tube (Chamberlain and others 2008) or lead to soluble Shh clusters after the proteolytic shedding of both lipid groups by the ADAM (a disintegrin and metalloproteinase) metalloprotease. A specialized class of actin-based filopodia with novel cytoskeletal features is also able to transport Shh from producing cells as shown not yet in the developing CNS, but in limb bud (Sanders and others 2013). Box 1. The origin of the name Hedgehog derives from the “spiked” phenotype of the cuticle of the Hh Drosophila mutant. In 1980, Nusslein-Volhard and Wieschaus identified mutations in the Hh gene in their large-scale screen for mutations altering the development of the fruit fly larval body plan. Drosophila Hh DNA was cloned in the early 1990s, while the components of the Hh signal transduction pathway have been first identified using Drosophila genetics. Whereas Dhh is the closest homolog to Drosophila Hh, Shh and Ihh are more closely related to one another than they are to fly Hh. The proteins able to bind Hh in Drosophila comprise Ptc and the co-receptors Ihog and Boi. In contrast Hip was not identified in invertebrates. Drosophila Smo display quite low overall sequence homology with mammalian Smo. However, self-interaction domains, phosphoregulatory motifs, and regulated conformational changes are conserved. The main difference is that phosphorylated Smo translocates to the plasma membrane in Drosophila or to the plasma membrane of the primary cilium in vertebrates. Indeed, the majority of Drosophila cells do not have primary cilia. In Drosophila, Ci is the single zinc finger transcription factor involved in the transcriptional response of Hh. Unlike vertebrate Smo, no small molecules are known to bind Drosophila Smo, which is not inhibited by sterol depletion. It was recently proposed that Drosophila Smo may be regulated differently from the vertebrate protein despite Ptc conservation. The cysteine rich domain is absolutely required for Drosophila Smo function, may be because it displays a critical role in stabilizing the active conformation. Recent genetic experiments suggest that phospholipids could serve as the physiological molecules regulating Drosophila Smo activity (Briscoe and Thérond 2013; Robbins and others 2012).

The transduction of Hh signal classically involves a major receptor complex associating the 12-transmembrane protein, Patched (Ptc), and one of its co-receptors, including the cell surface immunoglobulin/fibronectin proteins Cdon and Boc, or the anchored plasma membrane protein Gas-1 (Izzi and others 2011) (Fig. 1). Another protein able to bind Shh is the glycoprotein Hedgehog interacting protein (Hip) which constitutes the physiological antagonist of Hh proteins (Chuang and others 2003). Once Shh binds to the receptor complex, the repression exerted by Ptc on the 7-transmembrane protein Smoothened (Smo) is relieved, resulting in accumulation of Smo on the cell surface and its conformational switch to an active form (Zhao and others 2007). If the subcellular localization of Smo to the plasma membrane is likely the primary target of Ptc function, Smo itself, related to the G protein–coupled receptors (GPCRs) and recently crystallized (Wang and others 2013), regulates Ptc since ubiquitin ligases of the Smurf family mediate Smo-dependent Ptc degradation (Huang and others 2013). In Shh-responding cells, a complex signaling cascade is triggered. It involves the inhibition of Gli transcription factor (Gli1-3) processing into their transcriptional repressor forms together with the accumulation of the activator forms and finally, to the activation of target genes as previously described in several reviews (Barakat and others 2010; Briscoe and Thérond 2013; Chen and Jiang 2013; Robbins and others 2012). Gli1 is classically used as a convenient readout for the pathway activation whereas Gli2 and Gli3 function mainly as transcriptional activator (GliA) and repressor (GliR), respectively. The recently crystallized Suppressor of Fused, Su(Fu), interacts with the three Gli proteins at the level of critical residues to retain them in the cytosol (Zhang and others 2013b). New intracellular actors are continuously uncovered such as the cytoskeletal protein Zyxin which can bind to and inhibit Gli1 activity (Martynova and others 2013). This transduction pathway, so-called “canonical” differs from the “non-canonical” mechanisms, which involve Shh components without any Gli-mediated transcription. Non-canonical Shh signaling can itself be subdivided into mechanisms not requiring Smo (type I) and those downstream of Smo that do not require Gli factors (type II). Non-canonical type II signaling downstream of Smo notably involves Gi proteins whereas non-canonical type I signaling can be notably mediated through Ptc (Robbins and others 2012). Most Shh components can be detected in the primary cilium, an organelle present in nearly all vertebrate cells that extends into the extracellular environment and constitutes a sensor able to transmit information from the environment. Itself Shh has been identified close to the cilium base in the neural tube (Chamberlain and others 2008). On ligand binding to Ptc, simultaneous removal of


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Ptc and localization of Smo to the cilia occur. Smo trafficking is tightly regulated. This involves the Kif3a kinesin motor protein, the arrestin β2 GPCR regulator (Kovacs and others 2008), the Arl13b small GTPase (Larkins and others 2011) and the integrin-linked kinase (Barakat and others 2013a), leading to GliA formation (Kovacs and others 2008; Rohatgi and others 2007). Recently, the constitutive activity of the ciliary GPCR, GPR161, was found to be crucial for maintaining at the cilium base, a high localized Protein Kinase A (PKA) activity. The latter is aimed at inhibiting Shh signaling at baseline unless Smo is stimulated and leads to PKA exit from the cilium (Mukhopadhyay and others 2013). A second pool of PKA not concentrated at the cilium and responding to stimulation by non-ciliary GPCR, may serve to adjust the signaling level or even may completely stop the pathway by inhibition of Gli2 translocation into the cilium (Niewiadomski and others 2013). Moreover, a pool of Smo located outside the primary cilium is currently proposed to mediate Shh-dependent non-transcriptional chemotactic response (Bijlsma and others 2012). This finding indicates that Smo activity is not restricted to the cilia and highlights another level of complexity for type II noncanonical pathways related to Smo activation according to the requirement (type IIa) or not (type IIb) of the primary cilia.

Shaping the CNS via the Canonical Gli-Dependent Shh Pathway: Cellular Identity In the developing CNS, Shh is one of the major locally expressed signaling molecules that are critical for spatial patterning of the neuroepithelium (Cohen and others 2013). Shh is produced by both the notochord and floorplate at the ventral midline of the neural tube (Fig. 2A). Its availability is notably regulated by the extracellular matrix protein called Emilin 3, recently described in the Zebrafish notochord (Corallo and others 2013). Shh forms a ventral to dorsal gradient that is interpreted by neural tube progenitors to establish patterns of expression of homeodomain and bHLH transcription factors (TF). Such expression patterns allow the division of the ventral neural tube into 5 discrete dorsoventral domains of progenitors, each of which will generate specific subtypes of motoneurons and interneurons during the earliest stage of neurogenesis (E9.5-E10.5). The primary cilium is essential for the transduction of Shh signaling (Huangfu and others 2003) and the temporal deletion of the cilia protein Arl13b revealed the crucial role of Gli3 activity (Su and others 2012). Another control mechanism is the ligand-dependent feedback inhibition of Shh signaling governed by Ptc1, Ptc2 (the Ptc1 homologue) and Hip (Holtz and others 2013). The next stage (E11.5-E12.5) is the generation

of oligodendrocyte precursors (OPCs) derived from the Olig2-expressing pMN domain producing motoneurons before switching to oligodendrocytes (OLs). The switch likely depends on sulfatase 1, a regulator of the HSPG sulfation “code” (Touahri and others 2012), or on the Shh homolog, Ihhb, as reported in Zebrafish (Chung and others 2013). Even though some aspects of astrocyte specification taking place at the latest stage (E15.5) can occur in the absence of Shh, the morphogen is proposed to influence astrocytes by regulating the secreted calcium-binding protein S100. Finally, Shh signaling is also required for the proper formation of the ependymal zone lining the central canal of the developing spinal cord (Yu and others 2013). If the concentration and duration of Shh exposure likely dictate the precise position, number, and arrangement of cells (Balaskas and others 2012), live imaging in Zebrafish revealed that cells of the neural tube actively sort to their correct positions only after their disordered specification by Shh (Xiong and others 2013). The role of Shh signaling has also been investigated throughout the developing brain (Fuccillo and others 2006; Sousa and Fishell 2010). The sources of Shh for the developing forebrain are the mesendoderm and diencephalon between E8.5 and E9.5, and later on the medial ganglionic eminence (MGE), the preoptic area, the amygdala, the hypothalamus and the zona limitans interthalamica (ZLI). Shh signaling is first required for the patterning of ventral-most telencephalic regions and then for the generation of both neuronal and glial cell types. The tracing of Shh-expressing or responding cells in the embryonic forebrain suggest that distinct temporal Shh gradients may be responsible for the generation of distinct neural subtypes (Wada and others 2012). Shh and Gli3 act antagonistically for specification of most telencephalic progenitors as reported, for instance, in the hypomorphic mouse mutant Polydactyly Nagoja (Magnani and others 2010). OL and ventrally born gabaergic populations are generated in a Shh-dependent manner from the ventral ganglionic eminences. In the medial amygdala, a part of the limbic system mediating distinct aspects of emotional and behavioral processing, Shh-expressing and -responding progenitors generate three functionally distinct classes of neurons (Carney and others 2010). In the ventral diencephalon, Shh signal was first associated with the ventral separation of the eye fields, the patterning of the retina, optic chiasm and optic stalk. Shh is critical for the proliferation and patterning of hypothalamic neural progenitors which will give rise to the two most caudal regions, the mammillary and tuberal regions of the hypothalamus, a central regulator of critical homeostatic physiological processes such as temperature regulation, food intake, and circadian rhythms. At E9.5-E12.5, Shhexpressing progenitors give rise to neurons, astrocytes, and also to tanycytes, a specialized glial cell type in the


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Figure 2. Canonical Shh pathway and control of cellular identity in the CNS. (A) Shh secreted from the notochord (nc) and floorplate (fp) acts at distance in a concentration-dependent manner (red triangle) in the ventral neural tube. Shh induces the generation of interneurons and motoneurons at E10 from domains V0-V3 and MN, respectively. The latter also produces oligodendrocytes at E12. Ventral (V) to dorsal (D) orientation is indicated. (B) In the postnatal cerebellum, Shh is secreted by the Purkinje cells (red). At the earliest stages, Shh induces the proliferation of granule cell precursors (green) and the differentiation of Bergmann glia (blue). Shh is also involved in establishment and maintenance of the prospective white matter (PWM) containing interneuron and astrocyte precursors (purple) and promotes the proliferation of oligodendrocyte precursors (black). EGL, external granular layer; GL, granular layer; IGL, internal granular layer; ML, molecular layer; PCL, Purkinje layer; WM, white matter. (C) Shh maintains the adult neurogenic areas, including the subventricular (SVZ, left) and subgranular zones (SGZ, right). Red cells reflect Shh-expressing cells. Shh is also present in the cerebrospinal fluid within the ventricles. SVZ (a) and SGZ (b, c) enlargements are shown. Cells comprising the SVZ are astrocyte-like neural stem cells (B cells, dark green), transient-amplifying cells (C cells, light green) and neuroblasts (A cells, yellow to brown reflecting different progenitor microdomains). B/C cells in the SVZ and NSC in the SGZ are Shh-responding cells via the canonical pathway (Gli1+). (a′) indicates that Shh is required for the generation of specific neuronal progeny migrating to specific layers of the olfactory bulb (OB). E, ependymal cell; LV, lateral ventricle; N, neuron; Nb, neuroblast. (D) Regionally distinct astrocyte populations express Gli1 (green) in the adult forebrain as shown in the cortex (Cx), globus pallidus (GP), hypothalamus (HPT), thalamus (Th) unlike in the caudate putamen (CPu). Shh-expressing neurons (red) are detected in most areas expressing Gli1+ astrocytes. The magnification indicates that postnatal interruption of Shh signaling by Smo removal in astrocytes induces mild reactive gliosis in the cortex.


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hypothalamic median eminence. The most rostral preoptic region receives mostly glia from an independent, rostral, and late-appearing Shh-expressing domain (Alvarez-Bolado and others 2012). Regarding the pituitary gland, a master endocrine organ, Shh first derived from the oral ectoderm is implicated, via Gli2, in the specification and proliferation of early pituitary progenitors prior to the closure of Rathke pouch (the oral ectoderm part from which the gland develops). Then, diencephalon-derived Shh is required to control pituitary development through the regulation of the Bone morphogenetic protein and Fibroblast growth factor family (Wang and others 2010). In more caudal regions of the developing brain, such as the ventral midbrain (vMB) and hindbrain, Shh is required through Gli2 to induce before E11 many, but not all ventral cell types and through Gli3 to maintain Fgf8 expression. In this regard, the most studied role of Shh is its implication in two major regulatory loops controling the neurogenic niche enriched with progenitors of dopaminergic neurons. The first loop involves the transcription factors Foxa2 and Nato3. The second one relies on the Wnt/β catenin pathway, the persistent activation of which in early progenitors perturbs the cell cycle progression of these cells and antagonizes Shh expression. Shh also induces activation of Nurr1 and tyrosine hydroxylase (TH), important for the specification of postmitotic dopaminergic neurons (Nissim-Eliraz and others 2013). The robust expression of Shh signaling components in the neurogenic niche for dopaminergic progenitors at E8-E8.5 is then shifted to more lateral domains at E9.5-E12.5 in agreement with the phenotype of conditional mutants deleting Smo in vMB progenitors showing a transient reduction in dopamine neurons at E10.5, and then defects in neurons of the red nucleus, oculomotor nucleus, and raphe nuclei (Tang and others 2013). Intriguingly, Shh- and Gli1- expressing cells are basically the same cell populations that sequentially turn on Gli1 and then Shh expression. This tightly regulated dynamic is required to regulate the cell cycle status of dopaminergic progenitors and ultimately influence their final distribution in the ventral tegmental area and substantia nigra (Hayes and others 2013). At a more caudal level, Shh is required for the specification of most cell populations in the hindbrain rhombomer 1 such as in the tegmental nuclei (Moreno-Bravo and others 2013) and also for vascular outgrowth in the hindbrain choroid plexus (Nielsen and Dymecki 2010). A unique feature in the developing brain compared with the neural tube is the role of Shh in the specification of dorsal regions. The thalamus which notably relays sensory information to corresponding cortical areas is remarkable because of its proximity to the ZLI, a dorsal Shh source by E10.5. Diencephalon ventral midline and

ZLI both contribute to Shh gradient and are respectively responsible for Shh mitogenic role and establishment of Shh-dependent thalamic progenitor domains in a way reminding the situation observed in the neural tube (Epstein 2012). GliA and GliR each specify pattern and size of specific sets of thalamic nuclei, but do not show reciprocal antagonism. Consistently, their joint abolition does not rescue the wild type phenotype (Haddad-Tovolli and others 2012). The neocortex development takes place during late gestation stage at E13.5-E18.5. Progenitors expand by self-renewal in the ventricular zone (VZ), and generate intermediate progenitor cells (IPCs). Endogenous Ptc mediates the broad effects of Shh on both the transition from VZ progenitors to IPCs and the activation of IPC proliferation (Shikata and others 2011). Its conditional inactivation in nestin+ progenitors induces lamination defects and led to show the required interplay between Shh and Notch signaling for neocortical neurogenic divisions (Dave and others 2011). Consistently, Shh modulates the epidermal growth factor receptor (EGFR)dependent proliferation of radial glial cells through EGFR transactivation (Reinchisi and others 2013). Moreover, in the human developing cortex, Shh promotes the generation and maintenance of Olig2+ OPC (Ortega and others 2013). Shh is also essential for the development of the cerebellar cortex (Fig. 2B). Delivered through a transventricular route, the morphogen first induces the proliferation of cerebellar radial glia and neural progenitors derived from the germinal VZ in embryo (Huang and others 2010). Then, in the earliest postnatal stage, Purkinje neurons secrete Shh which stimulates in the external granule cell layer (EGL) the proliferation of granule cell precursors (GCPs) derived from the rhombic lip through a primary cilium-dependent mechanism. Moreover, Shh promotes the maturation of Bergmann glia and stimulates the proliferation of OPC both derived from the VZ (Bouslama-Oueghlani and others 2012; Spassky and others, 2008). Finally, Purkinje neuron–derived Shh also regulates the proliferation of postnatally born gabaergic inhibitory interneurons and astrocytes which are generated in a transient germinal compartment of the prospective white matter in the nascent cerebellum (Flemming and others 2013). Complete loss of Hh-dependent GCP proliferation occurs in the absence of all three Shh coreceptors Boc, Cdon, and Gas1 (Izzi and others 2011). Similarly, a specific class of HSPG expressed by cerebellar GCP and located adjacent to the primary cilia, also constitutes Shh co-receptors for this effect (Witt and others 2013). In Bergmann glia cells, the association of Ptc with the GPCR Gpr37l1, identified as a modulator of primary ciliogenesis, is likely implicated in the functional interaction of these cells with Purkinje neurons and GCP to maintain their proliferation (Marazziti and others


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Ferent and Traiffort 2013). In contrast, other signals such as vitronectin counteract Shh mitogenic effects for controling OPC proliferation (Bouslama-Oueghlani and others 2012). Recent advances in adult neurogenesis have highlighted the capacity of the brain to generate new neurons throughout adult life which may contribute alternatively to tissue repair or tumorigenesis. The two main neurogenic areas in the brain are the subventricular zone (SVZ) of the lateral ventricle (LV) and the subgranular zone (SGZ) of the dentate gyrus, which provide new neurons to the olfactory bulb (OB) and hippocampal dentate gyrus, respectively (Fig. 2C). Shh is required for progenitor cell maintenance in these telencephalic stem cell niches (Machold and others 2003). In both areas, neural stem cells (NSCs) during late gestation and slow-cycling NSC in adulthood are Shh-responsive (Ahn and Joyner 2005). Conditional removal of Smo function from the adult NSC niches shows that Shh signaling is required for astrocyte-like NSC maintenance and proliferation of their direct progeny, the transit-amplifying cells (Balordi and Fishell 2007). Loss-of-function studies using the adenovirus-mediated transfer of Hip in the adult mouse brain indicates that Shh is a retention factor restraining exit of neuroblasts out of the SVZ (Angot and others 2008). More recently, non-attenuated GliR was found as a primary inhibitor of adult NSC function in SVZ whereas only little requirement for GliA function exists in stimulating SVZ neurogenesis (Petrova and others 2013). Remarkably, Shh is selectively produced by a small group of adult ventral forebrain neurons close to the SVZ and is both necessary and sufficient for the specification of adult ventral NSC (Ihrie and others 2011; Merkle and others 2014). Besides the OB, the corpus callosum also receives a few progenitors newly generated in the SVZ and differentiating to the OL lineage in part via the decrease of Su(Fu) expression induced by the TF Sox10 (Pozniak and others 2010). In the SGZ, Smo ablation in nestin+ cells (Machold and others 2003), or impairment of primary cilia (Han and others 2008) highly decrease the generation of new neurons. Hippocampal neurogenesis depends on continued Shh signaling. In the postnatal stages, the neuronal sources of Shh are found locally in the hilar cells or at distance in cells located in the enthorhinal cortex and projecting to the hippocampus (Li and others 2013a). Besides being a regulator of astrocyte-like NSC, Shh is also active in non-NSC astrocytes of the mature forebrain in vivo (Fig. 2D). Regionally distinct subsets of astrocytes receive Shh signaling likely derived from neurons indicating Shh involvement in neuron–astrocyte communication (Garcia and others 2010). Moreover, the precise titration of GliR levels by Shh is also critical for maintaining cortical astrocyte function as suggested by the astrocyte partial gliosis resulting from an increase in Gli3R (Petrova and others 2013).

Shaping the Brain via the NonCanonical Shh Pathway: Axon Guidance, Connectivity, and Activity Besides its role in the specification of progenitor cells described above, Shh pathway is also implicated in the wiring of the neural network and the modulation of its activity. The first step of these processes is axonal guidance, allowing neurons to reach their correct targets and make functional connections. Shh belongs to a novel nonconventional family of guidance cues besides the classical molecules, including Slits, semaphorins, netrins, and ephrins (Yam and Charron 2013). The first evidences of the guidance properties of Shh were shown by genetic manipulations, using the mouse developing neural tube as a model. At E11.5, just after the major wave of progenitor cell specification in the ventral part of the neural tube, Shh gradient is still used to guide the commissural axons through the Boc receptor from the dorsal to the ventral developing spinal cord (Charron and others 2003; Okada and others 2006) (Fig. 3A1). Since attraction of the axons toward Shh is fast in vitro and does not require the Gli transcription factors, the process has been related to a noncanonical Shh pathway. The intracellular molecular pathway underlying Shh-mediated axon guidance is attributed to the activity of the Src-family-kinases (SFKs) (Yam and others 2009). Indeed, SFKs are phosphorylated in a polarized manner in the growth cones of the turning axons and their pharmacological inhibition is even able to steer away growing axons (Fig. 3A4). How the spatial Shh gradient information is transmitted into the growth cone to activate SFKs, and how SFK phosphorylation mediates turning are still open questions. The originality of Shh signaling in axon guidance is that it can mediate both attraction and repulsion. After commissural axons have reached the floorplate, they cross the midline, turn anteriorly along the antero-posterior axis and switch their response to Shh from attraction to repulsion (Fig. 3A2). At this time-point, the axons are repelled anteriorly with the highest Shh concentration being posterior. The molecular mechanisms responsible for the switch appear different depending on the model. In the chick neural tube, the switch is related to the binding of Shh to HSPG Glypican1, inducing the Hip receptor expression (Wilson and Stoeckli 2013) previously identified as the receptor mediating the repulsion of postcrossing commissural axons, without requiring the Smo transducer (Bourikas and others 2005). However, Hip deficient mice do not show any projection defects and Smo is necessary for the repulsive turning of the mouse commissural neurons. In this case, the repulsive action of Shh has been attributed to the high level of expression of the 14-3-3 proteins in postcrossing commissural neurons (Yam and others 2012). Shh-related axon guidance has also been proposed for the establishment of dopaminergic


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Figure 3. Non-canonical Shh pathway regulates assembly and activity of the CNS. (A1) Precrossing commissural neurons are attracted from the dorsal to the ventral neural tube by floorplate (fp)-derived Shh. Boc and Smo mediate Shh activity. (A2) At the midline, the neurons switch 14-3-3 expression from low to high levels and are subsequently repulsed in a Smo dependent manner by the Shh gradient. Anterior (A) to posterior (P) orientation is indicated. (A3) At the level of the optic chiasm, ipsilateral retinal ganglion cells (RGCs) are repelled by Shh activity mediated by Boc in a Smo-dependent manner. (A4, right) Shh gradient activates Src-family-kinases (SFK) and induces their polarized distribution within the growth cone of pre-commissural neurons in the rodent neural tube. (A4, left) Shh activates the protein kinase C α (PKCα) and the integrin-linked kinase (ILK) to repulse RGC growth cones in the chick optic chiasm. (B1) shows 3 neuronal circuits controlled by Shh: (1) In the cerebral cortex, Shh expression in corticofugal projecting neurons in layer V directs synapse formation with Boc-expressing neurons in layers II/III. (2) Shh+ neurons of the globus pallidus (GP) project onto subthalamic nucleus (STN) neurons expressing the Ptc receptor and are proposed to inhibit the electrical activity of STN neurons. (3) Ventral midbrain (vMB) dopaminergic neurons expressing tyrosine hydroxylase (TH+) provide Shh to cholinergic (Ach+) and fast-spiking gabaergic (GABA+) interneurons in the caudate putamen (CPu), which secrete back the glial cell line–derived neurotrophic factor (GDNF). (B2) shows the main electrophysiological activities described for ShhN at the presynaptic (top) and postsynaptic (bottom) levels: (1) Shh can be released under depolarizing conditions in transfected cell lines. (2) In cultures of hippocampal neurons, Shh promotes enlarged presynaptic profiles with big synaptic vesicles (blue) and a high incidence of mitochondria (brown). Shh also increases the frequency of spontaneous miniature excitatory postsynaptic currents suggesting increased neurotransmitter release probability. (3) ShhN application onto slices comprising neuronal networks at the level of the tractus solitarius or subthalamic nuclei modifies the electrical activity of postsynaptic neurons contributing to their homeostasis.


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Ferent and Traiffort projections from the midbrain to the rostral targets during development (Hammond and others 2009). In the telencephalon, the primary cilium of migrating MGE cells transduces Shh signal expressed in their migratory pathway through a mechanism involving Ptc and Smo to orient their migration toward the cortex. However, the mechanisms by which Shh influences the organization of the microtubule cytoskeleton and the subcellular distribution of the endomembrane system in the leading process of MGE cells, is still unknown (Baudoin and others 2012). Shh also has a repulsive influence on the projections of the retinal ganglion cells (RGCs), which express the ligand by themselves (Sanchez-Arrones and others 2013; Trousse and others 2001). At the level of the optic chiasm, most of the RGCs send their projections to the contralateral side of the brain but a small number still project ipsilateraly. These non-crossing axons are repelled by Shh expressed in the chiasm (Fig. 3A3). The repellant activity is mediated by Boc, a particularly interesting observation since Boc is responsible for attraction in the neural tube (Fabre and others 2010). In the case of chick RGC, the repulsion relies on a cellular transduction that involves the protein kinase Cα and integrin-linked kinase (Fig. 3A4), as their pharmacological or genetic blockade leads to a decrease in Shh-mediated RGC growth cone collapse and repulsion (Guo and others 2012). Shh is transported anterogradely in the RGC axons, from the retina to the superior colliculus, as previously shown by tracing experiments in the adult brain using in vivo incorporation of radiolabeled methionine (Traiffort and others 2001). In RGC primary cultures, Shh is identified as a punctiform signal associated with dendrites, axons and terminals. These signals can move along neuronal processes in both directions and are associated with synaptic vesicles (Beug and others 2011). These data suggest that Shh signaling is implicated in neural activity of the visual system, but several evidences illustrate that components of the Shh pathways are also found in the whole adult CNS and may thus play a significant role in shaping and/or maintaining neuronal connectivity (Traiffort and others 2010). In the adult hippocampal neurons, Shh is present in both pre- and postsynaptic terminals, in dendrites and is often associated with vesicular structures, including dense-cored vesicles, synaptic vesicles and endosomes (Petralia and others 2011). Smo transducer has been associated with synaptic vesicles in the mossy fiber axonal terminals of the hippocampus (Traiffort and others 2010). In young developing neurons, Ptc and Smo are clustered at growth cones, while they are concentrated in dendrites, spines, and postsynaptic sites in mature neurons. Remarkably, the subcellular location of Ptc was also reported in close vicinity to autophagosomes found in normal hippocampal mature neurons. Consistently, hippocampal neurons can respond

to Shh signaling by up-regulating autophagy activity, a highly conserved degrading process that removes damaged or unnecessary proteins and organelles (Pampliega and others 2013). These data suggest that once the wiring of the brain is settled, the Shh pathway may still have an important role in synaptic function (Fig. 3B1 and 3B2). Actually, high concentrations of potassium and heparin stimulate the secretion of Shh in PC6 neuronal cell lines, showing an activity-dependent release (Beug and others, 2011). When Shh is added to the media of primary cultured hippocampal neurons, their terminals show a swelling, an increase in the size of synaptic vesicles and a significant increase in the frequency of spontaneous miniature excitatory postsynaptic currents providing evidence of Shh role in presynaptic terminals (Mitchell and others 2012). Shh also regulates the electrical activity of neurons from the nucleus tractus solitarius and the subthalamic nucleus. Sub-nanomolar concentrations of Shh are sufficient to specifically inhibit the neuronal activity in a few minutes in brain slices containing these neurons and also to induce a burst of activity in the nucleus tractus solitarius (Traiffort and others 2010). The link between neuronal activity and Shh can also be seen in the developing spinal cord. Smo-dependent Ca2+ spikes were detected at the neuronal primary cilium after Shh stimulation (Belgacem and Borodinsky 2011). All these evidences suggest that Shh may modulate synaptic activity but what can be the neuronal circuitry relying on this pathway in the brain is still unknown. The cerebral cortex is a known source of Shh that has been recently attributed to neurons localized primarily to layers 4/5 and to a lesser extent to layer 2 (Garcia and others 2010). Genetic depletion of Shh in these cells results in reduced synapse formation and dendritic growth (Harwell and others 2012). Neurons from upper layers express Boc and send either ipsilateral or contralateral projections through the corpus callosum to the Shhproducing cells. Although these data highlight a cortical circuitry depending on the Shh pathway, no functional behavior has yet been linked to the activity of these specific cortical neurons. In contrast, depleting Shh in dopaminergic neurons of the substantia nigra also identified as a source of Shh, led to a locomotion phenotype comparable to the one observed in animal models of Parkinson’s disease. The phenotype is associated with adult-onset degeneration of dopaminergic, cholinergic, and gabaergic neurons of the mesostriatal circuit and suggests that Shh maintains homeostasis in this circuit (Gonzalez-Reyes and others 2012). Altogether these observations pave the way to a whole new dimension in the field of Shh research that considers the mechanisms of the diverse non-canonical pathways and their role in brain wiring, synaptic formation and function. A major remaining challenge is the precise link between the pathway assembling and the modulation of the neural network, the elaboration of a


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The Neuroscientist

Table 1. Main Congenital CNS Disorders Targeting Directly or Indirectly Shh Signaling Components. Name

Type of Shh Dysregulation

Holoprosencephaly

Mutations in Shh, Ptch1,Cdon, Gas1, and Gli2

Smith–Lemli–Opitz syndrome

Impairments in Smo and Ptc signaling consecutive to deficient synthesis of cholesterol Overexpression of Ptc related to the triplication of the gene encoding for the amyloid precursor protein Mutations in Gli2, Shh, and Hip

Down syndrome

Hypopituitarism

Major Clinical or Histological Symptoms in the CNS

References

More or less severe defects in midline cleavage of the forebrain and craniofacial midline anomalies Global intellectual disability and large spectrum of psychomotor development

Hong and Krauss (2012), Roessler and Muenke (2010)

Intellectual disability related to widespread neurogenesis reduction

Currier and others (2012)

Pituitary hormone deficiencies

Cohen (2012), Flemming and others (2013) Cohen (2010)

Cooper and others (2003), DeBarber and others (2011)

Greig cephalopolysyndactyly Pallister–Hall syndrome

Mutation in Gli3

Macrocephaly

Mutation in Gli3

Cohen (2010)

Opitz–Kaveggia–Lujan– Fryns

Disruption of a subunit of the RNA polymerase II transcriptional Mediator, Med12, which disrupt the regular constraint it exerts on Gli3 Reduced levels of Shh expression in the hypothalamus Impaired processing of Gli3

Hypothalamus harmatoma (tissue elements normally found at that site) Agenesis/dysgenesis of the corpus callosum, macrocephaly, and craniofacial dysmorphisms Hypothalamic, pituitary, and eye defects

Zhao and others (2012)

Brain midline and facial abnormalities Multisystemic disorders, including complex malformation of the cerebellum and brainstem

Putoux and others (2011)

Brain malformation, including occipital encephalocele

Thomas and others (2012)

Septo-optic dysplasia Hydrolethalus/ acrocallosal syndromes Joubert/Meckel syndromes

Mohr–Majewski syndrome

Impairment of Shh-dependent granule cell precursor proliferation consecutive to mutations in genes encoding ciliary proteins Abnormal processing of Gli3 consecutive to the disruption of the ciliary Transition Zone Protein Tctn3

functional output and the incriminated components to pathological conditions.

Shh-Associated Pathologies The major implication of Shh signaling in brain development is obviously consistent with the existence of numerous congenital anomalies and the development of tumors associated with the dysregulation of the pathway. Many genetic disorders targeting directly or indirectly Shh signaling components have been reported (Cohen 2010). Table 1 presents a non-exhaustive list of such disorders. Holoprosencephaly (HPE) and Down syndrome (DS)

Zhou and others (2012)

Aguilar and others (2012)

have been the focus of the most recent studies. HPE is the most common malformation of the human forebrain, with an incidence of up to 1:250 during embryogenesis and a wide severity spectrum, ranging from incompatibility with extra-uterine life to isolated midline facial differences. The clinical outcome of an individual carrying an inactivating Shh pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors (Hong and Krauss 2012). In a consistent manner, the Boc mutant enhances the severity of the HPE spectrum of Cdon mutant whereas Ptc heterozygosity decreases the penetrance of HPE in Cdon mutant mice. In addition, a


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Ferent and Traiffort dramatic synergy exists between Cdon mutation and ethanol administration in mice. The disruption of the pathway may also be the consequence of mutations found in other signaling pathways. For instance, Tgif mutations contributes to HPE phenotype in mouse as a consequence of Shh pathway disruption in the forebrain and Gli3 upregulation throughout the neural tube (Taniguchi and others 2012). DS, because of triplication of chromosome 21, is among the most frequent genetic causes of intellectual disability. The widespread neurogenesis reduction underlying the numerous neurological disabilities of DS has been related to Shh defect via Ptc overexpression. The latter is likely the result of increased levels of the amyloid precursor protein known to regulate neurogenesis and neurite length through Shh pathway (Trazzi and others 2013). The Ts65Dn mouse strain recapitulates not only some major brain structural and behavioral phenotypes of DS, including reduced size and cellularity of the cerebellum, but also learning deficits associated with the hippocampus. This model led to demonstrate that a single exposure of mutant animals on the day of birth to the small-molecule Smo agonist, SAG, is sufficient to normalize cerebellar morphology and restore learning ability associated with hippocampal function suggesting a possible direction for therapeutic intervention in patients (Currier and others 2012; Das and others 2013). Inappropriate activation of Shh signaling has been related to medulloblastoma, the most common malignant brain tumor of childhood, and to glioma, the most common primary tumors in the adult brain (Li and others 2009; Northcott and others 2012). Medulloblastoma arise from deregulated cerebellar development linked in particular to mutations in Ptc, Su(Fu) or Smo. The tumor was initially associated with Gorlin syndrome, an inherited disorder characterized by Ptc-inactivating mutations. Cells targeted by oncogenic Shh signaling have been initially restricted to the GCP lineage, although more recently, a population of nestin-expressing progenitors residing in the deep part of the EGL were found to exhibit more severe genomic instability, which could also potentially give rise to tumors (Li and others 2013b). The Smo antagonist, GDC-0449 from Genentech Company, was used in a clinical trial, in a patient affected by a metastatic medulloblastoma refractory to multiple therapies. Despite its initial therapeutic efficiency, drug resistance rapidly occurred, because of an acquired Smo mutation disrupting the ability of GDC-0449 to bind Smo (Ng and Curran 2011; Northcott and others 2012). Besides Smo, several other possible therapeutic targets are currently being identified inciting to develop novel approaches such as agents disrupting the primary cilia (Barakat and others 2013b) or blocking the death dependence receptor Neogenin1 characterized as a Shh downstream effector in neural precursor proliferation (Milla and others 2014).

Endothelial cells and astrocytes were proposed as a Shhproducing microenvironment in the perivascular niche of mouse glioma models. In human oligodendroglioma and astrocytoma, a neuronal source for Shh was also suggested. Putative glioma cancer stem cells could derive from endogenous Gli1+ cells, including those present in the stem cell niches (Li and others 2009). Together with the existence of glioma cancer stem cells, the resistance of gliomas to current therapies, and the role of Gli signaling in regulating expression of stemness genes and selfrenewal of these cells, molecules able to block Gli signaling should offer new therapeutic possibilities (Ng and Curran 2011). Moreover, a combination of inhibitors of Shh and PI3K signaling pathways, which have now entered clinical trials were shown to result in mitotic catastrophe and tumor apoptosis, markedly reducing the growth of specific types of glioblastomas in vitro and in vivo (Gruber Filbin and others 2013). Many experimental data suggest that activation of the Shh signaling pathway may constitute a potential therapeutic approach for diverse CNS degenerative pathologies (Traiffort and others 2010). This was first shown for various models of Parkinson’s disease, a model of facial nerve axotomy and a model of neonatal mouse brain injury induced by glucocorticoid administration (for review, Traiffort and others, 2010). More recently, Shh was also proposed to induce neurogenesis, angiogenesis, and oligodendrogenesis, which are major brain repair processes during stroke recovery (He and others 2013; Zhang and others 2013a). The protein is endowed with antioxidative effects in the model of amyotrophic lateral sclerosis based on overexpression of G93A superoxide dismutase 1 (SOD1) mutant (Peterson and Turnbull 2012). Finally, Shh signaling modulation is proposed as a potential novel approach for demyelinating diseases (Ferent and others 2013a; Traiffort and others 2010). On lysolecithin-mediated focal demyelination of the corpus callosum, Shh secreted by cells of the OL lineage, is a required factor to improve remyelination and decrease the inflammatory microenvironment (Ferent and others 2013b). Consistently, Sox17 promotes the generation of OL cell lineage by preventing Gli2 expression decrease in demyelinating lesions (Ming and others 2013). Shhmediated OL differentiation involves the modulation of OL transcriptome likely by decreasing histone acetylation (Wu and others 2012). Strikingly, at the level of the blood-brain barrier, perivascular astrocytes involved in CNS homeostasis secrete Shh which acts on adjacent blood-brain barrier–endothelial cells and provides a barrier-promoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attacks, as occurs in multiple sclerosis (Alvarez and others 2011). Shh is considered as an important regulator of specific astrocyte populations in the normal (Garcia and others 2010) and


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The Neuroscientist

Figure 4. Canonical and non-canonical Shh pathways drive distinct types of developmental and maintenance processes in the CNS.

injured (Sirko and others 2013) brain. Attenuation of Shh signaling in postnatal astrocytes by targeted removal of Smo results in up-regulation of GFAP labeling and cellular hypertrophy that characterizes astrogliosis in the cortex (Fig. 2D). Moreover, Shh constitutes the signal that acts directly on reactive astrocytes to trigger the mechanisms leading these cells to form self-renewing and multipotent neurospheres and thus to display a stem cell response which will promote the repair process (Sirko and others 2013). All these observations highlight the need for developing specific and potent pharmacological agents in order to manipulate the Shh pathway. Most of the time, the pharmacological target is the Smo transducer. Several in vivo experiments and even clinical assays reveal promising outcomes. Nevertheless, the selectivity of the small molecules toward the canonical or non-canonical pathways which involve Smo, is likely a crucial point. Similarly, the existence of ciliary and non-ciliary pools of Smo may be important to consider in order to evaluate the consequences of Smo inhibition or activation in the whole organism. These considerations open the way to the identification of relevant alternative targets among which the transcription factors Gli (Ng and Curran 2011), Shh-Cdon interaction which may trigger Cdon-dependent apoptosis (Delloye-Bourgeois and others 2013), the nucleocytoplasmic distribution of Su(Fu) (Tariki and others 2013) or the enzyme transferring palmitoyl to Hh (Petrova and others 2014). Further solid evidence should now be provided regarding the potential clinical benefit for the use of such molecules.

Concluding Remarks Hh signaling has yet provided fundamental insights into several areas of developmental and cell biology and will undoubtedly deliver many other unexpected observations. While the canonical pathway seems to be linked more to the proliferation and differentiation of neural progenitors, the non-canonical pathways are mostly implicated in the network formation and activity (Fig. 4). The simple existence of such a variety of different molecular pathways linked to the binding of only one ligand opens many questions. For instance, how does the cell understand which transduction mechanism should be activated when the binding of Shh occurs? Even at the binding level, the complexity of the receptors is intriguing. Canonical or non-canonical: Which way to go? Moreover, what type of behaviors may rely on the different Shh activity-dependent neural networks in the brain? Addressing these issues is crucial for understanding the mechanism resulting in the dysregulation of the pathway and for developing therapeutic approaches that will target the medical consequences of such dysregulations. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.


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Ferent and Traiffort References Aguilar A, Meunier A, Martinovic J, Bonniere M, Attie-Bitach, Encha-Razavi F, and others. 2012. Analysis of human samples reveals imparired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome. Proc Natl Acad Sci USA 109(42):16951–6. Ahn S, Joyner AL. 2005. In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature 437(7060):894–7. Alvarez-Bolado G, Paul FA, Blaess S. 2012. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions. Neural Dev 7:4. Alvarez JI, Dodelet-Devillers A, Kebir H, Ifergan I, Fabre PJ, Terouz S, and others. 2011. The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334(6063):1727–31. Angot E, Loulier K, Nguyen-Ba-Charvet KT, Gadeau AP, Ruat M, Traiffort E. 2008. Chemoattractive activity of sonic hedgehog in the adult subventricular zone modulates the number of neural precursors reaching the olfactory bulb. Stem Cells 26(9):2311–20. Balaskas N, Ribeiro A, Panovska J, Dessaud E, Sasai N, Page KM, and others. 2012. Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube. Cell 148(1-2):273–84. Balordi F, Fishell G. 2007. Mosaic removal of hedgehog signaling in the adult SVZ reveals that the residual wild-type stem cells have a limited capacity for self-renewal. J Neurosci 27(52):14248–59. Barakat B, Yu L, Lo C, Vu D, De Luca E, Cain JE, and others. 2013a. Interaction of smoothened with integrin-linked kinase in primary cilia mediates Hedgehog signalling. EMBO Rep 14(9):837–44. Barakat MT, Humke EW, Scott MP. 2010. Learning from Jekyll to control Hyde: Hedgehog signaling in development and cancer. Trends Mol Med 16(8):337–48. Barakat MT, Humke EW, Scott MP. 2013b. Kif3a is necessary for initiation and maintenance of medulloblastoma. Carcinogenesis 34(6):1382–92. Baudoin JP, Viou L, Launay PS, Luccardini C, Espeso Gil S, Kiyasova V, and others. 2012. Tangentially migrating neurons assemble a primary cilium that promotes their reorientation to the cortical plate. Neuron 76(6):1108–22. Belgacem YH, Borodinsky LN. 2011. Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord. Proc Natl Acad Sci U S A 108(11): 4482–7. Beug ST, Parks RJ, McBride HM, Wallace VA. 2011. Processing-dependent trafficking of Sonic hedgehog to the regulated secretory pathway in neurons. Mol Cell Neurosci 46(3):583–96. Bijlsma MF, Damhofer H, Roelink H. 2012. Hedgehogstimulated chemotaxis is mediated by smoothened located outside the primary cilium. Sci Signal 5(238):ra60. Bourikas D, Pekarik V, Baeriswyl T, Grunditz A, Sadhu R, Nardo M, and others. 2005. Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat Neurosci 8(3):297–304.

Bouslama-Oueghlani L, Wehrle R, Doulazmi M, Chen XR, Jaudon F, Lemaigre-Dubreuil Y, and others. 2012. Purkinje cell maturation participates in the control of oligodendrocyte differentiation: role of sonic hedgehog and vitronectin. PLoS One 7(11):e49015. Briscoe J, Thérond PP. 2013. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14(7):416–29. Carney RS, Mangin JM, Hayes L, Mansfield K, Sousa VH, Fishell G, and others. 2010. Sonic hedgehog expressing and responding cells generate neuronal diversity in the medial amygdala. Neural Dev 5:14. Chamberlain CE, Jeong J, Guo C, Allen BL, McMahon AP. 2008. Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning. Development 135(6):1097–106. Chamoun Z, Mann RK, Nellen D, von Kessler DP, Bellotto M, Beachy PA, and others. 2001. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science 293(5537):2080–4. Charron F, Stein E, Jeong J, McMahon AP, Tessier-Lavigne M. 2003. The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 113(1):11–23. Chen Y, Jiang J. 2013. Decoding the phosphorylation code in Hedgehog signal transduction. Cell Res 23(2):186–200. Chuang PT, Kawcak T, McMahon AP. 2003. Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung. Genes Dev 17(3):342–7. Chung AY, Kim S, Kim E, Kim D, Jeong I, Cha YR, and others. 2013. Indian hedgehog B function is required for the specification of oligodendrocyte progenitor cells in the zebrafish CNS. J Neurosci 33(4):1728–33. Cohen M, Briscoe J, Blassberg R. 2013. Morphogen interpretation: the transcriptional logic of neural tube patterning. Curr Opin Genet Dev 23(4):423–8. Cohen MM Jr. 2010. Hedgehog signaling update. Am J Med Genet A 152A(8):1875–914. Cohen LE. 2012. Gli2 mutations as a cause of hypopituitarism. Pediatr Endocrinol Rev 9(4):706–9. Cooper MK, Wassif CA, Krakowiak PA, Taipale J, Gong R, Kelley RI, and others. 2003. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 33(4):508–13. Corallo D, Schiavinato A, Trapani V, Moro E, Argenton F, Bonaldo P. 2013. Emilin3 is required for notochord sheath integrity and interacts with Scube2 to regulate notochordderived Hedgehog signals. Development 140(22):4594–601. Currier DG, Polk RC, Reeves RH. 2012. A Sonic hedgehog (Shh) response deficit in trisomic cells may be a common denominator for multiple features of Down syndrome. Prog Brain Res 197:223–36. Das I, Park JM, Shin JH, Jeon SK, Lorenzi H, Linden DJ, and others. 2013. Hedgehog agonist therapy corrects structural and cognitive deficits in a Down syndrome mouse model. Sci Transl Med 5(201):201ra120.


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Dave RK, Ellis T, Toumpas MC, Robson JP, Julian E, Adolphe C, and others. 2011. Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors. PLoS One 6(2):e14680. DeBarber AE, Eroglu Y, Merkens LS, Pappu AS, Steiner RD. 2011. Smith-Lemli-Opitz syndrome. Expert Rev Mol Med 13:e24. Delloye-Bourgeois C, Gibert B, Rama N, Delcros JG, Gadot N, Scoazec JY, and others. 2013. Sonic Hedgehog promotes tumor cell survival by inhibiting CDON pro-apoptotic activity. PLoS Biol 11(8):e1001623. Eaton S. 2008. Multiple roles for lipids in the Hedgehog signalling pathway. Nat Rev Mol Cell Biol 9(6):437–45. Epstein DJ. 2012. Regulation of thalamic development by sonic hedgehog. Front Neurosci 6:57. Fabre P, Shimogori T, Charron F. 2010. Segregation of ipsilateral retinal ganglion cell axons at the optic chiasm requires the Shh receptor Boc. J Neurosci 30(1):266–75. Ferent J, Ruat M, Traiffort E. 2013a. Investigation of the proteolipid protein promoter activity during demyelination and repair. Differentiation 85(4-5):182–9. Ferent J, Zimmer C, Durbec P, Ruat M, Traiffort E. 2013b. Sonic hedgehog signaling is a positive oligodendrocyte regulator during demyelination. J Neurosci 33(5):1759–72. Flemming GM, Klammt J, Ambler G, Bao Y, Blum WF, Cowell C, and others. 2013. Functional characterization of a heterozygous GLI2 missense mutation in patients with multiple pituitary hormone deficiency. J Clin Endocrinol Metab 98(3):E567–75. Fuccillo M, Joyner AL, Fishell G. 2006. Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development. Nat Rev Neurosci 7(10):772–83. Garcia AD, Petrova R, Eng L, Joyner AL. 2010. Sonic hedgehog regulates discrete populations of astrocytes in the adult mouse forebrain. J Neurosci 30(41):13597–608. Gonzalez-Reyes LE, Verbitsky M, Blesa J, Jackson-Lewis V, Paredes D, Tillack K, and others. 2012. Sonic hedgehog maintains cellular and neurochemical homeostasis in the adult nigrostriatal circuit. Neuron 75(2):306–19. Gruber Filbin M, Dabral SK, Pazyra-Murphy MF, Ramkissoon S, Kung AL, Pak E, and others. 2013. Coordinate activation of Shh and PI3K signaling in PTEN-deficient glioblastoma: new therapeutic opportunities. Nat Med 19(11):1518–23. Guo D, Standley C, Bellve K, Fogarty K, Bao ZZ. 2012. Protein kinase C-α and integrin-linked kinase mediate the negative axon guidance effects of Sonic hedgehog. Mol Cell Neurosci 50(1):82–92. Haddad-Tovolli R, Heide M, Zhou X, Blaess S, Alvarez-Bolado G. 2012. Mouse thalamic differentiation: gli-dependent pattern and gli-independent prepattern. Front Neurosci 6:27. Hammond R, Blaess S, Abeliovich A. 2009. Sonic hedgehog is a chemoattractant for midbrain dopaminergic axons. PLoS One 4(9):e7007. Han YG, Spassky N, Romaguera-Ros M, Garcia-Verdugo JM, Aguilar A, Schneider-Maunoury S, and others. 2008. Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells. Nat Neurosci 11(3):277–84.

Harwell CC, Parker PR, Gee SM, Okada A, McConnell SK, Kreitzer AC, and others. 2012. Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation. Neuron 73(6):1116–26. Hayes L, Ralls S, Wang H, Ahn S. 2013. Duration of Shh signaling contributes to mDA neuron diversity. Dev Biol 374(1):115–26. He QW, Xia YP, Chen SC, Wang Y, Huang M, Huang Y, and others. 2013. Astrocyte-derived sonic hedgehog contributes to angiogenesis in brain microvascular endothelial cells via RhoA/ROCK pathway after oxygen-glucose deprivation. Mol Neurobiol 47(3):976–87. Holtz AM, Peterson KA, Nishi Y, Morin S, Song JY, Charron F, and others. 2013. Essential role for ligand-dependent feedback antagonism of vertebrate hedgehog signaling by PTCH1, PTCH2 and HHIP1 during neural patterning. Development 140(16):3423–34. Hong M, Krauss RS. 2012. Cdon mutation and fetal ethanol exposure synergize to produce midline signaling defects and holoprosencephaly spectrum disorders in mice. PLoS Genet 8(10):e1002999. Huang S, Zhang Z, Zhang C, Lv X, Zheng X, Chen Z, and others. 2013. Activation of Smurf E3 ligase promoted by smoothened regulates hedgehog signaling through targeting patched turnover. PLoS Biol 11(11):e1001721. Huang X, Liu J, Ketova T, Fleming JT, Grover VK, Cooper MK, and others. 2010. Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development. Proc Natl Acad Sci U S A 107(18):8422–7. Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV. 2003. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426(6962):83–7. Ihrie RA, Shah JK, Harwell CC, Levine JH, Guinto CD, Lezameta M, and others. 2011. Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity. Neuron 71(2):250–62. Izzi L, Levesque M, Morin S, Laniel D, Wilkes BC, Mille F, and others. 2011. Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shhmediated cell proliferation. Dev Cell 20(6):788–801. Kovacs JJ, Whalen EJ, Liu R, Xiao K, Kim J, Chen M, and others. 2008. β-arrestin-mediated localization of Smoothened to the primary cilium. Science 320(5884):1777–81. Larkins CE, Aviles GD, East MP, Kahn RA, Caspary T. 2011. Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins. Mol Biol Cell 22(23):4694–703. Li G, Fang L, Fernandez G, Pleasure SJ. 2013a. The ventral hippocampus is the embryonic origin for adult neural stem cells in the dentate gyrus. Neuron 78(4):658–72. Li P, Du F, Yuelling LW, Lin T, Muradimova RE, Tricarico R, and others. 2013b. A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity. Nat Neurosci 16(12):1737–44. Li Z, Wang H, Eyler CE, Hjelmeland AB, Rich JN. 2009. Turning cancer stem cells inside out: an exploration of glioma stem cell signaling pathways. J Biol Chem 284(25):16705–9.


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Ferent and Traiffort Machold R, Hayashi S, Rutlin M, Muzumdar MD, Nery S, Corbin JG, and others. 2003. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 39(6):937–50. Magnani D, Hasenpusch-Theil K, Jacobs EC, Campagnoni AT, Price DJ, Theil T. 2010. The Gli3 hypomorphic mutation Pdn causes selective impairment in the growth, patterning, and axon guidance capability of the lateral ganglionic eminence. J Neurosci 30(41):13883–94. Marazziti D, Di Pietro C, Golini E, Mandillo S, La Sala G, Matteoni R, and others. 2013. Precocious cerebellum development and improved motor functions in mice lacking the astrocyte cilium-, patched 1-associated Gpr37l1 receptor. Proc Natl Acad Sci U S A 110(41):16486–91. Martynova NY, Ermolina LV, Ermakova GV, Eroshkin FM, Gyoeva FK, Baturina NS, and others. 2013. The cytoskeletal protein Zyxin inhibits Shh signaling during the CNS patterning in Xenopus laevis through interaction with the transcription factor Gli1. Dev Biol 380(1):37–48. Merkle FT, Fuentealba LC, Sanders TA, Magno L, Kessaris N, Alvarez-Buylla A. 2014. Adult neural stem cells in distinct microdomains generate previously unknown interneuron types. Nat Neurosci 17(2):207–14. Milla LA, Arros A, Espinoza N, Remke M, Kool M, Taylor MD, and others. 2014. Neogenin1 is a Sonic Hedgehog target in medulloblastoma and is necessary for cell cycle progression. Int J Cancer 134(1):21–31. Ming X, Chew LJ, Gallo V. 2013. Transgenic overexpression of Sox17 promotes oligodendrocyte development and attenuates demyelination. J Neurosci 33(30):12528–42. Mitchell N, Petralia RS, Currier DG, Wang YX, Kim A, Mattson MP, and others. 2012. Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons. J Cell Sci 125(Pt 18):4207–13. Moreno-Bravo JA, Perez-Balaguer A, Martinez-Lopez JE, Aroca P, Puelles L, Martinez S, and others. 2013. Role of Shh in the development of molecularly characterized tegmental nuclei in mouse rhombomere 1. Brain Struct Funct. Epub Mar 14. doi:10.1007/s00429-013-0534-6. Mukhopadhyay S, Wen X, Ratti N, Loktev A, Rangell L, Scales SJ, and others. 2013. The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 152(1-2):210–23. Ng JM, Curran T. 2011. The Hedgehog’s tale: developing strategies for targeting cancer. Nat Rev Cancer 11(7):493–501. Nielsen CM, Dymecki SM. 2010. Sonic hedgehog is required for vascular outgrowth in the hindbrain choroid plexus. Dev Biol 340(2):430–7. Niewiadomski P, Zhujiang A, Youssef M, Waschek JA. 2013. Interaction of PACAP with Sonic hedgehog reveals complex regulation of the hedgehog pathway by PKA. Cell Signal 25(11):2222–30. Nissim-Eliraz E, Zisman S, Schatz O, Ben-Arie N. 2013. Nato3 integrates with the Shh-Foxa2 transcriptional network regulating the differentiation of midbrain dopaminergic neurons. J Mol Neurosci 51(1):13–27. Northcott PA, Jones DT, Kool M, Robinson GW, Gilbertson RJ, Cho YJ, and others. 2012. Medulloblastomics: the end of the beginning. Nat Rev Cancer 12(12):818–34.

Nusslein-Volhard C, Wieschaus E. 1980. Mutations affecting segment number and polarity in Drosophila. Nature 287(5785):795–801. Okada A, Charron F, Morin S, Shin DS, Wong K, Fabre PJ, and others. 2006. Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature 444(7117):369–73. Ortega JA, Radonjic NV, Zecevic N. 2013. Sonic hedgehog promotes generation and maintenance of human forebrain Olig2 progenitors. Front Cell Neurosci 7:254. Palm W, Swierczynska MM, Kumari V, Ehrhart-Bornstein M, Bornstein SR, Eaton S. 2013. Secretion and signaling activities of lipoprotein-associated hedgehog and non-sterol-modified hedgehog in flies and mammals. PLoS Biol 11(3):e1001505. Pampliega O, Orhon I, Patel B, Sridhar S, Diaz-Carretero A, Beau I, and others. 2013. Functional interaction between autophagy and ciliogenesis. Nature 502(7470):194–200. Peterson R, Turnbull J. 2012. Sonic hedgehog is cytoprotective against oxidative challenge in a cellular model of amyotrophic lateral sclerosis. J Mol Neurosci 47(1):31–41. Petralia RS, Wang YX, Mattson MP, Yao PJ. 2011. Sonic hedgehog distribution within mature hippocampal neurons. Commun Integr Biol 4(6):775–7. Petrova E, Matevossian A, Resh MD. 2014. Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma. Oncogene. Epub Jan 27. doi:10.1038/onc.2013.575. Petrova R, Garcia AD, Joyner AL. 2013. Titration of GLI3 repressor activity by sonic hedgehog signaling is critical for maintaining multiple adult neural stem cell and astrocyte functions. J Neurosci 33(44):17490–505. Porter JA, Young KE, Beachy PA. 1996. Cholesterol modification of hedgehog signaling proteins in animal development. Science 274(5285):255–9. Pozniak CD, Langseth AJ, Dijkgraaf GJ, Choe Y, Werb Z, Pleasure SJ. 2010. Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression. Proc Natl Acad Sci U S A 107(50): 21795–800. Putoux A, Thomas S, Coene KL, Davis EE, Alalnay Y, Ogur G, and others. 2011. KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes. Nat Genet 43(6):601–6. Reinchisi G, Parada M, Lois P, Oyanadel C, Shaughnessy R, Gonzalez A, and others. 2013. Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation. Front Cell Neurosci 7:166. Robbins DJ, Fei DL, Riobo NA. 2012. The Hedgehog signal transduction network. Sci Signal 5(246):re6. Roessler E, Muenke M. 2010. The molecular genetics of holoprosencephaly. Am J Med Genet C Semin Med Genet 154C(1):56–61. Rohatgi R, Milenkovic L, Scott MP. 2007. Patched1 regulates hedgehog signaling at the primary cilium. Science 317(5836):372–6. Sanchez-Arrones L, Nieto-Lopez F, Sanchez-Camacho C, Carreres MI, Herrera E, Okada A, and others. 2013. Shh/ Boc signaling is required for sustained generation of ipsilateral projecting ganglion cells in the mouse retina. J Neurosci 33(20):8596–607.


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The Neuroscientist

Sanders TA, Llagostera E, Barna M. 2013. Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature 497(7451):628–32. Shikata Y, Okada T, Hashimoto M, Ellis T, Matsumaru D, Shiroishi T, and others. 2011. Ptch1-mediated dosagedependent action of Shh signaling regulates neural progenitor development at late gestational stages. Dev Biol 349(2):147–59. Sirko S, Behrendt G, Johansson PA, Tripathi P, Costa M, Bek S, and others. 2013. Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected]. Cell Stem Cell 12(4):426–39. Sousa VH, Fishell G. 2010. Sonic hedgehog functions through dynamic changes in temporal competence in the developing forebrain. Curr Opin Genet Dev 20(4):391–9. Spassky N, Han YG, Aguilar A, Strehl L, Besse L, Laclef C, and others. 2008. Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool. Dev Biol. 317(1):246–59. Su CY, Bay SN, Mariani LE, Hillman MJ, Caspary T. 2012. Temporal deletion of Arl13b reveals that a mispatterned neural tube corrects cell fate over time. Development 139(21):4062–71. Tang M, Luo SX, Tang V, Huang EJ. 2013. Temporal and spatial requirements of Smoothened in ventral midbrain neuronal development. Neural Dev 8:8. Taniguchi K, Anderson AE, Sutherland AE, Wotton D. 2012. Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway. PLoS Genet 8(2):e1002524. Tariki M, Wieczorek SA, Schneider P, Banfer S, Veitinger S, Jacob R, and others. 2013. RIO kinase 3 acts as a SUFUdependent positive regulator of Hedgehog signaling. Cell Signal 25(12):2668–75. Thomas S, Legendre M, Saunier S, Bessières B, Alby C, Bonnière M, and others. 2012. TCTN3 mutations cause Mohr-Majewski syndrome. Am J Hum Genet 91(2): 372–8. Touahri Y, Escalas N, Benazeraf B, Cochard P, Danesin C, Soula C. 2012. Sulfatase 1 promotes the motor neuron-tooligodendrocyte fate switch by activating Shh signaling in Olig2 progenitors of the embryonic ventral spinal cord. J Neurosci 32(50):18018–34. Traiffort E, Angot E, Ruat M. 2010. Sonic Hedgehog signaling in the mammalian brain. J Neurochem 113(3):576–90. Traiffort E, Moya KL, Faure H, Hassig R, Ruat M. 2001. High expression and anterograde axonal transport of aminoterminal sonic hedgehog in the adult hamster brain. Eur J Neurosci 14(5):839–50. Trazzi S, Fuchs C, Valli E, Perini G, Bartesaghi R, Ciani E. 2013. The amyloid precursor protein (APP) triplicated gene impairs neuronal precursor differentiation and neurite development through two different domains in the Ts65Dn mouse model for Down syndrome. J Biol Chem 288(29):20817–29. Trousse F, Marti E, Gruss P, Torres M, Bovolenta P. 2001. Control of retinal ganglion cell axon growth: a new role for Sonic hedgehog. Development 128(20):3927–36.

Wada Y, Yamauchi K, Murakami F, Tanabe Y. 2012. Temporally- and spatially regulated generation of distinct descendants by sonic hedgehog-expressing progenitors in the forebrain. Dev Neurobiol 72(8):1099–113. Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W, and others. 2013. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497(7449):338–43. Wang Y, Martin JF, Bai CB. 2010. Direct and indirect requirements of Shh/Gli signaling in early pituitary development. Dev Biol 348(2):199–209. Wilson NH, Stoeckli ET. 2013. Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner. Neuron 79(3):478–91. Witt RM, Hecht ML, Pazyra-Murphy MF, Cohen SM, Noti C, van Kuppevelt TH, and others. 2013. Heparan sulfate proteoglycans containing a glypican 5 core and 2-O-sulfoiduronic acid function as Sonic Hedgehog co-receptors to promote proliferation. J Biol Chem 288(36):26275–88. Wu M, Hernandez M, Shen S, Sabo JK, Kelkar D, Wang J, and others. 2012. Differential modulation of the oligodendrocyte transcriptome by sonic hedgehog and bone morphogenetic protein 4 via opposing effects on histone acetylation. J Neurosci 32(19):6651–64. Xiong F, Tentner AR, Huang P, Gelas A, Mosaliganti KR, Souhait L, and others. 2013. Specified neural progenitors sort to form sharp domains after noisy Shh signaling. Cell 153(3):550–61. Yam PT, Charron F. 2013. Signaling mechanisms of non-conventional axon guidance cues: the Shh, BMP and Wnt morphogens. Curr Opin Neurobiol 23(6):965–73. Yam PT, Kent CB, Morin S, Farmer WT, Alchini R, Lepelletier L, and others. 2012. 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing. Neuron 76(4): 735–49. Yam PT, Langlois SD, Morin S, Charron F. 2009. Sonic hedgehog guides axons through a noncanonical, Src-familykinase-dependent signaling pathway. Neuron 62(3):349–62. Yu K, McGlynn S, Matise MP. 2013. Floor plate-derived sonic hedgehog regulates glial and ependymal cell fates in the developing spinal cord. Development 140(7):1594–604. Zhang R, Chopp M, Zhang ZG. 2013a. Oligodendrogenesis after cerebral ischemia. Front Cell Neurosci 7:201. Zhang Y, Fu L, Qi X, Zhang Z, Xia Y, Jia J, and others. 2013b. Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci. Nat Commun 4:2608. Zhao L, Zevallos SE, Rizzoti K, Jeong Y, Lovell-Badge R, Epstein DJ. 2012. Disruption of SoxB1-dependent Sonic Hedgehog expression in the hypothalamus causes septooptic dysplasia. Dev Cell 22(3):585–96. Zhao Y, Tong C, Jiang J. 2007. Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450(7167):252–8. Zhou H, Spaeth JM, Kim NH, Xu X, Friez MJ, Schwartz CE, and others. 2012. MED12 mutations link intellectual disability syndromes with dysregulated GLI3-dependent Sonic Hedgehog signaling. Proc Natl Acad Sci U S A 109(48):19763–8.


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