Primary Cilium and Sonic Hedgehog Signaling During Neural Tube Patterning Role of GPCRs and Second Messengers Kasturi Pal, Saikat Mukhopadhyay Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas 75390 Received 24 February 2014; revised 9 May 2014; accepted 22 May 2014

ABSTRACT: The ventral neural tube in vertebrates is patterned by a gradient of sonic hedgehog (Shh) secreted from the notochord and floor plate. Forward genetic screens first pointed to the role of the primary cilium in ventral neural tube patterning. Further research has shown that most components of the Shh pathway localize to or shuttle through the primary cilium. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA)- and cilium-dependent manner. Recent work suggests that the orphan G-protein-coupled receptor (GPCR) Gpr161 localizes to cilia, and functions INTRODUCTION In this review, we discuss our current understanding of the role of signal transduction pathways important in sonic hedgehog (Shh) signaling, especially in the context of vertebrate neural tube development. We begin by describing the role of Shh in patterning of the ventral neural tube. Next, we discuss the discovery of the role of primary cilium in Shh-dependent neural tube patterning. After providing a quick overview of the current understanding of the molecular mechanisms that regulate Shh signaling in the context of the primary cilium, we provide a detailed description of PKA-dependent Gli repressor (GliR) processing. We Correspondence to: S. Mukhopadhyay (saikat.mukhopadhyay@ utsouthwestern.edu). Contract grant sponsor: UT Southwestern Medical Center and first-time tenure-track faculty recruitment funds from CPRIT (R1220; SM). Ó 2014 Wiley Periodicals, Inc. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/dneu.22193

as a negative regulator of Shh signaling by determining Gli processing via cAMP signaling. The primary cilium also functions as a signaling compartment for calcium in the Shh pathway. A better understanding of the role of the cilium as a signaling compartment, and the interplay of second messenger systems that regulate PKA activation and Gli amplification during signaling is critical for deciphering the role of Shh during development, neuronal differentiation, and tumorigenesis. VC 2014 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2014

Keywords: hedgehog; primary cilium; G-proteincoupled receptor; protein kinase A; neural tube

next discuss the role of the novel ciliary G-proteincoupled receptor (GPCR), Gpr161 in GliR processing during neural tube development. We also describe other GPCRs that impinge upon global PKA activity in regulating the output of Shh signaling. Next, we consider the role of cilia in compartmentalization of calcium signals during Shh signaling. Other reviews have extensively discussed the role of the primary cilium and Hh signaling during neuronal development (Goetz and Anderson, 2010; Louvi and Grove, 2011). Here, we focus on the interplay of GPCRs and second messenger pathways that regulate PKA activation and Gli amplification during Shh signaling and in the pathogenesis of Shh-dependent tumors such as medulloblastoma and basal cell carcinoma (BCC).

ROLE OF SONIC HEDGEHOG IN VERTEBRATE NEURAL TUBE PATTERNING The vertebrate central nervous system is derived from the neural plate, a sheet of columnar epithelial 1

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Figure 1 Shh-mediated embryonic neural tube patterning. Left, the Shh morphogen gradient is initially established by secretion from the notochord (N). Eventually, the cells of FP replace the notochord as the major source of Shh. Shh acts in a time- and concentration-dependent manner to specify five distinct domains of cell fate along the neural tube (p3, pMN, p1, p2, and p0). Spatially expressed genetic markers are known to exist for each of these domains, which have been represented on the right. While Shh-induced markers are expressed more ventrally, Shh-repressed markers are more dorsally located in areas of lowest Shh concentration. Right, the transcriptional gene regulatory network (GRN) for interpretation of Shh gradient by cells of the developing neural tube (stimulatory responses are marked as arrows, and inhibitory responses are in red). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

tissue in the dorsal region of the developing embryo. At the end of gastrulation, the neural plate invaginates to form the neural tube, which eventually undergoes dorso-ventral (D-V) patterning to generate distinct zones of neural progenitor cells. The key pathways that establish these discrete cellular zones are Shh-, Wnt-, and bone morphogenetic protein (BMP)-dependent. While Wnt and BMP are engaged in specifying the dorsal fates, a Shh morphogen gradient is involved in ventral fate specification (Yamada et al., 1993; Chiang et al., 1996). The ventrally located notochord is the primary source of graded Shh. In later stages of development, the notochord regresses and is replaced by the neural floor plate as the major source of localized Shh gradient. The Shh gradient initiates ventral patterning of neural tube, giving rise to six different domains of neural progenitors: floor plate (FP), p3, pMN, p2, p1, and p0 (Fig. 1). Tight patterning of these progenitor domains is essential for generating distinctive neuronal subtypes such as motor neurons and interneurons (Ericson et al., 1997). In the absence of Shh signaling, ventral neural cell fates are lost (Chiang et al., 1996). The progenitors acquire different spatial identities along the D-V axis of the neural tube due to a complex interplay of transcription factors, which are expressed in response to variations in Shh levels, as well as the duration for which they are exposed to the morphogen. Na€ıve explants of neural tube Developmental Neurobiology

exposed to Shh leads to sequential expression of oligodendrocyte transcription factor 2 (Olig2) and NK2 homeobox 2 (Nkx2.2), with Olig2 preceding Nkx2.2 expression. Nkx2.2 induction requires a higher concentration of Shh than Olig2. On the contrary, Shh inhibits Pax7, whose expression domain is restricted to the extreme dorsal cells of the neural tube. Thus higher concentrations and duration of exposure to Shh ensures more ventral fate (Dessaud et al., 2007) (Fig. 1). Reporter assays carried out in the intermediate neural plate explants show that this temporal and concentration-dependent expression of transcription factors is due to progressive desensitization of cells in response to sustained high concentration of Shh signaling. The adaptation results due to expression of Patched-1 (Ptch1), which inhibits signaling in absence of the ligand. Ptch1 is a 12 transmembrane domain protein that is the major receptor for hedgehog (Hh) ligands, and a transcriptional target of the Shh pathway. Thus, prolonged Shh signaling leads to enhanced Ptch1 expression in areas of highest concentration of the morphogen, suppressing further signaling by the pathway (Dessaud et al., 2007). Alternatively, in a non-cell autonomous way, Ptch1 expression leads to localized binding of Shh to cells overexpressing Ptch1, thus restricting the availability of the former in the more dorsal domains of the neural tube (Chamberlain et al., 2008). A negative feedback loop thus transduces an extracellular morphogen

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concentration into varying levels of intracellular transcriptional activity. A clear picture of how neural progenitors convert the extracellular dynamic Shh gradient to spatial expression of neuronal markers comes from tracking downstream Shh signaling in vivo. Using mathematical modeling, a transgenic reporter system, and monitoring Ptch1 expression levels in vivo, it has been established that spatial and temporal changes in Shh gradient switches on a transcriptional network comprising of Olig2, Nkx2.2, and Pax6 (Fig. 1). The mechanism by which the neural progenitor cells respond to the external Shh gradient is determined by the rewiring of this gene regulatory network (GRN), and is not a direct consequence of external changes in the morphogen gradient. The ability of the GRN to constantly modulate itself in response to perturbations in Shh signaling ensures a buffering action and makes cells insensitive to fluctuations. This, in turn, results in imprinting of a memory of the signal or “hysteresis” (Balaskas et al., 2012). In addition, stochasticity arising during early developmental patterning can be corrected by positional sorting of cells later (Xiong et al., 2013).

WHAT ARE PRIMARY CILIA? The neural tube epithelium is pseudostratified and hair like projections called primary cilia project from their apical surface into the central canal. The nonmotile primary cilia are widely present in quiescent cells in our body (except in cells from myeloid and lymphoid lineages) (Rosenbaum and Witman, 2002). Structurally, the primary cilium has a central core of nine microtubule doublets (910), which is templated from the mother centriole with associated pericentriolar material (called the basal body) (Fig. 2). The assembly and disassembly of the primary cilium is also integrated into the cell cycle, with assembly occurring during the G1/G0 phase, and disassembly before mitosis during the G2 phase (Kobayashi and Dynlacht, 2011). The axoneme is surrounded by the ciliary membrane, which is an extension of the contiguous plasma membrane. A specified region at the base of the cilium (called the transition zone) results in compartmentalization of components in the cilia (Reiter et al., 2012). An adjacent region of the plasma membrane (called the ciliary pocket) is rich in endocytosis components, while primary cilia are typically devoid of endosomes (Rohatgi and Snell, 2010). Primary cilia are dynamic structures, and are assembled by an active and conserved process called intraflagellar transport (IFT), first described in the Chlamydo-

Figure 2 Structure of the primary cilium. The microtubular axoneme of the primary cilium arises from the basal body. The transition zone is located at the junction of the basal body and the axoneme allowing restricted access to and from the cilia. This zone constitutes of Y-linkers that attaches the microtubule doublets to the membrane. IFT is crucial for ciliary assembly and protein turnover in the cilia. It involves bidirectional movement of IFT particles mediated by molecular motors along the microtubule-based axoneme in the cilia. IFT particles are biochemically composed of IFT-A and IFT-B complexes. Kinesin II is the major anterograde motor that propels movement of IFT particles with cargo molecules towards the microtubule plus ends. The cytoplasmic dynein 2 motor directs retrograde transport from the cilia tips back to the cilia base. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

monas in the laboratory of Joel Rosenbaum (Kozminski et al., 1993). IFT consists of trains of multipolypeptide particles, which move continuously along axonemal microtubules powered by anterograde and retrograde motors. IFT particles are organized into two complexes, called complex A (IFT-A) and complex B (IFT-B) (Scholey, 2008; Bhogaraju et al., 2013). Anterograde transport of these particles is mediated by kinesin II, whereas retrograde transport is powered by the dynein 2 motor (Rosenbaum and Witman, 2002). As IFT is a dynamic and Developmental Neurobiology

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bidirectional process, it also helps in removal and recycling disassembled materials (Rosenbaum and Witman, 2002). The primary cilia are now considered as vital sensory organelles for detection and transmission of a broad range of chemical and mechanical signals. Signaling mediated by the primary cilia plays fundamental roles in cellular differentiation, polarity, and cell cycle control (Wallingford and Mitchell, 2011). Not surprisingly, defects in cilia result in a heterogeneous group of newly described diseases known as “ciliopathies” that frequently present with brain malformations, neural tube defects, retinopathy, renal and hepatic cysts, polydactyly, bone deformities, mental retardation, and obesity (Hildebrandt et al., 2011).

DISCOVERY OF THE ROLE OF CILIA IN Shh SIGNALING In vertebrates, the primary cilia are at the focal point of the Shh signaling pathway during ventral neural tube patterning. The first link between this organelle and Shh signaling was revealed through a mouse forward genetic screen in Kathryn Anderson’s lab (Garcia-Garcia et al., 2005). Mouse mutants that affected the IFT machinery, including the IFT-B complex and IFT motors, exhibited loss of the ventral cell types specified by high levels of Shh (Huangfu et al., 2003; Huangfu and Anderson, 2005). Subsequently, most of the components of the Shh pathway have been found to localize to the cilia, or to shuttle through this compartment (Fig. 3). This includes Ptch1, a receptor for Hh ligands (Goodrich et al., 1997; Rohatgi et al., 2007), and Smoothened (Smo), a frizzled-family GPCR (Zhang et al., 2001; Corbit et al., 2005). In the absence of the ligand Shh, Ptch1 is localized to cilia, and represses the downstream effector of the pathway, Smo, from accumulating in the cilia (Rohatgi et al., 2007). Furthermore, the fulllength zinc-finger transcription factor Gli3 undergoes “limited” proteolysis to get converted to the repressor form (Gli3R), which keeps the pathway in an “off” state (see next section) (Wang et al., 2000; Humke et al., 2010; Wen et al., 2010). Shh binding to Ptch1 activates and retains Smo in the cilia, while Ptch1 is removed from the cilia (Corbit et al., 2005; Rohatgi et al., 2007). Activation of Smo also prevents processing of Gli3R, and results in formation of Gli2/3 into active forms (GliA) (Humke et al., 2010; Wen et al., 2010). The Gli2/3 proteins are also seen accumulating at the tips of the cilia upon Shh treatment (Haycraft et al., 2005; Kim et al., 2009; Wen et al., 2010). Finally, GliA formation leads to transcription Developmental Neurobiology

of the downstream target genes (Gli1, Ptch1). Among the Gli proteins, Gli1 functions exclusively as an activator (Bai and Joyner, 2001). Although both Gli2 and Gli3 have repressor domains, in the neural tube Gli3 is the major repressor, and Gli2 is the primary activator (Ding et al., 1998; Matise et al., 1998; Persson et al., 2002).

MECHANISM OF Gli PROCESSING BY PROTEIN KINASE A Partial proteolytic processing and complete degradation of Gli2/3, the terminal effectors of the Shh signaling pathway, is intricately regulated in the absence or presence of Shh, respectively. In the absence of Shh, Gli3 is processed into N-terminal Gli3R form, while in the presence of Shh, its completely proteolysed after formation of GliA. In the “off” state Gli2/3 full length (Gli2/3FL) proteins are phosphorylated at the C terminus by protein kinase A (PKA) (Tempe et al., 2006; Niewiadomski et al., 2013), the major negative regulator of Shh signaling pathway (Tuson et al., 2011). This triggers further phosphorylation events by casein kinase 1 (CK1) and glycogen synthase kinase 3b (GSK3b) (Tempe et al., 2006). These multisite phosphorylations establish binding sites for btransducing repeat containing protein (b-TrCP), an Fbox protein of the SCF (SKP1-Cul1-F-box) E3 ubiquitin ligase complex (Jiang and Struhl, 1998; Tempe et al., 2006; Pan and Wang, 2007) (Fig. 4). Following ubiquitination at specific residues, Gli2/3 undergoes limited proteolysis at the C-terminus, which removes the activator domain (Pan and Wang, 2007). The residual N-terminal repressor domain of the protein is translocated to the nucleus where it represses expression of Shh target genes. In presence of Shh, the initial phosphorylation trigger by PKA is lost, preventing further proteolytic processing. The fulllength proteins are converted into an enigmatic state called “active” Gli2/3 (Gli2/3A) proteins, and are translocated to the nucleus where they promote transcription of downstream targets of the Shh pathway. The formation of active Gli2/3 proteins could involve complete dephosphorylation of the PKA sites at the C-terminus, and phosphorylation (by an unknown kinase) in the N-terminus (Niewiadomski et al., 2014). In the nucleus, Gli3A is ubiquitinated by the SPOP-Cul3 ubiquitin ligase complex and is fully degraded by the proteasome (Wang et al., 2010). The conversion of Gli3FL to Gli3A or Gli3R also depends on its association with Suppressor of Fused (SuFu), another negative regulator of the Shh pathway (similar to PKA) (Svard et al., 2006; Wang et al., 2010).

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Figure 3 GPCRs and second messengers in regulation of Shh signaling. Left, in absence of Shh (“off” state), the 12-transmembrane protein Patched (Ptch1) prevents trafficking of the frizzled family GPCR, Smoothened (Smo) to the cilia. The ciliary localized rhodopsin family GPCR, Gpr161 is Gascoupled, and causes increased cAMP levels upon constitutive activity. Ciliary pools of Gpr161 could be activating PKA at close proximity to the cilia, resulting in processing of full length Gli3 to the inactive Gli3R, which represses transcription of downstream targets. Middle, in the presence of Shh (“on” state), the Shh receptor Ptch1 is removed from the cilia, which results in Smo activation and retention in the cilia. Gpr161 is also removed from the cilia, inhibiting further PKA activation. This terminates Gli3 processing into Gli3R, and the Gli2 activator transcribes downstream hedgehog targets (Gli1, Ptch1). Shh signaling in cerebellar progenitor neurons can be further modulated by other GPCRs, such as PAC1. In presence of the ligand PACAP, the receptor signals to globally activate PKA and prevent excessive Shh signaling. This can inhibit downstream signaling, even in the presence of Shh. The heterodimeric Ca21 channel Pkd1l1-Pkd2l1 located in the cilium can also positively regulate Shh signaling. Right, in postmitotic neurons, Shh-induced Ca21 spike activity mediates postmitotic differentiation. Activated Smo induces localized IP3 transients in the cilium, which are synchronized with intracellular Ca21 spikes. Ca21 levels depend on IP3-induced release from intracellular stores, and activation of TRPC1 and voltage-gated channels (Ca21voltage) (Belgacem and Borodinsky, 2011). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

When the Shh pathway is inactive, Gli3FL remains associated with SuFu, thereby sequestering Gli3 from the nucleus in the cytoplasm, and making it available for processing into Gli3R. The Sufu-Gli2/3 complex shuttles in and out of the cilia by the IFT machinery (Kim et al., 2009; Wen et al., 2010; Chen et al., 2011). However, in the presence of Shh, the association between Sufu and Gli3 is lost. This results in accumulation of Gli3 proteins in the ciliary tips, and aids in the conversion of Gli3FL to Gli3A and nuclear entry (Humke et al., 2010; Chen et al., 2011). The third member of the Gli family, Gli1 is the major downstream transcriptional target of the Shh pathway. Unlike Gli2/3, Gli1 functions only as a transcription activator. Recently, it has been demonstrated that the atypical protein kinase C i/k (aPKCi/ k) can positively regulate Gli1 activity, especially in

the context of BCC. aPKCi/k functions downstream of Smo to phosphorylate Gli1, and is also a transcriptional target of the Shh pathway. This positive feedback loop helps Gli1 achieve maximal DNA binding capacity and transcription. Deletion or pharmacological inhibition of aPKCi/k leads to loss of Shh signaling and proliferation in BCC (Atwood et al., 2013).

HOW IS PKA ACTIVATED DURING Gli PROCESSING? Paradoxical Effects of IFT-A Mutants in Shh Signaling The PKA-dependent processing of Gli3 into Gli3R is dependent on the cilia (Wen et al., 2010; Tuson Developmental Neurobiology

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Figure 4 Functional domains in Gli3. The Gli3 protein has a centrally located DNA binding domain with five C2H2 zinc-finger repeats, which is flanked on both sides by repressor and activation domains. Phosphorylation sites for Gli3 processing are shown below. Gli3 phosphorylation is initiated by PKA, followed by glycogen synthase kinase 3 b (GSK3b) and casein kinase 1 (CK1). This establishes binding sites for the b-TrCP/Cul1 E3 ubiquitin ligase, followed by ubiquitination at specific lysine residues (shown in red letters inside Gli3). Subsequently, limited proteolysis of the C-terminus by the proteasome, till it reaches the putative restriction site (marked by a dashed line), yields the repressor form (Gli3R). The processing of Gli3 by PKA is cilia-dependent. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

et al., 2011). PKA is activated by an increase in cAMP levels either by adenylyl cyclases, which are primarily activated upon ligand binding to Gascoupled GPCRs, or by inhibition of cAMP phosphodiesterases. The paradoxical effects of the IFT-A complex (one of the IFT complexes), with respect to other ciliary mutants on Shh signaling in the neural tube provided initial clues to the discovery of factors that regulate PKA-dependent Gli3 processing. The IFT-B complex is implicated in anterograde IFT (as mutants have phenotypes similar to lack of kinesin II, the anterograde motor), whereas the IFT-A complex is mainly thought to regulate retrograde transport (as mutants have defects similar to lack of the retrograde IFT motor dynein 2 that cause axonemal bulges due to loss of protein clearance from cilia) (Rosenbaum and Witman, 2002). As mentioned earlier, mutations in the IFT motor proteins and IFT-B complex result in loss of ventral cell patterning in neural tube (Huangfu et al., 2003; Huangfu and Anderson, 2005). This can be attributed to the fact that disruption of IFT (either anterograde or retrograde) leads to severe defects in ciliary organization (Rosenbaum and Witman, 2002). On the contrary, the role of the retrograde IFT module, IFT-A in neural tube patterning is more complex. IFT-A is a multiprotein complex with a “core” complex (comprising of Ift144, Ift140 and Ift122), and Ift139, Ift121 and Ift43 functioning as peripheral subunits (Mukhopadhyay et al., 2010; Behal et al., 2012). In null mutations of IFT-A genes (Ift139alien and Ift122sopb) and in a hypomorphic allele of Ift144 (Ift144twt), there is dorsal expansion of the FP, V3 progenitors and motor neuron domains (Tran et al., 2008; Ocbina et al., Developmental Neurobiology

2011; Qin et al., 2011; Liem et al., 2012). In a severe loss-of-function mutant allele of Ift144 (Ift144dmhd) or in cases of disruption of two IFT-A subunits, resulting in severe disruptions of ciliary structure, similar expansion of the motor neuron domains (requiring intermediary Shh signaling) is observed (Tran et al., 2008; Ocbina et al., 2011; Qin et al., 2011; Liem et al., 2012). This paradoxical increase in Shh signaling implicates that IFT-A may have additional preciliary functions in addition to its established role in retrograde IFT. Similar to IFT-A mutants, mutations in the tubby family protein, Tulp3 also demonstrate enhanced Shh signaling in the caudal neural tube (Norman et al., 2009; Patterson et al., 2009). Apart from its canonical role in retrograde trafficking, IFT-A “core” was shown to interact with Tulp3, and thereby traffic it into the cilia. Tulp3, in turn, traffics rhodopsin family GPCRs, through the N-terminal IFT-A- and C-terminal phosphoinositidebinding domains (Mukhopadhyay et al., 2010). These results strongly implicated a Tulp3/IFT-A-mediated rhodopsin family GPCR as a negative regulator of Shh signaling.

Role of the Novel GPCR, Gpr161 in Neural Tube Patterning Gpr161, a constitutively active orphan GPCR has been recently identified as a negative regulator of the Shh pathway (Mukhopadhyay et al., 2013). Trafficking of this GPCR to the primary cilia is mediated by Tulp3 and the IFT-A complex. In situ hybridization in mouse embryos showed low levels of ubiquitous

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expression of this receptor in E8.5–E9.5 stages. By mid-gestation, it is predominantly expressed in the neural tube, and in the later stages in the brain, spinal cord, dorsal ganglia, and the hind limb (Mukhopadhyay et al., 2013). A hypomorphic spontaneous allele of Gpr161 (vl; vacuolated locus) causes spina bifida (Matteson et al., 2008). A null Gpr161 mouse knock out results in mid-gestation lethality by E10.5 (Mukhopadhyay et al., 2013). The null mutant embryos demonstrated craniofacial defects (with loss of dorsal telencephalon markers Foxg1 and Gli3), loss of limb buds, and failure of neural tube closure at caudal ends. Increased Shh signaling was apparent throughout the rostro-caudal extent of the neural tube. This was determined by detecting elevated transcript/protein levels of Gli1/Ptch1 in knock out embryos. Furthermore, Gpr161 mutant embryos showed ectopic expression of Gli1 and Ptch1 in more dorsal domains of neural tube, although the overall Shh levels were similar to the wild type. These embryos showed a more “ventralized” neural tube, as indicated by a more dorsal expansion of the FP, p2, pMN, and p3 progenitor markers, reminiscent of Sufu (Svard et al., 2006; Wang et al., 2010) and PKA knock out embryos (Tuson et al., 2011).

Role of Gpr161 in Gli3 Processing Gpr161 knock out mutants exhibit defects in Gli3 processing, suggesting that it could be important in regulating this process. Genetic epistatic analysis of double mutants suggested that the Gli3 processing defects in Gpr161 mutants are cilia-dependent, and occurs independent of Smo. The Gpr161 phenotype is similar to both PKA and Sufu mutants; however, the effects of Sufu on the Shh pathway occur independent of cilia (Chen et al., 2009; Jia et al., 2009; Humke et al., 2010), suggesting that the effects of Gpr161 on Gli3 processing is primarily mediated by PKA activation. In the absence of a known ligand, inducible overexpression of Gpr161 suggested that it signals by increasing cAMP levels in a Gas–coupled manner. Interestingly, in presence of Shh, Gpr161 is removed from the cilia, preventing its activity. Thus, its possible that ciliary pools of Gpr161 result in increased cAMP levels in cilia (Mukhopadhyay et al., 2013) (Fig. 3), activating PKA in close proximity, such as at the basal body (Barzi et al., 2010; Tuson et al., 2011). Interestingly, mouse Gnas (Gas) knock out embryos are embryonic lethal by E9.5 with open neural tube and cardiac defects (Regard et al., 2013). The E9.5 knockout embryos also show up-regulation of Hh targets, and reduction of Wnt signaling targets. The neural tube in these embryos shows increased

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Hh signaling, as apparent from dorsal expansion of the ventral domains marked by Nkx2.2 and Hh expression, and loss of the dorsal Pax6 domain. Thus, Gas functions as a negative regulator of Hh signaling in the neural tube. Gas has been recently reported to be present in mammalian ciliary proteome (Ishikawa et al., 2012), and the effect of Gas in Shh signaling could be mediated by both ciliary and nonciliary pools.

ROLE OF OTHER GPCRs IN REGULATING Shh SIGNALING Like Gpr161, cAMP levels can be enhanced in the developing neural tube, by binding of the neuropeptide pituitary adenylyl cyclase activating peptide (PACAP) to its receptor PAC1 (Pisegna and Wank, 1993). PAC1 receptor is highly expressed in the developing murine neural tube, particularly in the FP and the dorsal areas from E10.5 onward. The mRNA transcripts for the peptide ligand localizes in the differentiating neurons of the neural tube. Exogenous application of PACAP to cultured neuroepithelial cells from E10.5 embryos triggers cAMP signaling and also reduces Gli1 transcript levels (Waschek et al., 1998). This rises the possibility that cAMP signaling by PACAP functions to antagonize Shh in the developing neural tube to promote dorsal fate specification. Apart from being the morphogen that facilitates neural tube patterning, Shh secreted by purkinje neurons promote proliferation of the outer cerebellar granular neuron precursors (cGNP) (Wallace, 1999; Wechsler-Reya and Scott, 1999). It appears that the primary cilia is essential for Shh-mediated regulation of cGNP division, as loss of cllia leads to cerebellar hypoplasia (Chizhikov et al., 2007). Mutations resulting in overactivation of the Shh pathway results in medulloblastoma (Goodrich et al., 1997; Taylor et al., 2002). The cilium plays a regulatory role in pathogenesis of Shh-dependent medulloblastoma and BCC. When the trigger for the uncontrolled proliferation is constitutively active Smo (SmoM2) (Xie et al., 1998), genetic disruption of cilia rescues the tumor phenotype. In contrast, loss of cilia in tumor models with constitutively active Gli2 enhances tumor growth, presumably due to lack of cilia-dependent formation of Gli3R (Han et al., 2009; Wong et al., 2009). It appears that fine-tuning of the Shh pathway is essential for striking a balance between growth and overproliferation. It has been shown that PACAP inhibits Shh-dependent cell proliferation in cGNP Developmental Neurobiology

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(Nicot et al., 2002), possibly by regulating global activation of PKA (Niewiadomski et al., 2013). This additional mode of PKA activation can serve as a “brake,” which can fine tune the Shh signaling pathway, especially in preventing hyper-activation of the Shh pathway during proliferation of cGNPs (Fig. 3). Thus, PACAP acts as a tumor suppressor in murine medulloblastoma (Lelievre et al., 2008). PACAP also shows 68% sequence similarity with another peptide neurotransmitter, vasoactive intestinal polypeptide (VIP). VIP can bind to secretin family Gas-coupled receptor receptors VPAC1 and VPAC2 (Gozes et al., 1995; Vaudry et al., 2000). During embryogenesis, maternal VIP induces embryonic growth by acting directly on receptors that are mostly localized to the FP and adjacent neural tube (Gressens et al., 1993; Hill et al., 1994). VPAC2 has also been shown to localize in the primary cilia of hypothalamic neurons, and in glial cells (Soetedjo et al., 2013). However, the role of VIP-dependent PKA activation during the Shh pathway is not known. Other GPCRs known to localize to the neuronal cilia are somatostatin receptor 3 (Sstr3) (Handel et al., 1999), dopamine receptors D1R and D2R (Marley and von Zastrow, 2010), melanin concentrating hormone receptor 1 (MCHR1) (Berbari et al., 2008a), serotonin receptor 6 (5HTR6) (Hamon et al., 1999), and neuropeptide Y receptors NPY2R and NPY5R (Loktev and Jackson, 2013a). The BBSome complex (Nachury et al., 2007) regulates GPCR trafficking to the cilia (Berbari et al., 2008b; Jin et al., 2010; Sun et al., 2012; Loktev and Jackson, 2013b). However, the Bbs knock out mice do not exhibit neural tube defects, and the role of these GPCRs in neuronal development is not clear. Thus far, we have only discussed the role of GPCRs signaling by Gas in neural tube development, as they can modulate Shh signaling though cAMPdependent PKA activation. A recent screen for novel inhibitors of Hh signaling downstream of Smo identified GPR39 as the target of a series of cyclohexylmethyl aminopyrimidine chemotype compounds (CMAPs). CMAPs activated GPR39 to stimulate Gaq-/Gai-coupled and b-arrestin–mediated signaling pathways that resulted in MAP kinase activation, and inhibition of Gli signaling (Bassilana et al., 2014). GPCR signaling by the Gai pathway can also play vital roles in neural tube development. It has been reported, that Protease activated receptor 1 (Par1) and Protease activated receptor 2 (Par2) double knock out mice show caudal neural tube defects, exencephaly and spina bifida. Signaling by the GaiRac1 pathway in the PAR2 expressing cells of the surface ectoderm contributes to neural tube closure, Developmental Neurobiology

as conditional knock down of these effectors leads to neural tube defects (Camerer et al., 2010).

ROLE OF Ca21 IN THE Shh PATHWAY Ciliary Ca21 During Shh Signaling Recent work using a ciliary fluorophore for measuring in vivo changes in Ca21 concentrations has shown that basal [Ca21] in the ciliary compartment is higher than cytoplasm. Changes in ciliary Ca21 concentration occur without substantially affecting global cytoplasmic levels. The increased ciliary Ca21 levels can be attributed to the heteromeric transient receptor potential (TRP) channel comprising of Pkd1l1-Pkd2l1, which is ciliary localized (Fig. 3). Primary mouse embryonic fibroblasts isolated from Pkd2l12/2 mice show significantly reduced Gli1 expression and Gli2 trafficking to ciliary tips upon activation of the Shh pathway (DeCaen et al., 2013; Delling et al., 2013). Currently, the role of ciliary Ca21 in Shh signaling is unclear, but speculative models include the role of Ca21-binding IFT-B complex proteins (e.g., Ift25) (Bhogaraju et al., 2011) that regulate trafficking of Shh pathway components into the cilia (Keady et al., 2012). Similarly, the ciliarylocalized Pkd1l1-Pkd2 channel determines left-right asymmetry during early embryonic morphogenesis and is expressed in the crown cells surrounding the node, and ciliary function in crown cells could involve either mechanosensory or chemosensory modalities (Field et al., 2011; Yoshiba et al., 2012).

Ca21-Mediated Noncanonical Hh Signaling Apart from specification of neuronal progenitors along the D-V axis of the neural tube by establishing an intricate network of transcription factors, Shh can also contribute in modulating the electrical activity in spinal neurons independently of transcription, by Ca21 and IP3 signaling during later development (Belgacem and Borodinsky, 2011). Shh signals can be acutely harnessed by postmitotic neurons of the developing ventral neural tube to generate intracellular Ca21 spikes. This phenomenon is dependent on Smo, as overexpression of a constitutively active form SmoM2 (Xie et al., 1998) enhances the Ca21 spike activity. While treatment with Smo agonist SAG recapitulates the Shh action, the Smo antagonist cyclopamine (Chen et al., 2002) abolishes this effect. As Smo has been indicated to signal by Gai (Riobo et al., 2006; Ogden et al., 2008), pertussis toxin

Neural Tube Patterning

mediated inhibition of Gai suppresses the Shhdependent increase in Ca21. Shh signaling induces localized IP3 transients in the cilium, synchronized with Ca21 spikes, suggesting that Shh-induced Ca21 spikes depend on IP3-induced Ca21 release from intracellular stores. The authors propose that Shhmediated activation of Smo in the cilia leads to activation of phospholipase C by the Gbc subunits of the G-protein heterotrimer resulting in increased IP3 levels. This triggers opening of IP3 receptor (IP3R)-operated intracellular Ca21 stores. Furthermore, increased Ca21 levels positively regulate TRPC channels in the cilia to reinforce the Shh-mediated Ca21 spikes, by mediating entry from extracellular sources. In accordance with this model, IP3R localizes at the ciliary base, whereas Gai and TRPC1 are localized to the cilia (Fig. 3). Ca21 spike activity regulates neurotransmitter specification in developing neurons (Marek et al., 2010; Swapna and Borodinsky, 2012). Shh signaling recapitulates the Ca21-dependent effect on GABAergic specification, and changes in Ca21 spike activity prevent SAG- or cyclopamine-induced effects on the neuronal phenotypes (Belgacem and Borodinsky, 2011). These results suggest a novel role of Shhinduced Ca21 spike activity during postmitotic differentiation (Belgacem and Borodinsky, 2011).

OUTSTANDING QUESTIONS AND FUTURE DIRECTIONS The discovery of the critical role of primary cilia in the vertebrate Shh pathway, and the elucidation of the key molecules that shuttle through the cilia during signaling has provided us with a broad overview of the organization of the Shh pathway in this subcellular compartment. The stage is now set for elucidating the functional role of these signaling molecules in cilia. This directly impinges on understanding the key role of the cilium in functioning as a signaling compartment in cellular pathways, and as a means to compartmentalize second messengers in a subcellular domain that maintains communication with the external environment. The signaling mechanisms that underlie cilia-mediated PKA activation in the Shh pathway are going to be interesting areas of future research, and would require precise tools for studying localized signaling events during Shh signaling. The precise subcellular location of PKA-mediated Gli processing is also currently unknown, and would be important in understanding the coordinated sequence of events that serve as the basis for the functioning of the cilium in this pathway. The role of Ca21 in Shh signaling, both during patterning and in postmitotic

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neuronal specification is intriguing, and further studies are required to determine the downstream effectors in these processes. Finally, discovering novel GPCR-regulated mechanisms important in Hh signaling in different tissues helps in understanding the role of Hh-regulated pathways during normal development and in the pathogenesis of Hh-dependent tumors. The authors declare that they do not have any potential sources of conflict of interest.

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