Cellular signalling by primary cilia in development, organ function and disease

Linking IFT to human disease

The above-described studies of IFT in Chlamydomonas set the stage for a landmark study which revealed that a hypomorphic mutation in the mouse Tg737 gene, which encodes an orthologue of the Chlamydomonas IFT-B polypeptide IFT88, causes ciliary loss and autosomal recessive (AR) PKD ^{79, 80}. This study provided the first evidence that defective primary cilia can lead to disease in mammals, and was supported by prior work in C. elegans demonstrating that homologs of the human autosomal dominant (AD) PKD1 and PKD2 gene products, the transmembrane proteins polycystin (PC) 1 and PC2, localize to neuronal sensory cilia and regulate male mating behaviour ⁸¹. In agreement with these observations, PC1 and PC2 were found to localize to primary cilia in mammalian cells such as those of the renal collecting duct ^82–84. The physiological importance of the polycystins is underscored by the fact that mutations in the corresponding genes lead to ADPKD — a disease characterized by the adult-onset development of kidney and liver cysts ^{85, 86}. Furthermore, mutations in PKD2 have been linked to left-right laterality defects in vertebrates, including humans (add refs: PMID 12062060 and PMID 21719175). It is generally accepted that proper functioning of PC1 and PC2 relies on their appropriate targeting to the ciliary compartment, but the precise mechanism by which the polycystins function at cilia to prevent cyst formation and control left-right patterning during development is a matter of intense debate ⁸⁵. For many years, the prevailing hypothesis was that PC1 and PC2 comprise a mechanosensitive receptor-Ca²⁺ channel complex, which is activated by fluid flow ^{87, 88}. This hypothesis was challenged ^{89, 90}, and it was subsequently suggested that PC2, independently of PC1, forms a homotetrameric K⁺ and Na⁺-conducting ciliary ion channel that is potentiated by intraciliary Ca^{2+ 91–93}. A report of the near-atomic-resolution structure of the human PC1-PC2 complex by single-particle cryo-electron microscopy, however, strongly favours a model in which PC1 and PC2 function together in regulating cation transport ⁹⁴. Nevertheless, despite important advances, the molecular mechanisms by which cilia and polycystins cooperate to regulate development and tissue homeostasis remain incompletely understood (discussed elsewhere⁸⁵). Further work in many different organisms, including mouse and human, has substantiated the link between primary cilia and kidney disease, and revealed a requirement for IFT and cilia in a range of other disease-relevant pathways (Figure 1).

[H2] Ciliary coordination of HH signalling

Most components of canonical HH signalling are dynamically associated with cilia. In addition to PTCH1 removal, SMO enrichment and association of GLI2, GLI3 and SUFU with ciliary tips, the microtubule-associated atypical kinesin, KIF7, also becomes enriched in ciliary tips during activation of HH signalling ^{136, 137}, where it. functions as both a positive and negative regulator of SHH signalling by modifying ciliary architecture ^{136, 137}. GPR161 is also removed from primary cilia in a SMO-dependent and β-arrestin-dependent manner following activation of HH signalling ^{28, 138}. β-arrestins are adaptor proteins that are recruited to the proximal C-terminus of GPR161 in a manner dependent on G protein-coupled receptor kinase 2 (GRK2)¹³⁸. GRK2 and GRK3 also transduce high level SHH signals through mechanisms independent of GPR161¹³³. Depletion of Inpp5e (which encodes a phosphoinositide 5-phosphatase responsible for removing the 5-phosphate from PI(4,5)P₂), leads to accumulation of PI(4,5)P₂ in cilia and increased steady-state levels of TULP3, IFT-A and TULP3-dependent cargo such as GPR161 and PC2 ^{139, 140}. In addition, removal of GPR161 from cilia upon activation of the SHH pathway is impaired in Inpp5e-knockout cells ^{139, 140}, irrespective of levels of the pathway activator SMO suggesting that a number of factors are responsible for the dynamic regulation of HH signalling components in cilia.

Activation of PKA in different subcellular regions is mediated by local cAMP production and anchoring of PKA to A-kinase anchoring proteins (AKAPs) ¹⁴¹. The distal C-tail of GPR161 has a conserved amphipathic helix that directly binds to type I PKA regulatory subunits ¹⁴² (Figure 2). Since the type I PKA regulatory subunit alpha localizes to cilia ¹⁴³, direct coupling of this subunit to PKA in this compartment might enhance the ability of GPR161 to activate PKA via constitutive cAMP signalling. nine adenylyl cyclases that regulate downstream cAMP signalling, at least three — ADCY3, ADCY5, and ADCY6 — are localized to cilia ^143–145. In particular, overexpression of ADCYC5 and ADCYC6 partially represses the HH pathway in the developing chicken neural tube ¹⁴⁶. However, factors that regulate trafficking of adenylyl cyclases to cilia are currently unknown, and the role of such factors in SHH signalling has not been established.

Ciliary GPCR signalling on central neurons

Most neurons in the mammalian brain possess primary cilia. The importance of neuronal cilia is highlighted by the fact that ciliopathies are associated with numerous neuropathologies, including anatomical abnormalities and neuropsychiatric disorders ¹⁶⁵. Neuronal cilia are enriched for certain GPCRs and downstream effector proteins ^{152, 166, 167}, suggesting they act as specialized signalling hubs. The importance of GPCR ciliary localization is underscored by the fact that mouse models of the ciliopathy BBS show dysregulation of GPCR ciliary localization. Specifically, deletion of BBS proteins leads to failure of somatostatin receptor subtype 3 (SSTR3), melanin-concentrating hormone receptor 1 and neuropeptide Y (NPY) receptor subtype 2 (NPY2R) to localize to neuronal cilia, whereas, dopamine receptor 1 accumulates in neuronal cilia ^{73, 74, 168}. In addition, tubby mice, which carry a mutation in the gene encoding the TUB protein and display ciliopathy phenotypes, exhibit defective localization of a number of GPCRs in neuronal cilia ^{74, 169}. Thus, defects in localization of GPCRs to neuronal cilia likely disrupt ciliary signalling and contribute to ciliopathy phenotypes.

Numerous studies have implicated ciliary GPCR signalling in both neural development and function. Inhibitory interneurons originate in the telencephalon from where they migrate to numerous brain regions and integrate into local neural circuits to regulate the balance of excitatory and inhibitory inputs. Disruption of interneuronal circuits has been linked to neurodevelopmental disorders such as schizophrenia, autism and intellectual disabilities ¹⁷⁰. Interestingly, conditional disruption of ARL13B, which is a GTPase required for proper ciliary signalling, in post-migratory interneurons perturbs circuit development in the mouse striatum ¹⁷¹. Specifically, loss of ARL13B in interneurons results in reduced dendritic and axonal complexity, disrupted synaptic connectivity, and functional deficits in synaptic activity ¹⁷¹. Interneurons lacking ARL13B also show disrupted ciliary calcium dynamics and a dramatic reduction in ciliary localization of SSTR3 ¹⁷¹. Intriguingly, expression of SSTR3 in ARL13B-deficient interneuronal cilia rescues the morphological and synaptic connectivity defects, whereas expression of a non-ciliary form of ARL13B with normal GTPase activity does not rescue the developmental defects in ARL13B-deficient interneurons ¹⁷¹, suggesting that SSTR3 signalling within interneuronal cilia is critical for proper inhibitory network construction and function.

Ciliopathies are also associated with cognitive deficits. SSTR3 localizes to neuronal cilia in the hippocampus, a region of the brain that is important for learning and memory. Treatment of mouse hippocampal neurons with the SSTR3 ligand, somatostatin, stimulates recruitment of endogenous β-arrestin into SSTR3-positive cilia, leading to a rapid β-arrestin-2-dependent decrease in the ciliary SSTR3 ¹⁶⁷, suggesting that ciliary export of activated SSTR3 is mediated by β-arrestin-2. In support of this finding, ciliary export of activated SSTR3 in cultured renal epithelial cells also requires β-arrestin-2 ^{28, 172}. Interestingly, mice lacking SSTR3, ADCY3, β-arrestin-2 or cilia in the hippocampus all show similar deficits in learning and memory ^173–176, implicating SSTR3 ciliary signalling in proper learning and memory.

GPCR signalling in neuronal cilia also contributes to the regulation of metabolic homeostasis. The first hint that cilia affect food intake and energy metabolism arose from the observation that some ciliopathies are associated with obesity. This connection was further supported by studies showing that conditional disruption of cilia in adult mice causes hyperphagia and obesity ^{177, 178}. More recent studies have provided important mechanistic insights into the metabolic neural circuits that are affected by GPCR ciliary signalling. The Gα_s-coupled GPCR melanocortin 4 receptor (MC4R) is an important component of the neurocircuitry that regulates food intake and energy expenditure. In fact, mutations in MC4R are the most common cause of monogenic obesity in humans, underlying up to 6% of early-onset or severe cases of adult obesity ¹⁷⁹. A 2018 study showed that MC4R localizes to cilia on neurons in the mouse paraventricular nucleus of the hypothalamus — a region of the brain that is important for the regulation of energy homeostasis and metabolism ¹⁸⁰. Interestingly, some MC4R mutations associated with obesity in humans impair ciliary localization of MC4R. Moreover, inhibition of ciliary cAMP signalling specifically in cilia on MC4R-expressing neurons of the paraventricular nucleus led to increased food intake and weight gain in mice ¹⁸⁰. Thus, impaired ciliary signalling of MC4R could be a common cause of syndromic and non-syndromic obesity in humans.

NPY also has an important role in the central regulation of food intake and energy expenditure ¹⁸¹. NPY2R localizes to cilia on hypothalamic neurons in mice ⁷⁴. Importantly, BBS mutant mice that lack NPY2R-positive cilia are obese and do not respond to NPY2R ligand ⁷⁴, suggesting that receptor ciliary localization is required for ligand-dependent signalling in vivo. Moreover, quantification of NPY2R-mediated cAMP signalling in a ciliated cell line revealed that ligand treatment produced a stronger signal in cells with a cilium than in non-ciliated cells ⁷⁴. Thus, NPY2R signalling is seemingly enhanced within the cilium. Similar to SSTR3, ligand treatment results in a decrease in NPY2R ciliary localization. NPY2R binds poorly to β-arrestin ¹⁸²; however, using live-cell imaging in a ciliated renal epithelial cell line, Nager et al ²⁸ revealed that activated NPY2R accumulates at the ciliary tip and is released in extracellular vesicles called ectosomes ²⁸ (Figure 3), raising the fascinating possibility that ciliary GPCR signals might be transmitted between cells. Interestingly, Nager et al also showed that agonist-dependent ciliary export of heterologously-expressed SSTR3 in inner medullary collecting duct cells lacking either BBSome function or β-arrestin-2, involved active recruitment of SSTR3 to the ciliary tip from where a considerable fraction was released in ectosomes ²⁸. These findings further support a role for the BBSome and β-arrestin-2 in GPCR ciliary export and led to the suggestion that loss of GPCR localization on neuronal cilia in BBS mutant mice ⁷³ might result from constitutive ectocytosis, rather than a defect in GPCR trafficking to cilia. However, this proposal would suggest that hippocampal neurons that lack β-arrestin-2 would also lack ciliary localization of SSTR3, yet ciliary localization of SSTR3 is not affected in β-arrestin-2-deficient hippocampal neurons¹⁶⁷. Why SSTR3 would be constitutively ectocytosed from cilia on BBS mutant neurons, whereas D1 accumulates in cilia on BBS mutant neurons even in the presence of agonist, is unclear ¹⁶⁸. Additional studies are therefore required to define the precise roles of the BBSome in GPCR trafficking to and from neuronal cilia.

WNT signalling: the TZ and the basal body

Dvl, β-catenin and several members of the β-catenin destruction complex have been shown to localize to the ciliary base and available evidence suggests that they can be modulated by components of the MKS and NPHP modules, which are protein complexes of the TZ, which forms a prominent part of the ciliary gate that regulate proteins entering and exiting the cilium (PMID: 27770015). For example, in the absence of a primary cilium, the MKS module component Jouberin (JBN, also known as AHI1) promoted WNT and β-catenin signalling by facilitating nuclear localization of β-catenin ²⁰⁵. The primary cilium induces depletion of nuclear and cytoplasmic JBN and thereby represses the effect of JBN on β-catenin. Ciliary JBN facilitates the ciliary localization of β-catenin following WNT3A stimulation and thereby negatively affects WNT signalling ²⁰⁶. JBN therefore has a dual function in regulating WNT–β-catenin signalling, depending on the presence or absence of the primary cilium. Another study showed that the cystic kidney and abnormal brain development phenotypes of mice deficient in JBN are largely caused by dysregulated WNT–β-catenin signalling ²⁰⁷. The transmembrane protein TMEM67 is another key component of the MKS module that recruits the tyrosine-protein kinase transmembrane receptor ROR2 to the TZ. Together TMEM67 and ROR2 respond to WNT5A to activate RHOA, modulate the actin cytoskeleton and promote epithelial branching morphogenesis; these proteins also partially facilitate WNT5A-mediated repression of the canonical pathway ²⁰⁸. In addition to MKS module components and NPHP2 (discussed above), evidence suggests that other NPHP module components including NPHP3 ²⁰⁹, NPHP4 ²¹⁰ and RPGRIP1L (PMID 22927466, ²¹²) modulate the WNT pathway through DVL. Similarly to NPHP2, NPHP3 and NPHP4 restrain DVL-mediated WNT–β-catenin activation in cell culture, and when depleted, induce WNT–PCP defects in vivo (PMID 18371931 and PMID 21498478). In addition, NPHP4 was reported to stabilize the E3 PHD finger domain E3 ubiquitin ligase JADE1 and promote its nuclear translocation ²¹³. JADE1 is a negative regulator of WNT–β-catenin signalling that ubiquitinates cytoplasmic and nuclear β-catenin irrespective of its phosphorylation status, thereby promoting its proteasomal degradation ²¹⁴. In ciliated cells, JADE1 is also localized at the TZ and the basal body, probably through its interaction with NPHP4. Thus, NPHP4, in addition to its negative effect on DVL, also negatively regulates β-catenin levels through JADE1. Similarly, RPGRIP1L regulates WNT–PCP pathways by modulating DVL levels ^{212, 215}. RPGRIP1L was reported to regulate the proteasome function in a cilia and basal body-dependent manner ²¹¹. Mouse embryonic fibroblasts deficient in RPGRIP1L exhibit impaired proteasomal activity at the basal body — a phenotype that can also be induced by depletion of the 19S proteasome subunits PSMD2, PSMD3 and PSMD4, all of which localize at the TZ and basal body. Indeed a key manner by which cilia might regulate WNT pathway components is through direct proteasomal degradation ^{212, 216, 217} (PMID: 24691443). Cilia proteomics have identified several proteasomal subunits that bind to BBSome subunits ²¹⁸, and depletion of BBS4 in cultured cells (which results in both canonical and WNT–PCP defects in zebrafish) reduces proteasomal activity and leads to an accumulation of cytoplasmic and nuclear β-catenin ²¹⁶.

Thus, available data suggest that various proteins in the TZ and basal body might act as a processing platform to which various WNT signalling components are recruited for post-translational modification and/or elimination through the basal body-associated proteasome pathway. However, it is important to remember that many TZ proteins may have extra-ciliary functions ²¹⁹ and further work is needed to decipher and distinguish their ciliary and non-ciliary functions and the context in which they regulate the WNT pathways.

Ciliary PDGFRα signalling

Two isoforms of PDGF receptors exist (PDGFRα and PDGFRβ), which function as either homodimers or heterodimers to control diverse cellular and developmental processes. Mutations in the genes expressing PDGFRα or its specific ligand, PDGF-AA, are associated with kidney and liver pathophysiologies, gastrointestinal stromal tumours, glioblastoma and a variety of other cancers ²³⁹. PDGFRα is principally localized to primary cilia ^{225, 240–243}, although non-ciliary localizations for this isoform have been reported for specific cell types ^243–245. PDGFRβ is primarily located and activated outside the cilium ^{240, 241}, but can induce AURKA-mediated disassembly of the primary cilium in cultured fibroblasts and RPE-1 cells through activation of the PLCγ pathway upon stimulation with PDGF-DD ²⁴⁶. In fibroblasts, ciliary PDGFRα signalling activates the PI3K–AKT and MEK1/2–ERK1/2–RSK pathways to control directional cell migration, which is achieved by targeting the Na⁺/H⁺ exchanger 1, NHE1, to the leading edge of the migrating cells, which project their cilium towards the leading edge and parallel to the path of motility ^{240, 247–249}. Consequently, defects in the formation of primary cilia by depletion of IFT-B proteins such as IFT88 and IFT172 block PDGFRα activation ^{247, 250}, rendering the cells unable to respond and move towards a gradient of PDGF-AA ²⁴⁷. In this context, AKT was shown to be activated at the ciliary base in complex with NPHP2 in a PDGF-AA-dependent manner ²⁵¹; these interactions could provide a platform for cross-talk between PCP and PDGFRα signalling pathways in regulating directional cell migration, although further work will be required to understand the potential interaction between these two signalling systems at the level of the cilium. In addition, knockdown of the serine/threonine-protein kinase Ataxia telangiectasia and RAD3-related protein (ATR), which underlies Seckel syndrome ²⁵², is associated with ciliary shortening and reduced responsiveness to PDGF-AA in cultures of fibroblasts ²⁵³, and depletion of the gene that encodes the ciliary TZ NPHP module component, RPGRIP1L, in mouse embryos leads to ventricular septal defects associated with loss of localization, which display cardiac ventricular septal defects ²⁵⁴ is associated with loss of localization of PDGFRα to primary cilia and reduced expression of the PDGFRα target gene, Hifα, in cardiac ventricular tissue ²⁴³. The latter observation supports the general concept that that dysfunctional cardiac cilia cause congenital heart defects ²⁵⁵.

Further studies have revealed diverse mechanisms that regulate ciliary targeting of PDGFRα and balance the output of PDGF-AA-mediated signalling. IFT20 appears to have a central role in this context by stabilizing the RING-finger domain family of CBL E3 ubiquitin ligases, c-CBL and CBL-b, which are tumour suppressor proteins that target active PDGFRs for internalization and degradation ²⁵⁶; dysfunctional CBL E3 ubiquitin ligases are associated with cancer development ²⁵⁷. Upon PDGF-AA stimulation CBL proteins are recruited to the primary cilium to mediate the ubiquitination and internalization of PDGFRα for feedback inhibition of signalling, whereas in IFT20-depleted cells, which lack the cilium, PDGFRα mislocalizes to the general cell surface, where PDGF-AA-mediated signalling is greatly overactivated due to autoubiquitination and proteasomal degradation of the CBL proteins ²⁵⁸. Similarly, co-depletion of c-CBL and CBL-b leads to reduced levels of IFT20 in combination with mislocalization and overactivation of PDGFRα ²⁵⁸. These results suggest the existence of a biochemical and functional relationship between IFT20 and CBL proteins in ciliary receptor sorting and modulation of PDGFRα signalling, which when defective underlies tumorigenic signalling in various tissues ²³⁹. Indeed, expression of the oncogenic mutant PDGFRα Asp842Val, which localizes to the Golgi ²³⁹ and is the most common PDGFRα mutation in gastrointestinal stromal tumours ²⁵⁹, promotes ciliary disassembly and cell proliferation by phenocoyping PDGF-DD-mediated signalling ²⁴⁶. Furthermore, rapamycin-mediated inhibition of mTOR signalling, which is upregulated in cells deficient in both c-CBL and CBL-b ²⁶⁰, rescues PDGF-AA–mediated signalling in IFT88 and IFT172 mutant fibroblasts that are devoid of primary cilia ²⁵⁰, although further studies are required to understand the relationship between aberrant mTOR signalling and mislocalization of PDGFRα in the context of ciliary signalling and tumorigenesis. Finally, INPP5E, was suggested to control ciliary PDGFRα signaling by inhibiting PDGF-mediated AKT activation^{261, 262, 263}. These findings could indicate that INPP5E contributes to regulation of the balanced output of PDGF-AA-mediated AKT signalling at the cilium and that ciliopathies associated with mutations in INPP5E, such as Joubert syndrome, are partly linked to defects in this pathway.

Consequences of TGFβ/BMP signalling

Increasing body of evidence indicates a prominent role for the primary cilium in co-organizing the balanced output of TGFβ/BMP signalling. The first report of active TGFβ signalling in the primary cilium was based on studies in cultured mouse and human fibroblasts in which TGFβ-RI and TGFβ-RII were shown to localize along and at the tip of the cilium ²⁶⁸. Upon ligand stimulation, the receptors accumulated at the ciliary base region, which is enriched in SMAD4, after which the receptors were internalized by clathrin-mediated endocytosis at the ciliary pocket to activate SMAD2/3 ²⁶⁸ (Figure 6). Similarly, TGFβ signalling was associated with activation of ERK1/2 at the ciliary base, but in contrast to SMAD2/3, TGFβ1-induced phosphorylation of this MAP kinase seemed to occur independently of clathrin-mediated endocytosis ²⁶⁸. These findings indicate that the primary cilium uses diverse mechanisms to fine-tune the output of canonical and non-canonical TGFβ signalling to control varied cellular responses, which could be relevant for processes such as those related to heart development, where TGFβ-dependent differentiation of mouse cancer stem cells and human embryonic stem cells into cardiomyocytes is associated with the temporal accumulation of TGFβ receptors and activation of SMAD2/3 at the ciliary base ²⁶⁸. TGFβ and BMP receptors also localize to primary cilia in other cell types ^{241, 269–273} to regulate mouse heart development in vivo ²⁷³, migration of human bone mesenchymal stem cells in bone remodelling assays ²⁷⁰ and osteogenic differentiation in vitro and bone formation in vivo ^{269, 271, 274}. Primary cilia were also identified as major regulators of TGFβ-mediated activation of R-SMADs in the differentiation of human adipose progenitors into myofibroblasts ²⁷⁵. Further, endothelial primary cilia, which function as flow sensors in the vasculature and contribute to blood–brain barrier integrity ^276–279, counteract endothelial-to-mesenchymal transition in a process that is associated with reduced canonical TGFβ signalling in the endocardial cushion area ²⁸⁰, whereas endothelial primary cilia cooperate with BMP signalling to stabilize vessel connections in the developing mouse retina ²⁸¹. More directly, shortening of primary cilia by TGFβ signalling ^282–284 is associated with epithelial-to-mesenchymal transition in mouse kidney epithelial cells ²⁸⁴ and impairment of mechanosensation and maturation in human osteoblasts ²⁸³.

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Figure 6.

Overview of ciliary TGFβ/BMP signalling.

Receptors of the transforming growth factor beta (TGFβ) and bone morphogenic protein (BMP) family are recruited to the primary cilium to activate receptor (R)–SMAD transcription factors (SMAD2/3 and SMAD1/5/8) partly via internalization of active receptors by clathrin-mediated endocytosis (CME) at the ciliary pocket. Activated R-SMADs form a trimeric complex with the co-SMAD, SMAD4, which translocate to the nucleus for targeted gene expression. TGFβ may activate ERK1/2 in the cilium independently of CME. RAB11-mediated recycling of TGFβ receptors to the primary cilium may be regulated by the subdistal appendage protein, CEP128. Feedback inhibition of ciliary TGFβ/BMP signalling is under the control of the inhibitor SMAD, SMAD7, as well as the E3 ubiquitin ligase, SMURF1. Ciliary TGFβ receptors may further stimulate the hedgehog pathway component, smoothened (SMO), leading to activation of GLI transcription factors. Abbreviations: TGFβ-RI/II, TGFβ receptors I and II; BMP-RI/II, BMP receptors I and II; GLI-A: activator form of GLI transcription factors; ERK1/2: extracellular signal–regulated kinase 1/2; EE: early endosome.

The importance of the cilium in coordinating TGFβ/BMP signalling is underscored by the fact that additional sets of proteins, which regulate receptor trafficking and mechanisms in feedback inhibition, are enriched at the cilia-centrosome axis (Figure 6). RAB11 ²⁸⁵, which is responsible for endosomal recycling of TGFβ receptors ²⁸⁶, is recruited to the cilium in a process that is mediated by the subdistal appendage protein, CEP128 ²⁸⁷. Consequently, loss of CEP128 leads to impaired phosphorylation of R-SMADs, which in zebrafish is associated with defective organ development ²⁸⁷. Conversely, feedback inhibitors of TGFβ/BMP signalling, including SMAD7 and SMURF1 ^{265, 288}, localize to the ciliary base ^{268, 273} and restrain excessive signalling from the primary cilium. SMAD7 has been proposed to interact with TGFβ receptors to limit ARL6-mediated ciliary localization of the receptors and suppress tumour cell migration and invasion by restricting cross-talk between TGFβ receptors and SMO in HH signalling ²⁷². In this context, hamartin (also known as tuberous sclerosis complex protein 1, TSC1, which originally was found to form a heterodimeric complex with TSC2 that negatively regulates mTOR signaling, is required for TGFβ-induced phosphorylation of SMAD2/3 and subsequent expression of GLI2 and WNT5A in an mTOR-independent manner ²⁸⁹, signifying an additional layer of cross-talk between multiple signaling pathways at the primary cilium. Finally, the E3 ubiquitin ligase SMURF1 negatively regulates phosphorylation of SMAD1/5/8 at primary cilia to control cell-type specification during in vivo heart development, which in Smurf1^−/− mouse embryos is associated with delayed outflow tract septation ²⁷³.