GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

Permanent deconstruction of intracellular primary cilia in differentiating granule cell neurons.

Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While essential during early developmental stages, the fate of granule cell cilia is unknown. Here, we provide nanoscopic resolution of ciliary dynamics in situ by studying developmental changes in granule cell cilia using large-scale electron microscopy volumes and immunostaining of mouse cerebella. We found that many granule cell primary cilia were intracellular and concealed from the external environment. Cilia were disassembed in differentiating granule cell neurons in a process we call cilia deconstruction that was distinct from pre-mitotic cilia resorption in proliferating progenitors. In differentiating granule cells, ciliary loss involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Cilia did not reform from the docked centrioles, rather, in adult mice granule cell neurons remained unciliated. Many neurons in other brain regions require cilia to regulate function and connectivity. In contrast, our results show that granule cell progenitors had concealed cilia that underwent deconstruction potentially to prevent mitogenic hedgehog responsiveness. The ciliary deconstruction mechanism we describe could be paradigmatic of cilia removal during differentiation in other tissues.

Context-dependent ciliary regulation of hedgehog pathway repression in tissue morphogenesis.

A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization-especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes-is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened, the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

Translational regulation of δ-tubulin through its 5'-untranslated region.

BACKGROUND: δ-tubulin - a member of tubulin superfamily, is found in a subset of eukaryotes including human where it has a role in centriole maturation. The mutation in the gene results in a disorganized microtubule triplet arrangement leading to formation of defective centriole. Since centriole maturation is a periodic event, it will be interesting to see if δ-tubulin is also regulated in a cell cycle dependent manner. METHODS AND RESULTS: In this regard we show that the abundance of δ-tubulin mRNA remains unchanged throughout the cell cycle. However, the protein level varies periodically with a significantly higher expression in S-phase, implying regulation at the level of translation. Sequence analysis establishes the presence of a 90-base long conserved region, including a consensus motif of nine residues in the 5´-untranslated region (5´-UTR) of δ-tubulin transcript. The deletion analysis of the conserved region using luciferase reporter assay system confirms its strong inhibitory effect on translation. Interestingly, microtubule associated protein 4 (MAP4) is found to interact specifically with the 90-base long conserved region in the 5´-UTR and possibly responsible, at least partially, for the translation inhibitory activity of the UTR. Remarkably, MAP4 interacts with δ-tubulin in a periodic manner at protein level also. CONCLUSION: The results reported here show that δ-tubulin protein expression is regulated at posttranscriptional level and strongly suggest the role of MAP4 in modulation of both abundance and function of δ-tubulin.

Human IFT-A complex structures provide molecular insights into ciliary transport.

Intraflagellar transport (IFT) complexes, IFT-A and IFT-B, form bidirectional trains that move along the axonemal microtubules and are essential for assembling and maintaining cilia. Mutations in IFT subunits lead to numerous ciliopathies involving multiple tissues. However, how IFT complexes assemble and mediate cargo transport lacks mechanistic understanding due to missing high-resolution structural information of the holo-complexes. Here we report cryo-EM structures of human IFT-A complexes in the presence and absence of TULP3 at overall resolutions of 3.0-3.9 Å. IFT-A adopts a "lariat" shape with interconnected core and peripheral subunits linked by structurally vital zinc-binding domains. TULP3, the cargo adapter, interacts with IFT-A through its N-terminal region, and interface mutations disrupt cargo transport. We also determine the molecular impacts of disease mutations on complex formation and ciliary transport. Our work reveals IFT-A architecture, sheds light on ciliary transport and IFT train formation, and enables the rationalization of disease mutations in ciliopathies.

Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia.

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

A pathogenic variant of TULP3 causes renal and hepatic fibrocystic disease.

Patient variants in Tubby Like Protein-3 (TULP3) have recently been associated with progressive fibrocystic disease in tissues and organs. TULP3 is a ciliary trafficking protein that links membrane-associated proteins to the intraflagellar transport complex A. In mice, mutations in Tulp3 drive phenotypes consistent with ciliary dysfunction which include renal cystic disease, as part of a ciliopathic spectrum. Here we report two sisters from consanguineous parents with fibrocystic renal and hepatic disease harboring a homozygous missense mutation in TULP3 (NM_003324.5: c.1144C>T, p.Arg382Trp). The R382W patient mutation resides within the C-terminal Tubby domain, a conserved domain required for TULP3 to associate with phosphoinositides. We show that inner medullary collecting duct-3 cells expressing the TULP3 R382W patient variant have a severely reduced ability to localize the membrane-associated proteins ARL13b, INPP5E, and GPR161 to the cilium, consistent with a loss of TULP3 function. These studies establish Arginine 382 as a critical residue in the Tubby domain, which is essential for TULP3-mediated protein trafficking within the cilium, and expand the phenotypic spectrum known to result from recessive deleterious mutations in TULP3.

Cilia-Localized Counterregulatory Signals as Drivers of Renal Cystogenesis.

Primary cilia play counterregulatory roles in cystogenesis-they inhibit cyst formation in the normal renal tubule but promote cyst growth when the function of polycystins is impaired. Key upstream cilia-specific signals and components involved in driving cystogenesis have remained elusive. Recent studies of the tubby family protein, Tubby-like protein 3 (TULP3), have provided new insights into the cilia-localized mechanisms that determine cyst growth. TULP3 is a key adapter of the intraflagellar transport complex A (IFT-A) in the trafficking of multiple proteins specifically into the ciliary membrane. Loss of TULP3 results in the selective exclusion of its cargoes from cilia without affecting their extraciliary pools and without disrupting cilia or IFT-A complex integrity. Epistasis analyses have indicated that TULP3 inhibits cystogenesis independently of the polycystins during kidney development but promotes cystogenesis in adults when polycystins are lacking. In this review, we discuss the current model of the cilia-dependent cyst activation (CDCA) mechanism in autosomal dominant polycystic kidney disease (ADPKD) and consider the possible roles of ciliary and extraciliary polycystins in regulating CDCA. We then describe the limitations of this model in not fully accounting for how cilia single knockouts cause significant cystic changes either in the presence or absence of polycystins. Based on available data from TULP3/IFT-A-mediated differential regulation of cystogenesis in kidneys with deletion of polycystins either during development or in adulthood, we hypothesize the existence of cilia-localized components of CDCA (cCDCA) and cilia-localized cyst inhibition (CLCI) signals. We develop the criteria for cCDCA/CLCI signals and discuss potential TULP3 cargoes as possible cilia-localized components that determine cystogenesis in kidneys during development and in adult mice.

Studying Hedgehog Signaling During Mouse Neural Tube Development.

The identity of ventral neural progenitors in the neural tube is largely dependent on Hedgehog (Hh) signaling. Variations in staining patterns are excellent indicators of aberrant Hh signaling. Here we describe the basic protocol to stain for progenitor populations based on transcription factor expression. We also provide an overview of ciliary and centrosomal staining in the neural tube.

Wnt1 Lineage Specific Deletion of Gpr161 Results in Embryonic Midbrain Malformation and Failure of Craniofacial Skeletal Development.

Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO (Gpr161 f/f; Wnt1-Cre) fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis.

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner.

The role of compartmentalized signaling in primary cilia during tissue morphogenesis is not well understood. The cilia localized G protein-coupled receptor, Gpr161, represses hedgehog pathway via cAMP signaling. We engineered a knock-in at the Gpr161 locus in mice to generate a variant (Gpr161mut1), which was ciliary localization defective but cAMP signaling competent. Tissue phenotypes from hedgehog signaling depend on downstream bifunctional Gli transcriptional factors functioning as activators or repressors. Compared to knockout (ko), Gpr161mut1/ko had delayed embryonic lethality, moderately increased hedgehog targets, and partially down-regulated Gli3 repressor. Unlike ko, the Gpr161mut1/ko neural tube did not show Gli2 activator-dependent expansion of ventral-most progenitors. Instead, the intermediate neural tube showed progenitor expansion that depends on loss of Gli3 repressor. Increased extraciliary receptor levels in Gpr161mut1/mut1 prevented ventralization. Morphogenesis in limb buds and midface requires Gli repressor; these tissues in Gpr161mut1/mut1 manifested hedgehog hyperactivation phenotypes-polydactyly and midfacial widening. Thus, ciliary and extraciliary Gpr161 pools likely establish tissue-specific Gli repressor thresholds in determining morpho-phenotypic outcomes.

Genome wide comparison of Leishmania donovani strains from Indian visceral leishmaniasis and para-kala-azar dermal leishmaniasis patients.

Visceral leishmaniasis (VL) or Kala-azar, primarily caused by Leishmania donovani, is a major health concern in many countries including India. Growing unresponsiveness among the parasites toward the available drugs is alarming, and so, it is necessary to decipher the underlying mechanism of such development for designing new therapeutics. Moreover, even after successful treatment, some VL patients develop apparently harmless skin lesions known as post-kala-azar dermal leishmaniasis (PKDL) which may serve as a reservoir of the parasite in the transmission cycle. Furthermore, recent reports of para-kala-azar dermal leishmaniasis (para-KDL) cases having PKDL manifestation with concomitant VL, emphasize the necessity of more attention to address complex nature of the parasite for eradicating the disease effectively. In the present study, whole genome sequencing is performed with sodium stibogluconate (SSG) sensitive and resistant L. donovani strains along with SSG sensitive para-KDL strains, derived from the clinical isolates of Indian patients to identify the genomic variations among them. Notably, the analyses of chromosome somy values and genome wide mutation profile in the coding regions reveal distinct clustering of the para-KDL strains with 24 genes being mutated uniquely in this group. Such distinguishing genomic changes among the para-KDL strains could be significant for the parasites to become dermatotropic. Overall, the study reveals a possible correlation of the development of SSG resistance and the transition towards the manifestation of PKDL with chromosome aneuploidy and non-synonymous genetic variations in the coding sequences of the L. donovani strains from Indian patients.

Interface controlled thermal resistances of ultra-thin chalcogenide-based phase change memory devices.

Phase change memory (PCM) is a rapidly growing technology that not only offers advancements in storage-class memories but also enables in-memory data processing to overcome the von Neumann bottleneck. In PCMs, data storage is driven by thermal excitation. However, there is limited research regarding PCM thermal properties at length scales close to the memory cell dimensions. Our work presents a new paradigm to manage thermal transport in memory cells by manipulating the interfacial thermal resistance between the phase change unit and the electrodes without incorporating additional insulating layers. Experimental measurements show a substantial change in interfacial thermal resistance as GST transitions from cubic to hexagonal crystal structure, resulting in a factor of 4 reduction in the effective thermal conductivity. Simulations reveal that interfacial resistance between PCM and its adjacent layer can reduce the reset current for 20 and 120 nm diameter devices by up to ~ 40% and ~ 50%, respectively. These thermal insights present a new opportunity to reduce power and operating currents in PCMs.

Developmental and regenerative paradigms of cilia regulated hedgehog signaling.

Primary cilia are immotile appendages that have evolved to receive and interpret a variety of different extracellular cues. Cilia play crucial roles in intercellular communication during development and defects in cilia affect multiple tissues accounting for a heterogeneous group of human diseases called ciliopathies. The Hedgehog (Hh) signaling pathway is one of these cues and displays a unique and symbiotic relationship with cilia. Not only does Hh signaling require cilia for its function but the majority of the Hh signaling machinery is physically located within the cilium-centrosome complex. More specifically, cilia are required for both repressing and activating Hh signaling by modifying bifunctional Gli transcription factors into repressors or activators. Defects in balancing, interpreting or establishing these repressor/activator gradients in Hh signaling either require cilia or phenocopy disruption of cilia. Here, we will summarize the current knowledge on how spatiotemporal control of the molecular machinery of the cilium allows for a tight control of basal repression and activation states of the Hh pathway. We will then discuss several paradigms on how cilia influence Hh pathway activity in tissue morphogenesis during development. Last, we will touch on how cilia and Hh signaling are being reactivated and repurposed during adult tissue regeneration. More specifically, we will focus on mesenchymal stem cells within the connective tissue and discuss the similarities and differences of how cilia and ciliary Hh signaling control the formation of fibrotic scar and adipose tissue during fatty fibrosis of several tissues.

Ubiquitin Tunes Hedgehog in Matters of the Heart.

Oligogenic inheritance makes the etiology of developmental diseases challenging to determine. In this issue of Developmental Cell, Kong et al., 2020 identify members of a membrane-tethered ubiquitin complex that attenuates Hedgehog signaling strength and genetically interact to regulate digit number, body patterning, and cardiac development.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning.

Lone-Pair Electron-Driven Thermoelectrics at Room Temperature.

Identifying materials with good electron transport and poor thermal transport properties for thermoelectric applications has been challenging. Here we report a series of new materials including Tl3TaSe4 and Tl3VS4 with promising thermoelectric properties giving thermoelectric figure of merit, zT ≈ 0.8 at room temperature using first-principles calculations. This high zT stems from the high electrical conductivity and ultralow thermal conductivity (κ). We calculate κ ≈ 0.1-0.2 W/m·K from a phonon Boltzmann's transport equation and κ ≈ 0.3-0.4 W/m·K from the two-channel model. Low phonon group velocities due to weakly bonded Tl atoms and strong anharmonicity associated with s2 lone electrons pair give rise to such a low κ in these systems.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Tulp3 Regulates Renal Cystogenesis by Trafficking of Cystoproteins to Cilia.

Polycystic kidney disease proteins, polycystin-1 and polycystin-2, localize to primary cilia. Polycystin knockouts have severe cystogenesis compared to ciliary disruption, whereas simultaneous ciliary loss suppresses excessive cyst growth. These data suggest the presence of a cystogenic activator that is inhibited by polycystins and an independent but relatively minor cystogenic inhibitor, either of which are cilia dependent. However, current genetic models targeting cilia completely ablate the compartment, making it difficult to uncouple cystoprotein function from ciliary localization. Thus, the role of cilium-generated signaling in cystogenesis is unclear. We recently demonstrated that the tubby family protein Tulp3 determines ciliary trafficking of polycystins in kidney collecting duct cells without affecting protein levels or cilia. Here, we demonstrate that embryonic-stage, nephron-specific Tulp3 knockout mice developed cystic kidneys, while retaining intact cilia. Cystic kidneys showed increased mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), mTOR, and persistently high cyclic AMP (cAMP) signaling, suggesting contribution of multiple factors to cystogenesis. Based on kidney-to-body-weight ratio, cystic index, and epithelial proliferation in developing tubules or cysts, the severity of cystogenesis upon Tulp3 deletion was intermediate between that caused by loss of polycystin-1 or cilia. However, concomitant Tulp3 loss did not inhibit cystogenesis in polycystin-1 knockouts, unlike ciliary disruption. Interestingly, ciliary trafficking of the small guanosine triphosphatase (GTPase) Arl13b, loss of which causes cystogenic severity similar to ciliary loss, was reduced prior to cyst initiation. Thus, we propose that cystogenesis in Tulp3 mutants results from a reduction of ciliary levels of polycystins, Arl13b, and Arl13b-dependent lipidated cargoes. Arl13b might be the ciliary factor that represses cystogenesis distinct from polycystins.