Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis.

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Regulatory mechanisms of cytoneme-based morphogen transport.

During development and tissue homeostasis, cells must communicate with their neighbors to ensure coordinated responses to instructional cues. Cues such as morphogens and growth factors signal at both short and long ranges in temporal- and tissue-specific manners to guide cell fate determination, provide positional information, and to activate growth and survival responses. The precise mechanisms by which such signals traverse the extracellular environment to ensure reliable delivery to their intended cellular targets are not yet clear. One model for how this occurs suggests that specialized filopodia called cytonemes extend between signal-producing and -receiving cells to function as membrane-bound highways along which information flows. A growing body of evidence supports a crucial role for cytonemes in cell-to-cell communication. Despite this, the molecular mechanisms by which cytonemes are initiated, how they grow, and how they deliver specific signals are only starting to be revealed. Herein, we discuss recent advances toward improved understanding of cytoneme biology. We discuss similarities and differences between cytonemes and other types of cellular extensions, summarize what is known about how they originate, and discuss molecular mechanisms by which their activity may be controlled in development and tissue homeostasis. We conclude by highlighting important open questions regarding cytoneme biology, and comment on how a clear understanding of their function may provide opportunities for treating or preventing disease.

Analysis of Dispatched Protein Processing and Sonic Hedgehog Ligand Release.

The 12-pass transmembrane protein Dispatched (DISP) is essential for Sonic Hedgehog (SHH) release from ligand-producing cells and is indispensable for establishment of the SHH morphogen gradient during tissue patterning. Regulatory events controlling DISP release of SHH are not yet fully characterized. We recently demonstrated that DISP is cleaved by FURIN proprotein convertase at a conserved site in its first extracellular loop. Mutation of the cleavage site attenuates DISP-mediated SHH release, which indicates that Furin cleavage is a positive step toward DISP protein maturation. In this chapter, we present a ligand release/retention protocol that allows for the analysis of DISP cleavage, DISP-mediated release of SHH ligand from producing cells, and secretion-dependent signal induction in target cells.

SPOP and CUL3 Modulate the Sonic Hedgehog Signal Response Through Controlled Degradation of GLI Family Transcription Factors.

The speckle-type POZ protein (SPOP) functions as a guardian of genome integrity and controls transcriptional regulation by functioning as a substrate adaptor for CUL3/RING-type E3 ubiquitin ligase complexes. SPOP-containing CUL3 complexes target a myriad of DNA-binding proteins involved in DNA repair and gene expression, and as such, are essential modulators of cellular homeostasis. GLI transcription factors are effectors of the Hedgehog (HH) pathway, a key driver of tissue morphogenesis and post-developmental homeostasis that is commonly corrupted in cancer. CUL3-SPOP activity regulates amplitude and duration of HH transcriptional responses by controlling stability of GLI family members. SPOP and GLI co-enrich in phase separated nuclear droplets that are thought to serve as hot spots for CUL3-mediated GLI ubiquitination and degradation. A similar framework exists in Drosophila, in which the Hedgehog-induced MATH (meprin and traf homology) and BTB (bric à brac, tramtrack, broad complex) domain containing protein (HIB) targets the GLI ortholog Cubitus interruptus (Ci) for Cul3-directed proteolysis. Despite this functional conservation, the molecular mechanisms by which HIB and SPOP contribute to Drosophila and vertebrate HH signaling differ. In this mini-review we highlight similarities between the two systems and discuss evolutionary divergence in GLI/Ci targeting that informs our understanding of how the GLI transcriptional code is controlled by SPOP and CUL3 in health and disease.

Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex.

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. The cytoneme morphogen transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signaling thresholds are achieved. How morphogens are transported along and deployed from cytonemes, how quickly a cytoneme-delivered, receptor-dependent signal is initiated, and whether these processes are conserved across phyla are not known. Herein, we reveal that the actin motor Myosin 10 promotes vesicular transport of Sonic Hedgehog (SHH) morphogen in mouse cell cytonemes, and that SHH morphogen gradient organization is altered in neural tubes of Myo10-/- mice. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid, receptor-dependent signal response that occurs within seconds of ligand delivery. This activity is dependent upon a novel Dispatched (DISP)-BOC/CDON co-receptor complex that functions in ligand-producing cells to promote cytoneme occurrence and facilitate ligand delivery for signal activation.

During development, cells must work together and talk to each other to build the organs and tissues of the growing embryo. To communicate precisely with long-distance targets, cells can project a series of thin finger-like structures known as cytonemes. Cells use these miniature highways to exchange cargo and signals, such as the protein sonic hedgehog (SHH for short). Alterations to the way SHH is exchanged during development predispose to cancer and lead to disorders of the nervous system. Yet, the mechanisms by which cytonemes work in mammals remain to be fully elucidated. In particular, it is still unclear how the structures start to form, and how the proteins are loaded and transported from one end to another. A 'molecular motor' called myosin 10, which can carry cargo along the internal skeleton of cells, may be involved in these processes. To find out, Hall et al. used fluorescent probes to track both myosin 10 and SHH in mouse cells, showing that myosin 10 carries SHH from the core of the signal-producing cell to the tips of cytonemes. There, the protein is passed to the target cell upon contact, triggering a quick response. SHH also appeared to be more than just passive cargo, interacting with another group of proteins in the signal-emitting cell before reaching its target. This mechanism then encourages the signalling cells to produce more cytonemes towards their neighbours. SHH is crucial during development, but also after birth: in fact, changes to SHH transport in adulthood can also disrupt tissue balance and hinder healing. Understanding how healthy tissues send this signal may reveal why and how disease emerges.

Dispatching Sonic Hedgehog: Molecular Mechanisms Controlling Deployment.

The Hedgehog (Hh) family of morphogens direct cell fate decisions during embryogenesis and signal to maintain tissue homeostasis after birth. Hh ligands harbor dual lipid modifications that anchor the proteins into producing cell membranes, effectively preventing ligand release. The transporter-like protein Dispatched (Disp) functions to release these membrane tethers and mobilize Hh ligands to travel toward distant cellular targets. The molecular mechanisms by which Disp achieves Hh deployment are not yet fully understood, but a number of recent publications provide insight into the complex process of Hh release. Herein we review this literature, integrate key discoveries, and discuss some of the open questions that will drive future studies aimed at understanding Disp-mediated Hh ligand deployment.

Cleavage activates dispatched for Sonic Hedgehog ligand release.

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is absolutely required for signaling to long-range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. Herein, we reveal vertebrate Dispatched is activated by proprotein convertase-mediated cleavage at a conserved processing site in its first extracellular loop. Dispatched processing occurs at the cell surface to instruct its membrane re-localization in polarized epithelial cells. Cleavage site mutation alters Dispatched membrane trafficking and reduces ligand release, leading to compromised pathway activity in vivo. As such, convertase-mediated cleavage is required for Dispatched maturation and functional competency in Hedgehog ligand-producing cells.

Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation.

Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.

A fixation method to preserve cultured cell cytonemes facilitates mechanistic interrogation of morphogen transport.

During development, extracellular cues guiding cell fate determination are provided by morphogens. One mechanism by which morphogens are proposed to traverse extracellular space is by traveling along specialized filopodia called cytonemes. These cellular highways extend between signal-producing and -receiving cells to enable direct morphogen delivery. Although genetic studies support cytoneme involvement in morphogen transport, mechanistic insight into how they are regulated is limited owing to technical challenges associated with performing cell biological analysis of the delicate filopodial structures. Here, we introduce a fixation method whereby cultured cell cytonemes can be preserved for imaging studies, allowing investigation of cytoneme regulation using standard cell biological techniques. Using this method, we examined Hedgehog-containing cytonemes and identified a role for the Hedgehog deployment protein Dispatched in cytoneme stabilization. We demonstrate that Hedgehog and Dispatched colocalize in cytonemes, and that cholesterol-modified Hedgehog acts through Dispatched to increase cytoneme occurrence. Live imaging suggests that this occurs through Dispatched-mediated slowing of cytoneme retraction rates. Dispatched-induced cytoneme modulation was recapitulated in wing imaginal discs of transgenic Drosophila, providing evidence that cultured cell cytoneme analysis is predictive of in vivo functionality.

Sonic Hedgehog Activates Phospholipase A2 to Enhance Smoothened Ciliary Translocation.

The G protein-coupled receptor Smoothened (Smo) is the signal transducer of the Sonic Hedgehog (Shh) pathway. Smo signals through G protein-dependent and -independent routes, with G protein-independent canonical signaling to Gli effectors requiring Smo accumulation in the primary cilium. The mechanisms controlling Smo activation and trafficking are not yet clear but likely entail small-molecule binding to pockets in its extracellular cysteine-rich domain (CRD) and/or transmembrane bundle. Here, we demonstrate that the cytosolic phospholipase cPLA2α is activated through Gßγ downstream of Smo to release arachidonic acid. Arachidonic acid binds Smo and synergizes with CRD-binding agonists, promoting Smo ciliary trafficking and high-level signaling. Chemical or genetic cPLA2α inhibition dampens Smo signaling to Gli, revealing an unexpected contribution of G protein-dependent signaling to canonical pathway activity. Arachidonic acid displaces the Smo transmembrane domain inhibitor cyclopamine to rescue CRD agonist-induced signaling, suggesting that arachidonic acid may target the transmembrane bundle to allosterically enhance signaling by CRD agonist-bound Smo.

Contributions of Noncanonical Smoothened Signaling During Embryonic Development.

The Sonic Hedgehog (Shh) signaling pathway is active during embryonic development in metazoans, and provides instructional cues necessary for proper tissue patterning. The pathway signal transducing component, Smoothened (Smo), is a G protein-coupled receptor (GPCR) that has been demonstrated to signal through at least two effector routes. The first is a G protein-independent canonical route that signals to Gli transcriptional effectors to establish transcriptional programs specifying cell fate during early embryonic development. The second, commonly referred to as the noncanonical Smo signal, induces rapid, transcription-independent responses that are essential for establishing and maintaining distinct cell behaviors during development. Herein, we discuss contributions of this noncanonical route during embryonic development. We also highlight important open questions regarding noncanonical Smo signal route selection during development, and consider implications of noncanonical signal corruption in disease.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies.

Dataset for phenotypic classification of genetic modifiers of smoothened and Hedgehog.

This data article includes supporting information for the research article entitled "The Small GTPase Rap1 is a Modulator of Hedgehog Signaling" [1]. Drosophila wing phenotypes induced by expression of a dominant negative Smoothened (Smo) mutant were cataloged into five distinct classes. Class distributions observed following expression of dominant negative Smo in control and sensitized backgrounds were quantified to serve as references for strength of phenotypic modification. Shifts in class distribution of Hedgehog (Hh) wing phenotypes resulting from introduction of loss-of-function alleles of select Ras family G protein genes and the Hh pathway regulators Fused and Suppressor of Fused are shown.

Smoothened Regulation: A Tale of Two Signals.

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the signal transducer of the developmentally and therapeutically relevant Hedgehog (Hh) pathway. Although recent structural analyses have advanced our understanding of Smo biology, several questions remain. Chief among them are the identity of its natural ligand, the regulatory processes controlling its activation, and the mechanisms by which it signals to downstream effectors. In this review, we discuss recent discoveries from multiple model systems that have set the stage for solving these mysteries. We focus on the roles of distinct Smo functional domains, post-translational modifications, and trafficking, and conclude by discussing their contributions to signal output.

The small GTPase Rap1 is a modulator of Hedgehog signaling.

During development, the evolutionarily conserved Hedgehog (Hh) morphogen provides instructional cues that influence cell fate, cell affinity and tissue morphogenesis. To do so, the Hh signaling cascade must coordinate its activity with other morphogenetic signals. This can occur through engagement of or response to effectors that do not typically function as core Hh pathway components. Given the ability of small G proteins of the Ras family to impact cell survival, differentiation, growth and adhesion, we wanted to determine whether Hh and Ras signaling might intersect during development. We performed genetic modifier tests in Drosophila to examine the ability of select Ras family members to influence Hh signal output, and identified Rap1 as a positive modulator of Hh pathway activity. Our results suggest that Rap1 is activated to its GTP-bound form in response to Hh ligand, and that the GTPase exchange factor C3G likely contributes to this activation. The Rap1 effector Canoe (Cno) also impacts Hh signal output, suggesting that a C3G-Rap1-Cno axis intersects the Hh pathway during tissue morphogenesis.

Genetic evidence for a Smoothened-Gα(i) signaling axis in mammals.

In this issue of Science Signaling, Villanueva et al. report on the identification of heterotrimeric guanine nucleotide-binding protein (G protein) Gαi2 as an essential effector of Smoothened-mediated mammary epithelial cell proliferation. Through a series of in vivo experiments, the authors delineate a Smoothened-regulated Gli-independent Gαi signal that provides direct genetic evidence connecting Smoothened with Gαi in mice.

Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling.

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small-molecule modulators.