The Cytoneme Connection: Direct Long-Distance Signal Transfer During Development Laura González-Méndez, Ana-Citlali Gradilla,‡ and Isabel Guerrero‡

PRIMER The cytoneme connection: direct long-distance signal transfer during development Laura González-Méndez*, Ana-Citlali Gradilla*,‡ and Isabel Guerrero‡

ABSTRACT dimensional structures in embryonic tissues and give rise to a During development, specialized cells produce signals that distribute precise spatial pattern remains an unresolved issue. Another among receiving cells to induce a variety of cellular behaviors and significant challenge is to decipher how only some cells are organize tissues. Recent studies have highlighted cytonemes, a type targeted by a specific signal, especially in a naïve tissue with a of specialized signaling filopodia that carry ligands and/or receptor confluence of different morphogenetic signals. Furthermore, it is complexes, as having a role in signal dispersion. In this Primer, we necessary to understand how target cells are selected when signaling discuss how the dynamic regulation of cytonemes facilitates signal cells are either acquiring different fates or are physically separated transfer in complex environments. We assess recent evidence for by other tissues. Finally, and considering that most morphogens are the mechanisms for cytoneme formation, function and regulation, insoluble lipid-modified or integral transmembrane proteins, it is of and postulate that contact between cytoneme membranes promotes equal importance to understand how their movement through the signal transfer as a new type of synapse (morphogenetic synapsis). extracellular space is achieved. Taking all these points into account, Finally, we reflect on the fundamental unanswered questions related the simple diffusion of morphogens does not seem to be a viable to understanding cytoneme biology. mechanism for precise cell positional information. On the other hand, an alternative mode of distant signaling has KEY WORDS: Cell-cell communication, Signaling filopodia, been put forward, thanks to advances in imaging technology and Cytoneme, Morphogenetic synapses the availability of genetic tools, reporter genes and fluorescent-tagged components. This proposal demonstrates that cells can communicate Introduction at a distance through cellular membrane protrusions. In this Primer, Embryonic patterning is key to the development of complex we focus on signaling filopodia (using the term ‘cytonemes’ for multicellular organisms by establishing the body plan. Our simplicity) as a conduit for the morphogen spatial distribution that understanding of developmental patterning is based on the concept generates patterns of gene expression and cell differentiation. of positional information, which proposes that cells acquire positional Cytonemes act as ‘spatial communication guides’, allowing direct values and differentiate accordingly to give rise to specific spatial physical contact at distance between signal-sending and signal- patterns (Wolpert, 1969). In 1952, Alan Turing proposed a receiving cell membranes (Ramírez-Weber and Kornberg, 1999). mathematical reaction-diffusion system to establish a precise spatial They overcome the challenge of signal transport through tortuous pattern through the interaction of activator and repressor molecules, structures within the tissue, disperse membrane-bound signaling later named morphogens. Here, positional information is acquired molecules and can provide spatial restriction, as well as specificity, in through gradients of diffusible substances (Turing, 1952; Wolpert, signaling. Here, we examine recent progress and hypothesize that 1969; Crick, 1970) produced by a localized group of cells (organizers) synaptic-like processes could be a common mechanism for direct (Spemann and Mangold, 1924). Progress in developmental genetics, signal transfer. Finally, we also summarize remaining questions and molecular identification of biological morphogens, highlighted the regarding the cellular mechanisms involved in cytoneme formation, functional importance of morphogens for embryonic development guiding and dynamics, as well as the mechanisms for signal transfer (Driever and Nusslein-Volhard, 1988; Green and Smith, 1990). during contact with recipient cells. Indeed, several morphogenetic signal gradients that provide positional information in embryos, are presently known (reviewed by Rogers and Types of membrane protrusions Schier, 2011). However, increasing experimental evidence has Thin membrane protrusions are becoming increasingly relevant for challenged simple diffusion models (Kornberg, 2014; Wolpert, 2016). intercellular communication. They are found in different organisms Today, the most common theory for morphogen gradient and tissues, and are implicated in several functions (cell-cell dispersal integrates three parameters: morphogen diffusion from a communication during early development, cell migration, stem cell- localized source; the rate of morphogen production; and the rate of mediated homeostasis and regeneration) and in the progression of morphogen degradation. In addition, interaction of morphogens particular types of cancer (reviewed by Caviglia and Ober, 2018; with extracellular molecules also plays an active role in the Mattes and Scholpp, 2018) (see Table 1). These variable functional regulation of their transport (reviewed by Restrepo et al., 2014). and structural characteristics of cell protrusions have led to various However, how morphogens spread through complex three- terminologies (Fig. 1). Cell membrane protrusions are structurally defined depending on their closed- or open-ended tip, and on the presence of microtubules (MT) and/or an actin cytoskeleton Centro de Biologıá Molecular ‘Severo Ochoa’ (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain. (reviewed by Yamashita et al., 2018). Closed-ended actin-based *These authors contributed equally to this work structures for cell-cell communication have been called signaling filopodia or cytonemes (Fig. 1A). In addition to actin cytoskeleton, ‡Authors for correspondence ([email protected]; [email protected]) tubulin has also been observed restricted to the base of some

A.-C.G., 0000-0003-0992-2557; I.G., 0000-0001-6761-1218 cytonemes (Sanders et al., 2013; Stanganello et al., 2015). DEVELOPMENT

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A Closed-ended actin-based protrusions ligand-receptor binding in a spatially restricted manner, with different extension and retraction velocities and lengths (see Table 1). These dynamics could allow the formation of adaptable Actin and specialized contact sites between juxtaposed membranes. Signal Until recently, it was difficult to functionally assess the role of protein cytonemes in cell signaling without affecting other cellular processes. New approaches using brief pulses of inhibitors or specifically targeting, using RNA interference, actin cytoskeleton B Closed-ended microtubule-based protrusions regulators in a short time-window allow for perturbing cytoneme formation without grossly disrupting overall cell morphology.

Tubulin These approaches have revealed a close correlation between cytoneme formation and signaling function (see Table 1). To date, cytonemes have been identified as signaling-specialized structures Signal protein in Drosophila and vertebrates (amphibians, chick, zebrafish and mouse), and in different developmental models such as during the formation of signaling gradients or in the differentiation of C Open-ended actin- and tubulin-based protrusions specialized cells in a pattern (see Table 1).

Role of cytonemes Actin Tubulin The presence of cytonemes is correlated with paracrine transport of fundamental signaling molecules, including Notch, Spitz (Spi)/ Signal Mitochondria Virus protein EGF, Branchless (Bnl)/FGF, Decapentaplegic (Dpp)/BMP, Wingless (Wg)/Wnt and Hedgehog (Hh)/Sonic hedgehog (Shh) (for references and details, see Table 1). Importantly, cytonemes have been described to be specifically associated with components Fig. 1. Types of membrane extensions for cell signaling. (A) Closed- of a particular signaling pathway, even when emerging from the ended, actin-based membrane extensions, known as signaling filopodia or same cells (Roy et al., 2011). In addition, cytonemes can transport cytonemes. They communicate with cells at a distance by transporting specific ligands and/or their receptors; they can contain microtubules at the base of the either the pathway ligand (or ligand complex) or the receptor (or protrusion. (B) Closed-ended microtubule-based membrane extensions, receptor complex), depending on whether they emanate from known as signaling MT-nanotubes. They transport specific receptors receiving or signal-producing cells, or from both, but always by reaching for the signal-sending cell. (C) Open-ended, actin-based achieve spatially restricted distribution of signals. protrusions, named filopodia tubes, bridges or conduits. They connect cytoplasm between cells and can contain microtubules in the case of Establishing morphogen gradients nanotubes or tunneling nanotubes (TNTs). Cytonemes generate the spatial distribution of morphogen molecules for paracrine signaling and can facilitate the graded MT-nanotubes are microtubule-based protrusions that contact cells dispersion of signals according to their extent and dynamics (see through closed-ended protrusions (Inaba et al., 2015) (Fig. 1B), Table 1, Fig. 2A). Cytoneme-mediated delivery of signaling ligands whereas tunneling nanotubes (TNTs) and membrane nanotubes has been shown for Wnt in zebrafish (Stanganello et al., 2015; (also known as cellular tubes, bridges or conduits) are actin-based Mattes et al., 2018), Hh in Drosophila and Sonic hedgehog (Shh) in open-ended connections, although in some cases they have also chick limb bud (Bischoff et al., 2013; Gradilla and Guerrero, 2013; been described to contain microtubules (Yamashita et al., 2018). Sanders et al., 2013; Chen et al., 2017; González-Méndez et al., TNTs and membrane nanotubes allow the exchange between cells of 2017). Conversely, cytonemes emanating from signal-receiving soluble cytoplasmic components, membrane-associated molecules, cells (see Table 1, Fig. 2B) have been shown to be important for the intracellular vesicles and even larger organelles (Rustom et al., 2004; graded distribution of the Drosophila FGF receptor homolog Gerdes and Carvalho, 2008; Rustom, 2016) (Fig. 1C). Thus, there is Breathless (Btl) (Du et al., 2018) in the developing air sac primordia an increasing number of reported intercellular communicating (ASP), a structure analogous to the human lung. filopodia with apparent structural and functional differences, and we are just beginning to understand the possible categories. In this Pattern formation Primer, we focus on signaling filopodia (cytonemes) and MT Cytonemes can also spatially distribute signals to differentiate nanotubes as close-ended protrusions for cell signaling. specific target cells within an otherwise apparently uniform field, e.g. to form a salt and pepper pattern. In Drosophila,signaling Discovery of cytonemes filopodia direct spatially biased Spitz/EGF enhanced signaling to Thin signaling filopodia were first observed during sea urchin determine the formation of a structural bract cell at the proximal side gastrulation (Miller et al., 1995). They localize at sites in which cell- of each mechanosensory organ in the leg, generating spatially cell signaling is known to take place, but no migration occurs, patterned cell fates (Peng et al., 2012). Similarly, signaling filopodia suggesting a function that is independent from cell migration. Later, direct spatially restricted lateral inhibition of Notch signaling to the term ‘cytoneme’ was coined to define the thin signaling generate the organized pattern of bristles in the notum (De Joussineau filopodia in Drosophila imaginal discs (Ramírez-Weber and et al., 2003; Cohen et al., 2010) (see Table 1). Cytonemes have also Kornberg, 1999). Here, cytonemes act as connectors to mediate been observed during butterfly wing patterning, which suggests that the interaction of the receptor cells with ligand-producing cells, signal delivered by cytonemes might determine the characteristic allowing for specific signal transduction between distant cells. color patterns (Ohno and Otaki, 2015; Iwasaki et al., 2017); indeed,

Importantly, cytonemes are highly dynamic structures that permit such patterns are not explained by signal diffusion alone (Iwata et al., DEVELOPMENT

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A 2018). In addition, cytoneme-mediated communication promotes the pigmented stripe pattern in zebrafish. Here, cytoneme-like ‘ ’ Signaling contacts protrusions called airinemes connect pigmented cells precursors at a distance to facilitate Delta/Notch-mediated signaling, which contributes to the clearance of pigmented cells from the developing Distance from source interstripe and the consolidation of the stripes (Hamada et al., 2014; Eom et al., 2015; Eom and Parichy, 2017) (see Table 1).

Directed morphogenesis Cytonemes can also coordinate morphogenesis of physically separated tissues, bypassing intermediate areas. The development of the ASP in Drosophila is one such example (see Table 1). Cytonemes emanating from the ASP allow the ASP cell membranes to contact the distant membranes of the ligand-producing cells of the wing disc. These contacts facilitate Dpp and FGF signal transfer, which are only produced in the wing disc and are received by the respective receptors at cytonemes of the ASP, promoting both cell B a Ligand-carrier cytonemes migration and patterning (Roy et al., 2014; Du et al., 2018; Huang et al., 2019; Sohr et al., 2019). In this system, cytonemes bypass intermediate tissues, such as the mesodermal layer, between the ASP and the imaginal disc for targeted signaling (Huang and Kornberg, 2015) (Fig. 4). In a similar process, signaling filopodia can also facilitate communication between immobile and migrating cells or moving cells during dynamic morphogenetic processes. It has recently been shown in vertebrates that cytonemes mediate long-distance cell contacts between epithelial and mesenchyme-like tissues b Receptor-carrier cytonemes to facilitate EphB3b-ephrin B1 signaling, which guarantees collective hepatoblast movement during asymmetric placement of liver and gut (Cayuso et al., 2016). See Table 1 for other examples of the role of coordinating morphogenesis by cytonemes in physically separated tissues.

Maintenance of the stem cell niche In addition to the coordination of developmental patterning, cytonemes are essential in the maintenance of restricted paracrine signaling between stem cells and their supportive niche (see Table 1). In the Drosophila ovary, cytonemes maintain the female c Ligand- and receptor-carrier cytonemes germline stem cell (GSC) niche by paracrine Hh signaling at short and long distances. Here, they enable the specificity of signal transport from the support cap cells to the adjacent population of niche cells, the escort cells, and activate the transcription of BMP family members: Dpp and Glass bottom boat (Gbb) (Rojas-Ríos et al., 2012). Similarly, protrusions defined as MT-nanotubes in the Drosophila testis restrict BMP signaling to maintain GSCs in an undifferentiated and self-renewing state. These protrusions extend from male GSCs and direct the Dpp receptor Tkv to the interior positions in the niche in order to bind the Dpp produced by hub cells, without influencing other non-stem cells nearby Fig. 2. Cytonemes coordinate cell signaling to establish developmental (Inaba et al., 2015). Paracrine signaling for stem cell niche patterning. (A) Signal-sending (red) and -receiving (blue) cells communicate maintenance has also been described for the intestinal stem cell with each other, extending cytonemes that emerge from the basolateral (ISC) niche in the Drosophila midgut. ISC cells are maintained by (mainly basal) surface of a developing epithelium. The signal-sending and the enteroendocrine signal Bursicon, which acts at distance on the distant receiving cells are highlighted with paler colors. Graded distribution of the signal (arrow) is given by the proportional number of signal-transferring Lgr2 G-protein-coupled receptor (Scopelliti et al., 2014); however, contacts according to the distance between cells. Additionally, cytonemes to date no mechanism for signal transfer has been reported. can also allow specialization of a single cell (blue) without affecting that of Interestingly, the Lgr2 vertebrate orthologs and stem cell markers neighboring cells. Thus, these mechanism of cellular communication enable Lgr4 and Lgr5 promote cell membrane protrusions that resemble patterning of tissues. (B) Cytoneme contact types. (a) Cytonemes emanating cytonemes in stem cell cultures in vitro (Snyder et al., 2015). Thus, from signal-producing cells contact with specific receptors, mainly at the basal future research might indeed further implicate cytoneme-mediated membrane of the receiving cell. (b) Cytonemes carrying specific receptors extend from receiving cells to capture the signal at the basal membrane of the signal delivery to restrict ISC populations, both during development signal-sending cell. (c) Cytonemes from both signal source and receptor cells and in regenerative processes. Another example is in the Drosophila contact with each other for signal transfer. hematopoietic organ, the lymph gland, in which a small cluster of DEVELOPMENT

3 PRIMER Development (2019) 146, dev174607. doi:10.1242/dev.174607 posterior signaling cells (PSC) controls the balance between first suggested in the sea urchin (Miller et al., 1995). More recently, multipotent prohemocytes and differentiating hemocytes. Here, several membrane-bound signaling proteins have been observed PSCs also exhibit extensive protrusions that project towards the trafficking through cytonemes. Indeed, the extracellular transport lymph gland and appear to be required for the maintenance of the of membrane-anchored morphogens has been associated with undifferentiated cell population, probably by mediating signaling extracellular particles, such as argosomes (Greco et al., 2001), between PSCs and hematopoietic progenitors (Mandal et al., 2007; lipoprotein particles (Panáková et al., 2005), exosomes (Gross et al., Fuwa et al., 2015). 2012; Koles et al., 2012; Beckett et al., 2013; Gradilla et al., 2014) and exosome-like particles (Danilchik et al., 2013; Matusek et al., 2014). Mechanism for cytoneme formation and function Association of extracellular vesicles (EVs) to cytonemes has been Basolateral cytoneme formation described for the Hh morphogen in Drosophila. In this case, In epithelial tissues, cytonemes emanate from specific membrane cytoneme-mediated Hh graded distribution is dependent on protein territories that have an intrinsic basolateral polarization (Fig. 2A). complexes involved in the formation of multivesicular bodies Furthermore, in Drosophila, basolateral positioning of the signaling (MVBs), which are necessary for the biogenesis of signaling EVs ligands Hh, Wg, Delta and Spz, as well as their reception processes, (Gradilla et al., 2014; Matusek et al., 2014; Vyas et al., 2014) have also been described (Steinhauer et al., 2013; Cohen et al., 2010; (Fig. 3). Moreover, live-cell imaging shows that vesicles containing Huang and Kornberg, 2015; Chen et al., 2017; González-Méndez Hh travel along cytonemes and mediate the transfer of signal et al., 2017). This subcellular localization implicates a mechanism or between linked cells (Gradilla et al., 2014; González-Méndez et al., mechanisms that drive both signaling components and machinery for 2017). A related example is that of the zebrafish ‘airinemes’, which the initiation of protrusion to the basolateral side (Fig. 3). In the deliver membranous vesicles containing the Notch ligand Delta Drosophila wing disc, an apico-basal activity gradient of the Rho from xanthoblast to distant dark pigmented cells or melanophores. GTPase Rac has been described to generally regulate filopodial This initiates Notch signaling, which contributes to melanophore polarization (Georgiou and Baum, 2010; Couto et al., 2017). This migration towards stripes and away from the interstripe space Rac gradient is regulated by adherent-junction (AJ) proteins, and (Eom et al., 2015). Interestingly, these vesicles also seem to have drives both the position and shape of epithelial filopodia. Likewise, a a role in the formation of ‘airinemes’ by mediating the association of vesicle-sorting mechanism has been described to transport signaling xanthoblasts surface blebs and moving macrophages. Motile ligands to the basolateral side (Callejo et al., 2011; Bilioni et al., macrophages engulf intact ‘airineme’ vesicles and drag the 2013; Steinhauer et al., 2013; Yamazaki et al., 2016; Sohr et al., vesicles and filaments from the Delta-producing cell as they 2019; reviewed by Gradilla and Guerrero, 2013; Guerrero and move away; in doing so, the macrophages pull and extend the Kornberg, 2014) (Fig. 3). However, the regulatory mechanisms for ‘airinemes’ from the bleb-contact sites to the melanophores for cytoneme cargo upload have yet to be determined and whether the direct signaling (Eom and Parichy, 2017). Other reported vesicle- vesicle-recycling mechanism could also contribute to cytoneme like puncta associated with cytonemes include the motor protein formation. In zebrafish, Wnt8a at the plasma membrane recruits myosin X, which is observed along cytonemes transporting transducer of CDC42-dependent assembly protein 1 (Toca-1) that Shh in chick limb bud (Sanders et al., 2013) and in Wnt- locally activates cytoneme nucleation (Ho et al., 2004; Stanganello bearing cytonemes in zebrafish (Stanganello et al., 2015). Wnt et al., 2015). Hence, intracellular trafficking of the Wnt ligand localization to EVs has also been shown in vertebrates and would be key for the spatial localization of membrane protrusion and Drosophila, although no link between this vesicle type and signaling. cytonemes has been reported (Gross et al., 2012; Koles et al., 2012; Beckett et al., 2013). Trafficking of signaling components along cytonemes Taken together, cytonemes can be envisioned as extensions of There are increasing reports of vesicles or dynamic puncta on membrane territories that couple mechanisms for signaling and cytonemes, which again support a general process of intracellular protrusion initiation. Intracellular vesicle trafficking has a role in the vesicle trafficking for localized signaling territories at membranes (see cellular localization of signaling mediated by cytonemes. Table 1). The use of protrusions as tracks for membrane vesicles was Cytonemes, in turn, can also serve as tracks for the long-distance

AJ Endosome Rac

MVB

EVs Rac activity Actin

Fig. 3. Basolateral polarization of cytoneme-mediated signaling. Mechanisms for either formation or loading of basal polarized cytonemes in epithelial cells are still not well characterized. However, an apico-basal gradient of Rac activity that directs filopodia formation towards the basal membranes has beendescribed in the Drosophila wing disc. In the same line, the basolateral secretion of Hedgehog (Hh) and Wingless (Wg) proteins (not shown) has also been defined, and is achieved via an MVB-mediated recycling mechanism from the apical to the basolateral side of the wing disc epithelium. Hh-containing vesicles are then transported by cytonemes; for Wg, signaling through cytonemes has not yet been described. AJ, adherent junction; MVB, multivesicular body; EVs, extracellular vesicles. DEVELOPMENT

4 PRIMER Development (2019) 146, dev174607. doi:10.1242/dev.174607 transport of vesicle-like structures (Fig. 3). In addition, latest reception (Huang and Kornberg, 2015) (Fig. 4C). Establishing the research has revealed sorting of the Drosophila FGF ligand Bnl Dpp gradient in the Drosophila wing disc also depends on towards the basal side of the wing disc-producing cells, where the cytonemes containing the receptor Thickvein (Tkv). These ASP-emanating cytonemes carrying the FGF receptor Btl make cytonemes extend from cells situated on both sides of the source contact for ligand uptake (Sohr et al., 2019). This ligand sorting territory containing Dpp from producer cells, but no specific contact mechanism requires an intracellular protein cleavage process, site has been defined (Fig. 4B) (Ramírez-Weber and Kornberg, which ensures there is sufficient ligand to sustain the cytoneme 1999). uptake required for appropriate graded dispersion and ASP Experiments using GFP reconstitution across synaptic partners morphogenesis (Sohr et al., 2019). However, further research is (GRASP) have defined precise physical contact sites of cytonemes needed to elucidate the various contributions of vesicle dynamics in from the Drosophila ASP. In the ASP, these cytonemes carrying cytoneme-mediated cell-to-cell communication. receptor complexes contact distant Dpp- and FGF-producing cells of the wing disc (Roy et al., 2014; Du et al., 2018) (Fig. 4D). On the Targeted signal delivery by cytonemes other hand, GRASP reconstitution experiments show that contacts Another key issue to address is how cytonemes recognize and at cytonemes from myoblasts carrying the Notch ligand Delta also contact the target cell for signal transfer. Several studies have contact the ASP, and vice versa, suggesting a possible cytoneme- explored this and have identified two types of contacts: the cytoneme interaction to activate Notch signaling (Huang and cytoneme-cell body contact (Stanganello et al., 2015) and the Kornberg, 2015) (Fig. 4C). cytoneme-cytoneme contact (González-Méndez et al., 2017). These For Hh distribution within Drosophila epithelia, GRASP indeed two types are also conditioned by whether cytonemes carry ligands revealed direct cytoneme-cytoneme contact sites between distant from ligand-producing cells or specific receptor complexes from producer and receptor cells all along their length (González-Méndez responding cells, or both (see Table 1). For example, in the zebrafish et al., 2017) (Fig. 4A). Similar contacts have been suggested for Shh embryo, the tips of cytonemes from Wnt8a-producing cells transfer signaling in the developing chick limb bud, where cytonemes from the ligand by contacting the cell body of responding cells Shh-producing mesenchymal cells interact at defined territories (Stanganello et al., 2015). Conversely, in Drosophila (Fig. 4A-D), along cytonemes that carry the Shh co-receptors and adhesion cytonemes carrying the Frizzled (Fz) receptor from the ASP cells molecules BOC and CDO (Sanders et al., 2013). In addition, the contact the Wg-producing cell bodies in the wing disc for signal Drosophila ortholog of CDO, Interference hedgehog (Ihog), is

A B Trachea Dpp Ptc Hh Wing disc A P

Wing disc Wing disc basal surface Tkv apical surface V

D D C Cross-section Dpp

ASP Tkv FGF

N FGFR ASP Dl Fz Myoblasts Myoblasts Wg Wing disc basal surface Wing disc basal surface

Key Ligand Hh Dpp FGF Wg DI

Receptor Ptc Tkv FGFR Fz N

Fig. 4. Drosophila wing disc: a remarkable model for cytoneme-mediated signaling. Cytoneme-mediated signaling in the Drosophila wing imaginal disc at the L3 larval stage. Colored regions show morphogen-producer cells: Decapentaplegic (Dpp, green), Hedgehog (Hh, red), Wingless (Wg, yellow), FGF (blue) and Delta (Dl, brown). (A) A cytoneme-mediated Hh signaling gradient is established at the basolateral side of the epithelium. Hh expressed in P compartment cells (red) is located in vesicles traveling along basal cytonemes to interact with cytonemes from A compartment cells (gray) containing the receptor Patched (Ptc). (B) A cytoneme-mediated Dpp signaling gradient in the wing disc is established at the apical side of the epithelium. Dpp is expressed in the central region of the wing disc and cells located at both sides respond to Dpp extending apical cytonemes containing the Thickvein (Tkv) receptor. (C) Cytoneme- mediated signaling between three different epithelial tissues: wing imaginal disc, air sac primordium (ASP) and myoblasts. Wing disc-associated myoblasts located between wing disc and ASP epithelia send cytonemes that carry the Frizzled (Fz) receptor to receive the Wg signal expressed in the imaginal disc cells. In addition, myoblasts extend cytonemes carrying the ligand Delta (Dl, brown) to interact with the Notch (N, white) receptor present in the air sac primordia (ASP) cells. (D) Cytoneme-mediated signaling directs ASP development. Signal-specific cyotenemes mediate Dpp or FGF signaling at the ASP. ASP cells extend cytonemes containing either the Tkv receptor to contact Dpp-expressing cells or the FGF receptor (Btl) to contact FGF (Bnl)-expressing cells in the wing disc. DEVELOPMENT

5 PRIMER Development (2019) 146, dev174607. doi:10.1242/dev.174607 detected in several contact points along overlapping cytonemes. step to the secretion of Hh in exosomes, which could take place These contact points might work as specialized sites for Hh specifically at contact points between cytonemes from signal reception and contain other components of the Hh reception producer and receptor cells (Gradilla et al., 2014) (Fig. 5). complex, such as Patched (Ptc) and the glypicans Dally and Dally- Supporting this theory, the contact sites at membranes along like (Dlp) (González-Méndez et al., 2017). As Hh ligand is cytonemes revealed annular structures that resemble specialized membrane bound, such contacts might also define the subcellular presynaptic membrane swellings (González-Méndez et al., 2017), location of proteolytic processing or shedding of anchored Hh, termed synaptic boutons, where vesicles are released at the end of either from cytoneme membranes or from exovesicles released at the neurons during synapsis. Interestingly, both the Hh ligand and its site; this would allow the release of active morphogen only in receptor, Ptc, and co-receptor, Ihog, are localized in these annular response to a precise signal (reviewed by Manikowski et al., 2018). structures (González-Méndez et al., 2017). Moreover, by using the GRASP technique it has been suggested that the size of the contact A synaptic-like signaling model for cytoneme function between cytonemes or between cytonemes and the target cell is A signal transfer mechanism similar to a neuronal synapsis has been comparable with that of immune and neuronal synapses (Roy et al., hypothesized (Kornberg and Roy, 2014; Roy et al., 2014; Chen 2014). Furthermore, synaptic transfer of morphogenetic signals has et al., 2017; González-Méndez et al., 2017; Huang et al., 2019). A also been observed at Drosophila larval neuromuscular junctions, ‘synapse’ is an intercellular communication mechanism that implies where Wg is transported across synapses by vesicles released from close cell-cell specialized contact, which allows for specific targeted presynaptic cells (Korkut et al., 2009). signaling and implies the intracellular localization of presynaptic If signal transmission is confined to the cytoneme-mediated and postsynaptic molecular machinery in specific membrane areas. contact site(s) between sending and recipient cells, signaling must Synaptic-like signaling through cytonemes shares these general then be dependent on the frequency and stability of these contacts characteristics by facilitating direct contact, even at several cell (Fig. 2A). Therefore, it is crucial to identify the molecules responsible diameters of distance. Furthermore, the ligand presentation complex for establishing these synaptic-like contacts. To date, proteins could be compared with the presynaptic molecules, and the exposed to the cellular surface, including the adhesion molecule receptor/receptor complex with the postsynaptic molecules in Ihog, have been reported to be modifiers of cytoneme dynamics specialized areas; thus, the term ‘morphogenetic synapsis’ has (Bischoff et al., 2013; González-Méndez et al., 2017). Such proteins been coined (reviewed by Kornberg, 2017). could enable cytonemes to interact with surrounding cells, sensing As in synapses, MVBs containing Hh move along cytonemes in both the extracellular matrix (ECM) and cell membranes. Indeed, Drosophila epithelia. These MVBs are the immediately previous ASP cytoneme extensions use Dally and Dally-like (Dlp) glypicans

Endosome Cytoskeleton

Lysosome

Receptor complex Endocytosis Receptor

Glypicans Adhesion Morphogen proteins

Exocytosis

Signaling contacts MVB

Cytoskeleton

Fig. 5. The ‘morphogenetic synapse’ model. Cytoneme-mediated signal transfer at specific contact sites can be conceived as a synapse-like process between signal-producer and -receptor cells. Adhesion proteins and glypicans from both membranes mediate this close cell-to-cell contact and facilitate the binding of signal to its receptor, either from cytoneme membranes (top) or from exovesicles released at the site (bottom). The model describes the synaptic- like process involved in Hh signaling. Hh travels in multivesicular bodies (MVBs) along cytonemes and is released in exosomes to interact at the contact sites with the receptor Patched (Ptc) in the receiving cytonemes. The Hh co-receptors Ihog (shown as adhesion proteins) and the glypican Dally-like (Dlp) are also recruited for ligand/receptor interaction (not shown). Inset shows a detail of synapse-like contact between ligand-carrying cytonemes and receiving cytonemes. DEVELOPMENT

6 PRIMER Development (2019) 146, dev174607. doi:10.1242/dev.174607 as substrates for growth, and are also dependent on laminin-activated receptor Ror2 (Mattes et al., 2018). PCP activation leads to the integrins (Roy et al., 2014; Huang and Kornberg, 2015). In addition, activation of actin polymerization processes, which are dependent synaptic adhesion proteins such as Neuroglian, Capricious and on the small GTPase Cdc42, followed by outgrowth of a subset of Neuroligin2 are required for establishing functional contacts between new Wnt8a-carrying cytonemes, which facilitate more Wnt8a ASP cytonemes and wing disc ligand-sending cells (Roy et al., 2014; transport to activate canonical Wnt signaling at a distance. This Huang and Kornberg, 2015; Huang et al., 2019). Therefore, it is feedback loop also regulates Wnt cytoneme formation in zebrafish conceivable that these membrane and ECM proteins play a role in neural plate patterning and fibroblasts, and in human embryonic cytoneme guidance as well as in the recognition and establishment of kidney and gastric cancer cells (Mattes et al., 2018). physical contact with the distant cell. Interestingly, very recent In stem cells, there is also evidence of signal reception mediated research suggests that both the cytoneme establishment and their by cytonemes, which in turn regulate cytoneme extension. For contact in the ASP are influenced by glutamate-mediated signaling, example, Hh cytonemes within the stem cell niche of the adult which produces intracellular calcium influx waves. This work can Drosophila ovary extend until they receive the ligand; impairment even distinguish a requirement for distinct sets of presynaptic of signal reception causes cytonemes to extend until ligand is (Synaptotagmine 1, Synaptobrevin, Vesicle Glutamate Transporter, reached and wild-type signaling is activated (Rojas-Ríos et al., etc.) and postsynaptic (Synaptotagmine 4, Glutamate Receptor II) 2012). In this way, the GSC niche responds to insufficient Hh regulators for cytoneme-mediated Dpp signaling (Huang et al., signaling, which suggests an ability of cellular niches to respond to 2019). challenging physiological conditions. However, in the wing In summary, increasing evidence shows that the cytoneme imaginal disc and abdominal histoblasts, both cytoneme connection resembles a synaptic contact, supporting cell-cell formation and dynamics appear to be independent of Hh signal signaling in diverse biological contexts (Fig. 5). Keeping in mind reception, because the protrusions form and dynamically extend in marked differences regarding the complexity of molecules involved, the absence of either Hh ligand or its receptor, Ptc (González- contact duration, which in the case of neuronal synapsis can last up Méndez et al., 2017). Thus, there are still unanswered questions to months, or the link to a propagating electrical impulse, some key regarding the fundamental mechanisms for initial cytoneme features are shared, such as cell polarization, directed secretion for formation and its links to pathway specificity and adaptability. communication, and specialized membrane domains containing specific receptors and cell adhesion molecules at the contact sites. Conclusions Increasing evidence shows that cells master distant communication Regulation of cytonemes through feedback loops through filopodial protrusions. These structures can direct the Another intriguing issue is how the spatial and temporal patterning of tissues, and facilitate both graded and selective establishment of cytonemes is regulated to generate diversity, e.g. distribution of signaling molecules. In addition, filopodial to generate different signaling gradient shapes for different tissue protrusions serve as connectors of distant associated tissues and morphologies. In addition, several signaling pathways can co-exist have a pivotal role both during the development of and in the in the same tissues, such as in the ASP and the wing disc in maintenance of adult tissue homeostasis. However, the distinction Drosophila (Fig. 4). Supporting the hypothesis that specific between different protrusions that perform cell-cell communication cytomemes exist for each signaling pathway, recent work is not completely clear; the current classifications might indeed proposes self-regulatory mechanisms for cytoneme-mediated change as further knowledge is acquired. signaling, in which activation of a signaling pathway fuels the Key issues to address include, among many others: understanding regulation of protrusions carrying its own pathway components directionality of targeting during cytoneme formation and their (Du et al., 2018; Mattes et al., 2018). dynamic extension; how cytonemes intercalate between non target In the Drosophila ASP, genome-edited constructs for the FGF cells either within a tissue or to reach physically separated tissues; and ortholog Bnl (Bnl:GFP) and FGF receptor ortholog Btl (Btl:Cherry) how the signaling pathway specificity of cytonemes is achieved. have shown that FGF moves specifically from the wing disc- Cytonemes are indeed cytoskeleton-driven extensions of specialized producing cells to the receiving cytonemes in the ASP (Fig. 4D), membrane territories, which could couple the presence of signaling forming a long-range concentration gradient that adapts to the components, cytoneme formation and probably even the factors developing ASP-specific shapes (Du et al., 2018; Sohr et al., 2019). needed for contact. All these components are most probably Interestingly, this adaptation involves two targets of the pathway, dependent on intracellular vesicular trafficking, although further cut and pointed-P1, which respond to either low or high levels of the research is needed to confirm this hypothesis. ligand FGF, respectively. In turn, these transcription factors have Regarding the cytoneme contact for information exchange, this antagonistic effects: Pointed-P1 enhances the number and length of might be a synapse-like process. Neurons, axons and dendrites are FGF-receiving cytonemes, while Cut acts to repress protrusions. extended to signal through synapses with diverse distant target cells Thus, the gradient is a consequence of initial protrusions exposed to (chemical synapses). Various types of synapses also exist for the high levels of signaling close to the source, which in turn enhances vertebrate immune system cells (immune synapses), which either cytoneme contact and signaling that will further promote protrusion synapse with each other or with tumor or infected cells (reviewed by extension that maintains high signaling levels. In contrast, lower Alcover et al., 2016). Interestingly, recent research has shown that levels of signaling will repress protrusion extension, which will human immunodeficiency virus can propagate by inducing, in translate into lower levels of signaling over time (Du et al., 2018). addition to TNT, filopodia formation between infected and A second feedback loop mechanism has been described for Wnt uninfected cells (Sherer and Mothes, 2008); this is also signaling where cytoneme transport of the zebrafish ligand Wnt8a considered to be a synaptic process (virological synapses) acts in both paracrine and autocrine signaling. For paracrine (reviewed by Sewald et al., 2016; Dufloo et al., 2018). The signaling, Wnt8a activates the canonical Wnt pathway (Stanganello morphogenetic synapsis (reviewed by Kornberg, 2017) could be et al., 2015), while it triggers the alternative planar cell polarity envisioned as part of an evolutionarily conserved mechanism for

(PCP) pathway through autocrine signaling by binding the co- cellular communication that supports cell-cell signaling in diverse DEVELOPMENT

7 PRIMER Table 1. Structural and functional characteristics of cytonemes described in different organisms Cytoneme Signaling pathway Organism Tissue type cargo Vesicle association Function Structural components Length Dynamics References (A) Drosophila Drosophila Wing disc and Ligand Hh ligand Hh in motile Morphogen gradient Actin (dependent on capping Up to 40 μmin In histoblasts: lifetime Bischoff et al. (2013); abdominal carrier Hh signaling exovesicles and formation for wing protein α, SCAR, Pico, wing disc; 3.1-10.9 min; Gradilla et al. epithelia MVBs along Hh- and abdomen Diaphanous and Filamin) 8.5 μmupto extension at (2014); González- sending cytonemes patterning 12.1 μmin 1.5-5.0 μm/min; Méndez et al. histoblasts retraction at (2017) 1.2-5.2 μm/min Drosophila Wing disc and Receptor Ptc receptor Ptc receptor in motile Morphogen gradient Actin (dependent on capping Up to 40 μmin In histoblasts: lifetime Chen et al. (2017); abdominal carrier Hh signaling puncta along Hh- formation for wing protein α, SCAR, Pico, wing disc; 3.2-11.6 min; González-Méndez epithelia receiving cytonemes patterning Diaphanous and Filamin) 8.5 μmupto extension at et al. (2017) 12.1 μmin 1.6-5.8 μm/min; histoblasts retraction at 1.2-4.2 μm/min Drosophila Wing disc Receptor Tkv receptor Tkv receptor in motile Morphogen gradient Actin 20.8 μm, up to Unmeasured Ramirez-Weber and epithelium carrier Decapentaplegic puncta along Dpp- formation for wing 80 μm Kornberg (1999) (Dpp) signaling receiving cytonemes patterning Drosophila ASP Receptor Btl receptor Btl receptor (FGFR) Signaling across Actin (dependent on Up to 40 μm Unmeasured Roy et al. (2014); Du epithelium carrier Bnl signaling and Bnl ligand (FGF) tissues and Diaphanous and on the et al. (2018); in motile puncta morphogen gradient cell-adhesion proteins Huang et al. along Bnl-receiving formation for Neuroglian and Neuroligin (2019); Sohr et al. cytonemes dynamic air sac 2) (2019) patterning Drosophila ASP Receptor Tkv receptor Tkv receptor (BMPR) in Signaling across Actin (dependent on Up to 40 μm Unmeasured Roy et al. (2014); epithelium carrier Decapentaplegic motile puncta along tissues Diaphanous, on the cell- Huang et al. (Dpp) signaling Dpp (BMP)-receiving adhesion proteins (2019) cytonemes Neuroglian and Neuroligin 2, and on glutamate signaling proteins GluRII and Syt4) Drosophila Eye disc Receptor EGFR receptor EGFR in motile puncta Signaling across Actin <30 μm Unmeasured Roy et al. (2011) epithelium carrier Spi signaling along Spi-receiving tissues doi:10.1242/dev.174607 dev174607. 146, (2019) Development cytoneme Drosophila Myoblasts Receptor Fz receptor Fz receptor in motile Signaling intermediary Actin (dependent on 25 μm Unmeasured Huang and Kornberg (flight carrier Wg signaling puncta along Wg across tissues for Diaphanous and on the (2015) muscle (Wnt)-receiving signal turnover cell-adhesion protein progenitors) cytoneme Neuroglian) Drosophila Myoblasts Ligand Dl ligand Dl ligand in motile Signaling intermediary Actin (dependent on 25 μm Unmeasured Huang and Kornberg (flight carrier Notch signaling puncta along Dl- across tissues for Diaphanous and on the (2015) muscle receiving cytoneme signal turnover cell-adhesion protein progenitors) Neuroglian) Drosophila Thorax (notum Ligand Dl ligand Undefined Cell specialization by Actin (dependent on Rac and Up to 10 μm Lifetime: >500 s De Joussineau et al. cuticle) carrier Notch signaling lateral inhibition for SCAR) (2003); Cohen bristle organization et al. (2010); Hadjivasiliou et al. (2016) Drosophila Leg disc Ligand Spi ligand Undefined Cell specialization for Actin and possibly Up to 9.5 μm Unmeasured Peng et al. (2012) epithelium carrier Spitz Spi signaling bract cell microtubules (marked by differentiation Jupiter); dependent on Diaphanous Continued 8

DEVELOPMENT Table 1. Continued PRIMER Cytoneme Signaling pathway Organism Tissue type cargo Vesicle association Function Structural components Length Dynamics References Drosophila Developing Ligand Hh ligand Undefined GSC niche Actin (dependent on 0.25-1.11 μm Unmeasured Rojas-Rios et al. ovary carrier Hedgehog Hh maintenance Diaphanous and Wasp) (2012) signaling Drosophila Testis Receptor Tkv receptor Tkv receptor in motile Stem cell niche Microtubules sensitive to Up to 4 μm Unmeasured Inaba et al. (2015) carrier Dpp signaling puncta along Dpp- maintenance colcemid receiving cytonemes Drosophila Lymph gland Unknown Unknown Undefined Stem cell niche Actin Unmeasured Unmeasured Mandal et al. (2007); maintenance Fuwa et al. (2015) (B) Others Sea urchin Embryo Unknown Unknown Beaded appearance Signaling across Actin and some microtubules Up to 80 μm Extend at 10 μm/min Miller et al. (1995) gastrulation physical barriers for patterning Zebrafish Embryo neural Ligand Wnt8a ligand Wnt8a ligand in motile Morphogen gradient Actin (dependent on CDC42) Up to 50 μm 110 nm/s to 240 nm/s Stanganello et al. plate carrier Wnt signaling puncta along Wnt8a- formation for embryo and tubulin at the base (2015); Mattes sending cytonemes patterning et al. (2018) Zebrafish Embryo Unknown Unknown Undefined Signaling across Actin, tubulin (only in the 60 μmupto Extension: 3.4 μm/min Caneparo et al. blastula physical barriers proximal region of the 380 μm (2011) bridge) Zebrafish Epidermis Ligand Dl ligand Dl ligand in Cell specialization and Actin and possibly 60 μm, up to Extension at 4 μm/min, Hamada et al. (pigment carrier Notch signaling membranous color pattern microtubules (marked by 250 μm up to 12 μm/min; (2014); Eom et al. cells) vesicles move along formation tubulin); dependent on retraction at 2 μm/ (2015); Eom and Dl-sending CDC42, sensitive to min, up to 10 μm/min Parichy (2017) cytonemes nocodazole Chicken Embryo Receptor Fz receptor Fz receptor in motile Signaling across Actin and possibly Up to 20 μm Extension at 53.76 nm/ Sagar et al. (2015) somites carrier Wnt signaling puncta along Wnt- physical barriers microtubules (marked by s; retraction at receiving cytoneme Tubulin); dependent on 68.80 nm/s Rac1, Fascin and Cofilin Chicken Limb bud Receptor Shh ligand Shh ligand in motile Morphogen gradient Actin, tubulin at the base 34 μm, up to Extend at 150 nm/s Sanders et al. (2013) carrier Hh signaling puncta along Shh- formation for limb 150 μm sending cytonemes patterning

Chicken Limb bud Ligand Cdo and Boc co- Cdo and Boc co- Morphogen gradient Actin, tubulin at the base Up to 150 μm Extend at 150 nm/s Sanders et al. (2013) doi:10.1242/dev.174607 dev174607. 146, (2019) Development carrier receptors receptors in motile formation for limb Hh signaling puncta along Shh- patterning receiving cytonemes Mouse Embryo Unknown Unknown Motile vesicle-like Signaling across Actin, sensitive to Up to 34.6 μm Unmeasured Salas-Vidal and blastocyst bulges along physical barriers cytochalasin Lomeli (2004) filopodia Human Stem cell line Unknown Unknown Undefined Stem cell niche Actin (dependent on Lgr4 and Up to 80 μm Unmeasured Snyder et al. (2015) maintenance Lgr5 receptors) (protrusions are restored within 1 h of cytochalasin treatment) ASP, air sac primordium; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; Bnl, Branchless; Boc, Brother of CDO; Btl, Breathless; Cdo, Cam-related/downregulated by oncogenes; Dl, Delta; Dpp, Decapentaplegic; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; Fz, Frizzled; GSC, germline stem cell; Hh, Hedgehog; MVB, multivesicular body; Ptc, Patched; SCAR, Suppressor of cAMP receptor; Shh, Sonic hedgehog; Spi, Spitz; Tkv, Thick-Vein; Wg, Wingless. 9

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The Cytoneme Connection: Direct Long-Distance Signal Transfer During Development Laura González-Méndez*, Ana-Citlali Gradilla*,‡ and Isabel Guerrero‡

The Cytoneme Connection: Direct Long-Distance Signal Transfer During Development Laura González-Méndez, Ana-Citlali Gradilla,‡ and Isabel Guerrero‡