Signaling by morphogen proteins controls many aspects of development, homeostasis, and disease (Garcia et al., 2018; Tabata and Takei, 2004; Taipale and Beachy, 2001). These signaling proteins are released from cells that produce them, and they distribute across the tissues they target, forming concentration gradients that induce signal transduction and activate gene expression in a concentration-dependent manner. The importance of regulation by morphogen gradients to growth, cell fate, and patterning underlies the imperative to understand how morphogens disperse across tissues.

For more than a century, it was assumed that morphogens spread across tissues by passive diffusion in extracellular space (either ‘free’ or ‘restricted’), and both experimental observations and theoretical modeling have been offered in support (Rogers and Schier, 2011). Spreading morphogen proteins have been proposed to exist in various forms, including as multimeric complexes or encapsulated in lipoprotein particles, exosomes, or micelles (Christian, 2012). Implicit in these models are the ideas that signaling is proportional to amounts of signaling proteins produced by designated groups of cells, and that release creates an extracellular pool of signaling protein that distributes in extracellular fluid in ways that are dependent on interactions with substances that are encountered or until they are removed from the pool by degradation or by receptor-mediated absorption. The pool is assumed to be formed by constitutive release from producing cells.

An alternative mechanism of dispersion is direct exchange at cell-cell contacts, and the contrasts with diffusion-based modes of dissemination have been reviewed extensively (Gradilla and Guerrero, 2013; Kornberg, 2017; Kornberg, 2016; Roy and Kornberg, 2015). Specialized filopodia called cytonemes are conduits that transport and transfer signaling proteins to target cells at synaptic contacts (González-Méndez et al., 2020; González-Méndez et al., 2017; Kornberg, 2016). Evidence for the essential role of cytonemes in morphogen signaling includes many identified genetic conditions that impair cytonemes, reduce cytoneme contacts, and compromise signaling. Although their fine structure is not fully understood, cytonemes contain actin filaments, ribosomes, and proteins that confer voltage sensitivity, calcium dependence, and glutamatergic activity (Huang et al., 2019; Junyent et al., 2020; Wood et al., 2021). These features, as well as the close apposition of pre- and postsynaptic membranes at cytoneme synapses, are also characteristic of neuronal glutamatergic synapses. Neuronal synapses have another key feature—signaling is titrated by frequency and quantity of neurotransmitter release from synaptic vesicles, and by efficiency of neurotransmitter clearance from the synaptic gap (Blakely and Edwards, 2012). It is not known if cytoneme synapses also store morphogen signaling proteins in synaptic vesicles and if morphogen signaling at cytoneme synapses is also dependent on regulated release and uptake.

Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) are evolutionarily conserved morphogen signaling proteins that have been implicated in organogenesis and stem cell maintenance, and their misregulation in mammals has been linked to inherited diseases and cancers (Briscoe and Thérond, 2013; Morikawa et al., 2016; Nusse and Clevers, 2017). In the columnar cells of the Drosophila wing imaginal disc, Hh is expressed specifically and uniformly by posterior (P) compartment cells (Figure 1A). In the wing blade primordium of the wing disc, Hh released by P compartment cells is taken up by anterior (A) compartment cells within 30 μm (10 cells) of the anterior/posterior (A/P) compartment border. Transfers of Hh from the P to A compartment cells are cytoneme-dependent (Bischoff et al., 2013; Chen et al., 2017). Hh in the A compartment distributes to form a concentration gradient that induces signal transduction and activates expression of target genes in partially overlapping stripes (Callejo et al., 2011; Chen et al., 2017). These domains of expression reflect graded responses to Hh, from highest and ‘short-range’ (engrailed [en], patched [ptc], and dpp) to lowest and ‘long-range’ (cubitus interruptus [ci]). The spatial relationships of these domains are reproducible, with single-cell resolution.

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Amount of Hh mRNA.

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Figure 1—source data 2

Ptc band width.

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In the wing blade primordium, cells that express dpp form a stripe of 6–8 cells adjacent to the A/P compartment border (Teleman and Cohen, 2000). wg is expressed in a two cell-wide stripe that is orthogonal to the Dpp stripe and straddles the dorsal/ventral (D/V) compartment border (Neumann and Cohen, 1997). Both Dpp and Wg disperse to form concentration gradients on both sides of their respective stripes of expressing cells. In the wing disc, transport of Dpp is cytoneme-mediated (Huang and Kornberg, 2015; Roy et al., 2014) and although the role of cytonemes in Wg dispersion have not been investigated, Wnt signaling in zebrafish is cytoneme-mediated (Stanganello et al., 2015; Stanganello and Scholpp, 2016).

Here, we asked whether the distributions of Hh, Wg, and Dpp in cells of the wing disc are regulated in ways that might be analogous to distributions of glutamate at chemical neuronal synapses. More than 99% of glutamate in the brain is stored in presynaptic terminals and is active only after release in titrated amounts, but it is not known whether uptake of signaling proteins at cytoneme synapses is regulated. To investigate this question, we asked if the Hh, Wg, and Dpp distributions are dependent and proportional to the amount produced. We also asked if the three target tissues that respond to disc-produced Hh take up Hh from a common pool. Our data show that the delivery of signaling proteins to target cells is regulated with respect to both amount and destination.

Protein secretion is characterized as either constitutive, such that synthesis and discharge are linked and concurrent, or regulated, such that proteins are made and stored until release is stimulated (Kelly, 1985). Antibody-producing lymphocytes are examples of constitutive secretory cells (Holodick et al., 2010). Examples of cells that regulate secretion include endocrine pancreatic cells that discharge hormones into the circulatory system, and neurons that pass signals to target cells at chemical synapses. The work reported here demonstrates that delivery of Hh, Dpp, and Wg to target cells is regulated and is not dependent on the amount of protein produced or on constitutive release—that differences in production as much as 4× for Hh, 2× for Dpp, and 7× for Wg did not change the amount of uptake or signal transduction. This work also reports that delivery of Hh to a tissue is not influenced by the complexity or size of the target fields—that the presence or absence of Hh-receiving tracheal cells and myoblasts was of no consequence to Hh signaling in the same region of the wing disc. The conclusion we draw is that the contours of Hh, Dpp, and Wg distributions are determined by controlled exchanges between producing and receiving cells, not by constitutive release from producing cells. This is an important, defining feature of the process that disperses these proteins across tissues.

Diffusion models of morphogen gradient formation are based on the idea that producing cells create a pool of secreted, extracellular signaling protein whose distributions are determined by interactions with extracellular components that non-producing cells contribute—such as receptors, ECM proteins, and negative regulators that absorb or bind passively diffusing protein (Madamanchi et al., 2021; Stapornwongkul and Vincent, 2021). Although these models assume that morphogen proteins are released constitutively from producing cells, our findings that the amount of signaling protein in a target field is a small fraction of the amount produced and is not proportional to production, do not explicitly invalidate them. Factors contributed by non-producing cells in the signaling domain might, in theory, feedback to compensate for variations in levels of protein in an extracellular pool. However, measures of expression of negative regulators of signal transduction for Hh (shf), Dpp (brk, sog, pent, cv-2), and Wg (Notum) detected no changes in the genotypes we analyzed that had different gene copy numbers (Figure 6).

In late third instar larvae, the distal portion of the tracheal ASP commingles with mesenchymal myoblasts on the basal surface of the wing disc anterior to the A/P compartment border (Figure 1A). All the ASP, myoblast, and disc cells that are within approximately 60 μm of the Hh-expressing, P compartment disc cells activate Hh signaling (Hatori and Kornberg, 2020). Thus, despite the cell cycle, shape, constitution, and fate differences between the cells in this microenvironment, the primary determinant of Hh signaling appears to be distance from producing cells. If Hh were released into the extracellular space within this microenvironment and if the ASP, myoblast, and disc cells shared available Hh, we might expect that the distance over which Hh spreads and the extent of Hh signaling would depend on the number of recipient cells in this Hh target field. It does not, genetic conditions that reduced the number of cells in the target field did not increase the number of remaining cells in the microenvironment that activated Hh signaling (Figure 8). This result shows that in the microenvironment, Hh uptake is not determined by either production levels, cell type, or number of other cells that also take up Hh. This finding is not consistent with the idea that Hh populates a shared extracellular pool from which different cells draw, a central tenet of diffusion models. Our interpretation is that this finding is consistent with cytoneme-mediated signaling and the idea that dissemination of Hh involves direct cell-to-cell exchanges between one producing and one receiving cell. Our results suggest that these cell-to-cell exchanges are regulated and are insensitive to modest increases or decreases in Hh production. The ~20× excess in production relative to amounts transferred to target cells may buffer the system and ensure that Hh is not limiting for the process that regulates its transfer. And because the exchanges are restricted to interactions between single pairs of cells, they are insensitive to increases or decreases in the number of other cells in the target field.

The idea that morphogen gradients form by diffusion originated before morphogens were discovered to be signaling proteins, and the first attempt to model gradient formation based on chemical and physical principles assumed that they were small organic molecules that diffuse freely into and out of cells (Crick, 1970). Protein moving in an extracellular environment prior to receptor-mediated uptake has been modeled with more complex mathematics (Lander, 2007; Madamanchi et al., 2021), but diffusion-based dissemination is still without direct evidence. Studies of morphogen proteins expressed at physiological levels and in normal conditions that have been interpreted as supporting diffusion-based dissemination have used methods that do not distinguish cell-free protein from cell-bound protein. In addition, these studies have not been carried out in conditions in which cytonemes could be imaged (Stapornwongkul and Vincent, 2021).

Diffusion-based dissemination has been inferred indirectly from distributions of signaling proteins in normal and mutant contexts. Mutants with defective heparan sulfate proteoglycans that do not distribute morphogens normally are examples, but it has yet to be established whether the observed effects were due to inhibition of protein movement in extracellular space (Bishop et al., 2007; Häcker et al., 2005; McGough et al., 2020; Mii and Takada, 2020) or to inhibition of cytoneme function (Bischoff et al., 2013; González-Méndez et al., 2017). Diffusion-based dissemination has also been inferred from responses to protein released from an implanted bead loaded with protein (Meyers and Martin, 1999) or from a micropipette (de la Torre et al., 1997), and from the behavior of protein spreading from sites of ectopic overexpression (Nowak et al., 2011). However, the protein in these experiments may not disperse by normal routes. In our experiments, Hh release was not gated if Hh was produced at excessively high levels by cells that were genetically engineered for overexpression, in contrast to Hh protein produced at close to normal levels (Figure 1). We suggest that protein released from beads, pipets, or overexpressing cells may not reflect the normal state.

Studies that have examined the robustness of morphogen signaling systems to variations in either production or response (Li et al., 2018; Zhang et al., 2020).

Evidence for cytoneme-mediated transfer of signaling proteins at cell-cell contacts is both genetic and histologic. Cytonemes defective for proteins that provide essential functions to neuronal synapses such as the cell adhesion protein Capricious, the calcium-binding protein Synaptotagmin-4, and potassium rectifying channel Irk-2 do not make normal numbers of functional synaptic contacts, do not disseminate signaling proteins, and are signaling deficient (Huang et al., 2019; Roy et al., 2014). Hh, Dpp, and Bnl/FGF are visible moving along cytonemes that extend from producing cells and link with receiving cells, and are also visible after uptake colocalized with their respective receptors in cytonemes that extend from receiving cells and link to producing cells (Capdevila and Guerrero, 1994; Du et al., 2018; Huang et al., 2019; Roy et al., 2014; Torroja et al., 2004). Thus, the evidence for cytoneme-based dissemination is direct and strong.

The process that distributes signaling proteins into concentration gradients that decline with increasing distance from source cells appears to have several components. One regulates the number of cytonemes that link producing and receiving cells. In the wing disc and ASP systems, the number of cytonemes linking producing and receiving cells correlates with signaling strength—cells far from signal sources and low signaling have fewer cytonemes than do cells close to signal sources and high signaling (Du et al., 2018; Roy et al., 2014). For Branchless/FGF signaling in the ASP, positive feedback that increases the number of relatively short cytonemes of cells that have high signaling levels and are close to source cells, and negative feedback that decreases the number of relatively long cytonemes in cells that have lower signaling levels and are farther from source cells. This system of regulation contributes to the formation of a spatial gradient of cytoneme number and concentration gradient of Branchless/FGF (Du et al., 2018). Similar feedback systems may sculpt the cytoneme gradients that disperse Hh and Dpp. Another regulatory mechanism involves feedback responses to signaling. Hh signaling, for example, enhances expression of Ptc, a receptor that binds and sequesters Hh, thereby reducing Hh dispersion and shaping the contour and extent of the Hh gradient (Chen and Struhl, 1996; Li et al., 2018).

A third regulatory mechanism is reported here—the gating which releases constant amounts of Hh, Dpp, and Wg independently of levels of production. Although we did not identify the rate-limiting step or steps that set these amounts, we made several observations that are relevant. We found that the amount of Hh detected at the basal surface of producing cells was constant and independent of level of production, suggesting that placement at the basal membrane is gated. This idea is consistent with the observation that levels of extracellular Hh were less than total Hh in these cells, but the possibilities remain that release from producing cells and/or uptake by receiving cells may also be regulated. Our investigations of cytoneme biology have been guided by known features of neurons and neuronal signaling, and we have identified many features of cytoneme-mediated signaling that are analogous. These include spatially-specific signaling at synapses that link cells at distances of <40 nm (Roy et al., 2014), synaptic localization of proteins such as the voltage-gated calcium channel and Synaptotagmin, essential roles for the glutamate receptor and glutamate transporter, and trans-synaptic stimulation of calcium transients (Huang et al., 2019). And calcium-dependent release of glutamate is essential for both cytoneme-mediated signaling and for glutamatergic excitatory neuronal synapses. It remains for further investigations to determine how morphogen proteins are released from producing cells and are exposed to receiving cells, and if protein transfers at cytoneme synapses are controlled.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Ryo Hatori

   Cardiovascular Research Institute University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2224-5802

2. Brent M Wood

   Cardiovascular Research Institute University of California, San Francisco, San Francisco, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Guilherme Oliveira Barbosa

   Cardiovascular Research Institute University of California, San Francisco, San Francisco, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5881-0896

4. Thomas B Kornberg

   Cardiovascular Research Institute University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   tkornberg@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6879-7066

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