Research Article

Cytoneme-Mediated Contact-Dependent Transport of the Drosophila Decapentaplegic Signaling Protein

Science  21 Feb 2014:
Vol. 343, Issue 6173,
DOI: 10.1126/science.1244624

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Structured Abstract

Introduction

In multicellular organisms, morphogen signaling proteins move from “signaling centers” where they are produced to target cells whose growth and patterning they regulate. Whereas much progress has been made identifying and characterizing signaling proteins such as the transforming growth factor–β family member Decapentaplegic (Dpp), which is produced in the Drosophila wing imaginal disc, the mechanisms that disperse signaling proteins remain controversial. We characterized Dpp signaling in a system in which cytonemes, a specialized type of filopodia implicated in long-distance signaling, could be imaged, and in which movement of signaling proteins and their receptors could be followed.

Embedded Image

Cytonemes take up and transport morphogens. Micrograph showing a tracheal branch marked with mCherry overlying a wing disc expressing Dpp (tagged with green fluorescence). Cytonemes extend from the medial region of the branch to Dpp-expressing disc cells, and from the tip of the branch toward FGF-expressing disc cells. Dpp has been taken up and transported by the cytonemes that contact Dpp-expressing cells.

Methods

We expressed fluorescence-tagged forms of proteins that function in morphogen signaling to monitor Dpp in signal-producing cells, its receptor in signal-receiving cells, and proteins and cell structures that participate in trafficking of signaling proteins. Signaling was characterized in live, unfixed tissue as well as by immunohistochemistry, and under conditions of both gain- and loss-of-function genetics.

Results

Cells that received Dpp and activated Dpp signal transduction extended cytonemes that directly contacted Dpp-producing cells. The contacts were characterized by relative stability and membrane juxtaposition of less than 15 nm. Cytonemes that contained the Dpp receptor in motile puncta also contained Dpp taken up from Dpp-producing cells. In contrast, a different set of cytonemes that contacted fibroblast growth factor (FGF)–producing cells contained the FGF receptor but did not take up Dpp. The cytonemes were reduced in number and length in genetic loss-of-function conditions for diaphanous, which encodes a formin; for neuroglian, which encodes an L1-type cell adhesion molecule; and for shibire, which encodes a dynamin. Cytonemes were present in loss-of-function conditions for capricious, which encodes a leucine-rich repeat cell adhesion protein, but these cytonemes failed to contact Dpp-producing cells. Signaling was abrogated in all these conditions that created defective cytonemes, although the signal-producing cells were not compromised. The mutant conditions were not lethal to the affected cells, and the mutant cells retained competence to autocrine signaling.

Discussion

This work describes cytonemes that receive and transport signaling proteins from producing cells to target cells, and shows that cytoneme-mediated signal exchange is both contact-dependent and essential for Dpp signaling and normal development. Contact-mediated signal exchange and signaling are also the hallmarks of neurons—an analogy that extends to the functional requirements for the diaphanous, neuroglian, shibire, and capricious genes by both neurons and epithelial cells. Discoveries of cytonemes in many cell types and in many organisms suggest that contact-mediated signaling may be a general mechanism that is not unique to neurons.

Morphogen Pipeline

Developmental effects of morphogens are often thought to result from release of such signaling proteins from a cell, which then diffuse away to act by binding to receptors on distant target cells. But evidence is accumulating that another mechanism exists for such communication. Endothelial cells in the fruit fly have long, skinny extensions that reach away from cells for long distances, and these “cytonemes” can take up morphogens from adjacent cells. A key experiment to support a signaling role of such structures would be to show that disruption of cytonemes disrupts signal transduction. Roy et al. (p. 10.1126/science.1244624, published online 2 January; see the Perspective by Rørth) provide such evidence and conclude that the fly morphogen known as decapentaplegic (a relative of transforming growth factor.β) must be transported through cytonemes to promote proper development of the trachea.

Abstract

Decapentaplegic (Dpp), a Drosophila morphogen signaling protein, transfers directly at synapses made at sites of contact between cells that produce Dpp and cytonemes that extend from recipient cells. The Dpp that cytonemes receive moves together with activated receptors toward the recipient cell body in motile puncta. Genetic loss-of-function conditions for diaphanous, shibire, neuroglian, and capricious perturbed cytonemes by reducing their number or only the synapses they make with cells they target, and reduced cytoneme-mediated transport of Dpp and Dpp signaling. These experiments provide direct evidence that cells use cytonemes to exchange signaling proteins, that cytoneme-based exchange is essential for signaling and normal development, and that morphogen distribution and signaling can be contact-dependent, requiring cytoneme synapses.

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