GPI-anchored FGF directs cytoneme-mediated bidirectional contacts to regulate its tissue-specific dispersion

ARTICLE https://doi.org/10.1038/s41467-022-30417-1 OPEN GPI-anchored FGF directs cytoneme-mediated bidirectional contacts to regulate its tissue-specific dispersion 1234567890():,; Lijuan Du 1, Alex Sohr 1,2, Yujia Li 1 & Sougata Roy 1✉ How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and promotes target-specific cytoneme contacts and contact-dependent FGF release. FGF-source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through cell-adhesion-molecule (CAM)-like FGF-FGFR binding. Contact-mediated FGF-FGFR interactions induce bidirectional responses in ASP and source cells that, in turn, polarize FGF-sending and FGF-receiving cytonemes toward each other to reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion. 1 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA. 2Present address: Division of Cell and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA. ✉email: NATURE COMMUNICATIONS | (2022)13:3482 | https://doi.org/10.1038/s41467-022-30417-1 | www.nature.com/naturecommunications 1 ARTICLE D NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30417-1 uring development, intercellular communication of morphogens is critical for embryonic cells to determine their positional identity, directionality, and interactions in an organized pattern to sculpt tissue. These conserved families of secreted morphogens/signals, such as fibroblast growth factor (FGF), Hedgehog (Hh), Wingless (Wg)/Wnt, epidermal growth factor (EGF), and decapentaplegic (Dpp—a bone morphogenetic protein (BMP) homolog), act away from their sources and, upon binding to receptors, activate gene regulatory pathways to induce functions in recipient cells1,2. Strikingly, each signal and signaling pathway can generate a wide range of cell types and organizations in diverse contexts3. Understanding how signals might inform cells of their positional identity, directionality, and interactions and organize these functions in diverse tissue-specific patterns is critical to understanding morphogenesis. The discrete tissue-specific organization of morphogen signaling is known to be dependent on the ability of signal-receiving cells to selectively sense and respond to a specific signal3. In contrast, traditional models predict that the signal presentation from the source via free secretion and extracellular diffusion is a non-selective process. However, recent advances in microscopy revealed that both signal-producing and receiving cells could extend signaling filopodia named cytonemes and selectively deliver or receive signals through cytoneme–cell contact sites4–9. Essential roles of cytonemes or cytoneme-like filopodia have been discovered in many vertebrate and invertebrate systems and are implicated in most signaling pathways, including Hh, Dpp, FGF, EGF, Ephrin, and Wnt under various contexts4–18. The prevalence and similarities of these signaling filopodia suggest that the polarized target-specific morphogen exchange through filopodial contacts is an evolutionarily conserved signaling mechanism. These findings bring along a paradox - not only do signals instruct cells and organize discrete cellular patterns, but cells also control the patterns of signal presentation and reception by organizing the distribution of cytonemes and cytoneme contacts6,9. This interdependent relationship of signals and signaling cells through cytonemes, however, would require precise spatiotemporal coordination between cytoneme contact formation and signal release. We started the current investigation with the premise that a better understanding of the processes that produce cytoneme contacts and control contact-driven signal release is essential to understanding morphogenesis. We asked: (1) How do cytonemes recognize a specific target cell and form signaling contacts? (2) How are secreted signals controlled for polarized target-specific release, exclusively at the cytoneme contact sites? (3) Do cytoneme contact formation and signal release spatiotemporally coordinate with each other? If so, how? To address these questions, we focused on the inter-organ dispersion of a Drosophila FGF, Branchless (Bnl), during the development of the wing imaginal disc-associated air-sac primordium (ASP)19,20. Bnl is expressed in a discrete group of wing disc cells, and it induces morphogenesis of the tubular ASP epithelium that expresses the Bnl receptor, Breathless (FGFR/ Btl)9,19,21. Epithelial cells at the ASP tip extend polarized Btlcontaining cytonemes to contact Bnl-producing wing disc cells and directly take up Bnl in a contact- and receptor-dependent manner5,9. The formation of Bnl-specific polarity and contacts of ASP cytonemes are self-sustained by Bnl-signaling feedbacks9. Consequently, Bnl reception and signaling via cytonemes can precisely adapt and dynamically coordinate with ASP growth. With increasing distance from the Bnl-source, ASP cells extend gradually fewer polarized Bnl-receiving cytonemes, leading to the emergence of asymmetric Bnl dispersion and signaling patterns within the ASP9. However, how ASP cytonemes might recognize the bnl-source for signaling contacts, and, on the other hand, how 2 Bnl producing cells might both inhibit free Bnl secretion and facilitate Bnl release selectively at the cytoneme contact sites are unknown. Here we report that Bnl is post-translationally modified by the addition of a glycosylphosphatidylinositol (GPI) moiety, which anchors Bnl to the outer leaflet of its source cell membrane. We provide evidence that the GPI anchoring of Bnl enables Bnl source cells to selectively present the signal to Btl-expressing cells through cytonemes, and that the cell adhesion molecule (CAM)like22–28 Btl–Bnl interactions coordinate bidirectional matchmaking of cytonemes for contacts. Importantly, although the GPI anchor inhibits free Bnl secretion, it promotes contact-mediated tissue-specific Bnl release for long-range patterning. These findings suggest that while cytonemes are critical for organizing tissue-specific Bnl signaling, the GPI-anchored Bnl programs the spatiotemporal distribution of cytoneme contacts to self-regulate its dispersion. Results The reciprocal polarity (...truncated)