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The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts. Fruit fly larvae undergo a remarkable physical transformation to become an adult fly. During this transformation, the tissues in the larvae change into the structures found in the adult. For example, the adult wings, flight muscles, and other structures needed for coordinated flight form from a pair of disc-like tissues called the wing imaginal discs. For these structures to develop correctly, the cells in the wing imaginal discs need to receive coordinated instructions about what types of cells they need to become. Within the wing discs, finger-like projections called cytonemes link specific cells together to allow signal molecules to move between the cells; this controls the development of the wing disc itself as well as structures called dorsal air sacs, which supply oxygen to the flight muscles in the adult fly. However, it is not known if cytonemes allow the exchange of signal molecules between cells involved in the formation of other structures needed for flight. Here, Huang and Kornberg investigated the role of cytonemes in the development of the flight muscles in fruit flies. The experiments reveal that cells called myoblasts—which will later become the flight muscle cells—form two sets of cytonemes with other cells. One set connects the myoblasts to cells in the developing air sac, which allows a signal protein called Delta to signal from the myoblasts into the air sac cells. The other set of cytonemes connects the myoblasts to wing disc cells. This enables another signal molecule called Wingless, which is produced in wing disc cells, to move into the myoblasts and block the production of Delta. Huang and Kornberg's findings reveal a new role for cytonemes in coordinating the development of the flight muscles and the dorsal air sacs. A future challenge will be to understand how individual cytonemes are able to connect to specific cells.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development – up to nine embryos at a time. The DaXi single-objective light-sheet microscope achieves fast, high-quality imaging of large volumes. DaXi’s design allows increased scanning range without sacrificing imaging speed or quality, multiview imaging and versatile sample mounting.

Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C . elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.

Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, we analyzed EGFR and RET Drosophila tumor models and tested several genetic loss-of-function conditions that impair cytoneme-mediated signaling. Neuroglian, capricious, Irk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. We observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. We also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and we determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. Our results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy.

An infrared fluorescent protein based on a new monomeric scaffold is described in this paper and is tested for protein fusion in cells and in vivo. Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

Drosophila dorsal air sac development depends on Decapentaplegic (Dpp) and Fibroblast growth factor (FGF) proteins produced by the wing imaginal disc and transported by cytonemes to the air sac primordium (ASP). Dpp and FGF signaling in the ASP was dependent on components of the planar cell polarity (PCP) system in the disc, and neither Dpp- nor FGF-receiving cytonemes extended over mutant disc cells that lacked them. ASP cytonemes normally navigate through extracellular matrix (ECM) composed of collagen, laminin, Dally and Dally-like (Dlp) proteins that are stratified in layers over the disc cells. However, ECM over PCP mutant cells had reduced levels of laminin, Dally and Dlp, and whereas Dpp-receiving ASP cytonemes navigated in the Dally layer and required Dally (but not Dlp), FGF-receiving ASP cytonemes navigated in the Dlp layer, requiring Dlp (but not Dally). These findings suggest that cytonemes interact directly and specifically with proteins in the stratified ECM. The embryos of animals develop in a controlled manner that ensures that their tissues and organs form properly and at the right time. These processes depend on molecules called morphogens that are distributed throughout the embryo in specific ways and that are dispersed via extensions that protrude from the surfaces of cells. These extensions, called cytonemes, transport the morphogens across the distances that separate cells and transfer these molecules to target cells via direct contact. However, it was not known how cytonemes navigate to their targets. The fruit fly Drosophila is commonly used to investigate how animals develop organs and tissues. Previous studies have shown that the development of one of the fly’s organs – the air sac primordium –relies on morphogens transported by cytonemes.Now, Huang and Kornberg reveal that these cytonemes navigate to their targets by using the composition of the mesh-like framework – referred to as the extracellular matrix – that surrounds animal tissues as a guide. Further experiments showed that the extracellular matrix between the cells that produce the morphogens and the cells of the air sac primordium is roughly arranged into layers. These layers contain different molecules and the cytonemes navigate within specific layers. These findings reinforce the idea that the extracellular space is organized and regulated, and show that the extracellular matrix is essential for developmental signaling. Future challenges include understanding how the layers of the extracellular matrix form and how information is encoded in these layers for the cytonemes to decipher as they navigate to their targets.

Bicoid (Bcd) protein distributes in a concentration gradient that organizes the anterior/posterior axis of the Drosophila embryo. It has been understood that bcd RNA is sequestered at the anterior pole during oogenesis, is not translated until fertilization, and produces a protein gradient that functions in the syncytial blastoderm after 9–10 nuclear divisions. However, technical issues limited the sensitivity of analysis of pre-syncytial blastoderm embryos and precluded studies of oocytes after stage 13. We developed methods to analyze stage 14 oocytes and pre-syncytial blastoderm embryos, and found that stage 14 oocytes make Bcd protein, that bcd RNA and Bcd protein distribute in matching concentration gradients in the interior of nuclear cycle 2–6 embryos, and that Bcd regulation of target gene expression is apparent at nuclear cycle 7, two cycles prior to syncytial blastoderm. We discuss the implications for the generation and function of the Bcd gradient. As an embryo develops, a single cell transforms into a collection of different types of cells. One protein that is crucial for this process in fruit fly embryos is Bicoid. Thirty years ago, scientists discovered that Bicoid protein is concentrated at the head end of the embryo and gradually decreases in amount towards the rear end. This concentration gradient of Bicoid protein organizes the embryo body and regulates the expression of many genes, thus directing the cells to develop different identities. Several assumptions had been made about how this gradient is established. It was thought that in the unfertilized egg, the mRNA molecules that will be translated to produce Bicoid proteins are stored in an inactive state in the region of the egg that later develops into the embryo’s head. In the embryo, the mRNA molecules were believed to remain in the head region while being translated, with the newly formed proteins then gradually spreading from this site to create the Bicoid gradient. It was also thought that no Bicoid proteins are stored in the unfertilized egg. However, no known methods were sensitive enough to investigate these assumptions. Now, using newer and more sensitive methods, Ali-Murthy and Kornberg show that Bicoid protein is present in the unfertilized fruit fly egg in the same region as the mRNA molecules that make Bicoid. Furthermore, the Bicoid gradient forms when the embryo has fewer than 32 nuclei, much earlier in development than previously thought. The Bicoid protein also does not appear to spread passively towards the rear of the embryo, but is transported in a more orchestrated manner. Overall, Ali-Murthy and Kornberg’s results suggest that the early fruit fly embryo is more organized and actively regulated than had been previously understood. This paves the way for further studies that use sensitive techniques to investigate this early stage of development.

Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. Here we show that in response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling. Tracheal remodeling is a key step during Drosophila metamorphosis. Here they report that tracheal progenitor cells are activated through Yorkie-dependent proliferation and migration, which is induced by metabolic deficit and insulin signaling.

We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII.Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by L-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.

Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. We investigated dispersion of Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc. We discovered that delivery of Hh, Wg, and Dpp to their respective targets is regulated. We found that <5% of Hh and <25% of Wg are taken up by disc cells and activate signaling. The amount of morphogen that is taken up and initiates signaling did not change when the level of morphogen expression was varied between 50 and 200% (Hh) or 50 and 350% (Wg). Similar properties were observed for Dpp. We analyzed an area of 150 μm×150 μm that includes Hh-responding cells of the disc as well as overlying tracheal cells and myoblasts that are also activated by disc-produced Hh. We found that the extent of signaling in the disc was unaffected by the presence or absence of the tracheal and myoblast cells, suggesting that the mechanism that disperses Hh specifies its destinations to particular cells, and that target cells do not take up Hh from a common pool.