Sougata Roy

Specificity of Drosophila Cytonemes for Distinct Signaling Pathways

Signaling protein receptors are segregated into different cell protrusions in Drosophila cells. Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. We investigated the specificity of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. We conclude that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme.

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

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. 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. Transfer of signaling proteins along long filopodia is required for proper development in the fruit fly. [Also see Perspective by Rørth] 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.

Cytonemes as specialized signaling filopodia

Development creates a vast array of forms and patterns with elegant economy, using a small vocabulary of pattern-generating proteins such as BMPs, FGFs and Hh in similar ways in many different contexts. Despite much theoretical and experimental work, the signaling mechanisms that disperse these morphogen signaling proteins remain controversial. Here, we review the conceptual background and evidence that establishes a fundamental and essential role for cytonemes as specialized filopodia that transport signaling proteins between signaling cells. This evidence suggests that cytoneme-mediated signaling is a dispersal mechanism that delivers signaling proteins directly at sites of cell-cell contact.

Communicating by touch – neurons are not alone

Long-distance cell-cell communication is essential for organ development and function. Whereas neurons communicate at long distances by transferring signals at sites of direct contact (i.e., at synapses), it has been presumed that the only way other cell types signal is by dispersing signals through extracellular fluid--indirectly. Recent evidence from Drosophila suggests that non-neuronal cells also exchange signaling proteins at sites of direct contact, even when long distances separate the cells. We review here contact-mediated signaling in neurons and discuss how this signaling mechanism is shared by other cell types.

Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome

Blocking a BMP signaling pathway may ameliorate neurological defects in patients with fragile X syndrome. BMP signaling underlies fragile X syndrome Fragile X syndrome (FXS) is a heritable cognitive disability and autism spectrum disorder caused by loss of expression of the gene encoding the RNA binding protein FMRP. FMRP suppresses the translation of various mRNAs. Kashima et al. found that the transcript encoding the bone morphogenetic protein type II receptor (BMPR2) was a critical FMRP target that contributed to the neuronal morphology seen in FXS. Loss of FMRP in fruitfly and mice increased the abundance of BMPR2 and activated a downstream kinase LIMK1 in neurons; increased BMPR2 abundance and cofilin phosphorylation (suggesting increased LIMK1 activity) were also detected in postmortem brain tissue from FXS patients. Reducing signaling through the BMPR2-LIMK1 pathway suppressed abnormal dendritic spine growth in FXS model mice, suggesting that this might be a therapeutic option for FXS patients. Epigenetic silencing of fragile X mental retardation 1 (FMR1) causes fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. FXS correlates with abnormal synapse and dendritic spine development, but the molecular link between the absence of the FMR1 product FMRP, an RNA binding protein, and the neuropathology is unclear. We found that the messenger RNA encoding bone morphogenetic protein type II receptor (BMPR2) is a target of FMRP. Depletion of FMRP increased BMPR2 abundance, especially that of the full-length isoform that bound and activated LIM domain kinase 1 (LIMK1), a component of the noncanonical BMP signal transduction pathway that stimulates actin reorganization to promote neurite outgrowth and synapse formation. Heterozygosity for BMPR2 rescued the morphological abnormalities in neurons both in Drosophila and in mouse models of FXS, as did the postnatal pharmacological inhibition of LIMK1 activity. Compared with postmortem prefrontal cortex tissue from healthy subjects, the amount of full-length BMPR2 and of a marker of LIMK1 activity was increased in this brain region from FXS patients. These findings suggest that increased BMPR2 signal transduction is linked to FXS and that the BMPR2-LIMK1 pathway is a putative therapeutic target in patients with FXS and possibly other forms of autism.

Paracrine signaling mediated at cell-cell contacts

Recent findings in several organ systems show that cytoneme‐mediated signaling transports signaling proteins along cellular extensions and targets cell‐to‐cell exchanges to synaptic contacts. This mechanism of paracrine signaling may be a general one that is used by many (or all) cell types in many (or all) organs. We briefly review these findings in this perspective. We also describe the properties of several signaling systems that have previously been interpreted to support a passive diffusion mechanism of signaling protein dispersion, but can now be understood in the context of the cytoneme mechanism.

Transcriptional Analysis of the Principal Cell Division Gene, ftsZ, of Mycobacterium tuberculosis

Multiple promoters drive the expression of the principal cell division gene, ftsZ, in bacterial systems. Primer extension analysis of total RNA from Mycobacterium tuberculosis and a Mycobacterium smegmatis transformant containing 1.117 kb of the upstream region of M. tuberculosis ftsZ and promoter fusion studies identified six ftsZ transcripts and their promoters in the ftsQ open reading frame and ftsQ-ftsZ intergenic region. The presence of multiple promoters reflects the requirement to maintain a high basal level of, or to differentially regulate, FtsZ expression during different growth conditions of the pathogen in vivo.

Direct Delivery Mechanisms of Morphogen Dispersion

Cytonemes facilitate the formation of morphogen gradients by transferring signaling proteins from producing to receiving cells. This Presentation focuses on how morphogen signaling proteins disperse across developmental fields. Although the steady-state distributions of morphogen signaling proteins have been described well in a number of contexts, the mechanisms that generate these distributions have remained uncertain. Results presented here show that these proteins transfer from producing to target cells at points of direct contact, even when the producing and target cells are not immediate neighbors.

Developmental compartments in the larval trachea of Drosophila

The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 revealed that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein–expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks. DOI: http://dx.doi.org/10.7554/eLife.08666.001

The ftsZ Gene of Mycobacterium smegmatis is expressed Through Multiple Transcripts

The principal essential bacterial cell division gene ftsZ is differentially expressed through multiple transcripts in diverse genera of bacteria in order to meet cell division requirements in compliance with the physiological niche of the organism under different environmental conditions. We initiated transcriptional analyses of ftsZ gene of the fast growing saprophytic mycobacterium, Mycobacterium smegmatis, as the first step towards understanding the requirements for FtsZ for cell division under different growth phases and stress conditions. Primer extension analyses identified four transcripts, T1, T2, T3, and T4. Transcriptional fusion studies using gfp showed that the respective putative promoter regions, P1, P2, P3, and P4, possessed promoter activity. T1, T2, and T3 were found to originate from the intergenic region between ftsZ and the upstream gene, ftsQ. T4 was initiated from the 3’ portion of the open reading frame of ftsQ. RT-PCR analyses indicated co-transcription of ftsQ and ftsZ. The four transcripts were present in the cells at all growth phases and at different levels in the cells exposed to a variety of stress conditions in vitro. T2 and T3 were absent under hypoxia and nutrient-depleted stationary phase conditions, while the levels of T1 and T4 remained unaffected. These studies showed that ftsZ gene expression through multiple transcripts and differential expression of the transcripts at different growth phases and under stress conditions are conserved in M. smegmatis, like in other Actinomycetes.