High-Resolution Live Imaging of Axonal RNP Granules in Drosophila Pupal Brain Explants.

Dynamic and local adjustments of the axonal proteome are observed in response to extracellular cues and achieved via translation of axonally localized mRNAs. To be localized, these mRNAs must be recognized by RNA binding proteins and packaged into higher-order ribonucleoprotein (RNP) granules transported along axonal microtubules via molecular motors. Axonal recruitment of RNP granules is not constitutive, but rather regulated by external signals such as developmental cues, through pathways yet to be identified. The Drosophila brain represents an excellent model system where to study the transport of RNP granules as it is triggered in specific populations of neurons undergoing remodeling during metamorphosis. Here, we describe a protocol enabling live imaging of axonal RNP granule transport with high spatiotemporal resolution in Drosophila maturing brains. In this protocol, pupal brains expressing endogenous or ectopic fluorescent RNP components are dissected, mounted in a customized imaging chamber, and imaged with an inverted confocal microscope equipped with sensitive detectors. Axonal RNP granules are then individually tracked for further analysis of their trajectories. This protocol is rapid (less than 1 hour to prepare brains for imaging) and is easy to handle and to implement.

Live-Imaging of Axonal Cargoes in Drosophila Brain Explants Using Confocal Microscopy.

Live-imaging of axonal cargoes within central nervous system has been a long-lasting interest for neurobiologists as axonal transport plays critical roles in neuronal growth, function, and survival. Many kinds of cargoes are transported within axons, including synaptic vesicles and a variety of membrane-bound and membrane-less organelles. Imaging these cargoes at high spatial and temporal resolution, and within living brains, is technically very challenging. Here, we describe a quantitative method, based on customized mounting chambers, allowing live-imaging of axonal cargoes transported within the maturing brain of the fruit fly, Drosophila melanogaster. With this method, we could visualize in real time, using confocal microscopy, cargoes transported along axons. Our protocol is simple and easy to set up, as brains are mounted in our imaging chambers and ready to be imaged in about 1 h. Another advantage of our method is that it can be combined with pharmacological treatments or super-resolution microscopy.

A stochastic framework to model axon interactions within growing neuronal populations.

The confined and crowded environment of developing brains imposes spatial constraints on neuronal cells that have evolved individual and collective strategies to optimize their growth. These include organizing neurons into populations extending their axons to common target territories. How individual axons interact with each other within such populations to optimize innervation is currently unclear and difficult to analyze experimentally in vivo. Here, we developed a stochastic model of 3D axon growth that takes into account spatial environmental constraints, physical interactions between neighboring axons, and branch formation. This general, predictive and robust model, when fed with parameters estimated on real neurons from the Drosophila brain, enabled the study of the mechanistic principles underlying the growth of axonal populations. First, it provided a novel explanation for the diversity of growth and branching patterns observed in vivo within populations of genetically identical neurons. Second, it uncovered that axon branching could be a strategy optimizing the overall growth of axons competing with others in contexts of high axonal density. The flexibility of this framework will make it possible to investigate the rules underlying axon growth and regeneration in the context of various neuronal populations.

The Secret Life of RNA: Lessons from Emerging Methodologies.

The last past decade has witnessed a revolution in our appreciation of transcriptome complexity and regulation. This remarkable expansion in our knowledge largely originates from the advent of high-throughput methodologies, and the consecutive discovery that up to 90% of eukaryotic genomes are transcribed, thus generating an unanticipated large range of noncoding RNAs (Hangauer et al., 15(4):112, 2014). Besides leading to the identification of new noncoding RNA species, transcriptome-wide studies have uncovered novel layers of posttranscriptional regulatory mechanisms controlling RNA processing, maturation or translation, and each contributing to the precise and dynamic regulation of gene expression. Remarkably, the development of systems-level studies has been accompanied by tremendous progress in the visualization of individual RNA molecules in single cells, such that it is now possible to image RNA species with a single-molecule resolution from birth to translation or decay. Monitoring quantitatively, with unprecedented spatiotemporal resolution, the fate of individual molecules has been key to understanding the molecular mechanisms underlying the different steps of RNA regulation. This has also revealed biologically relevant, intracellular and intercellular heterogeneities in RNA distribution or regulation. More recently, the convergence of imaging and high-throughput technologies has led to the emergence of spatially resolved transcriptomic techniques that provide a means to perform large-scale analyses while preserving spatial information. By generating transcriptome-wide data on single-cell RNA content, or even subcellular RNA distribution, these methodologies are opening avenues to a wide range of network-level studies at the cell and organ-level, and promise to strongly improve disease diagnostic and treatment.In this introductory chapter, we highlight how recently developed technologies aiming at detecting and visualizing RNA molecules have contributed to the emergence of entirely new research fields, and to dramatic progress in our understanding of gene expression regulation.

Live imaging of axonal transport in Drosophila pupal brain explants.

Axonal transport is essential for the initial growth, maintenance and synaptic plasticity of axons, and altered axonal transport has been observed in different models of neurodegenerative pathologies. Dissecting the mechanisms underlying axonal transport in developing or degenerating brains requires dynamic imaging of axonal cargo movement in living samples. Whereas methods exist to image axonal transport in Drosophila larval neurons, they are not suitable to follow this process during metamorphosis, when brains undergo extensive remodeling. Here we present a simple method that enables confocal imaging of both fast and slow axonal transport in Drosophila pupal brain explants. We describe how to prepare chambers adapted for live imaging, how to maintain brain explants under physiological conditions and how to monitor and quantitatively analyze the movement of fluorescently labeled cargoes. This protocol requires minimal equipment and is ideally suited for experiments that combine genetics, optogenetics and pharmacological approaches. The brains can be prepared for image acquisition in 1.5 h, and the protocol can be performed easily in any fly laboratory.

Imp promotes axonal remodeling by regulating profilin mRNA during brain development.

Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues [1-4]. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches [5]. Here, we identify the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting [6-12]. We show that whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, we demonstrate that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. Our study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation.

Principles and roles of mRNA localization in animal development.

Intracellular targeting of mRNAs has long been recognized as a means to produce proteins locally, but has only recently emerged as a prevalent mechanism used by a wide variety of polarized cell types. Localization of mRNA molecules within the cytoplasm provides a basis for cell polarization, thus underlying developmental processes such as asymmetric cell division, cell migration, neuronal maturation and embryonic patterning. In this review, we describe and discuss recent advances in our understanding of both the regulation and functions of RNA localization during animal development.

Expression of Slit and Robo genes in the developing mouse heart.

Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart.

The fabulous destiny of the Drosophila heart.

For the last 15 years the fly cardiovascular system has attracted developmental geneticists for its potential as a model system of organogenesis. Heart development in Drosophila indeed provides a remarkable system for elucidating the basic molecular and cellular mechanisms of morphogenesis and, more recently, for understanding the genetic control of cardiac physiology. The success of these studies can in part be attributed to multidisciplinary approaches, the multiplicity of existing genetic tools, and a detailed knowledge of the system. Striking similarities with vertebrate cardiogenesis have long been stressed, in particular concerning the conservation of key molecular regulators of cardiogenesis and the new data presented here confirm Drosophila cardiogenesis as a model not only for organogenesis but also for the study of molecular mechanisms of human cardiac disease.

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation.

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis.

Dynamics of the basement membrane in invasive epithelial clusters in Drosophila.

The basement membrane (BM) represents a barrier to cell migration, which has to be degraded to promote invasion. However, the role and behaviour of the BM during the development of pre-invasive cells is only poorly understood. Drosophila border cells (BCs) provide an attractive genetic model in which to study the cellular mechanisms underlying the migration of mixed cohorts of epithelial cells. BCs are made of two different epithelial cell types appearing sequentially during oogenesis: the polar cells and the outer BCs. Here, we show that the pre-invasive polar cells undergo an unusual and asymmetrical apical capping with major basement membrane proteins, including the two Drosophila Collagen IV alpha chains, Laminin A and Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical transcytosis mechanism that involves the small GTPase Drab5. Apical capping is transient and is followed by rapid shedding prior to the initiation of BC migration, suggesting that the apical cap blocks migration. Consistently, non-migratory polar cells remain capped. We further show that JAK/STAT signalling and recruitment of outer BCs are required for correct shedding and migration. The dynamics of the BM represents a marker of migratory BC, revealing a novel developmentally regulated behaviour of BM coupled to epithelial cell invasiveness.

The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis.

In mammals, the JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is activated in response to cytokines and growth factors to control blood cell development, proliferation and cell determination. In Drosophila, a conserved JAK/STAT signaling pathway controls segmentation in embryos, as well as blood cell development and other processes in larvae and adults. During embryogenesis, transduction of the Unpaired [Upd; also known as Outstretched (Os)] ligand through the JAK/STAT pathway requires Domeless, a putative membrane protein with distant homology to vertebrate type I cytokine receptors. We have isolated domeless (dome) in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, we show that dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, the Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. We also show that dome functions downstream of upd and that both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, our data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis.