@article {69622, title = {A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.}, journal = {Nat Methods}, year = {2024}, month = {2024 Sep 20}, abstract = {

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7{\texttimes} increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca concentration using fluorescence lifetime imaging microscopy (FLIM).

}, issn = {1548-7105}, doi = {10.1038/s41592-024-02411-6}, url = {https://doi.org/10.1038/s41592-024-02411-6}, author = {Farrants, Helen and Shuai, Yichun and Lemon, William C and Monroy Hernandez, Christian and Zhang, Deng and Yang, Shang and Patel, Ronak and Qiao, Guanda and Frei, Michelle S and Plutkis, Sarah E and Grimm, Jonathan B and Hanson, Timothy L and Tomaska, Filip and Turner, Glenn C and Stringer, Carsen and Keller, Philipp J and Beyene, Abraham G and Chen, Yao and Liang, Yajie and Lavis, Luke D and Schreiter, Eric R} } @article {68587, title = {Automated reconstruction of whole-embryo cell lineages by learning from sparse annotations.}, journal = {Nature Biotechnology}, volume = {41}, year = {2023}, month = {2023 Jan 01}, pages = {44-49}, abstract = {

We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8\% of cell lineages spanning 1 h, as compared to 31.8\% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs.

}, keywords = {Animals, Blastocyst, Cell Lineage, Embryo, Mammalian, Mice, Microscopy}, issn = {1546-1696}, doi = {10.1038/s41587-022-01427-7}, author = {Malin-Mayor, Caroline and Hirsch, Peter and Guignard, Leo and McDole, Katie and Wan, Yinan and Lemon, William C and Kainmueller, Dagmar and Keller, Philipp J and Preibisch, Stephan and Funke, Jan} } @article {68313, title = {Automated reconstruction of whole-embryo cell lineages by learning from sparse annotations.}, journal = {Nature Biotechnology}, year = {2022}, month = {2022 Sep 05}, abstract = {

We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8\% of cell lineages spanning 1 h, as compared to 31.8\% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs.

}, issn = {1546-1696}, doi = {10.1038/s41587-022-01427-7}, author = {Malin-Mayor, Caroline and Hirsch, Peter and Guignard, Leo and McDole, Katie and Wan, Yinan and Lemon, William C and Kainmueller, Dagmar and Keller, Philipp J and Preibisch, Stephan and Funke, Jan} } @article {67627, title = {In~vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {Cell Reports}, volume = {35}, year = {2021}, month = {2021 Jun 22}, pages = {109284}, abstract = {

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1\ μM to 10\ mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109284}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {65624, title = {Live-cell imaging in the era of too many microscopes.}, journal = {Current Opinion in Cell Biology}, volume = {66}, year = {2020}, month = {2020 May 26}, pages = {34-42}, abstract = {

At the time of this writing, searching Google Scholar for {\textquoteright}light-sheet microscopy{\textquoteright} returns almost 8500 results; over three-quarters of which were published in the last 5 years alone. Searching for other advanced imaging methods in the last 5 years yields similar results: {\textquoteright}super-resolution microscopy{\textquoteright} (\>16 000), {\textquoteright}single-molecule imaging{\textquoteright} (almost 10 000), SPIM (Single Plane Illumination Microscopy, 5000), and {\textquoteright}lattice light-sheet{\textquoteright} (1300). The explosion of new imaging methods has also produced a dizzying menagerie of acronyms, with over 100 different species of {\textquoteright}light-sheet{\textquoteright} alone, from SPIM to UM (Ultra microscopy) to SiMView (Simultaneous MultiView) to iSPIM (inclined SPIM, not to be confused with iSPIM, inverted SPIM). How then is the average biologist, without an advanced degree in physics, optics, or computer science supposed to make heads or tails of which method is best suited for their needs? Let us also not forget the plight of the optical physicist, who at best might need help with obtaining healthy samples and keeping them that way, or at worst may not realize the impact their newest technique could have for biologists. This review will not attempt to solve all\ these problems, but instead highlight some of the most recent, successful mergers between biology and advanced imaging technologies, as well as hopefully provide some guidance for anyone interested in journeying into the world of live-cell imaging.

}, issn = {1879-0410}, doi = {10.1016/j.ceb.2020.04.008}, author = {Lemon, William C and McDole, Katie} } @article {64866, title = {In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {bioRxiv}, year = {2019}, month = {2019 Mar 8}, abstract = {Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 {\textmu}M to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.}, doi = {10.1101/571422}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {64842, title = {Metabolic regulation of developmental cell cycles and zygotic transcription.}, journal = {Current Biology}, volume = {29}, year = {2019}, month = {2019 Mar 01}, pages = {1193-8}, abstract = {

The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2019.02.028}, author = {Djabrayan, Nareg J-V and Smits, Celia M and Krajnc, Matej and Stern, Tomer and Yamada, Shigehiro and Lemon, William C and Keller, Philipp J and Rushlow, Christine A and Shvartsman, Stanislav Y} } @article {49487, title = {A practical guide to adaptive light-sheet microscopy.}, journal = {Nature Protocols}, volume = {13}, year = {2018}, month = {2018 Oct 26}, pages = {2462{\textendash}2500}, abstract = {

We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope. The protocol describes (i) the mechano-optical implementation of the adaptive imaging hardware, including technical drawings for all custom microscope components; (ii) the algorithms and software library for automated adaptive imaging, including the pseudocode and annotated source code for all software modules; and (iii) the execution of adaptive imaging experiments, as well as the configuration and practical use of the AutoPilot framework. Setup of the adaptive imaging hardware and software takes 1-2 weeks each. Previous experience with light-sheet microscopy and some familiarity with software engineering and building of optical instruments are recommended. Successful implementation of the protocol recovers near diffraction-limited performance in many parts of typical multicellular organisms studied with light-sheet microscopy, such as fruit fly and zebrafish embryos, for which resolution and signal strength are improved two- to fivefold.

}, issn = {1750-2799}, doi = {10.1038/s41596-018-0043-4}, author = {Royer, Lo{\"\i}c A and Lemon, William C and Chhetri, Raghav K and Keller, Philipp J} } @article {49012, title = {A preferred curvature-based continuum mechanics framework for modeling embryogenesis.}, journal = {Biophysical Journal}, volume = {114}, year = {2018}, month = {2018 Jan 23}, pages = {267-77}, abstract = {

Mechanics plays a key role in the development of higher organisms. However, understanding this relationship is complicated by the difficulty of modeling the link between local forces generated at the subcellular level and deformations observed at the tissue and whole-embryo levels. Here we propose an approach first developed for lipid bilayers and cell membranes, in which force-generation by cytoskeletal elements enters a continuum mechanics formulation for the full system in the form of local changes in preferred curvature. This allows us to express and solve the system using only tissue strains. Locations of preferred curvature are simply related to products of gene expression. A solution, in that context, means relaxing the system{\textquoteright}s mechanical energy to yield global morphogenetic predictions that accommodate a tendency toward the local preferred curvature, without a need to explicitly model force-generation mechanisms at the molecular level. Our computational framework, which we call SPHARM-MECH, extends a 3D spherical harmonics parameterization known as SPHARM to combine this level of abstraction with a sparse shape representation. The integration of these two principles allows computer simulations to be performed in three dimensions on highly complex shapes, gene expression patterns, and mechanical constraints. We demonstrate our approach by modeling mesoderm invagination in the fruit-fly embryo, where local forces generated by the acto-myosin meshwork in the region of the future mesoderm lead to formation of a ventral tissue fold. The process is accompanied by substantial changes in cell shape and long-range cell movements. Applying SPHARM-MECH to whole-embryo live imaging data acquired with light-sheet microscopy reveals significant correlation between calculated and observed tissue movements. Our analysis predicts the observed cell shape anisotropy on the ventral side of the embryo and suggests an active mechanical role of mesoderm invagination in supporting the onset of germ-band extension.

}, doi = {10.1016/j.bpj.2017.11.015}, author = {Khairy, Khaled and Lemon, William C and Amat, Fernando and Keller, Philipp J} } @article {48891, title = {A general method to fine-tune fluorophores for live-cell and in vivo imaging.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Oct}, pages = {987-994}, abstract = {

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

}, keywords = {Animals, Brain, Cell Line, Coloring Agents, Drosophila, Image Processing, Computer-Assisted, Larva, Mice, Microscopy, Fluorescence, Photochemical Processes, Staining and Labeling}, issn = {1548-7105}, doi = {10.1038/nmeth.4403}, author = {Grimm, Jonathan B and Muthusamy, Anand K and Liang, Yajie and Brown, Timothy A and Lemon, William C and Patel, Ronak and Lu, Rongwen and Macklin, John J and Keller, Philipp J and Ji, Na and Lavis, Luke D} } @article {47987, title = {Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms.}, journal = {Nature Biotechnology}, volume = {34}, year = {2016}, month = {2016 Oct 31}, pages = {1267-78}, abstract = {

Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish (Danio rerio) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.

}, issn = {1546-1696}, doi = {10.1038/nbt.3708}, author = {Royer, Lo{\"\i}c A and Lemon, William C and Chhetri, Raghav K and Wan, Yinan and Coleman, Michael and Myers, Eugene W and Keller, Philipp J} } @article {47267, title = {Real-time three-dimensional cell segmentation in large-scale microscopy data of developing embryos.}, journal = {Developmental Cell}, volume = {36}, year = {2016}, month = {2016 Jan 25}, pages = {225-40}, abstract = {

We present the Real-time Accurate Cell-shape Extractor (RACE), a high-throughput image analysis framework for automated three-dimensional cell segmentation in large-scale images. RACE is 55{\textendash}330 times faster and 2{\textendash}5 times more accurate than state-of-the-art methods. We demonstrate the generality of RACE by extracting cell-shape information from entire Drosophila, zebrafish, and mouse embryos imaged with confocal and light-sheet microscopes. Using RACE, we automatically reconstructed cellular-resolution tissue anisotropy maps across developing Drosophila embryos and quantified differences in cell-shape dynamics in wild-type and mutant embryos. We furthermore integrated RACE with our framework for automated cell lineaging and performed joint segmentation and cell tracking in entire Drosophila embryos. RACE processed these terabyte-sized datasets on a single computer within 1.4\ days. RACE is easy to use, as it requires adjustment of only three parameters, takes full advantage of state-of-the-art multi-core processors and graphics cards, and is available as open-source software for Windows, Linux, and Mac\ OS.

}, doi = {10.1016/j.devcel.2015.12.028}, author = {Stegmaier, Johannes and Amat, Fernando and Lemon, William C and McDole, Katie and Wan, Yinan and Teodoro, George and Mikut, Ralf and Keller, Philipp J} } @article {47814, title = {Whole-animal imaging with high spatio-temporal resolution.}, journal = {Proceedings of SPIE}, volume = {9720}, year = {2016}, month = {2016 Jul 18}, pages = {97200R}, abstract = {

We developed isotropic multiview (IsoView) light-sheet microscopy in order to image fast cellular dynamics, such as cell movements in an entire developing embryo or neuronal activity throughput an entire brain or nervous system, with high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To achieve high temporal resolution and high spatial resolution at the same time, IsoView microscopy rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. In a post-processing step, these four views are then combined by means of high-throughput multiview deconvolution to yield images with a system resolution of <= 450 nm in all three dimensions. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 μm and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional (spatially anisotropic) light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, such as lattice lightsheet microscopy or diSPIM, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

}, isbn = {978-162841954-2}, issn = { 0277786X}, doi = {10.1117/12.2212564}, author = {Chhetri, Raghav and Amat, Fernando and Wan, Yinan and H{\"o}ckendorf, Burkhard and Lemon, William C and Keller, Philipp J} } @article {47082, title = {Efficient processing and analysis of large-scale light-sheet microscopy data.}, journal = {Nature Protocols}, volume = {10}, year = {2015}, month = {2015 Oct 2}, pages = {1679-96}, abstract = {

Light-sheet microscopy is a powerful method for imaging the development and function of complex biological systems at high spatiotemporal resolution and over long time scales. Such experiments typically generate terabytes of multidimensional image data, and thus they demand efficient computational solutions for data management, processing and analysis. We present protocols and software to tackle these steps, focusing on the imaging-based study of animal development. Our protocols facilitate (i) high-speed lossless data compression and content-based multiview image fusion optimized for multicore CPU architectures, reducing image data size 30{\textendash}500-fold; (ii) automated large-scale cell tracking and segmentation; and (iii) visualization, editing and annotation of multiterabyte image data and cell-lineage reconstructions with tens of millions of data points. These software modules are open source. They provide high data throughput using a single computer workstation and are readily applicable to a wide spectrum of biological model systems.

}, doi = {10.1038/nprot.2015.111}, author = {Amat, Fernando and H{\"o}ckendorf, Burkhard and Wan, Yinan and Lemon, William C and McDole, Katie and Keller, Philipp J} } @article {45457, title = {Light sheet-based imaging and analysis of early embryogenesis in the fruit fly.}, journal = {Methods in Molecular Biology}, volume = {1189}, year = {2015}, month = {2015}, pages = {79-97}, abstract = {

The fruit fly is an excellent model system for investigating the sequence of epithelial tissue invaginations constituting the process of gastrulation. By combining recent advancements in light sheet fluorescence microscopy (LSFM) and image processing, the three-dimensional fly embryo morphology and relevant gene expression patterns can be accurately recorded throughout the entire process of embryogenesis. LSFM provides exceptionally high imaging speed, high signal-to-noise ratio, low level of photoinduced damage, and good optical penetration depth. This powerful combination of capabilities makes LSFM particularly suitable for live imaging of the fly embryo.The resulting high-information-content image data are subsequently processed to obtain the outlines of cells and cell nuclei, as well as the geometry of the whole embryo tissue by image segmentation. Furthermore, morphodynamics information is extracted by computationally tracking objects in the image. Towards that goal we describe the successful implementation of a fast fitting strategy of Gaussian mixture models.The data obtained by image processing is well-suited for hypothesis testing of the detailed biomechanics of the gastrulating embryo. Typically this involves constructing computational mechanics models that consist of an objective function providing an estimate of strain energy for a given morphological configuration of the tissue, and a numerical minimization mechanism of this energy, achieved by varying morphological parameters.In this chapter, we provide an overview of in vivo imaging of fruit fly embryos using LSFM, computational tools suitable for processing the resulting images, and examples of computational biomechanical simulations of fly embryo gastrulation.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-1164-6_6}, author = {Khairy, Khaled and Lemon, William C and Amat, Fernando and Keller, Philipp J} } @article {47116, title = {Whole-animal functional and developmental imaging with isotropic spatial resolution}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Oct 26}, pages = {1171-8}, abstract = {

Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5 μm and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

}, doi = {10.1038/nmeth.3632}, author = {Chhetri, Raghav K and Amat, Fernando and Wan, Yinan and H{\"o}ckendorf, Burkhard and Lemon, William C and Keller, Philipp J} } @article {46984, title = {Whole-central nervous system functional imaging in larval Drosophila.}, journal = {Nature Communications}, volume = {6}, year = {2015}, month = {2015 Aug 11}, pages = {7924}, abstract = {

Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. We image both brain and ventral nerve cord, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, we predict functional connections between CNS regions and reveal neurons in the brain that identify type and temporal state of motor programs executed in the ventral nerve cord.

}, doi = {10.1038/ncomms8924}, author = {Lemon, William C and Pulver, Stefan R and H{\"o}ckendorf, Burkhard and McDole, Katie and Branson, Kristin M and Freeman, Jeremy and Keller, Philipp J} } @article {45866, title = {Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Apr 22}, pages = {e01699}, abstract = {

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01699}, author = {Zsch{\"a}tzsch, Marlen and Oliva, Carlos and Langen, Marion and De Geest, Natalie and Ozel, Mehmet Neset and Williamson, W Ryan and Lemon, William C and Soldano, Alessia and Munck, Sebastian and Hiesinger, P Robin and Sanchez-Soriano, Natalia and Hassan, Bassem A} } @article {6918, title = {Live imaging of nervous system development and function using light-sheet microscopy.}, journal = {Molecular Reproduction and Development}, volume = {82}, year = {2013}, month = {2015 Jul}, pages = {605-18}, abstract = {

In vivo imaging applications typically require carefully balancing conflicting parameters. Often it is necessary to achieve high imaging speed, low photo-bleaching, and photo-toxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage at the same time. Light-sheet microscopy provides good performance in all of these categories, and is thus emerging as a particularly powerful live imaging method for the life sciences. We see an outstanding potential for applying light-sheet microscopy to the study of development and function of the early nervous system in vertebrates and higher invertebrates. Here, we review state-of-the-art approaches to live imaging of early development, and show how the unique capabilities of light-sheet microscopy can further advance our understanding of the development and function of the nervous system. We discuss key considerations in the design of light-sheet microscopy experiments, including sample preparation and fluorescent marker strategies, and provide an outlook for future directions in the field.

}, issn = {1098-2795}, doi = {10.1002/mrd.22258}, author = {Lemon, William C and Keller, Philipp J} } @article {4142, title = {Functional dissection of a neuronal network required for cuticle tanning and wing expansion in Drosophila.}, journal = {The Journal of Neuroscience The Official Journal of the Society for Neuroscience}, volume = {26}, year = {2006}, month = {2006 Jan 11}, pages = {573-84}, abstract = {

A subset of Drosophila neurons that expresses crustacean cardioactive peptide (CCAP) has been shown previously to make the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Here we present evidence that CCAP-expressing neurons (NCCAP) consist of two functionally distinct groups, one of which releases bursicon into the hemolymph and the other of which regulates its release. The first group, which we call NCCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion that lie within the expression pattern of the enhancer-trap line c929-Gal4. We show that suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which we call NCCAP-R, consists of NCCAP neurons outside the c929-Gal4 pattern. Because suppression of synaptic transmission and protein kinase A (PKA) activity throughout NCCAP, but not in NCCAP-c929, also blocks tanning and wing expansion, we conclude that neurotransmission and PKA are required in NCCAP-R to regulate bursicon secretion from NCCAP-c929. Enhancement of electrical activity in NCCAP-R by expression of the bacterial sodium channel NaChBac also blocks tanning and wing expansion and leads to depletion of bursicon from central processes. NaChBac expression in NCCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. Our results suggest that NCCAP form an interacting neuronal network responsible for the regulation and release of bursicon and suggest a model in which PKA-mediated stimulation of inputs to normally quiescent bursicon-expressing neurons activates release of the hormone.

}, keywords = {Animals, Bacterial Proteins, Cyclic AMP-Dependent Protein Kinases, Drosophila melanogaster, Drosophila Proteins, Electroretinography, Ganglia, Gene Targeting, Genetically Modified, Invertebrate, Invertebrate Hormones, Nerve Net, Neurons, Neuropeptides, Phenotype, Pigmentation, Recombinant Fusion Proteins, Shaker Superfamily of Potassium Channels, Sodium Channels, Synaptic Transmission, Wing}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3916-05.2006}, author = {Luan, Haojiang and Lemon, William C and Peabody, Nathan C and Pohl, Jascha B and Zelensky, Paul K and Wang, Ding and Nitabach, Michael N and Holmes, Todd C and White, Benjamin H} }