Structural and developmental principles of neuropil assembly in C. elegans.

Neuropil is a fundamental form of tissue organization within the brain1, in which densely packed neurons synaptically interconnect into precise circuit architecture2,3. However, the structural and developmental principles that govern this nanoscale precision remain largely unknown4,5. Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation'6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy7,8 coupled with lineage-tracing and cell-tracking algorithms9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.

WormGUIDES: an interactive single cell developmental atlas and tool for collaborative multidimensional data exploration.

BACKGROUND: Imaging and image analysis advances are yielding increasingly complete and complicated records of cellular events in tissues and whole embryos. The ability to follow hundreds to thousands of cells at the individual level demands a spatio-temporal data infrastructure: tools to assemble and collate knowledge about development spatially in a manner analogous to geographic information systems (GIS). Just as GIS indexes items or events based on their spatio-temporal or 4D location on the Earth these tools would organize knowledge based on location within the tissues or embryos. Developmental processes are highly context-specific, but the complexity of the 4D environment in which they unfold is a barrier to assembling an understanding of any particular process from diverse sources of information. In the same way that GIS aids the understanding and use of geo-located large data sets, software can, with a proper frame of reference, allow large biological data sets to be understood spatially. Intuitive tools are needed to navigate the spatial structure of complex tissue, collate large data sets and existing knowledge with this spatial structure and help users derive hypotheses about developmental mechanisms. RESULTS: Toward this goal we have developed WormGUIDES, a mobile application that presents a 4D developmental atlas for Caenorhabditis elegans. The WormGUIDES mobile app enables users to navigate a 3D model depicting the nuclear positions of all cells in the developing embryo. The identity of each cell can be queried with a tap, and community databases searched for available information about that cell. Information about ancestry, fate and gene expression can be used to label cells and craft customized visualizations that highlight cells as potential players in an event of interest. Scenes are easily saved, shared and published to other WormGUIDES users. The mobile app is available for Android and iOS platforms. CONCLUSION: WormGUIDES provides an important tool for examining developmental processes and developing mechanistic hypotheses about their control. Critically, it provides the typical end user with an intuitive interface for developing and sharing custom visualizations of developmental processes. Equally important, because users can select cells based on their position and search for information about them, the app also serves as a spatially organized index into the large body of knowledge available to the C. elegans community online. Moreover, the app can be used to create and publish the result of exploration: interactive content that brings other researchers and students directly to the spatio-temporal point of insight. Ultimately the app will incorporate a detailed time lapse record of cell shape, beginning with neurons. This will add the key ability to navigate and understand the developmental events that result in the coordinated and precise emergence of anatomy, particularly the wiring of the nervous system.

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph polarize F-actin during embryonic morphogenesis by regulating the WAVE/SCAR actin nucleation complex.

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.

α-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells.

The BK channel, a voltage- and Ca(2+)-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian α-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel α-subunit, in body-wall muscle cells. CTN-1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of SLO-1. In body-wall muscle cells, CTN-1 coclusters with SLO-1 at regions of dense bodies, which are Z-disk analogs of mammalian skeletal muscle. In ctn-1 loss-of-function (lf) mutants, SLO-1 was mislocalized in body-wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn-1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo-1(gf);ctn-1(lf). These results suggest that CTN-1 plays an important role in BK channel function by mediating channel subcellular localization.

The branched actin nucleator Arp2/3 promotes nuclear migrations and cell polarity in the C. elegans zygote.

Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements. However, the role of actin nucleators in nuclear migration and positioning is poorly understood. We show that the branched actin nucleator, Arp2/3, affects nuclear movements throughout embryonic and larval development in C. elegans, including nuclear migrations in epidermal cells and neuronal precursors. In one-cell embryos the migration of the male pronucleus to meet the female pronucleus after fertilization requires Arp2/3. Loss of Arp2/3 or its activators changes the dynamics of non-muscle myosin, NMY-2, and alters the cortical accumulation of posterior PAR proteins. Reduced establishment of the posterior microtubule cytoskeleton in Arp2/3 mutants correlates with reduced male pronuclear migration. The UNC-84/SUN nuclear envelope protein that links the nucleus to the MT and actin cytoskeleton is known to regulate later nuclear migrations. We show here it also positions the male pronucleus. These studies demonstrate a global role for Arp2/3 in nuclear migrations. In the C. elegans one-cell embryo Arp2/3 promotes the establishment of anterior/posterior polarity and promotes MT growth that propels the anterior migration of the male pronucleus. In contrast with previous studies emphasizing pulling forces on the male pronucleus, we propose that robust MT nucleation pushes the male pronucleus anteriorly to join the female pronucleus.

Differential adhesion regulates neurite placement via a retrograde zippering mechanism.

During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.

Dynamics of peptidergic secretory granule transport are regulated by neuronal stimulation.

BACKGROUND: Peptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs) from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated. Sensory neurons also package opiate receptors in peptide-containing LDCVs, which is thought to be important in pain sensation. Since peptide granules cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides. RESULTS: A vector encoding soluble peptidylglycine alpha-hydroxylating monooxygenase fused to green fluorescent protein was constructed to address these questions in cultured primary peptidergic neurons of the trigeminal ganglion using time lapse confocal microscopy. The time course of release differs with secretagogue; the secretory response to depolarization with K+ is rapid and terminates within 15 minutes, while phorbol ester stimulation of secretion is maintained over a longer period. The data demonstrate fundamental differences between LDCV dynamics in axons and growth cones under basal conditions. CONCLUSIONS: Under basal conditions, LDCVs move faster away from the soma than toward the soma, but fewer LDCVs travel anterograde than retrograde. Stimulation decreased average anterograde velocity and increases granule pausing. Data from antibody uptake, quantification of enzyme secretion and appearance of pHluorin fluorescence demonstrate distributed release of peptides all along the axon, not just at terminals.

Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis.

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.

The type I membrane protein EFF-1 is essential for developmental cell fusion.

Multinucleate cells are widespread in nature, yet the mechanism by which cells fuse their plasma membranes is poorly understood. To identify animal fusogens, we performed new screens for mutations that abolish cell fusion within tissues of C. elegans throughout development. We identified the gene eff-1, which is expressed as cells acquire fusion competence and encodes a novel integral membrane protein. EFF-1 sequence motifs suggest physicochemical actions that could cause adjacent bilayers to fuse. Mutations in the extracellular domain of EFF-1 completely block epithelial cell membrane fusion without affecting other perfusion events such as cell generation, patterning, differentiation, and adhesion. Thus, EFF-1 is a key component in the mechanism of cell fusion, a process essential to normal animal development.

Depolymerization-driven flow in nematode spermatozoa relates crawling speed to size and shape.

Cell crawling is an inherently physical process that includes protrusion of the leading edge, adhesion to the substrate, and advance of the trailing cell body. Research into advance of the cell body has focused on actomyosin contraction, with cytoskeletal disassembly regarded as incidental, rather than causative; however, extracts from nematode spermatozoa, which use Major Sperm Protein rather than actin, provide at least one example where cytoskeletal disassembly apparently generates force in the absence of molecular motors. To test whether depolymerization can explain force production during nematode sperm crawling, we constructed a mathematical model that simultaneously describes the dynamics of both the cytoskeleton and the cytosol. We also performed corresponding experiments using motile Caenorhabditis elegans spermatozoa. Our experiments reveal that crawling speed is an increasing function of both cell size and anterior-posterior elongation. The quantitative, depolymerization-driven model robustly predicts that cell speed should increase with cell size and yields a cytoskeletal disassembly rate that is consistent with previous measurements. Notably, the model requires anisotropic elasticity, with the cell being stiffer along the direction of motion, to accurately reproduce the dependence of speed on elongation. Our simulations also predict that speed should increase with cytoskeletal anisotropy and disassembly rate.

Fusogenic activity of EFF-1 is regulated via dynamic localization in fusing somatic cells of C. elegans.

BACKGROUND: Many animal tissues form via fusion of cells. Yet in all instances of developmental cell fusion, the mechanism underlying fusion of plasma membranes remains poorly understood. EFF-1 is required for most somatic cell fusions in C. elegans, and misexpressed EFF-1 alters the normal pattern of fusing hypodermal cells. However, the autonomous activity of EFF-1, the rules governing its specificity, and the mechanism of its action have not been examined. RESULTS: We show that EFF-1 acts as a cellular fusogen, capable of inducing fusion of virtually any somatic cells in C. elegans, yet targeted precisely to fusion-fated contacts during normal development. Misexpression of EFF-1 in early embryos causes fusion among groups of cells composed entirely of nonfusion-fated members. Measurements of cytoplasm diffusion in induced fusion events show that ectopic EFF-1 expression produces fusion pores similar to those in normal fusion events. GFP-labeled EFF-1 is specifically targeted to fusion-competent cell contacts via reciprocal localization to the touching membranes of EFF-1-expressing cells. EFF-1 function is also governed by intercellular barriers that prohibit cell fusion between distinct tissues. Analysis of mutant versions of EFF-1 indicates a novel mode of fusogenicity, employing neither a phospholipase active site nor hydrophobic fusion-peptide acting solely in pore formation. CONCLUSIONS: EFF-1 can confer potent fusogenic activity to nonfusing cell types. However, it is normally targeted only to fusion-fated cell borders via mutual interaction between EFF-1-expressing cells and relocalization to the plasma membrane. Because EFF-1 appears evolutionarily unique to nematodes, multiple mechanisms may have evolved for controlled plasma-membrane fusion in development.

Measurement of muscle disease by quantitative second-harmonic generation imaging.

Determining the health of muscle cells by in vivo imaging could impact the diagnosis and monitoring of a large number of congenital and acquired muscular or cardiac disorders. However, currently used technologies are hampered by insufficient resolution, lack of specificity, or invasiveness. We have combined intrinsic optical second-harmonic generation from sarcomeric myosin with a novel mathematical treatment of striation pattern analysis, to obtain measures of muscle contractile integrity that correlate strongly with the neuromuscular health of mice suffering from genetic, acquired, and age-related decline in skeletal muscle function. Analysis of biopsies from a pilot group of human volunteers suggests a similar power in quantifying sarcopenic changes in muscle integrity. These results provide the first strong evidence that quantitative image analysis of sarcomere pattern can be correlated with physiological function, and they invite the application of SHG imaging in clinical practice, either in biopsy samples or via microendoscopy.

Optical imaging of cell fusion and fusion proteins in Caenorhabditis elegans.

Cell fusion is a very dynamic process in which the entire membrane and cellular contents of two or more cells merge into one. Strategies developed to understand the component processes that make up a full fusion event require imaging to be performed over a range of space and time scales. These strategies must cover detection of nanometer-sized pores, monitoring cytoplasmic diffusion and the dynamic localization of proteins that induce fusion competence, and three-dimensional reconstruction of multinucleated cells. Caenorhabditis elegans' small size, predictable development, and transparent body make this organism optimal for microscopic investigations. In this chapter, focus is placed on light microscopy techniques that have been used thus far to study developmental fusion events in C. elegans and the insights that have been gained from them. There is also a general overview of the developmental timing of the cell fusion events. Additionally, several protocols are described for preparing both fixed and live specimens at various developmental stages of C. elegans for examination via optical microscopy.

Ultrastructural imaging of cell fusion in Caenorhabditis elegans.

Caenorhabditis elegans is a well-established model system particularly suited for studying cell-cell fusion because of its highly predictable and rapid development and its known cell lineage. This chapter focuses on understanding the ultrastructural components of cell fusion through the use of transmission electron microscopy (TEM). Published TEM studies have described the initial demonstration of syncytial cells in the worm, the vesiculation of the bilayers between cells during widening of the normal fusion aperture, and the appearance of microfusion intermediates in the membranes of cells with fusion-defective mutations. Capturing events observed in embryos on the light microscope and preserving the integrity of cellular membranes for examination by TEM require some special considerations that differ from many ultrastructural studies of cells. The principles of different techniques for TEM and details of protocols that have been used to investigate cell fusion in the nematode are discussed in this chapter.

Quantitative assays for cell fusion.

Cell fusion would seem to be obviously recognizable upon visual inspection, and many studies employ a simple microscopic fusion index to quantify the rate and extent of fusion in cell culture. However, when cells are not in monolayers or when there is a large background of multinucleation through failed cytokinesis, cell-cell fusion can only be proven by mixing of cell contents. Furthermore, determination of the microscopic fusion index must generally be carried out manually, creating opportunities for unintended observer bias and limiting the numbers of cells assayed and therefore the statistical power of the assay. Strategies for making assays dependent on fusion and independent of visual observation are critical to increasing the accuracy and throughput of screens for molecules that control cell fusion. A variety of in vitro biochemical and nonbiochemical techniques have been developed to assay and monitor fusion events in cultured cells. In this chapter, we briefly discuss several in vitro fusion assays, nearly all based on systems of two components that interact to create a novel assayable signal only after cells fuse. We provide details for the use of one example of such a system, intracistronic complementation of beta-galactosidase activity by mutants of Escherichia coli lacZ, which allows for either cell-by-cell microscopic assay of cell fusion or quantitative and kinetic detection of cell fusions in whole populations. In addition, we describe a combination of gene knock-down protocols with this assay to study factors required for myoblast fusion.

Coherent and incoherent SHG in fibrillar cellulose matrices.

Second Harmonic Generation (SHG) microscopy probes the organization of tissue or material structure through morphological and polarization analyses. In terms of diagnostic or analytical potential, it is important to understand the coherent and incoherent aspects of the emission in highly scattering environments. It is also of fundamental importance whether the SHG polarization signatures are retained in such turbid media. We examine these issues for purified cellulose specimens, which, in analogy to structural proteins, comprise highly birefringent and chiral fibrillar structures. In these matrices we observe predominantly coherent forward directed emission as well as backwards contrast consisting of direct, coherent emission and an incoherent component arising from multiply scattered forward directed SHG. These processes display a pronounced depth dependence evidenced by changes in morphology as well in the measured forward-backwards ratio (F/B). Specifically, from regions near the surface the backwards channel displays small fibrils not present in the forward emission. In addition, at depths beyond one mean free path, the fibril morphologies become highly similar, suggesting the observed backwards contrast is also comprised of a component that arises from multiple scattering of the initially forward directed wave. The depth dependence of the forward to backward ratio is consistent with Monte Carlo simulations of photon diffusion based on the measured scattering coefficient mus of 75 cm-1 and anisotropy factor, g=0.94 at the SHG wavelength. Consistent with the experimental observations, these simulations indicate that the backwards channel becomes increasingly incoherent with increasing depth into the specimen. We also demonstrate that the polarization dependence of the SHG can be measured through 500 microm of thickness. Similarly, the SHG signal anisotropy is largely preserved through this depth with only a slight depolarization being observed.