Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Colonies of the aphidPseudoregma alexanderi produce morphologically-specialized first-instar larvae, termed soldiers, that defend the colony from predators. The environmental cues and physiological mechanisms governing soldier production are currently unknown. Here we present a morphometric study of soldiers and normal first-instar larvae ofP. alexanderi. Several morphological features (fore-leg length and width, hind-leg length, and horn length) plotted against body length display relationship that are similar to a sigmoidal curve. We found further support for an earlier finding that soldiers fall into two size categories, majors and minors, although both types of soldiers appear to follow the same allometry. The patterns of allometry in the soldier-producing aphids are very different from those found in other social insects and do not easily fit into the traditional categorization of allometries. We present two simple alternative models of soldier development as a framework for guiding future studies of the mechanisms of soldier production.

SUMMARY: High-resolution, three-dimensional (3D) imaging of large biological specimens generates massive image datasets that are difficult to navigate, annotate and share effectively. Inspired by online mapping applications like GoogleMaps, we developed a decentralized web interface that allows seamless navigation of arbitrarily large image stacks. Our interface provides means for online, collaborative annotation of the biological image data and seamless sharing of regions of interest by bookmarking. The CATMAID interface enables synchronized navigation through multiple registered datasets even at vastly different scales such as in comparisons between optical and electron microscopy. AVAILABILITY: http://fly.mpi-cbg.de/catmaid.

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist's perspective.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.

Hippocampal neurons can display reliable and long-lasting sequences of transient firing patterns, even in the absence of changing external stimuli. We suggest that time-keeping is an important function of these sequences, and propose a network mechanism for their generation. We show that sequences of neuronal assemblies recorded from rat hippocampal CA1 pyramidal cells can reliably predict elapsed time (15-20 s) during wheel running with a precision of 0.5 s. In addition, we demonstrate the generation of multiple reliable, long-lasting sequences in a recurrent network model. These sequences are generated in the presence of noisy, unstructured inputs to the network, mimicking stationary sensory input. Identical initial conditions generate similar sequences, whereas different initial conditions give rise to distinct sequences. The key ingredients responsible for sequence generation in the model are threshold-adaptation and a Mexican-hat-like pattern of connectivity among pyramidal cells. This pattern may arise from recurrent systems such as the hippocampal CA3 region or the entorhinal cortex. We hypothesize that mechanisms that evolved for spatial navigation also support tracking of elapsed time in behaviorally relevant contexts.

The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. The relationship between cell class and lineage is complex. Here we developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. We demonstrated the power of CLIn in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. We mapped 18 dopaminergic neurons from three distinct clusters to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. We also identified type II lineages that produce doublesex- and fruitless-expressing neurons and examined whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain. Blocking apoptosis in these lineages resulted in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism.