Retina-derived signals control pace of neurogenesis in visual brain areas but not circuit assembly.

Brain development is orchestrated by both innate and experience-dependent mechanisms, but their relative contributions are difficult to disentangle. Here we asked if and how central visual areas are altered in a vertebrate brain depleted of any and all signals from retinal ganglion cells throughout development. We transcriptionally profiled neurons in pretectum, thalamus and other retinorecipient areas of larval zebrafish and searched for changes in lakritz mutants that lack all retinal connections. Although individual genes are dysregulated, the complete set of 77 neuronal types develops in apparently normal proportions, at normal locations, and along normal differentiation trajectories. Strikingly, the cell-cycle exits of proliferating progenitors in these areas are delayed, and a greater fraction of early postmitotic precursors remain uncommitted or are diverted to a pre-glial fate. Optogenetic stimulation targeting groups of neurons normally involved in processing visual information evokes behaviors indistinguishable from wildtype. In conclusion, we show that signals emitted by retinal axons influence the pace of neurogenesis in visual brain areas, but do not detectably affect the specification or wiring of downstream neurons.

A single-cell resolution gene expression atlas of the larval zebrafish brain.

The advent of multimodal brain atlases promises to accelerate progress in neuroscience by allowing in silico queries of neuron morphology, connectivity, and gene expression. We used multiplexed fluorescent in situ RNA hybridization chain reaction (HCR) technology to generate expression maps across the larval zebrafish brain for a growing set of marker genes. The data were registered to the Max Planck Zebrafish Brain (mapzebrain) atlas, thus allowing covisualization of gene expression, single-neuron tracings, and expertly curated anatomical segmentations. Using post hoc HCR labeling of the immediate early gene cfos, we mapped responses to prey stimuli and food ingestion across the brain of freely swimming larvae. This unbiased approach revealed, in addition to previously described visual and motor areas, a cluster of neurons in the secondary gustatory nucleus, which express the marker calb2a, as well as a specific neuropeptide Y receptor, and project to the hypothalamus. This discovery exemplifies the power of this new atlas resource for zebrafish neurobiology.

Visual recognition of social signals by a tectothalamic neural circuit.

Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1-5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6-8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12-14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.

A Live-Cell Imaging Approach for Measuring DNA Replication Rates.

We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level.

VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation.

To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc), the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG). Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

Resolving new ultrastructural features of cytokinetic abscission with soft-X-ray cryo-tomography.

Mammalian cytokinetic abscission is mediated by the ESCRT membrane fission machinery. While much has been clarified on the topology and kinetics of abscission through high-resolution microscopy, key questions regarding the mechanism of abscission remain open. Here we apply cryogenic soft-X-ray tomography to elucidate new ultrastructural details in the intercellular membrane bridge connecting cells undergoing abscission. In particular, we resolve defined ring-like structures inside the midbody dark zone that have been inaccessible to EM, and identify membrane extrusions at the abscission sites. In cells at late stages of abscission we resolve a complex array of helical spirals, extending the structural information obtained by EM. Our results highlight the advantages of soft-X-ray tomography and emphasize the importance of using complementary approaches for characterizing cellular structures. Notably, by providing new structural data from intact cells we present a realistic view on the topology of abscission and suggest new mechanistic models for ESCRT mediated abscission.

A simple, straightforward correlative live-cell-imaging-structured-illumination-microscopy approach for studying organelle dynamics.

Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two-fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high-speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high-speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells.

Differential Inhibition of Human Atherosclerotic Plaque-Induced Platelet Activation by Dimeric GPVI-Fc and Anti-GPVI Antibodies: Functional and Imaging Studies.

BACKGROUND: Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. OBJECTIVES: This study sought to compare compounds interfering with platelet GPVI-atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. METHODS: Human atherosclerotic plaque-induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. RESULTS: GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc-free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. CONCLUSIONS: Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury.