Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells.

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

Using the NoiSee workflow to measure signal-to-noise ratios of confocal microscopes.

Confocal microscopy is used today on a daily basis in life science labs. This "routine" technique contributes to the progress of scientific projects across many fields by revealing structural details and molecular localization, but researchers need to be aware that detection efficiency and emission light path performance is of major influence in the confocal image quality. By design, a large portion of the signal is discarded in confocal imaging, leading to a decreased signal-to-noise ratio (SNR) which in turn limits resolution. A well-aligned system and high performance detectors are needed in order to generate an image of best quality. However, a convenient method to address system status and performance on the emission side is still lacking. Here, we present a complete method to assess microscope and emission light path performance in terms of SNR, with a comprehensive protocol alongside NoiSee, an easy-to-use macro for Fiji (available via the corresponding update site). We used this method to compare several confocal systems in our facility on biological samples under typical imaging conditions. Our method reveals differences in microscope performance and highlights the various detector types used (multialkali photomultiplier tube (PMT), gallium arsenide phosphide (GaAsP) PMT, and Hybrid detector). Altogether, our method will provide useful information to research groups and facilities to diagnose their confocal microscopes.

Super-resolution microscopy demystified.

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel).

The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.

Myosin VIIA is a marker for the cone accessory outer segment in zebrafish.

The accessory outer segment, a cytoplasmic structure running alongside the photoreceptor outer segment, has been described in teleost fishes, excluding the model organism zebrafish. So far, the function of the accessory outer segment is unknown. Here, we describe the ultrastructure of the zebrafish cone accessory outer segment by electron microscopy. Starting at the connecting cilium, the accessory outer segment runs parallel alongside the cone outer segment (COS). A thin plasma bridge connects the outer segment with the accessory outer segment, whose surface is enlarged by foldings and invaginations. Beside the morphological descriptions, we demonstrate that the Usher protein myosin VIIa (Myo7a) is a specific marker for the zebrafish cone accessory outer segment. Zebrafish cone photoreceptors possess a large and well-differentiated accessory outer segment, in which the unconventional motor protein Myo7a is highly enriched. The direct cytoplasmic contact with the COS as well as the surface enlargement of the accessory outer segment suggests an important role of this structure in transport and exchange of metabolites between the COS and the surrounding retinal pigment epithelium. In future studies of the outer retina, more attention should be paid to this often neglected structure.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation.

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

Acquisition speed comparison of microscope software programs.

Reliable software is a prerequisite for successful operation of a modern wide field fluorescence microscope. When used for live cell imaging, acquisition speed is of particular interest. This is both because biological processes can be highly-dynamic, and to avoid unnecessary photobleaching and phototoxicity of living samples. This article shows that besides the hardware (microscope) components themselves, the acquisition control software is an important influencing factor of speed performance. We tested and compared the speed performance of five different generic applications (Image-Pro Plus, MetaMorph, Micro-Manager, SlideBook, and Volocity) using typical experimental setups involving a single specific state-of-the-art fluorescence microscope configuration. The test measurements included multichannel experiments, z-stacking, burst acquisition, as well as combinations of these measurements with time-lapse acquisitions. The measured data provided values for guiding the testing and analysis of other microscope systems with similar configurations. Despite the identical hardware settings, significant and surprisingly large speed differences were evident among the various software applications. Additionally, no application was identifiable as the fastest in all tests. Our work pinpoints the importance of the control software in determining a system's "real" maximal imaging speed. The study could serve as basis for further tests, eventually influencing the system selection criteria for speed-sensitive applications.

Funduscopy in adult zebrafish and its application to isolate mutant strains with ocular defects.

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals.A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out.Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas.In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.

Visual acuity in larval zebrafish: behavior and histology.

BACKGROUND: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. RESULTS: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1 degrees ). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1 degrees ). CONCLUSION: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae.

Space-saving advantage of an inverted retina.

Vertebrate eyes are of the simple or camera type with a single optical system that creates an image on the retina in the back of the eye. There, the visual information is encoded as nervous signals by photoreceptors, processed by retinal neurons, and then sent to the brain via the optic nerve. Surprisingly at first sight, the retinal neurons are located between the lens and the light-sensitive parts of the photoreceptors. The tissue scatters some light, which leads to loss of light and image blur. The inverted retina has, therefore, long been regarded as inferior. Here, we provide evidence that the inverted retina actually is a superior space-saving solution, especially in small eyes. The inverted retina has most likely facilitated the evolution of image-forming eyes in vertebrates, and it still benefits especially small and highly visual species.

Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina.

Over the last 20 years, the zebrafish has become an important model organism for research on retinal function and development. Many retinal diseases do not become apparent until the later stages of life. This means that it is important to be able to analyze (gene) function in the mature retina. To meet this need, we have established an organotypic culture system of mature wild-type zebrafish retinas in order to observe changes in retinal morphology. Furthermore, cell survival during culture has been monitored by determining apoptosis in the tissue. The viability and excitability of ganglion cells have been tested at various time points in vitro by patch-clamp recordings, and retinal functionality has been assessed by measuring light-triggered potentials at the ganglion cell site. Since neurogenesis is persistent in adult zebrafish retinas, we have also monitored proliferating cells during culture by tracking their bromodeoxyuridine uptake. Reverse genetic approaches for probing the function of adult zebrafish retinas are not yet available. We have therefore established a rapid and convenient protocol for delivering plasmid DNA or oligonucleotides by electroporation to the retinal tissue in vitro. The organotypic culture of adult zebrafish retinas presented here provides a reproducible and convenient method for investigating the function of drugs and genes in the retina under well-defined conditions in vitro.

The zebrafish mutant lbk/vam6 resembles human multisystemic disorders caused by aberrant trafficking of endosomal vesicles.

The trafficking of intracellular vesicles is essential for a number of cellular processes and defects in this process have been implicated in a wide range of human diseases. We identify the zebrafish mutant lbk as a novel model for such disorders. lbk displays hypopigmentation of skin melanocytes and the retinal pigment epithelium (RPE), an absence of iridophore reflections, defects in internal organs (liver, intestine) as well as functional defects in vision and the innate immune system (macrophages). Positional cloning, an allele screen, rescue experiments and morpholino knock-down reveal a mutation in the zebrafish orthologue of the vam6/vps39 gene. Vam6p is part of the HOPS complex, which is essential for vesicle tethering and fusion. Affected cells in the lbk RPE, liver, intestine and macrophages display increased numbers and enlarged intracellular vesicles. Physiological and behavioural analyses reveal severe defects in visual ability in lbk mutants. The present study provides the first phenotypic description of a lack of vam6 gene function in a multicellular organism. lbk shares many of the characteristics of human diseases and suggests a novel disease gene for pathologies associated with defective vesicle transport, including the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, the Hermansky-Pudlak syndrome, the Chediak-Higashi syndrome and the Griscelli syndrome.

Evidence for RPE65-independent vision in the cone-dominated zebrafish retina.

An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11-cis-retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an all-trans to 11-cis-retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11-cis-retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all-trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11-cis-retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11-cis-retinal for cone vision.

Impaired retinal differentiation and maintenance in zebrafish laminin mutants.

PURPOSE: To characterize morphologic and physiological alterations in the retina of three laminin mutant zebrafish, bashful (bal, lama1), grumpy (gup, lamb1), and sleepy (sly, lamc1), which were identified in forward genetic screens and were found to be impaired in visual functions. METHODS: Mutant larvae were observed for defects in visual behavior by testing their optokinetic response (OKR). In addition, electroretinograms (ERG) were measured and retinal morphology was examined by standard histology, immunocytochemistry, TUNEL assay, and electron microscopy. RESULTS: Both, gup and sly showed no OKR at any light intensity tested, whereas bal embryos showed some remaining OKR behavior at more than 40% of contrast. Consistent with the OKR result, gup and sly did not show an ERG response at any light intensity tested, whereas bal mutants exhibited small a- and b-waves at high light intensities. All three laminin mutants showed altered ganglion cell layers, optic nerve fasciculations, and lens defects. Again, bal showed the least severe morphologic phenotype with no additional defects. In contrast, both, gup and sly, showed severe photoreceptor outer segment shortening and synapse alteration (floating ribbons) as well as increased cell death. CONCLUSIONS: Lamb1 and lamc1 chains play an important role in the morphogenesis of photoreceptors and their synapses. In contrast, lama1 is not involved in outer retina development.

A rat model of Parkinsonism shows depletion of dopamine in the retina.

The retinal dopamine (DA) deficiency is an important feature of the pathogenesis in Parkinson's disease (PD) visual dysfunction. Systemic inhibition of complex I (rotenone) in rats has been proposed as a model of PD. In this study, we investigated whether systemic inhibition of complex I can induce impairment of DA-ergic cells in the retina, similar to the destruction of retinal cells found in PD patients. Rotenone (2.5mg/kg i.p., daily) was administered over 60 days. Neurochemically, rotenone treated rats showed a depletion of DA in the striatum and substantia nigra (SN). In addition, the number of retinal DA-ergic amacrine cells was significantly reduced in the rotenone treated animals. This study is the first one giving highlight towards a deeper understanding of systemic complex I inhibition (rotenone as an environmental toxin) and the connection between both, DA-ergic degeneration in the nigrostriatal pathway, and in the DA-ergic amacrine cells of the retina.

Nyctalopin is essential for synaptic transmission in the cone dominated zebrafish retina.

The first synapse in the vertebrate visual system is the photoreceptor synapse between rod and cone photoreceptors and the second-order bipolar cells. Although mutations in the nyctalopin gene (NYX) in humans lead to congenital stationary night blindness (CSNB1), affecting synaptic transmission between both types of photoreceptors and ON-bipolar cells, the function of nyctalopin in cone-dominant animal models has not been studied. Because the larval zebrafish retina is cone-dominant, we isolated the zebrafish nyx ortholog and raised a polyclonal antibody against the protein. Nyctalopin is expressed postsynaptically in both synaptic layers of the retina. Functional disruption via morpholino antisense injection leads to characteristic defects in the electroretinogram and defects in visual contrast sensitivity. We therefore demonstrated that nyctalopin plays a similar role in retinal synapse function in the cone pathway as in the rod pathway, thereby creating a genetic model for CSNB1 and its effects on cone vision.

The Zebrafish fade out mutant: a novel genetic model for Hermansky-Pudlak syndrome.

PURPOSE: To characterize retinal morphology and visual system function in the zebrafish mutant fade out (fad) and to establish the mutant as a lower vertebrate model for Hermansky-Pudlak syndrome (HPS). METHODS: Retinal morphology of fad larvae was examined between 3 and 9 days postfertilization (dpf) by standard histology, transmission electron microscopy, and immunohistochemistry examination. Apoptotic cells were visualized by TdT-mediated dUTP nick-end labeling (TUNEL) staining. Visual system function was probed by electroretinography and behavioral assessment by optokinetic response measurements. Blood clotting was evaluated by time to occlusion testing of blood vessels as an arterial thrombosis assay. The chromosomal location of fad was determined by simple sequence-length polymorphism mapping. Genomic fragments of candidate genes were cloned by standard molecular techniques and mapped to the zebrafish genome by radiation hybrid mapping. RESULTS: Mutant fad larvae are hypopigmented and show structural defects in the outer retina. Melanosomes of these larvae in the retinal pigment epithelium are hypopigmented, generally smaller, and progressively reduced in number compared to nonmutant larvae. Progressive microvilli protrusions into the photoreceptor cell layer are not detectable, and photoreceptor outer segments get shorter and are misaligned. Photoreceptors subsequently undergo apoptosis, with a peak of cell death at 6 dpf. Electrical responses of the retina and visual performance are severely reduced. Blood clotting is prolonged in mutant fad larvae. Genomic mapping of fad reveals distinct genomic positions of the mutant gene from known human HPS genes. CONCLUSIONS: The fad mutant shows syndromic defects in pigmentation, outer retinal structure and function, and blood clotting. This syndrome is characteristic of Hermansky-Pudlak syndrome (HPS), making fad a novel genetic model of HPS. The gene does not cosegregate with the known human HPS genes, suggesting a novel molecular cause of HPS.

Time course and development of light adaptation processes in the outer zebrafish retina.

Retinomotor movements are morphological changes in the outer retina in response to changing light conditions. They can be separated into two components: Migration of pigment granules within the microvilli of the retinal pigment epithelium (RPE) and positional changes in photoreceptor cells. These positional changes optimize exposure of the cone and rod photoreceptors to light. The aim of this study was to analyze both the time course of retinomotor movements in the adult zebrafish and the maturation of these processes in the developing fish. We show that retinomotor movements are used as a dark/light adaptation mechanism in zebrafish. In adult zebrafish, melanin granules of the RPE migrate with constant speed and reach the fully light adapted (LA) state approximately after 1 h. In contrast, about two thirds of double cone outer segment movements are finished in 5 min, and are fully completed in 10 to 20 min. During development there are three crucial stages leading to mature retinomotor movements in response to light: at 5 dpf (days post fertilization) the migration of pigment granules begins, at 20 dpf the pigment granules condense in the apical part of the RPE microvilli, and at 28 dpf, concomitant with the functional maturation of rods, the double cones contract as in adult retinas.

Knockdown of cone-specific kinase GRK7 in larval zebrafish leads to impaired cone response recovery and delayed dark adaptation.

Phosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of grk7 in zebrafish and knocked down gene expression of grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.

Photoreceptor morphology is severely affected in the beta,beta-carotene-15,15'-oxygenase (bcox) zebrafish morphant.

The retinoic acid molecule, a vitamin A derivative, is of key importance for eye and photoreceptor development in vertebrates. Several studies have provided evidence that the ventral part of the retina is particularly susceptible to impairment in retinoid signalling during the period of its development. In zebrafish, targeted gene knockdown of beta,beta-carotene-15,15'-oxygenase (bcox), the key enzyme for vitamin A formation, provokes a loss of retinoid signalling during early eye development that results in microphthalmia at larval stages. Using this model, we analysed the consequences of this for the retinal morphology of the fish larvae in structural details. Our analyses revealed that rods and cones do not express photoreceptor specific proteins (rhodopsin, peanut agglutinin, zpr1) in the peripheral retina. The photoreceptors in the central retina showed shortened outer segments, and electron dense debris in their intermembranal space. The number of phagosomes was increased, and cell death was frequently observed in the outer nuclear layer. Furthermore, the number of Muller cells was significantly reduced in the inner nuclear layer. Thus, we found that the lack of retinoid signalling strongly effects photoreceptor development in the ventral and dorsal retina. In addition, shortened outer segments and cell death of the remaining photoreceptors in the central retina indicate that there is an ongoing need for retinoid signalling for photoreceptor integrity and survival at later developmental stages.