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

Управління трудовим потенціалом при створенні інноваційної продукції

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

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

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

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. (Invest Ophthalmol Vis Sci. 2006;47:4523-4531

Nuclear crowding and nonlinear diffusion during interkinetic nuclear migration in the zebrafish retina

Funder: Cambridge Commonwealth, European and International Trust; FundRef: http://dx.doi.org/10.13039/501100003343Funder: Natural Sciences and Engineering Research Council of Canada; FundRef: http://dx.doi.org/10.13039/501100000038Funder: Cambridge Philosophical Society; FundRef: http://dx.doi.org/10.13039/100013858An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant

Zebrafish dou yan mutation causes patterning defects and extensive cell death in the retina

The size of an organ is largely determined by the number of cells it contains, which in turn is regulated by two opposing processes, cell proliferation and cell death, however, it is generally not clear how cell proliferation and cell death are coordinated during development. Here, we characterize the zebrafish dou yan mi234 mutation that results in a dramatic reduction of retinal size and a disruption of retinal differentiation and lamination. The retinal size reduction is caused by increased retinal cell death in a non–cell-autonomous manner during early development. The phenotypic defect in dou yan mi234 arises coincident with the onset of retinal neurogenesis and differentiation. Interestingly, unlike many other small eye mutations, the mutation does not increase the level of cell death in the brain, suggesting that the brain and retina use different mechanisms to maintain cell survival. Identification and further study of the dou yan gene will enhance our understanding of the molecular mechanisms regulating retinal cellular homeostasis, i.e., the balance between cell proliferation and cell death. Developmental Dynamics 236:1295–1306, 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56024/1/21148_ftp.pd

Ontogeny of cone photoreceptor mosaics in zebrafish

Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a “cone mosaic.” Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish ( Danio rerio ) have four cone subtypes arranged into precise rows in the adult retina. Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish expressing green fluorescent protein (GFP) in their UV-sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a “mosaic metric” statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array; i.e., the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birth dating to show that the larval cone mosaic pattern remains as a distinct region within the adult retina and does not reorganize into the adult row pattern. In addition, the abundance of cone subtypes relative to other subtypes is different in this larval remnant compared with that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species. J. Comp. Neurol. 518:4182–4195, 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77982/1/22447_ftp.pd

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