Forward Genetic Analysis of Visual Behavior in Zebrafish

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders

Characterizing OXPHOS inhibitor-mediated alleviation of hypoxia using high-throughput live cell-imaging.

Acknowledgements: The authors thank the Radboudumc Technology Center Microscopy for use of their microscopy facilities.BACKGROUND: Hypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell's mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia. METHODS: B16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system. RESULTS: Hypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors. CONCLUSIONS: We successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy

Targeting neural circuitry in zebrafish using GAL4 enhancer trapping

We present a pilot enhancer trap screen using GAL4 to drive expression of upstream activator sequence (UAS)-linked transgenes in expression patterns dictated by endogenous enhancers in zebrafish. The patterns presented include expression in small subsets of neurons throughout the larval brain, which in some cases persist into adult. Through targeted photoconversion of UAS-driven Kaede and variegated expression of UAS-driven GFP in single cells, we begin to characterize the cellular components of labeled circuits

The <i>darl</i> Mutant Shows Retinotectal Mapping Deficits

<p>(A and B) The nasal-dorsal quadrant of the retina was labeled with DiO (green), and the temporal-ventral quadrant was labeled with DiD (magenta). In <i>darl^s327,</i> the ventral branch of the optic tract is missing (arrow). Scale bar is 100 μm.</p> <p>(C and D) Dorsal view of the tectum in the same larvae as in A and B. The ventral half of the <i>darl^s327</i> tectum is not innervated by the dorsal-nasal RGC axons. Anterior is to the left and ventral is to the bottom. Tectal neuropil is demarcated by the dotted line, based on DAPI counterstaining (blue). Scale bar is 50 μm.</p

Example of a Mutant with Abnormal Morphology of Cone Photoreceptors

<p>Photoreceptors in a retinal section stained with DAPI (A and B) and a marker for double cones, zpr1 (C and D) at 7 dpf in WT larva (A, C, and E) and <i>yoi^s121</i> mutant retina (B, D, and F). Merged images of DAPI (in green) and zpr1 (in magenta) are also shown (E and F). Both zpr1-positive and zpr1-negative cone photoreceptors in the mutant are “stumpy” when compared to those in the control retina (arrows). B, bipolar cells; C, cone photoreceptor cells; H, horizontal cells; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar is 10 μm.</p

Example of a Mutant with a Potential Defect in Light Adaptation

<p>OKR is plotted at several time points before and after dark treatment for 45 min. WT sibling larvae (<i>n</i> = 6) recover quickly from the dark pulse, while <i>nki^s136</i> mutants (<i>n</i> = 6) show reduced responsiveness for several minutes after return to the light. Average number of saccades to a constant motion stimulus is shown for each time point. Error bars indicate standard deviation.</p