Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Canonical Wnt/ß-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with ß-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

Live Imaging of Intracranial Lymphatics in the Zebrafish.

RATIONALE: The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. OBJECTIVE: Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. METHODS AND RESULTS: Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. CONCLUSIONS: This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.

High resolution imaging of the cortex isolated from sea urchin eggs and embryos.

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.