Анализ функций генов и визуализации реснички, созданного потока жидкости в Vesicle Купфера
Summary
Реснички генерируемых потоков жидкости в Vesicle Купфера (KV) управляет левой-правой паттерна рыбок данио эмбрионов. Здесь мы описываем технику, чтобы модулировать функции гена в частности в КВ клеток. Кроме того, мы показываем, как доставить флуоресцентные шарики в KV для визуализации потока жидкости.
Abstract
Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These ‘LR cilia’ generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-β superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer’s vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs—in combination with optical transparency and rapid development of the zebrafish embryo—make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.
Protocol
Representative Results
Discussion
Использование стадии конкретных инъекций целевой MO к DFC / KV клеточной линии является полезным подходом к изучению клеточных автономии функции гена и избежать плейотропным фенотипа, вызванные глобальным нокдаун гена. Тем не менее, эти инъекции могут быть технически сложным. Инъекции MO ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Мы благодарим Фионы Фоли за отличную поддержку лаборатории и рыбок данио помощи. Эта работа была поддержана AHA predoctoral стипендии для GW (11PRE5730027) и NHLBI гранты HJY (R01HL66292) и JDA (R01HL095690).
Materials
| Name of Reagent/Material | Company | Catalogue Number |
| Standard Control oligo-Lissamine tagged | Gene Tools, LLC | |
| Custom Rock2b morpholino oligo | Gene Tools, LLC | |
| Fluoresbrite Multifluorescent 0.5 micron Microspheres | Polysciences, Inc. | 24054 |
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