Published: March 31st, 2013
Cilia-generated fluid flow in Kupffer’s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.
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.
Overview of Stage-Specific Zebrafish Embryo Injections
Antisense morpholino oligonucleotides (MO), which bind to a targeted mRNA and disrupt protein expression from that transcript, are widely used in gene knockdown (loss-of-function) studies in zebrafish13,14. Gene Tools, LLC offers MOs that are tagged with either carboxyfluorescein (emits green fluorescence) or lissamine (emits red fluorescence) to detect MO in injected embryos using fluorescent microscopy. By injecting MO into the y.......
Stage-specific MO injections provide a useful approach to analyze gene function in specific compartments of the embryo. Figure 1 presents a flow chart of the injection strategies used to test gene function in DFC/KV cells and how to introduce fluorescent beads to visualize fluid flow in KV. The distribution of fluorescent MO in successful stage-specific injected embryos is shown schematically in Figures 1D-F and in live embryos in Figure 2. An unsuccessful MO injection, .......
Using stage-specific injections to target MO to the DFC/KV cell lineage is a useful approach to study cell-autonomy of gene function and avoid pleiotropic phenotypes caused by global gene knockdown. However, these injections can be technically challenging. Injection of MO between the 256-cell and 1,000-cell stages can result in three possible outcomes: 1) the MO remains aggregated at the injection site, 2) the MO diffuses throughout the yolk and enters DFC/KV cells or 3) the MO diffuses throughout the yolk and enters DFC.......
We thank Fiona Foley for excellent lab support and zebrafish care. This work was supported an AHA predoctoral fellowship to G.W. (11PRE5730027) and NHLBI grants to H.J.Y. (R01HL66292) and J.D.A. (R01HL095690).....
|Name of Reagent/Material
|Standard Control oligo-Lissamine tagged
|Gene Tools, LLC
|Custom Rock2b morpholino oligo
|Gene Tools, LLC
|Fluoresbrite Multifluorescent 0.5 micron Microspheres
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