Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Abstract

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions 1 . Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish [2][3][4][5][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 organs 7 . 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 stages 8,9 . During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo 10,11 . 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 hpf 8 . 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 cells 12 . 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 videomicroscopy 2 . 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. Video LinkThe video component of this article can be found at http://www.jove.com/video/50038/ Protocol Overview of Stage-Specific Zebrafish Embryo InjectionsAntisense 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 zebrafish 13,14 . Gene Tools, LLC offers MOs that are tagged with either carboxyfluorescein (emits green fluorescence) or ...

Small heat shock proteins Hspb7 and Hspb12 regulate early steps of cardiac morphogenesis

“…Unlike most embryonic cells, the DFCs retain cytoplasmic bridges with the yolk cell. Therefore, it is possible to deregulate gene expression specifically in these KV progenitors by targeting injections into the yolk syncytial layer (YSL) at the 1000-cell (1K) stage (Wang et al, 2013). At this point the YSL remains a syncytium that is non-contiguous with blastomeres of the embryo proper.…”

Cilia in vertebrate left–right patterning

“…Understanding the dynamics of flows inside the fish LRO (KV) has been more challenging due to its more sphere-like shape. Anticlockwise rotational flow patterns have been described in KV by tracking fluorescent microbeads using manual [34] or automated [35] approaches or by tracking laser-induced cellular debris [36]. Observations of flow dynamics indicated strong right-to-left flow in anterior region of the LRO and slower return right to left flow at posterior pole [31,37,38].…”

Sept6 Is Required for Ciliogenesis in Kupffer's Vesicle, the Pronephros, and the Neural Tube during Early Embryonic Development

“…At the 256-cell stage, the bridge between yolk and DFCs/KV cells is open while other bridges between yolk and embryonic cells are closed, so that tMO can enter the progenitors of the DFCs through the cytoplasmic bridge without entering most other embryonic cells (Fig. 7A) (61). WISH analysis using the probe of sox17, which marks the DFCs and endoderm cells during the blastula stage, indicated that DFC migration and cohesion were not affected ( Fig.…”

“…KV is a ciliated, fluid-filled organ in zebrafish that plays a critical role in nodal signaling and LR development (38,60,61). Because septins are known to localize at the base of primary cilia and affect cilium-based signaling in other vertebrate cells (13,25), we reasoned that sept6 may affect LR asymmetry in zebrafish by regulating ciliogenesis in KV.…”

“…Dorsal forerunner cells (DFCs)/KV cells are known to play a central role in the development of LR asymmetry (37)(38)(39)61). To determine whether sept6 affects LR asymmetry through these cells or other mechanisms, we injected embryos with sept6-tMO at the 256-cell stage.…”