Analysis of signaling pathways in zebrafish development by microinjection

Abstract

The zebrafish oocyte differs substantially from the zygote and cleavage-stage embryo with regard to the ease with which it can be microinjected with proteins or reagents that modify subsequent development. The objective of this chapter is to describe methods developed in this and other laboratories for microinjection and calcium imaging in the unfertilized zebrafish egg. Methods of immobilizing the oocyte include a holding chamber and a holding pipette. The holding chamber allows imaging of three or four oocytes simultaneously, while the holding pipette facilitates imaging of localized regions in the oocyte. Injection of calcium green dextran via holding chambers allowed detection of global changes in Ca2+ release following fertilization and development through early blastula stages. Injection and imaging with the holding pipette method allowed discrimination of calcium changes in the egg cortex from that in the central regions of the cell. The results demonstrate the highly localized nature of calcium signaling in the zebrafish zygote and the implications of this signaling for embryonic development.

Fig. 6.1

Design of an oocyte holding chamber. The row of holes approximately 800 μm in diameter were drilled along one edge of the plastic and openings were cut with a scalpel to admit the injection pipette (A). The cover was cut from a plastic coverslip as were guides to hold the cover on the insert but still allow it to move (B). Panel (C) shows the cover slid over the oocyte chambers to prevent their escape upon addition of aquarium water to initiate fertilization.

Fig. 6.2

Holding pipette used to immobilize a zebrafish oocyte. A typical holding pipette produced by flame-polishing of a glass capillary is used to pick up and immobilize an unfertilized oocyte. Magnification is indicated by the bar which represents 100 μm.

Fig. 6.3

Fertilization-induced changes in calcium green fluorescence. Zebrafish oocytes were injected with calcium green dextran and an inactive protein (glutathione-S-transferase) then imaged by confocal fluorescence microscopy before fertilization (left), or at 60 s post-insemination (right). The increase in calcium green is most apparent in the egg cortex at 60 s post-insemination. Magnification is indicated by the bar which represents 100 μm.

Fig. 6.4

Quantitation of fertilization-induced changes in calcium green fluorescence. Oocytes were injected with calcium green dextran and GST, then images were recorded after a 10-min recovery period. Fertilization was initiated by addition of a mixture of sperm and water at time 0 and images were recorded every 15 s. Fluorescence was quantitated by pixel intensity quantitation using Metamorph 6.1 software. The top panel represents global fluorescence measured from a cross-section of the entire zygote. The middle panel represent fluorescence measured within a circular region in the center of the zygote (central cytoplasm) comprising approximately 25% of the total cross-sectional area. The bottom panel represents fluorescence measured within an arc traced over the cortex (cortical cytoplasm) as near as possible to the micropyle. This arc comprised approximately 20% of the perimeter of the zygote.

Fig. 6.5

Calcium green fluorescence in the region of the micropyle. An unfertilized oocyte was picked up with a holding pipette and rotated until the micropyle was visible. The egg was then injected with calcium green dextran and fertilized as above. The images shown here were obtained at 50 s post-insemination and demonstrate the localized calcium green fluorescence in the region of the micropyle (arrows). Magnification is indicated by the bar which represents 100 μm.