HuC–eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones
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The field of axon guidance is taking advantage of the powerful genetic and imaging tools that are now available to visualise growth behaviour in living cells, both in vivo and in real time. We have developed a method to visualise individual neurons within the living zebrafish embryo which provides exceptional cellular resolution of growth cones and their filopodia. We generated a DNA construct in which the HuC promoter drives expression of eGFP. Injection of the plasmid into single cell fertilised zebrafish egg resulted in mosaic expression of eGFP in neurons throughout the developing embryo. By manipulating the concentration of injected plasmid, it was possible to optimise the numbers of neurons that expressed the construct so that individual growth cones could be easily visualised. We then used time-lapse high magnification widefield epifluorescence microscopy to visualise the growth cones as they were exploring their environment. Growth cones both near the surface of the embryo as well as deep within the developing brain of embryos at 20 h post fertilisation were clearly imaged. With time-lapse sequence imaging with intervals between frames as frequent as 1 s there was minimal loss of fluorescence intensity and the dynamic nature of the growth cones became evident. This method therefore provides high magnification, high resolution time-lapse imaging of living neurons in vivo and by use of widefield epifluorescence rather than confocal it is a relatively inexpensive microscopy method.
KeywordsAxon Filopodia Embryo Time-lapse Growth cone Telencephalon
This work was supported by grants from the Australian N.H.M.R.C. to JStJ and BK and from the Australian Research Council to B.K.
Movie 1. Time-lapse movie of eGFP-expressing Rohon-Beard axons interacting with surrounding unlabelled cells. Select frames are shown in Fig. 2. Frames were captured every 5 s. Time is shown in minutes and seconds (MPG 2,442 kb)
Movie 2. Time-lapse movie of growth cones of eGFP-expressing Rohon-Beard axons. Select frames are shown in Fig. 3. Frames were captured every 10 s; time sequence misses 3 frames between 4:40 and 5:20 and 2 frames between 8:30 and 9:00 due to adjustments to the microscope being made during acquisition which prevented images being captured. Time is shown in minutes and seconds (MPG 2,607 kb)
Movie 3. High magnification real time movie of a growth cone from an eGFP-expressing Rohon-Beard neuron. Frames were captured every second and are displayed every second (real time). The movie shows a short extract from the entire sequence; select frames from the entire sequence are shown in Fig. 4. Time is shown in minutes and seconds (MPG 10,269 kb)
Movie 4. Real time dynamic movement of an eGFP-expressing growth cone within the telencephalon. Frames were captured every 2 s and are displayed every 2 s (real time). The movie shows a short extract from the entire sequence; select frames from the entire sequence are shown in Fig. 5. Time is shown in minutes and seconds (MPG 16,905 kb)
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