We limited our study to CaP motor neurons within a 4-somite window around the cloaca in order to avoid morphological and functional variability that arise between cell types and along the rostro-caudal developmental wave.
Imaging was performed on a Roper confocal spinning disk head mounted on a Zeiss upright microscope, and acquisitions were done with a CoolSNAP HQ2 CDD camera (Photometrics, USA) through the MetaMorph software (Molecular Devices, USA). Embryos were anesthetized using 0.02% tricaine (MS-222, Sigma) diluted in egg water and embedded in 1% low melting-point agarose in a glass-bottom cell tissue culture dish (Fluorodish, World Precision Instruments, USA). Acquisitions were done using water immersion long working distance lenses, at 40x magnification (W DIC PL APO VIS-IR; 421,462–9900) for z-stack images of the whole tectum and at 63x magnification (W PL APO VIS-IR (421480–9900) for single plane time-lapse imaging of linear axonal segments, and for filopodia imaging. Acquisitions were done using the MetaMorph software (Molecular Devices) and resolution in z was set at 1um for stacks. Images were assembled and analyzed in ImageJ (NIH). 6dpf z- stacks taken in two frames were stitched together using the pairwise stitching function of the Stitching plugin [46 (link)].
Imaging was performed on a Roper confocal spinning disk head mounted on a Zeiss upright microscope, and acquisitions were done with a CoolSNAP HQ2 CDD camera (Photometrics, USA) through the MetaMorph software (Molecular Devices, USA). Embryos were anesthetized using 0.02% tricaine (MS-222, Sigma) diluted in egg water and embedded in 1% low melting-point agarose in a glass-bottom cell tissue culture dish (Fluorodish, World Precision Instruments, USA). Acquisitions were done using water immersion long working distance lenses, at 40x magnification (W DIC PL APO VIS-IR; 421,462–9900) for z-stack images of the whole tectum and at 63x magnification (W PL APO VIS-IR (421480–9900) for single plane time-lapse imaging of linear axonal segments, and for filopodia imaging. Acquisitions were done using the MetaMorph software (Molecular Devices) and resolution in z was set at 1um for stacks. Images were assembled and analyzed in ImageJ (NIH). 6dpf z- stacks taken in two frames were stitched together using the pairwise stitching function of the Stitching plugin [46 (link)].
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