Computational protocol: Targeted Pth4-expressing cell ablation impairs skeletal mineralization in zebrafish

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Protocol publication

[…] Embryos were mounted, imaged, and cells ablated as previously described []. Pth4:eGFP; Bactin2:mCherry zebrafish were visualized throughout development as described [] and the number of eGFP-expressing cells in the lateral hypothalamus was scored.For cell ablation experiments, Pth4:eGFP and Bactin2:mCherry adult fish were crossed and double positive embryos were selected for experiments. Pth4:eGFP+/Bactin2:mCherry+ embryos (n = 26 per experimental group) were mounted into the mold ventral side down to allow ablation of two subsets of neural bodies. Using confocal imaging, eGFP-expressing Pth4 cells were detected and specifically selected for ablation based on red fluorescence from the ubiquitous nuclear marker. Targeted laser ablation was done with a two-photon laser at 770 nm for 10 cycles at 65% power on a Zeiss LSM 710 inverted confocal microscope with an LD C-Apochromat 40x/1.1 W Corr objective and Zen 2009 software’s ‘Regions’ tool for cell selection. Each step of the ablation process (before ablation, during ablation of selected cells, and after ablation) was captured as a single plane image, and full z-stacks were captured at the beginning and immediately at the end for each experimental embryo. Imaged embryos were analyzed to determine degree of ablation and overall health of both targeted cells and surrounding untargeted cells.Two rounds of ablations were performed per experimental embryo at different time points (1 and 2 dpf). This protocol was chosen as the most efficient of several attempted iterations for a complete ablation process at each time point and to achieve low neuronal recovery during development. Afterwards, ablated and control larvae were captured as full z-stack images at 3 and 7 dpf to monitor recovery of the Pth4-expressing neurons. Images collected were analyzed using Imaris software (Bitplane) and exported as TIFF files. [...] Student´s t-test was used when two samples were compared and data is presented as mean ± SEM, with significance inferred from p values of 0.05 or less. Statistical analysis and figures were performed with PASW Statistics 18.0 and SigmaPlot 12.0, respectively. […]

Pipeline specifications

Software tools Imaris, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy
Organisms Danio rerio
Chemicals Phosphates