Figure 4.
Figure 4. Filopodia can be detected and quantified in sprouting endothelia in the developing zebrafish embryo using intravital imaging and FiloQuant. (A) Simplified cartoon of a zebrafish embryo. Insets represent magnified regions of interest (ROIs). In particular, endothelial tip cells in the sprouting intersegmental arteries are highlighted to indicate the region being imaged in 29 h postfertilization (hpf) transgenic embryos expressing GFP in the endothelium (Tg(fli1:EGFP)y1; roy−/−; mitfa−/−). (B and C) Transgenic zebrafish embryos (Tg(fli1:EGFP)y1; roy−/−; mitfa−/−) were treated with 1% DMSO or150 ng/ml latrunculin B from 25 hpf to 29 hpf. The embryos were then anesthetized and mounted in low-melting point agarose on glass-bottom dishes. Z stack images of the sprouting segmental arteries were obtained live using an SDC microscope (long working distance 63× water objective, CMOS camera). Representative maximal z projection and the z projection used for FiloQuant analyses are shown (B). In addition, ROIs (yellow square) used for FiloQuant analyses, magnified area (red squares), and filopodia detected using FiloQuant (magenta) are displayed. Bars: (main) 20 µm; (inset) 5 µm. Quantification of filopodia density and filopodia length using the semiautomated version of FiloQuant are displayed as Tukey box plots; C; DMSO, 15 embryos imaged; latrunculin B, 17 embryos imaged; filopodia density: DMSO, 60 ROIs analyzed; latrunculin B, 71 ROIs analyzed; filopodia length: DMSO, 2,232 filopodia measured; latrunculin B, 138 filopodia measured; ***, P < 5.49 × 10−25). The Tukey box plots represent the median and the 25th and 75th percentiles (interquartile range); points are displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t test (unpaired, two tailed, unequal variance). (D) FiloQuant (semiautomated; software 2) readouts of filopodia number were compared with manual analyses from a total of 54 images of sprouting endothelial tip cells from DMSO-treated embryos (related to Fig. 4, A–C). (E) FiloQuant readouts of filopodia length were compared with manual analyses in one image of a sprouting endothelial tip cell from a DMSO-treated embryo. Bars: (main) 20 µm; (inset) 5 µm. Red and yellow insets denote magnified areas and magenta highlights the filopodial protrusions detected by FiloQuant. Blue arrows point to examples of filopodia that were accurately measured using FiloQuant. Red arrows highlight filopodia that were assigned a shorter length by FiloQuant compared with manual analyses. Correlation between FiloQuant and manual analyses are displayed on the right where the blue and red arrows represent the same filopodia indicated in the corresponding images (75 filopodia measured).

Filopodia can be detected and quantified in sprouting endothelia in the developing zebrafish embryo using intravital imaging and FiloQuant. (A) Simplified cartoon of a zebrafish embryo. Insets represent magnified regions of interest (ROIs). In particular, endothelial tip cells in the sprouting intersegmental arteries are highlighted to indicate the region being imaged in 29 h postfertilization (hpf) transgenic embryos expressing GFP in the endothelium (Tg(fli1:EGFP)y1; roy−/−; mitfa−/−). (B and C) Transgenic zebrafish embryos (Tg(fli1:EGFP)y1; roy−/−; mitfa−/−) were treated with 1% DMSO or150 ng/ml latrunculin B from 25 hpf to 29 hpf. The embryos were then anesthetized and mounted in low-melting point agarose on glass-bottom dishes. Z stack images of the sprouting segmental arteries were obtained live using an SDC microscope (long working distance 63× water objective, CMOS camera). Representative maximal z projection and the z projection used for FiloQuant analyses are shown (B). In addition, ROIs (yellow square) used for FiloQuant analyses, magnified area (red squares), and filopodia detected using FiloQuant (magenta) are displayed. Bars: (main) 20 µm; (inset) 5 µm. Quantification of filopodia density and filopodia length using the semiautomated version of FiloQuant are displayed as Tukey box plots; C; DMSO, 15 embryos imaged; latrunculin B, 17 embryos imaged; filopodia density: DMSO, 60 ROIs analyzed; latrunculin B, 71 ROIs analyzed; filopodia length: DMSO, 2,232 filopodia measured; latrunculin B, 138 filopodia measured; ***, P < 5.49 × 10−25). The Tukey box plots represent the median and the 25th and 75th percentiles (interquartile range); points are displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t test (unpaired, two tailed, unequal variance). (D) FiloQuant (semiautomated; software 2) readouts of filopodia number were compared with manual analyses from a total of 54 images of sprouting endothelial tip cells from DMSO-treated embryos (related to Fig. 4, A–C). (E) FiloQuant readouts of filopodia length were compared with manual analyses in one image of a sprouting endothelial tip cell from a DMSO-treated embryo. Bars: (main) 20 µm; (inset) 5 µm. Red and yellow insets denote magnified areas and magenta highlights the filopodial protrusions detected by FiloQuant. Blue arrows point to examples of filopodia that were accurately measured using FiloQuant. Red arrows highlight filopodia that were assigned a shorter length by FiloQuant compared with manual analyses. Correlation between FiloQuant and manual analyses are displayed on the right where the blue and red arrows represent the same filopodia indicated in the corresponding images (75 filopodia measured).

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