Computational protocol: Visualizing Late Insect Embryogenesis: Extraembryonic and Mesodermal Enhancer Trap Expression in the Beetle Tribolium castaneum

Similar protocols

Protocol publication

[…] Eggs were collected over a 4-hour interval; age is given in hours after egg lay (hAEL) for the lower end of the range (“minimum age”). For these analyses, images were acquired every ten minutes with the DeltaVision microscope at 30°C. The motorized stage enabled the simultaneous recording of all three lines and the nuclear-GFP line, which was used for staging calibration. Film 1 recorded younger eggs over a 20-hour interval (4.8–24.8 hAEL). Film 2 overlapped with Film 1 by 6.9 hours in recording older eggs for 46 hours (17.9–64.2 hAEL). Together, Films 1 and 2 spanned embryogenesis from the uniform blastoderm stage (4.8 hAEL, 7% development) until after dorsal closure (64.2 hAEL, 89%). Two separate films were recorded due to the logistics of using a shared imaging facility, and to ensure a sufficient sample size for the older stages (as embryos mounted at the blastoderm stage are somewhat fragile, not all of these are able to hatch after three days of constant filming with the conditions used here). For each line, sample sizes ranged from 7 to 11 embryos per film (specified in , ). Where data were available, embryos from all four transgenic lines (nGFP, G12424, KT650, G04609) were used for calculations of stage age (). For quantification of the EGFP expression time course, time-lapse images were analyzed in ImageJ software (NIH), where the integrated density was measured on maximum intensity projection images for polygonal regions of interest that fully encompassed a given egg. The “relative GFP signal” represents the integrated density divided by the area of the region of interest, where plotted values show the mean±1 standard deviation. In plotting the results from Films 1 and 2 on the same graph, the slight reduction in relative EGFP signal in Film 2 reflects a small reduction in exposure time during acquisition compared to Film 1, an optimization for preventing signal saturation. For tracking yolk globules (2), the ImageJ plugin MTrackJ was used . […]

Pipeline specifications

Software tools ImageJ, MTrackJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Tribolium castaneum