Journal: Development (Cambridge, England)

Article Title: Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation

doi: 10.1242/dev.202775

Figure Lengend Snippet: FRAP demonstrates DL-LEXY rapidly transits in and out of the nucleus, even under blue light illumination. (A) Stills of an embryo that underwent FRAP in the dark (Dark), after bleaching (Dark, ROI Bleach) and after recovery (Dark, Recover). Images were false-colored using a rainbow colormap, and a Gaussian blur (r=4) was performed for display. The black circular ROI marks the region that was bleached, and the black square represents the area of the magnified view in B. (B) The same as in panel A except a magnified view. (C) Plots of the mean fluorescent intensity for the nucleus at the center of the black circular ROI. The different line and marker colors represent FRAP performed on three different embryos. The green shaded region marks time points when the DL-mCh-LEXY signal was bleached (∼1 min). The recovery after bleaching was fit to the displayed equation and estimates of β, which relates to the inverse of the recovery time, are displayed for each of three embryos. Estimates of I ss , which relates to the final steady state fluorescence levels, are 8628 a.u., 8337 a.u. and 8537 a.u. Estimates of α, which relates to the difference between the final steady state fluorescence levels and the fluorescence levels at the beginning of the recovery, are 7807 a.u., 7567 a.u., and 7238 a.u. (D) A similar experimental setup to A with different embryos, where only the first panel is in the dark and all other panels are with blue light applied. (E) The same as in panel D except a magnified view. (F) The same as C, except for embryos that underwent bleaching while blue light was applied. β estimates for each of three embryos are displayed. Estimates of I ss are 2781 a.u., 2702 a.u. and 2727 a.u. Estimates of α are 1622 a.u., 1574 a.u. and 1639 a.u. The mean β is significantly different between the dark and the light ( P =0.003, two-sample unpaired t -test). Ventral views of embryos are shown with the field of view positioned in the center of the trunk around the ventral-most point. t =0 indicates the start of imaging, at mid-nc14, as determined by the cellularization front having progressed to 50% of nuclei length.

Article Snippet: To quantify the gene expression patterns, first the ring of nuclei was segmented by using the MATLAB edge function and a Laplacian of Gaussian with a standard deviation of 20 for the filter on the DAPI or histone channel.

Techniques: Marker, Fluorescence, Imaging