Fig. 1

Zelda-binding sites provide quantitative temporal control of gene expression. a Schematic representation of the truncated version of the sna shadow enhancer (snaE) and the variants carrying different numbers and locations of extra Zelda (Zld)-binding sites (black stars represent canonical Zld-binding sites: CAGGTAG). Each enhancer controls a common minimal promoter (blue box, sna promoter) that transcribes a yellow gene with 24 MS2 repeats located at its 5’ end. b–g Transgenic embryos stained with a MS2 probe (red, panels b–g) and with a snail probe (green, b, d, f). Dorsal views at nc14, anterior to the left, scale bars represent 30μm. h–m Workflow for automatic tracking of single nucleus transcriptional activity. h, i Snapshot of a maximum intensity projected Z-stack from live imaging movie representing nuclei labeled with His2Av-mRFP transgene (h) and associated automatic segmentation, where each tracked nucleus is given a random color (i). j Maximum intensity projected Z-stack image extracted from a typical movie showing the transcriptional foci (white dot). k False-colored image, representing the segmented transcriptionally inactive nuclei (blue), active nuclei (red) and associated transcriptional foci (yellow dot). l Maximum intensity projected Z-stack image representing a typical gastrulation, used as a landmark to determine dorso-ventral (D/V) coordinates (yellow line). m Representative image exhibiting the spatial domain (grey, here 25μm surrounding the ventral furrow) defined by precise D/V coordinates. n–q Live imaging of transcriptional activity driven by the snaE upon increasing numbers of Zld-binding sites. Nuclei that displayed transcriptional activity were false-colored in yellow. Snapshots from maximum intensity projected Z-stack of live imaging movies are shown from nc11 to nc14. Scale bars represent 10μm. r Temporal coordination profiles during nc14. Synchrony curves were obtained by quantifying transcriptional activation during nc14 in a region of 50μm around the ventral furrow: snaE (6 movies, n = 970 nuclei) (grey curve), snaE + 1Zld5’ (4 movies, n = 700 nuclei) (light blue curve), snaE + 2Zld (5 movies, n = 723 nuclei) (dark blue curve), snaE + 3Zld (4 movies, n = 824 nuclei (purple curve)) and snaE-shadow (4 movies, n = 612) (yellow curve). Dashed line represents 50% of activation. The time origin is the end of mitosis (start of nc14). Error bars represent SEM