Supplementary Figure 4: Grainy head–mutant phenotypes in eye–antennal discs.

a, Cross scheme to generate grh^IM mutant clones in eye–antennal discs from wandering third-instar larvae. b,b′, Confocal images of eye–antennal discs with grh^IM mutant clones. Wild-type cells are marked with GFP and have Grh protein in their nuclei (red, b′), while the grh-mutant clones do not have GFP or detectable Grh protein (reproducible results for four discs each). Scale bars, 100 μm. c,d, Confocal images of eye–antennal discs; nuclei are stained with DAPI (blue) and wild-type cells are stained with GFP (green) and Dcp1, a marker of apoptosis (red, or white in the bottom panels) (reproducible results for three discs each). c,c′, Control with genotype ey^3.5-flp/ey^3.5-flp; Ubi-GFP/CyO. A regular but sparse pattern of apoptotic cells is observed across the disc (white, c′). d, Disc with Grh-mutant clones, with genotype ey^3.5-flp/ey^3.5-flp; FRT42 grh^IM/FRT42 Ubi-GFP. The non-GFP clones are homozygous mutant for grh (grh^IM). Loss of Grh in these clones is associated with increased levels of apoptotic cells, as visualized by Dcp1 expression (d′). e, Cross schemes for the control and largely grh^IM mutant eye–antennal discs that were used for ATAC–seq. The control discs have ubiquitous expression of Grh protein (red + blue) across the disc; in the grh-mutant disc, most cells do not have Grh protein (red) anymore (blue only) (reproducible results for five discs). f, Photographs of pupae originating from the grh^IM/cell-lethal cross; the top two pupae are controls, which appear normal and were alive or enclosed (n = 111). Pupae from animals with the grh^IM mutant discs are shown on the bottom row (zoomed-in view in f′). These grh-mutant animals have lethal defects during pupation. From the initial cross, one-third of animals are expected to be homozygous mutant for Grh; out of 203 pupae, 71 were dead and 132 were alive.